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Vasudevan Biochemistry 8th Edition
Vasudevan Biochemistry 8th Edition
BIOCHEM STRY
for Me dic al Students
..
SECTION A
Biochemical
Perspective to Medicine
Chapter at a Glance
The learner will be able to answer questions on the following topics:
0 History of biochemistry 0 Hydrophobic interactions
0 Ionic bonds 0 Principles of thermodynamics
0 Hydrogen bonding 0 Donnan membrane equilibrium
0~G)
TABLE 1.2: Bacterial and mammalian cells
Prokaryotic cell Eukaryotif_ ceJL __
.,
Size Small Large; 1doo to 10,000 times
Cell wall
!
Rigid Membrane of lipid bilayer V Transf13r of
Nucleus Not defined Well defined electrons
•.
- •------..
l
Organelles Nil Several; including mitochondria
and ~ samP<
0 Atom
,0 Positive ion
Sharingof
electrons
Y is called digestion or primary metab~ . After absorp-
tion, the small mole~s are further broken down and
oxidized to carbon dioxide. In this process, NADH or
FADH2 are generated. This is named as secondary or
intermediary metabolism. Finally, these reducing
equivalents enter the electron trans1Port chain in the mi-
Molecule
tochondria, where they are oxidized to water; in this pro-
Fig. 1.1: ~
cess energy is trapped as ATP. This is termed tertiary
Substrate
{A) Two hydrophobic
molecules surrounded
by water molecules
\
I
Enzyme (
----
Fig. 1.3: Ionic bonds used in p: o~ teractions
{B) When two
hydrophobic molecules
come together,
surrounding water
molecules are minimal
@ Hydrogen Bonds
These are formed by sharing of a hydrogen between
two electron donors. Hydrogen bonds result from elec- g . ·.4: Hydrophobic interaction
F~
trostatic attraction between an electronegative a\om and 1. . h . . t t
. attr ct1ve forces between c emIca1 groups in con ac .
a hydrogen atom that is anded covalently to a secon~ This force will drastically reduce, when the distance
electrol ~ive atom. a hydrogen atom forms . .
u-\.-\ 1..-c h H
a "CQ\Tal t 1ond with on Iy one ot er atom. owever,
between atoms Is increased. Although very weak, van der
. . .
--,,- Waals forces colh~ct1vely contribute maximum towards
hydrogen atom coyale~tl)wndedto a donor atom, mly
';/:==~
- =~~~r~~~=-=~;f.=~~~=,=:::;::: the stability of proltein structure, especially in preserving
_...... form an o ti , the hydrogen bo d
1 the nonpolar interior structure of proteins.
with an acceptor atom. In biological systems, both donors
\ and acceptors are usually n~ gen or oxJ,ife n atf m~ .
especially those atoms in amino (NH 2) and ht oxyl
WATER: THE UNIVERSAL SOLVENT
(OH) groups. Water constitutes :about 70 to 80 percent of the weight of
With regard to protein chemistry, hydrogen rel asing most cells. The hydrogen atom in one water molecule is
groups are -NH (imidazole, in dole, peptide); -OH ~ erine, attracted to a pair oIfelectrons in the outer shell of an oxygen
threonine) and -NH2 (arginine, lysine). Hydrogen accep- atom in an adjacent molecule. The structure of liquid water
ting groups are COO-(aspartic, glutamic) C=O (peptide); contains hydrogen-bonded networks (Fig . 1.5).
and S- S (disulphide). The DNA structure js roaiotaioed water is a polar molecule. Molecules with polar bonds
the
by hydrogen bonding between
residues. J
purine a1pyrimidine that can easily fornn hydrogen bonds wit~~ ~~r and can
dissolve in water are termed "hJ'.drophJ!$"ift11as immense
© Hydrophobic Interactions
Nonpolar groups have a tendency to associate with
hydrogen bondin~, capacity both with other molecules
and also the adjacent water molecules. This contributes
to cohesiveness o1'water. Water favors hydrophobic inter-
each other in an aqueous environment; this is referred actions and provides a basis for metabolism of insoluble
to as hydrophobic interaction . These are formed by substances.
interactions between QOnpolar hydrophobic side chains
by eliminating water mo lecules. The force that causes PRINCIPLES OF
hydrophobic molecules or nonpolar portions of molecules
to aggregate together rather than to dissolve in water
THERMODYNAMICS
is called the [ hydrophobicboiio} (Fig.1.4). This serves Thermodynamics is concerned with the flow of heat and
to hold lipophilic side chains of amino acids together. it deals with the relationship between heat and work.
Thus nonpolar molecules will have minimum exposure Bloenergetics, o r biochemical thermodynamics, is the
to water molecules. study of the energy changes accompanying biochemical
reactions. Biologi1cal systems use chemical energy to
Van der Waals Forces ...L
0 (, drive processes within living cells.
These are very weak forces of attraction between all
atoms, due to oscillating dipoles, described by the Dutch First Law of Thermodynamics
physicist Johannes van der Waals (1837- 1923). He was The total energy of a system, including its surround-
awarded Nobel Prize in 1910. These are short range ings, remains constant. Or, t.E = Q - W, where Q is the
Chapter 1: Biochemical Perspiective to Medicine 7
kj.~,k'
denotes a portion of the tbtal energy change in a
system that is available for doing work.
y_., vi·i'rr
- Hydrogen bond
For most biochemical reactions ,. \is seen that t.H is
nearly equal to t.E. So,ft.G = t.E - T ~YHence, t.G or free
energy of a system depends on the change in internal
J "'"' '"
energy and change in entropy of a system.
A{:Ai;J;?
Standard Free Energy Change
It is the free energy change under standard conditions.
It is designated as t.G0 • The standard conditions are
defined for biochemical reactions at a pH of 7 and 1 M
Fig. 1.5: Water molecules hydrogen bonded conce~OAraAEkl#ferentiated-by-;!!t"~ ming sign t.G0 '.
·11 is d ~eA,1{ f latec!J'.ia,'ijie.,,equitlbr~yn__ponstant. Actual
heat absorbed by the system and W is the work done. free e ~ b aA9es dei,ene-o~ ~tant and product.
Thi s is also called the law of conservation of energy. Most of the reversible metabolic reactions are near-
If heat is transformed into work, there is proportionality equilibrium reactions and therefore their t.G is nearly zero.
between the work obtained and the heat dissipated. The net rate of near-equilibrium reactions are effectively
A system is an object or a quantity of matter, chosen for regulated by the relative concentr,ation of substrates
observation. All other parts of the universe, outside the and products. The metabolic reactions that function far
boundary of the system, are called the surrounding. from equilibrium are irreversible. The velocities of these
reactions are altered by changes in1 enzyme activity. A
Second Law of Ttiermodynamics highly exer!;lonic reaction is irreversible and goes to com-
The total entropy of a system must increase if a pletion. Such a reaction that is part of a metabolic path-
process is to occur spontaneously. A reaction occurs way, confe f direction to the pathway and makes the entire
spontaneously if t.E is negative, or if the entropy of the pathwa irreversible
system increases. Entropy (S) is a measure of the degree Laws of thermodynamic have many applications in
of randomness or disorder of a system. Entropy becomes biology and biochemistry, such as study ~ TP hydro-
maximum in a system as it approaches true equilibrium. lysis@,~m9rane diffusion zyme catalysis as well as
Enthalpy is the heat content of a system and entropy A binding a@ !:9tein stabi!ity'. These laws have been
~ is that fraction of enthalpy w hich is not available to do used to explain hypothesis of origin of life.
\.!Y useful work. I
, - [ ~ l;-:o:-:s:-:e-::;
d;-s::'.y:--:-s:-:t~e-=
m=-=a--=p--=p-ro
_a_c
- ;h~e- s_ a_s
_ t,...a_,.e
- o-,-e_q_u
...,i"", Three Trpes of Reactions
~ Any system can spontaneously proceed from a A. A reaction can occur spontanHously when t.G is
state of low probability (ordered state) to a state of high negativa.. Then the- reactionJ xergonic. If t.G is
probability (disordered state). The entropy of a system of great magnitude, the reaction goes to completion
may decrease with an increase in that of the surround- and is essentially irreversible.
ings . The second law may be expressed in simple terms B. When t.G is zero, the system is at equilibrium.
as Q = T x t.S , where Q is the heat absorbed, Tis the C. For reactions where t.G is positive, an input of energy
absolute temperature and t.S is the change in entropy. is required to drive the reaction. Tl~e reaction is termed
as endergonic (Examples are gi1ven in Chapter 5).
Gibbs Free Energy Concept Similarly a reaction may be exot1 ennic (t.H is nega-
tive), isothennic (t.H is zero) or endothe ic (t.H is positive).
The term free energy is used to get an equation combin- Energetically unfavorable reaction may be driven
ing the first and second laws of thermodynamics. Thus, forward by coupling it with a favorablle reaction.
t.G = t.H - Tt.S, where t.G is the change in free energy, Glucose + Pi --. Glucose-6-phosphate (reaction 1)
t.H is the change in enthalpy or heat content of the system ATP + H O --. ADP + Pi (reaction 2)
and t.S is the change in entropy. The term free energy Glucose+ ATP-. Glucose-6-phosphate+ADP (3)
l 9\ b_~
8 Section A: Chemical Basis of Life
Chapter at a Glance
The learner will be able to answer questions on the foUowing topics:
, I
Nucleus
Transport mechanisms
Endoplasmic reticulum
Simple and facilitated diffusion
Golgi apparatus
Ion cha nnels
Lysosomes , p~-,.\~
Active transport
Mitochondria
Uni port, symport and antiport
Plasma membrane
Ribosome
Plasma
membrane
Mitochondrion - - -- -
Rough /\ Marker Enzymes
endoplasmic
reticulum Some enzymes are present in certain organelles only;
Nucleus such specific enzymes are called as marker enzymes ·
Chromatin
(Table 2.1 ). After centrifugation, the separated organelles
Nuclear pore
are identified by detection of marker enzymes in the
sample.
I NUCLEUS
It is the most prominen organelle of the cell. All cells
Fig. 2.1: A typical cell in the body contain nucleus, except mature RBCs in
r
Chapter 2: Subcellular Organelles and Cell Membranes 11
Fig. 2.3: M
tW~2, Erythrocytes do not contain mitochm1dria. The tail of
spermatozoa is fully packed with mitochondria. Mito-
chondria are the powerhouse of the cell, where energy
l.!:._YSOSOMES released from oxidation of food stuffs is trapped as
Discovered in 1950 by Christian de Duve (Nobel Prize chemical energy in the form of ATP (see Chapter 21 ). Meta-
.
1974), lysosomes are tiny organelles. Solid wastes of a bolic functions of mitochondria are shown in Table 2.2.
township are usually decomposed in incinerators. Inside Mitochondria have two membranes. The in~ mem-
a cell, such a process is taking place within the lysosomes. brane convolutes into folds or cristae (Fig. 2.3)~ inner
They arG gs of enzymp. Clinical applications of lyso- mitochondrial membrane contains the e~ es of el~ -
somes are shown in Box 2.1. tron transport chain (see Chapter 21 ). fluid matrilf
Endocytic vesicles and phagosomes are fused with contains the enzymes of citric acid cycle, urea cycle and
lysosome (primary) to form the secondary lysosome heme synthesis.
or digestive vacuole. Foreign particles are progressively Cytochrome P-450 system present in mitochondrial
digested inside these vacuoles. Completely hydrolyzed inner membrane is involved in stenoidogenesis (see
products are utilized by the cell. As long as the lysosomal Chapter 45). Superoxide dismutase is present in cytosol
Chapter 2: Subcellular Organelles and Cell Membranes 13
'll d itochondria Electron transport chain, ATP generation, TCA cycle, oefa oxidati'6n oH;li'.•y-a'/!id s, ke'tone body production, urea synthesis
(part), heme synthesis (part), gluconeogenesis (part), pyrimidine synthesis (part)
Cytosol Protein synthesis, glycolysis, glycogen metabolism, HMP shunt pathway, transaminations, fatty acid synthesis, cholesterol
synthesis, heme synthesis (part), urea synthesis (part), pyrimidine synthesis (part), purine synthesis
n
Fence with gates; gates open when
message is received
Nucleus Manager's office
Endoplasmic reticulum Conveyer belt of p roduct ion units Fluid Mosaic Model
Golgi apparatus Packing units
Lysosomes Incinerators The lipid bilayer was originally proposed by Davson and
Vacuoles Lorries carrying finished products
Danielle in 1935. Later, the structure of the biomem-
Mitochondria Power generating units
branes was described as a fluid mosaic model (Singer
and Nicolson, 1972).
and mitochondria (see Chapter 30). Mitochondria also The phospholipids are arranged in bilayers with the
contain specific DNA. The integral inner membrane pro- polar head groups oriented towards the extracellular side
teins, are made by mitochondrial protein synthesizing an~ e cytoplasmic side with a hydrophobic core (Fig.
machinery. However, the majority of proteins, especially 2.4~ / Choline co~ ining phospholipids are mainly in the
of outer membrane are synthesized under the control of external layer ar@e~3-01i~ e containing
cellular DNA. The division of mitochondria is under the phospholipids in the inner layer. Gerd Binnig and Heinrich
command of mitochondrial DNA. Mitochondrial ribo- Rohrer introduced the scanning electron microscopy
somes are different from cellular ribosomes. Antibiotics in 1981 by which the outer and inner layers of membranes
inhibiting bacterial protein synthesis do not affect cellular could be visualized separately. They were awarded
processes, but do inhibit mitochondrial protein biosyn- Nobel Priz~ 86l Eac~ leaflet is 25 A thick2with the
thesis (see Chapter 40).(Mitochondria play a role in · h.¥9 portjon 19 A and tail 15 A lbick The total thickness
tnggenng apoptosi~ (see Chapter 42). is about 50 to 80 A.
A summary of functions of organelles is given in The lipid bilayer shows free lateral movement of its
~ able 2.2 and Box 2.3. components, hence the membrane is said to be fluid in
nature. Fluidity enables the memb~ane to perform endo-
LASMA MEMBRANE cytosis and exocytosis. However, ttie cprnponents go
The plasma membrane separates the cell from the not freely move from inner to outer layer, or outer to inner
external environment. It has highly selective permeability layer (flip-flop movement is restrict d). During apoptosis
properties so that the entry and exit of compounds (programed cell death), flip-flop movement occurs. The
are regulated. The cellular metabolism is in turn influ- cholesterol content of the membrane alters the fluid-
enced and probably regulated by the membrane. The ity of the membrane. When cholesterol concentration
membrane is metabolically very active. The enzyme, increases, the membrane becomes less fluid on the outer
nucleotide phosphatase (5' nucleotidase) and alkaline surface, but more fluid in the hydrophobic core. The
phosphatase are seen on the outer art of cell mem- nature of the fatty acids also affects the fluid ity of the
brane; they are therefore calle ecto-en s membrane. Unsaturated cis fatty acids increase the
Membranes are mainly made up of lipids, proteins fluidity w hereas trans fatty acids decrease the fluidity.
and small amount of carbohydrates. The contents of these Sphingolipids and glycerophospholipids constitute the
compounds vary according to the nature of the mem- largest percentage of the lipid weight of biological mem-
brane. The carbohydrates are present as glycoproteins branes.
14 Section A: Chemical Basis of Life
. I
SOA
]2sA ]
Transmembrane
protein
Fig. 2.4A: The fluid mosaic model of membrane Fig. 2.4B: Proteins are anchored in membrane by different mecha-
nisms
@ cytoskeleton
Human body is supported by the skeletal system;
• • •
similarly the structure of a cell is maintained by the cyto-
skeleton present underneath the plasma membrane.
The cytoskeleton is responsible for the sbape pf the cell,
its nJ,QlilitY and chromosomal rnmt.em.ents during cell
division. ®
(I(') The cytoskeleton is ~.p,posed of m icrofilaments, Fig . 2.5: Facilitated diffusion. The carrier molecule exists in two
~ ntermediate filaments an~ microtubules, formi ng a net- con Formations
work within the cell. Microtubules contain alpha and betam D'!ae.i'ii~"'Tii;;iric'
tubulin with a diameter of 25 nm. They are essential fo sive ransport
formation of mitotic spindle and participate in exocytosis Simple Diffusion
and endocytosis. @.nca Blkalo1°5°sed as anticancer
drugs. iobibi11be farmatiori:ol..mi~ @ioole-byJD.tecfer- Solutes and gases enter into the cells passively. They are
iQ.Q-Witb..tb.e-assem~ rotu bu les and thus in hi bit ,-driven by the concentration gradient. The rate of entry
ell division. 'is proportional to the solubility of that solute in the hydro-
phobic core of the-membrane. Simple diffusion occurs
TRANSPORT MECHANISM
- --- from higher to lower concentration. This does not require
Thc(@rmeabilit,y of substaoc:d across cell membrane is · any energy. However, it is a very slow process.
degendeot theic sol! 1bility io lipid§ and not on their Diffusion of gases such a~ a[ CO2) NO ahd Cq:Occurs
molecular siz~-~ compounds are generally at a rate that is solely dependent upon c;~mcentration
impermeable ~ eq~ e carrier mediated transport. Qradients. Lipophilic molecules will also diffuse across
An important function of the membrane is to withhold membranes at a rate th at is directly proportional to tbe
unwanted molecules, while permitting entry of molecules SQlubjJjty pf the coropo1,od io th e n:ien:ibrane.
necessary for cellular metabolism. Transport mecha- F .. d D ·u .
nisms are classified into: { BC/11tate 111us1on
t ive transport This is a carrier mediated process. (Fig . 2.5). Important
A. Simple diffusion features of facilitated diffusion are:
B. Facilitated diff~~ ormones regulate the a. The carrier mechanism c~ ld b§Cg) turated which
number of carrier moleqfes_For example, glucose tr.aos- is simi.!slr...t~ of eQ2l!JleS.
port across membrane is by f~ involv- b. StructI1ra1ty similar sal! 1te !': can competjtjyel£lohjbjt r,....
ing a family of @llCbse fraosporters. Glucose transport the entry of the solutes. p1D~
is described in detail in Chapter 10. c. Facilitated diffusion can operate bi,YirectjonaUy.
C. Ion channels are s~cjaljzed carrier systoo:is. d. This mechanism does not reguirea;qergv but the
They allow pass! ; i ;of molecules in accordance with the rate of transport is more rapid than si_mple diffusion
@ ncentratioo gr,a_:_~ _ process.
2 transpolt e. The carriE!r molecules__Gfn exist in two conforma-
mps can drive molecules against the gradient tions, RI r and ~ fsf'ates. In ~ Rong st~. the
using energy. active sites are ex os d t · r, when the
fcu-tli~
E diffusion involving a family of ~ cose transe,ortfil:s.
Ion
Glucose transport is described in detail in Chapter 10. [ Membranes have special devices called ion channels
'-.:>~ c..~~ (see Fig. 2.9). Ion channels are tra11smembrane proteins
A UaP.orms · that allow the selective entry of various ions. Salient
They are water channels (Fig. 2.6) throug h which water features are enumerated in Box 2.4. These channels are
crosses the plasma membranes of cells. They QQ!l1wl the for qu.ic~ transport of electrolytes, such as~ ..$.
water cnnteoJ of cells. 'Agre.and_MacKinnof.l w~ war- and....Qr-. These are selective ion conductive
~G Nobel Prize for Chemistry in 2003 for their contribu- channels are specialized protein molecules that span~tie r&._
tions on aquaporins and ion channels. Diseases, such membranes. The channels generally remain closed, but in
as (oe hro enic diabetes insipid us due to impaired response to stimulus, they o~ allowing rapid flux of ions
fu nction of these chann Is. down the gradient. This may be compared to openjpg Q!
->
'ff t • ~ d -b d . th e gate of a cioeroa bo••se, when people rysb to enter
lien d I eren a ua onn are aescnoea in ru.iwar,s . . . . 11 .. •
d AQP pg) L ff t· -f in. Hence, this regulation Is named as gated . Such 10n
rme . oss o unc I0n o rena I . . .
_ . . . . channels are important f erve impulse pmpagation,
~quaponns is a~soci d wit~ sever~! dise~se ates; (i}ynaptic transmission a l: ecretion of biologically active
st
r;.e_du.ced ex,12ress,on, . 9 ~is associated with nephro- substances from the cells. Ion channels are different
enic diabetes insipidus NOi . :-.ira.m ion transport pumps described below.
Channe apathies are a group of disorders that
result fro a norm liti i r t · orming thej,Q[l A)..igand-Gated Channels
chan I to r ins. Channelopathies may be Ligand gated channels are o~ e-
acgu!!.;d or c~genital. ~ onge ital channelopathi~s may ~ - The bJnding o! a ligand to a receptor site O!l the
occur due to genetic · s in s~ . pot~fum, charme~..results in the.opening (or closi_ng) of the channel.
ch©rid e and ca~ channels. A few examples are The ligand may ~ ~tracellular sigoa ling molecule
Bartter sxndrome, myasthenia gravis, lo_!!9 and short OJ or an iotracellulrr: messJ nger. Clinical applications of
syndromes, cystic fibrosis {chloride channel), Liddle's channels are shown in Box 2.5.
Chapter 2: Subcellu/ar Organelles ancf Cell Membranes 17
a
Exterior
Peter Agre Roderick Jens Skou
Plasma
NP2003 MacKinnon NP 1997
membrane
b. 1949 NP 2003 b. 1956 b. 1918
•
sfeJase:) 40 - o -
Calcium channels:~ppropriate ti
Q ~_9alcium
·
exposed to ionophores, the ion gradient is dissipated.
- The ~ on..oLJtle.ion.grac:fum.U~es tb.fl proton
coannels are opened I sarcoplasmic reticulum gradient. The result is uncoupling ~of...oxidaiiv.e ph~p
membrane, leading to an ~ ~ ~ Q J be 1 ·on (see Chapter 21 ). '])\ :1'-
cy,to~QJ..oLtrius.ci~ ls. (~Alcilim e aooel 617iclffirs are f?J cD ~t\,el
1 l .,.._..
- therefore wid~ Y. ,u,sed in the 111F~ementotb.yp,;r1eD;!_Of1 ,. • e Transport \) .
Amelogeni~ m.ieio present in enamel of>teeth . . N, 1
U '-'->
has hydrophobic residues on the outside. A 27 amino The salient features of active transport are:
acid portion of amelog_e~ raJciuro cha~ ~ a. This form of transport requires energy. About {o~
Phosphorylation of a s@ resid the prot · of the total energy expenditure in a cell is useWr
the calcium channel. Calcium ions zoo rough and are the active transport system.
ot"tunneled to the mineralization front. e amelogenin is b. The active transport is unidirec1LI.orl.al
used for the formation of calcium hydroxy apatite crystals. c. It requires specialized integral proteins called trans,:
po
B)Voltage-Gated Channels d. The transport system is §.aiuralf~ at higher concen-
trations of solutes.
7 Voltage-gated channels (see Fig . 2.9) are opened by wem- e. The transporters are s.1isceQ!jble to iphibitioo by
brane depolarization. The channel is usually ~ in the specific organic or inorganic c,ompounds. General
gr~upd sti;!te. The membrane potential change (voltag! reaction is depicted in Figures :2.8 and 2.9 .
•
--
di!t,eceo.ce) switches the ion channel to open, lasting less
than ~5 !!!llli~G.Qn9s.
Sodium Pump I
In voltage-gated channels, the channels open or
close in response to changes in membrane potential. It is the best example for active transport. Cell has low
0'oJ1age-gated sodium channei~ and Qgitage-gated intracellular sodium; but concentration of gotassium
_eotassium chann~ are the common examples. These inside the cell is v~ 1intained by sodium-
are seen in gerye s!!,§..and are involved in the conduction potassium activated ATPase, £Jenerally called as
of nerve impulses. sodium pump. The ATPase is an integral protein of the
r
• 5
Cytoplasm
•
4 2
3
Extracellular fluid
Fig. 2.8: The sodium potassium pump. It brings sodium ions out of the cells and potassium ions into the cells. Black circle = sodium
ion; green square = potassium ion; pink circle = phosphate. (1) Cytoplasmic sodium ions (3 numbers) bind to the clhannel protein. This
favors phosphorylation of the protein along with hydrolysis of ATP; (2) Phosphorylation causes the protein to change conformation,
expelling the sodium ions across the membrane; (3) Simultaneously, extracellular potassium ions (2 numbers) bind to the carrier
protein. Potassium binding leads to release of phosphate group; (4) So, original conformation is restored~ (5) !Potassium ions are
released into the cytoplasm. The steps 1, 2, 3, 4 and 5 are repeated .
Extracellular
Simple Ion/water ATP-coupled •
diffusion channel Gated-ion channel Symporter Antiporter active-transport
DOD
DD
Mil M6 Mil M Oc°
DOD
DD
Intracellular
6 -Ions
~ } Macromolecules
Carrier Examples
ndOC1YtOsis
1) Simple no no Water
diffusion The selective or adsorptive pinocytosis is receptor-
i.) Facilitated yes no nil Glucose to RBCs mediated; also called as abso rptiv,e pinocytosis. Low
diffusion
density lipoprotein (LDL) is a good 13xample. LDL binds
:1) Primary yes yes directly Sodium pump
to the LDL receptor and the complex is later internalized.
active
The cytoplasmic side of these vesicles are coated with
4) Secondary
active
yes yes indi~ect"
'-._J
Glucose to intestine
filaments; mainly composed of ClathIrin. These are called
s) Ion
channels
yes no no Sodium channel Clathrin coated pits. Absorption of cholesterol by clathrin
coated pit is shown in Chapter 14. After the LDL-recepto r
22 Section A: Chemical Basis of Life
2-3. Which of the following cellular organelle can cause B. Membrane proteins are fixed and cannot move
autodigestion? C. Membrane contains bimolecular leaflet of lipids
A. Golgi bodies B. Lysosomes D. Permeability of drugs across cell membrane is
C. Microsomes D. Peroxisomes dependent on their solubility in water
2-4. All the following activities are taking place inside 2-15. Galactosyltransferase is a marke,r enzyme of:
mitochondria, except:
A. Cell membrane B. Mitochondria
A. Citric acid cycle
C. Lysosomes D. Golgi complex
B. Electron transport chain
2-16. ATP synthase is a marker enzyme present in:
C. Beta oxidation of fatty acid
D . De novo fatty acid synthesis A. Cytoplasm B. Mitochondria
2-5. All the following statements are true with regard to C . Lysosomes D. Golgi complex
biomembranes, except 2-17. All the following enzymes are p,resent in lysoso-
A. They are selectively permeable mes, except:
B. Have interior hydrophilic and exterior hydrophobic A. Alpha fucosidase
groups B . Arylsulfatase
C . Lipid bilayers contain phospholipids and cholesterol C . Beta glucuronidase
D. Carbohydrate residues are located on their surfaces D. Superoxide dismutase
2-6. The fluid mosaic model of membrane includes: 2-18. Golgi bodies are:
A. Cholesterol molecules in the membrane increase A. Involved in phagocytosis
stability of membrane B. Temporary storage sites for secretory products
B. Membrane proteins are fixed and cannot move C . Concerned with disposal of waste products
C . Membrane contains bimolecular leaflet of lipids D. Engaged in transport of molec1ules into mitochon-
D. Permeability of drugs across cell membrane is dria
dependent on their solubility in water 2-19. Which of the following is not syn1thesized in endo-
2-7. All the following lipids are present in cell membra- plasmic reticulum?
nes , except: A. Lipoproteins B. Glycoproteins
A. Lecithin B. Cholesterol C . RNA D . Ganglioside
C . Sphingomyelin D. Triacylglycerol 2-20. Which of the diseases listed bel<lw is a lysosomal
2-8. Fluidity of membranes depends on: storage disease?
A. Nature of fatty acids A. Zellweger syndrome
B. Concentration of proteins B. Inclusion cell disease
C. Glycosylation of proteins C. Pompe's disease
D. Membrane pumps D. Leber's neuropathy
2-9. Sodium-potassium pump is inhibited by: 2-21. Peroxisomes are:
A. Phlorizin B. Digoxin A . The site of biological oxidation
C . Valinomycin D. Dicoumarol B. The site of post-translational modifications
2-10. Which of the statements regarding active transport is C . Concerned with drug detoxification
incorrect? D. Involved in the production of free radicals
A. Requires carrier protein 2-22. Which are specialized membrane, structures?
B. Energy dependent A. Peroxisomes B. Desmosomes
C. Against concentration gradient C. Lysosomes D. Microsomes
D . Transport of water is an example 2-23. All the following are channel formers, except:
2-11 . All the following are channel formers, except: A. Gramicidin B. Amelogenin
A. Gramicidin B. Amelogenin C . Valinomycin D. Glutamate
C. Valinomycin D. Glutamate 2-24. Phagocytosis involves:
2-12. Symport system operates in the transport of: A. Respiratory burst
A. Bicarbonate B. Chloride B. Lipid peroxidation
C. Calcium D. Glucose C. Alpha oxidation
2-13. Glucose-6-phosphatase is a marker enzyme pre- D. Endosmosis
sent in: 2-25. Which of the following is a featur,e of endocytosis?
A . Cytoplasm B. Mitochondria A. Plasma membrane is invaginateid to form vesicles
C. Lysosomes D. Microsomes B. Cytoplasmic side of vesicle is coated by clathrin
2-14. The fluid mosaic model of membrane includes: filaments
A. Cholesterol molecules increase stability of mem- C. LDL uptake is by endocytosis
brane D. Heparin helps in this mechanism
Chapter 2: Subcellular Organelles and Cell Membranes 23
2-1 . What is the function of Golgi complex? 2-15. What are salient features of facilitated diffusion?
Maturation and processing of nascent proteins, glyco- It is carrier mediated. It does not require energy directly.
2-16. Give an example of facilitated transport.
sylation of proteins, secretion of newly synthesized pro-
Glucose transporters.
teins.
2-17. Give some examples of ion channels.
2-2. What is the function of endoplasmic reticulum? Ion channels for calcium, potassium and chloride.
Biosynthesis of proteins, drug metabolism, desaturation 2-18. What are ionophores?
of fatty acids. They are transport antibiotics which increase the
2-3. Where does protein synthesis takes place? permeability of membrane to ions, e.g., valinomycin,
On the walls of endoplasmic reticulum and also in cytosol. gramicidin.
2-4. What are cathepsins? 2-19. What are the salient features of active transport?
They are intracellular proteolytic enzymes. It requires transporters. It requires energy. Transport is
generally unidirectional.
2-5. What is the function of lysosomes?
2-20. Give examples of active transport systems.
They are bags of hydrolytic enzymes that bring about
Sodium pump, Calcium pump.
degradation of macromolecules. 2-21 . What is the importance of sodium pump?
2-6. What is lysozyme? Cell has low intracellular sodium; but concentration of
It is an enzyme present in external secretions. potassium inside the cell is high; this is maintained by
2-7. What is the most important function of mitochondria? sodium pump.
Generation of ATP. 2-22. What is the clinical significance of sodium pump?
2-8. What are the important metabolic events taking Digoxin increases the contractility of the cardiac mus-
place in mitochondria? cle, by inhibiting the sodium pump.
TCA cycle, Electron transport chain, Beta oxidation of 2-23. What is a uniport?
It carries single solute across the membrane.
fatty acids and urea cycle.
2-24. Give examples of uniport system.
2-9. What are ecto-enzymes? Glucose transporter (GluT2) operating in most of the
Enzymes seen on the outer part of cell membrane. cells is an example. Calcium pump is another example.
2-10. Give examples of ecto-enzymes. 2-25. What is co-transport?
Alkaline phosphatase, 5'nucleotidase. If transfer of one molecule depends on simultaneous
2-11. What is the structure of cell membrane? transfer of another molecule, it is called co-transport
system.
Fluid mosaic model.
2-26. What is symport?
2-12. What are characteristics of fluid mosaic model? In symport, the transporter carries two solutes in the
Membrane is composed of lipid bilayer. Phospholipids same direction across the membrane.
are arranged in bilayers with a hydrophobic core . 2-27. Give examples of symport.
2-13. What are the components of membrane that alter Sodium dependent glucose transporter (SGluT). Amino
the fluidity? acid transport is another example for symport.
Cholesterol and unsaturated fatty acids. 2-28. What is antiport system?
The antiport system carries two solutes or ions in
2-14. What are the different types of transport mecha-
opposite direction.
nisms? 2-29. Give examples of antiport.
Passive and active. Passive type is subclassified as Sodium potassium ATPase and ch loride bicarbonate
simple diffusion and facilitated diffusion. exchanger.
_ _ _ _ _Chapter 3
Amino Acids:
Structure and Properties
/, I I
Chapter at a Glance
The learner will be able to answer questions on the following topics:
Classification of amino acids based on structyre A-b Reactions due to carboxyl group (C...O)
B Based on side chain character -- Reactions due to amino group tN\:-4:i)
c Based on metaboljc fate CD Reactions of SH group {_S.1--\)
l> Based on nutritional regy jrements ,a~ Pepti de bond formation
.tifO lsoelectric prnnt
-
composed of a number of amino acids linked by peptide
bonds. nine (Fig. 3.8). (i--lPtt.J
Although about 500 amjno acjds occur in nature, B. Aromatic amino acids: Phenylalanine, Tyrosine
only are se~ uman bod . Most of the (Fig. 3.9). [f>T"]
amino acids (~ oline) a re O acid C. Heterocyclic amino acids: Trypto~han (Fig. 3. 10),
w hich means tnanhe amino group is attached to the Histidine (Fig. 3.11 ). L 11\J
same carbon atom to which the carboxyl group is D. lmino acid: Praline (Fig. 3.1 1). Cr]
attached (Fig. 3.1 ). E. Derived amino acids:
a. Derived amino acids found in proteins: After
the synthesis of proteins, some of the amino acids
are·~ d. e.g. ~ e (Fig. 3.12) and
are important components of
A. Aliphatic amino acids
collagen. G2 mma carboxylatjon of glutamjc acid
a. Monoamino monocarboxylic acids: residues of proteins is important for clotting
J. ~ e amino acids: Glycine, Alanine (Fig. 3.2)
t-' • Branched chain amino acids: ValiJ:le, Leu~ ~
WP-L-I] ~ - I 00-
l'!,(1'1).,
lsoleucine (Fig. 3.3)
t! · J:jyd?J'ISY- amino acids: § erine, Threonine 0.j
1
yH-NHt
(Fig. 3.4.)
r• Sulfdr-cootajning amino acids: Cysteine, Methio- R
nine (Fig . 3.5) (.Ctv') Fig. 3.1: General structure
Chapter 3: Amino Acids: Structure and Properties 25
1 coo-
l iH-NH;
-] n ',<? 1 0
r-NH; I
ffi jcoo- 1
I NH•3
CH-
~1-
j
CH--CH3 CH2
TH~
I I ~
H CH3 HUH CH2
I 3' I
CH3 CH3
Glycine Alanine
Gly; G Ala; A Valine Leucine lsoleucine
Val;V Leu; L lie; I
Fig. 3.2: - le amino acids Fig. 3.3: Blllflllil!l chain amino acids
.
1
Fl lf coo- 1 coo-
l n coo
I
I
,. +~
+
+ I • C H- NH3
+
.__C H-NH3
l1H-NH3 CH-NH3
I CH2 .. ,tik~ CH2
I
CH24 0H I CH,-@] CH2
I
CH3 § 0 CH 3
Serine; Ser; S Threonine; Thr; T Cysteine; Cys; c ~ Methionine; Met; M
160-NH2 I 1~00- 1
Asparagine; Asn; N Glutamine; Gin; Q Aspartic acid; As1p; D Glutamie acid; Glu; E
'.t
coo-
I
CH- NH3
+
a
0
6H- NH; I
IF\lb' CH- NHI
HO
' - - - - -- - '
H2
I
C
r-:1 +
I CH- NH 3
I 600- 6 00-
CH2
f3 TH2 L -
I Phenylalanine·; Phe; F Tyrosine; Tyr; Y ( ....,OL>-•v•...
CH2 y CH2
I I (Benzene group) (Phenol group)
H2 6 TH2 Fig. 3.9: Aromatic: amino acids
, NH c CH2
I •
C=NH2
I (}
I I ~HJ+
NH2
Fig. 3.8: mine a cids Fig: 3.10: . . . . (W) with indole group
26 Section A: Chemical Basis of Life
coo-
~CH2
~ 1,H-NH;
coo-
Histidine; His; H (imidazole group)
I
H2
H2-NH3
N H2
+
coo-
..
@ Aspartic acid
Glu tamic
Asp
Glu
D
E
Aliphatic, MADC
Aliphat ic, MADC
acid
Lysine Lys K Aliphatic, DBMC
@ Arginine
Phenyl
Arg
Phe
R
F
Alip hat ic, DBMC
Aromatic
alanine
Tyrosine Tyr y Aromatic Amino acids having non-polar side chains: These
Tryptophan Trp w Aro matic groups a~tli,1ydrophobic (water repel!_antLind lipo-
Hist idine His H Heterocyclic philic. Therefore, the parts of proteins made up of
Proline Pro p lminoacid these amino acids will be hydrophobic in nature.
(MAMC: Monoamino mono See Table 3.2 for classification based on side chain.
carboxylic acid). B. Amino acids having uncharged or non-ionic polar
side chains : These amino acids are i,_ydrophiliS, in
process (Fig. 3.12). In ribosomal proteins and in nature.
histones, amino acids are extensively methy- C. Amino ~cids · harged or~onic olar si e
l~ted and acetylated. chains hydlrophilic
b . Derived amino acids not seen in proteins Acidicarnino acids: They have a negative charge
(Non-protein amino acids): Some derived amino on the R group: Aspartic acid and Glutamic acid.
acids are ~en free in cells, e.g. 9!]ithine (Fig. (Tyrosine, is mildly acidic since it has a weakly
3.12), CJ.!rulline, ~ cys.J.e,jpe. These are ionisable phenolic hydroxyl group)
produced during the metabolism of amino acids. b. Basic amino acids: They have a positive charge
~ e may be considered as derived from on the R group: Lysine, Arginine and Histidine.
tyrosine.
c. Non-alpha amino acids: Gamma amino bu~
acid (GABA) ~ erived-ff8m-'glutamic acid. A. Purely ·& tc>genic: @u..9ne is purely ketogenic
~ - where amino group is in beta because it is converted to ketone bodies.
Chapter 3: Amino Acids: Structure and Properties 27
\
28 Section A: Chemical Basis of Life
I iso_electric point ,tJith NaoH; molecules ac.quire the atom and therefore, shows no optic;i° actiJity. All others
aQJ.Onic.Jom:i. When 50% of molecules.are anions, that are optically active.The mirror image forms produced
pH is calledfuK2:.twith--respect to NHJ The isoelectric with reference to the alpha carbon atom, are called..Q.a.lli:l
pH {pl) for m~
be calculated :
ino monocarboxylic amino acids can L isomers. The L-amino acids occur in nature and are
therefore called ~atural amino acids. ~ ~ ino acid~
•
Chapter 3: Amino Acids: Structure and Properties 29
- -
COOH
+OH-
coo- l
I +OH-
coo-
I
cHf NH 3• I
CH-E i] l
I +-- +-- CH-NH 2
CH2 + H• I + H• I
CH2
CH2
booH pK1 = 2.1 I pK3 = 3.9 pK2 = 9 .8
COOH coo-
J Ntt_charge n I ') Net c harge: -1 Net charg0
Fig. 3.15: Ionization of aspartic acid
HO - y -
I
CH2 0H
H ~
I
y-H IH
I
-i-61 - L -- +CO2 - - - -- --
R R Amid e Formation b'5 F-\C JDlc @
L-glyceraldehyde L-amino acid 0-amino acid The-CO OH group of dicarbo xylic amino acids (other
than alpha carbox yl ) can combin e with ammon ia to form
=~~,btiG~
Fig. 3.16: L- and D-amino acids
the corresponding amide. For examp le,
Asparti c acid + NH -+ Aspara gine
seen in small amoun ts in micr ~aA~ consti-
3
Glutam ic acid + NH -+ Gt utamin e (Fig.3. 18)
3
tuents Gµunicidin~ . P_g!y- These amides are also compo nents of protein
rm!Xin:Acti = \ n~ p linomyc,in, as well as bacterial structu re. The amide group of glutam ine seNes as the
cell wall ~ tidoglyc ans. D-amin o acids have multiple source of nitroge n for nucleic acid synthe sis.
...,,.--- - -
coo- coo-
coo-
l • l -E[l \.'"':'!I §
_Jpha k~o-
l
c:(g ,
CH-NH3 CH~
Glutamine
I "' . glutarate I
synthetase I CH Glutamate CH 2
( \. CH
I
2 I 2
CH2
I
CH2
CH I - I -
ATP ADP+ Pi 2
coo
I coo
c~
coo
Glutamine ) I
Glutamic acid
c Hf NH3•
I
I
Fig. 3.1 8: Formation of glutamine
CH 3
"d b t f
<>lnh<> <>minn
Th e ~ , .~...........,,...-,min .a6l can e rans err:$
nrn, , ... nf au.uµQ
at aJ)5aline eH
and serves as a mechanism for th ce
transport of carbon dioxide from tissues to the lungs by i. ~amf!!!_amino ric acid (GABA , a decivative
hemoglobin (see Chapter 23). of glutannic acid) and dopamine (derived from
Hb-N H +CO -+ Hb-NH --COO H (Carbamino-Hb)
2 2 tyrosine) are neurotransmitters.
Chapter 3: Amino Acids: Structl!lre and Properties 31
-
s.
6. Acidic amino acids have more than one CDOHgroup
and asic amino acids have more than one amino
group.
7.( Deriveq)amino acids may be found in proteins, or
15. Glycine ~
16. Decarboxylar
involving alpha
_ymmett.ic..carbon atom and there-
fore has no op ical activity.
nd amide formation are reactions
OH group.
they may - ds formed as 17. Transamination, oxidative deamination and forma-
~ ates. tion of carbamino compounds are reactions where
8. A functional classification based on the p.Q!fil_QJ gc04p takes part.
nonpol?r and ionized and non-ionized nature of
side chains is more useful.
9. Based on the metabolic fate, amino acids may be
-
18. Peptide bon¢?zare formed between the
group of ooe amino acid and the
ha amin
ha car
group of the next with elimination of a water mole-
glu~enic, ket~ nic or b~ . cule.
Chapter 3: Amino Acids: Struct,ure and Properties 33
3-1 . One of the amino acids listed below is not basic: 3-4. All the following are branched chain amino acids,
A. Arginine B. Histidine except:
C. Glutamine D. Lysine A. Valine B. Leucine
3-2. Which of the following amino acids has a hydroxyl C. lsoleucine D. Thre,onine
3-5. All the following are sulphur containing amino
group?
acids, except
A. Valine B. Threonine
A. Cysteine B. Methionine
C. Leucine D. Histidine C. Homocysteine D. Threonine
3-3. All the following amino acids are acidic, except: 3-6. Guanidinium group is present in:
A. Aspartic acid B. Tyrosine A. Arginine B. Tryp,tophan
C. Glutamic acid D. Threonine C. Histidine D. Prol1ine
34 Section A: Chemical Basis of Life
3-7. lndole ring is present in: 3-16. Nonessential amino acids:
A. Arginine B. Tryptophan A. Are not seen in tissue proteins
C. Histidine D. Proline B. Could be synthesized in the body
3-8. The following amino acids have hydrophobic side C. Have no role in body metabolism
chains, except: D. Have aromatic ring structure
A. Tyrosine B. Lysine
3-17. At isoelectric pH, the amino acids and proteins
C. Leucine Valine
show:
3-9. lmidazole ring is present in:
A. Maximum net charge
A. Arginine B. Tryptophan
C. Histidine D. Proline B. Maximum mobility in electric field
3-10. Pyrrolidine group is present in which of the fol- C. Maximum precipitability
lowing amino acids? D. Minimum buffering action
A. Arginine B. Tryptophan 3-18. This amino acid has 2 optically active centers (asy-
C. Histidine D. Praline mmetric carbon atoms):
3-11. Which of the following amino acids has a non- A. Glycine B. lsoleucine
polar side chain? C. Valine D. Tryptophan
A. Serine B. Valine 3-19. Enzymes are activated by phosphorylation of
C. Asparagine D. Threonine
which amino acid residue?
3-12. All the following are glucogenic amino acids, except.
A. Cysteine B. Serine
A. Glycine B. Serine
C. Glutamic acid D. Lysine
C. Leucine D. Aspartic acid
3-20. The amino acid which has maximum buffering
3-13. Which of the following amino acids has a net
positive charge at physiological pH? capacity at physiologic pH is:
A. Glutamic acid B. Lysine A. Arginine B. Alanine • I
;
3-1 . What are branched chain amino acids? 3-7. Which amino acids have hydrophobic side
Valine, Leucine and lsoleucine. chains?
3-2. What are hydroxy amino acids? Valine, Leucine, lsoleucine.
Serine and Threonine. 3-8. Give the names of aromatic amino acids. •
3-3. Name the sulphur containing amino acids. Phenylalanine and Tyrosine.
Cysteine and Methionine. 3-9. What are heterocyclic amino acids?
3-4. Name the acidic amino acids. Tryptophan and Histidine.
Aspartic acid and Glutamic acid. 3-10. Give an example of an imino acid.
3-5. What are the basic amino acids? Praline
Lysine and Arginine. 3-11 . Give examples of derived amino acids.
3-6. Which amino acid contains thioether bond ? Hydroxyproline, hydroxylysine. omithine, citrulline, homo-
Methionine. cysteine.
Chapter 3: Amino Acids: Structure and Properties 35
3-12. Arginine contains which special group? 3-30. Which amino acid has maximum buffering capacity
Guanidinium group at physiological pH?
3-13. Benzene group is present in which amino acid? Histidine.
Phenylalanine 3-31. Which amino acid is opticall}r inactive?
3-14. Phenol group is present in which amino acid? Glycine.
Tyrosine 3-32. What are the isomeric forms of amino acids?
3-15. Tryptophan contains what special group? D and L varieties.
3-33. What are natural amino acids,?
lndole group
Only L amino acids are seen in nature.
3-16. Which special group is present in Histidine?
3-34. What is decarboxylation of a1n amino acid?
lmidazole group.
The carboxyl group is removed from the amino acids
3-17. Name some hydrophobic amino acids.
to form the corresponding aminie.
Valine, Leucine and lsoleucine.
3-35. Give examples of decarboxylation reactions.
3-18. Pyrrolidine is present in which amino acid?
Histidine to Histamine; Tyrosine to Tyramine; Trypto-
Proline
phan to Tryptamine.
3-19. Hydrophobic bonds are formed in protein between
3-36. What is produced when glutamic acid is decar-
which amino acids?
boxylated?
Valine, leucine and isoleucine residues.
Gamma amino butyric acid or GABA.
3-20. What is the basis of classification of amino acids
3-37. What is glutamine?
into ketogenic and glucogenic?
Thal is the amide of glutamic aieid.
Ketogenic amino acids will enter into the metabolic
3-38. What is an amide?
pathway of fats, while glucogenic amino acids enter the
The extra carboxyl group (othor than alpha carboxyl)
pathway of glucose metabolism. can combine with ammonia to lform the corresponding
3-21 . Name a purely ketogenic amino acid. amide.
Leucine 3-39. What is transamination?
3-22. Name some glucogenic amino acids. The alpha amino group of amino acid can be transferred
Glycine; Serine; Aspartic acid. to alpha keto acid to form tile corresponding new
3-23. What are essential amino acids? amino acid and alpha keto acid .
They can not be synthesized in the body; and so, they 3-40. Give an example of transami1nation reaction.
are to be provided in the diet. Glutamic acid + pyruvic acid to alpha keto glutarate +
3-24. How many amino acids are essential? alanine
Eight amino acids are essential; two are semi-essential 3-41 . What is the biological signi1ficance of transamin-
and the rest 10 are nonessential. ation reaction?
3-25. Name any three essential amino acids. These are important for the int,er-conversion of amino
lsoleucine, Leucine, Threonine. acids. Nonessential amino acids are synthesized by
3-26. Is phenylalanine an essential amino acid? this process.
Yes. 3-42. What is the clinical significance of transami-
3-27. What about Tyrosine? nases?
Tyrosine is nonessential. Transaminases in blood are ele1vated in liver and heart
3-28. What is isoelectric point? diseases.
The pH at which the molecule carries no net charge is 3-43. What is the significance of SI-I groups in proteins?
called isoelectric point. The SH group of cysteine can lform a disulphide (S-S)
:; 3-29. What are the characteristic features of iso-electric bond with another cysteine residue. The two cysteine
pH? residues can connect two polypeptide chains by the
At isoelectric point the amino acid will carry no net formation of interchain disulphide bonds.
charge; there is no mobility in electrical field. Solubility 3-44. Phosphorylation is taking pllace on which amino
will be minimum. The tendency for precipitation will be acid?
maximum. Serine and Threonine.
_ _ _ _ _ Chapter 4
Proteins:
Structure and Function
Chapter at a Glance
The learner will be able to answer questions on the following topics:
A D Peptide bonds B D Sequence analysis (study of primary structure)
c Primary structure of proteins B D lsoelectric pH of proteins
C D Secondary structure e. D Precipitation reactions of proteins
C, D Tertiary st ructure Classification of proteins
C.., D Quaternary structure .,gl<'b Quantitative estimation of proteins
The word protein is derived from Greek word, "proteje_~" 0-50 amin acids is a peptide. By convention,
which means~ . As the name shows, the proteins peptide chains containing uiore than 50 aQJ,ino
are of paramount importance for biological systems. acids are called proteins~ Y
Out of the total dry body weight, ~ f In a tripeptide , there are 3 amino acids, but these 3
proteins. Proteins are used for body building; all the can be any of the total 20 amino acids. Thu6203 = 8000
maj~tructural and functional aspects of the body are different perro11ta1tjons ang carobinations..~ possible
carried out by protein molecules. Abnormality jn protein in a tripeptide. An ordinary protein having abour@_o
structure will lead to molecular diseases with profound amino acid~, will have f20100 different poss1b1~ . This
alterations in metabolic functions. number is more tt1an the total number of atoms present
Proteins contain Carbon, Hydrogen, Oxygen and in the whole univeirse. Thus, even though there are only
<_cjf>~ Nitrogen as the major components while Sulfur and Phos- 20 amino acids, by changing the sequence of com-
phorus are minor constituents. Nitrogen is characteristic
of proteins. On an average, the nitrogen content of
ordinary proteins is 16% by weight. All proteins are
polymers of a~ino acids. 1 rn~.,,.t,:;iil,
® ~lneAolds.,. Linked by Peptide Bond
Alpha carboxyl group of one amino acid reacts with Proteins have different levels of structural organization;
alpha amino group of another amino acid to form a primary, secondary, tertiary and quaternary. oJriv--J-
peptide bond o r CO-NH bridge (Fig. 4.1 ). (_ S"(ff) qrq~•.•ffl
Proteins are synthesized by polymerization of amin'I:? Primary Str FtU e ®
acids through peptide bonds. Two amino acids combined Sequence of mino Acids in.Proteins
to form a dipeptide; three amino acids form a tripep-
tide; four will make a tetrapeptide; a few amino acids Protein structure is studied as the primary, secondary,
together will make an oligopeptide; and combination of tertiary and quatemary levels (B9x 4.1). Primary structure
Chapter 4: Proteins: Structure and Function 37
Fig. 4.2: Peptide bond is a partial double bond (T°'oM) terminal (N-1terminal) end and the amino acid contri-
,~1',e.QQ -=- buting the alpha-amino group is named as the first
,.
denotes the number and sequence of amino acids amino acid. (Fig. 4.3). Usually the N-terminal amino
in the protein. The higher levels of organization are acid is written on the left hand side when the sequence
decided by . the primary structure. Each polypeptide of the protein is denoted. Incidentally, the biosynthesis
chain has a unique amino acid sequence decided by of the protein also starts from the amino terminal end.
the genes. The primary structure is maintained by the The other end of the polypeptide chain is the carboxy
covalent bonds of the peptide linkages (Fig. 4.1). terminal encl (C-terminal), where there is a free alpha
Students should have a clear concept of the term carboxyl group which is contributed by the last amino
"sequen ". S foll~ example: acid (Fig. 4.3,). All other alpha amino and alpha carboxyl
Gly - Ala - Val (1) groups are involved in peptide bond formation. Amino
GI - Val - Ala (2) acid resi eptides are named by changing
Both the tripepti es shown above contain the same the suffi '-in,e" to "- I' .g. Glycine to Glycyl.
amino acids; but their sequence is altered. When the H2-Gly-Ala-Val-COOH
sequence is changed 1 the peptide is also
different. In the above example, the amino group of glycine is
free; but carboxyl group of glycine is bonded with amino
Characteristics of a Peptide Bond group of alanine; the carboxyl group of alanine is, in
The peptide bond is a partial double turn, bonded with the amino group of valine; while the
bond. The ~N §ond ~s 'trans) in carboxyl grou of valine is fr e. Therefore this peptide is
nature andh ere i? ' no fw dom._gf named as €!;{c I-alan I-valin It is abbreviated as Gly-
rotati9n because of the partjai ctouble Ala-Val, or. simply as GAV.
bond character (Fig. 4.2). The distance
is 1.32A which is midway between single Branched and Circular Proteins
bond (1.49A) and double bond (1.27A). GN Generally th1:! polypeptide chains are linear. However
The S9! chains are free to rotate on Ramachandran branching points in the chains may be produced by
either side of the peptjde bond. The 1922-2001 ihterchain disulphide bridges. The covalent disulphide
angles of rotation, known as amachandran angles, bonds betwe,en different polypeptide chains in the same
therefore determine the spatial orientation of the peptide protein (i~:£bfil_~ 'f'rl'"'"""'"Lftions of the same ol e -
chain. (Dr GN Ramachandran diq pioneering work on the tide chain (infrachain re art of the rima structur
structural aspects of proteins during 1~50s and 1960s). · Rarely~ rrsteatr of the alpha COOH group, the
gamma carlt>oxyl group of glutamic acid may enter
Numbering of Amino Acids in Proteins into peptide bond formation, e.g. Glutathione (gamma-
In a polypeptide chain, at one end there will be one glutamyl-cysteinyl-glycine) (see Chapter 18). The term
free alpha amino group. This end is called the amino pseudopeptide is used to denote such a peptide bond
40 Section A: Chemical Basis of Life
H H O H H 0 Primary structure
I I R 11 I I R II
-..... N ....
· •c,..··c ..-.,..N , !
C'
... C ... _,....N
'N ·· i . C
, i _, C ...
C ...
is determined by the
sequence of amino acids
?
I R II I I R II I
~ ~ A1p~
helix Secondary structure
H ; 0 H .,_. occurs when the amino
0 H H 0 H H acids are linked by
II R I
.. c .. , _. N.. , c ,
I II R I
_.,....C. f .. N......_
I
C / y hydrogen bonds
•
H 0 H H 0 H is formed when alpha helices
and beta sheets are held
Fig . 4 .7: Structure of beta-pleated sheet together by weak interactions
Quaternary structure
chain. Heterodimer contains two different types of poly- consists of more than one
peptides as a functional unit. For example, 2 alpha-chains polypeptide chain
-
Transport Proteing hydro9en bonding and consequent weakness of collagen
Hemoglobin, the transporter of oxygen is a tetrameric (see Chapter 49). The quarter-staggered triple helical
protein (alpha 2, beta 2), w ith each monomer having structure of collagen · oonc.ibJo.Jc · ,;,.~;•1" , __ _ __
Chapter 4: Proteins: Structure and Function 43
redissolves in highly acidic or alkaline solutions. When 4. Transport proteins, e.g. hemoglobin, myoglobin, albu-
milk is curdled, the casein forms the white curd, because min, transferrin
..
agglutinates all RBCs and WBCs.
vi. Scleroproteins: They are insoluble in water, salt
solutions and organic solvents and soluble only in
Renal ration:
hot strong acids. They form supporting tissues,
native form is e.g. collagen of bone, cartilage and tendon; keratin
regen rated of hair, horn, nail and hoof.
Denatured protein;
Random coil structure; A ~ e ar apwt:
rl)y:proteiri
function is lost. Primary slrticture i intact.
Conjuga ed Proteins ( '3~~ h
is funct,onai.,:;-
They are combinations of protein with a non-protein part,
Fig. ~~\;,\\ -· called prosthetic group (Table 4.2). Conjugated proteins
may be classified as follows:
i. Albumins: They are soluble in water a d coagu- i. Glycoproteins: These are proteins combined
lated by heat. Human serum albumin ha a mole- with carbohydrates. (8ydroxyl groups of serine or
cular weight of {>9 000 e.g. lactalbumin o milk and threonin~ and Gmide groups of asparagine and
egg albumin. glutamine)orm linkages with carbohydrate residues.
ii. Globulins: These are insoluble in ater, but .§1,Qsd gr u12 and many serum proteins
soluble in diluw salt sofiiffiins. They are also a re glycoproteins. When the carbohydrate content
coagulated by heat, e.g. ei;m globuli , .§filld..m is more than 10% of the molecule, the viscosity is
Q.!Q.b.!Jjins, le.9umin of peas. correspondingly increased ; they are sometimes
Protamines: These are soluble in wat known as mucoproteins or proteoglycans.
acids. They ar not coagulated by hea ii. Lipoproteins: These are proteins loosely combi-
contain large n . ned with lipid components. They occur in _Qjood an.9
dues, and so are strongly b c. Hence they can cell membranes. Serum lipoproteins are des-
combine with other acidic proteins . .._
P_, ro....t,;;+...,.,.-.....-.~ cribed in Chapter 14.
is a common commercial pie iii. Nucleoproteins: These are proteins attached
insu lin. to nucleic acids, e.g. Histones. The DNA carries
iv. Prolamins : They are soluble in 70-80°/4 alcohol, negative charges, which combines with positively
but insoluble in pure water. They are rich in praline charged proteins.
Chapter 4: Proteins: Structure and Function 45
Heme
Histones DNA
Opsin 11 -cls-retinal
uccinate dehydrogenase Protein Riboflavin as FAD
Apoferritin Iron
Poor Proteins
Apoceruloplasmin Copper They l~ k in ma.2-Y essential amino acids and a diet
based on these proteins will not even sustain the original
iv. Chromoproteins: These are proteins with colored body weight. Zein from corn lacks tryptophan and lysine.
prosthetic groups. Hemoglobin (Heme, red); Flavo-
proteins (Riboflavin, yellow), Visual purple (Vitamin l ologically lmpo!1ant Pe~~tides (_Sm) c~
A, purple) are some examples of chromoproteins. When 10 or less number of amino acids are joined
v. Phosphoproteins: These contain phosphorus. together, it is called an oligopeptide. Some of them are
Casein of milk and vitellin of egg yolk are exam- biologically active. A few examples are given below:
pies. The phosphoric aci!i is e~!Q.!tle~ / 4._i. Thyrotropin releasing hormone (TRH) is a
gro~ Q serlQ_e.pnd threQOi._n.e residues of proteins . .::J... ~ ith th ce of Glu-His-Pro; but the
vi. Metalloproteins: They contain metal ions. Exam- I and Pro ar · #
pies are Hemoglobin C!f_an), C~ ochrome Ocon), Txro- ii. Glutathione is a'W~JW-111!:- It is gamma glutamyl
® s~ ~pey,;and Carbonic anhydrase (Zinc). cysteinyl glycine (see Chapter 118). It is involved in
~ ~emb(QneJnteg£ih' and is import
Derlvea Proteins keeping enz mes in active state:.
Disadvantage
It takes many days to get the result, and is unsuitable for
routine clinical work.
==·
are fo rmed. 14. Tertiary structure of a protein is the most thermo-
4. Nitrogen ,a~ tent of ordinary proteins is on the s;iv,pamical1Y,,,5.tab~ configuration.
1
average ~ /o by weight. 15. Quaternary structure is present only in certain pro-
5. Protein structure can be defined and studied at more than one polypeptide chain eg.
four levels viz. Primary, Secondary, Tertiary and
Quaternary. 16. Th~ tion of a protein is dependent on subunit
6. Proteins have the primary level of structure which interaction.
denotes the linear sequence of amjno acjds linked 17. Chemical properties of the proteins depend on the
by peptide bonds. colloidal nature of the particles in solution and the
7. The primary sequence is ge,petjcaUy determined @ lure of the side cha1Q);. This explains the precipi-
tation reactions and color reactions of proteins.
• and is unjgue and fixed f~ n produced
18. Solubility of a protein is dependent on the ionic
by a particular species of organism.
8. Primary structure determines the biological activity concentration of the medium. Hence, proteins may
be 'saJ!si.dJn.'_or_'sal!e.d...o.ut'.
of the protein. Alterations lead to loss of functional
19. Denaturation of protein results in l,9.s s of bjolog~ I
capacity. E.g . Si9Js!.e cell hemoglobin (HbS).
~ ty primary structure. Denaturation
9. The N terminal a~ o acid is the 1st amino acid
may be r · .
having a free ajgJJ§. NH2 gmup and t,ie C terminal
20. Proteins can be classified based on (i) Function)
amino acid, (the last amino acid) ha~ free alph~
(ii) Composition (iii) Shape and (iv) Nutritional value.
COOH group .
21 . Methods of protein estimation include~ orime
10. A protein having more t ne polypeptide chain, (Biuret and Lowry's metbo~ nd enzyme linked
like insulin, has interch
immunosorbent assa (ELIS~ l _ ... _.,
~-
Q~'c.c":~~o.,
4-1 . During denaturation, proteins will not lose their 4-9. Proteins can be precipitated by the following
structure, with regard to: methods, except:
A. Primary structure B. Secondary structure A. Adding alcohol and acetone
C. Tertiary structure D. Quaternary structure B. Saturating with ammonium sulphate
4-2. The characteristic features of the peptide bond C. Using salts of heavy metals
include all the following, except: D. Shifting the pH away from the iso electric point
A. Does not allow freedom of rotation 4-10. Denatured proteins:
B. It is a partial double bond A. Are soluble
C. Always has cis configuration B. Are difficult to digest
D. Absorbs UV light at 280 nm C. Are biologically inactive
4-3. The force maintaining the primary structure of a
D. Peptide bonds are broken
protein:
4-11. Which of the following is a simple protein?
A. Peptide bonds B. Hydrophobic forces
A. Casein B. Insulin
C. Hydrogen bonds D. Electrostatic (ionic) bonds
4-4. The forces maintaining the secondary, tertiary and
C. Hemoglobin D. Tyrosinase
•
4-12. In glycoproteins, the carbohydrate chains are com-
quaternary structures of a protein are the follow-
bined through glycosidic linkages with :
ing, except:
A. Electrostatic (ionic) bonds A. Hydroxyl groups of serine or threonine residues of
B. Hydrophobic forces proteins
C. Van der Waals forces B. Epsilon amino nitrogen of lysine residues of pro-
D. Peptide bonds teins
4-5. The amino acid which did not allow formation of C. Guanidium group of arginine residues of proteins
alpha-helix is: D. Phenol group of tyrosine residues of proteins
A. Glutamate B. Praline 4-13. The protein which does not answer the aldehyde
C. Tyrosine D. Histidine test is:
4-6. Tertiary structure of a protein describes: A. Hemoglobin
A. The sequence of amino acids B. Albumin
B. Location of disulphide bonds C. Casein
C. Amino terminal end amino acid D. Gelatin
D. The nature of protein folding 4-14. Proteins may be estimated by the following methods,
4-7. One of the following proteins does not have a qua-
except
ternary structure:
A. Biuret method
A. Albumin
B. Heat coagulation
B. Hemoglobin
C. Kjeldahl's digestion
C. Lactate dehydrogenase
D. lmmunoglobulin G D. Nephelometry
4-8. All the following reagents are used for Identifying 4-15. All the following are examples of tertiary structure
the first amino acid in a protein, except: of proteins, except:
A. Cyanogen bromide A. Alpha helix
B. Fluorodinitrobenzene B. Beta pleated sheet
C. Dansyl chloride C. Triple stranded helix
D. Phenyl isothiocyanate D. Peptide bonds
Chapter 4: Proteins: Structure and Function 49
4-16. Tertiary structure of a protein describes: 4-25. Study of linear sequence of amino acids is done by
A. Sequence of amino acids all techniques listed except
B. Location of disulphide bonds A. End group analysis
C. Amino terminal end amino acid B. Hydrolysis by proteolytic enzymes
D. The nature of protein folding C. Analyzing the content of each amino acid
4-17. Proteins may be denatured irreversibly by: D. Denaturing the protein
A. Adding urea 4-26. Different polypeptide chains are held together by:
B. Bringing to iso electric pH A. Peptide bonds
C. Heat coagulation B. Disulphide bonds
D. Reduction with mercaptoethanol C. Glycosidic bonds
4-18. Lectins are: D. Ester bonds
A. Animal proteins having specific amino acid binding 4-27. Primary structure decides:
site A. Rate of synthesis of protein
B. Antibody molecules acting against cells B. Biological activity of the protein
C. Plant proteins having specific carbohydrate bind- C. Rate of degradation of the protein
D. Effect of proteolytic enzymes on protein
ing site
4-28. Secondary and tertiary levels of protein structure
D. Blood proteins having a lecithin group
are dependent on:
4-19. Ultraviolet light at 280 nm is absorbed by which
A. Presence of disulfide bonds
component of proteins?
B. Primary structure
A. Peptide bonds
C. PH of the medium
B. Sulfhydryl group of cysteine
D. PK value of component amino acids
C. lndole ring of tryptophan
4-29. The protein having predominantly alpha helical
D. lmidazole ring of proline
structure is
4-20. The nature of the bond linking amino acids to each
A. Collagen B. Keratin
other is:
• A. Covalent
C. Fibroin D. Myoglobin
4-30. Which of the following is NOT true regarding the
B. Co-ordinate
tert.iary structure of proteins
C. Ionic A. It is a random coil structure
D. Hydrophobic B. Disulfide bonds are formed between any two cys-
4-21 . How many peptide bonds are present in gluta-
teine residues
thione? C. Position of disulfide bonds are predetermined and
A. 1 B. 2 fixed
C. 3 D. 4 D. Denaturation using reducing agents does not aff-
4-22. Basic difference between two polypeptides is in ect the disulfide bonds
the- 4-31 . Protein having a large number of disulfide bonds is:
A. Structural conformation A. Collagen
B. Primary sequence of amino acids B. Keratin
C. Number of side chains C. Hemoglobin
D. Number of hydrophobic bonds D. Albumin
4-23. Human insulin differs from bovine insulin in: 4-32. Which of the following proteins does not possess
A. Biological activity a quaternary structure?
B. Number of amino acids A. Myoglobin
C. Position of disulfide bonds B. Lactate dehydrogenase
D. Sequence of amino acids C. Hemoglobin
4-24. A covalent bond between the alpha carboxyl group D. lmmunoglobulin M
of one amino acid and alpha amino group of the 4-33. Which of the following is NOT true regarding hem-
neighboring amino acid is called oglobin?
A. Cis double bond A. Has 4 independent subunits
B. lsopeptide bond B. Each subunit has one heme residue
C. Pseudopeptide bond C. Each subunit can bind one molecule of oxygen
D. Peptide bond D. All four subunits are similar
50 Section A : Chemical Basis of Life
4-24. What are the features of isoelectric point? 4-35. How are proteins classified on physical basis?
At the pl value, the proteins will not migrate in an elec- Simple proteins, conjugated proteins and derived pro-
trical field ; solubility, buffering capacity and viscosity teins.
will be minimum and precipitation will be maximum. 4~6. Give examples of simple prote•ins.
4-25. What is the isoelectric pH of human albumin? Albumins, Globulins, Protamines, Prolamins, Lectins,
It is 4.7. Scleroproteins.
4-26. How proteins are precipitated from solution? 4-37. Give examples of scleroprotei1ns.
Any factor which neutralizes the charge or removes Collagen of bone, cartilage and tendon; keratin of hair.
water of hydration will cause precipitation of proteins.
4 .38. W hat are conjugated proteins',?
4-27. How is albumin precipitated?
Combinations of protein with a non-protein part, called
By Full saturation of ammonium sulphate.
prosthetic group.
4-28. What about gobulins?
4-39. How are conjugated group subclassified?
Globulins are precipitated by half saturation of ammo-
Glycoproteins, lipoproteins, nucleoproteins, chromo-
nium sulphate.
proteins. phosphoproteins and rnetalloproteins.
4-29. Give example of isoelectric precipitation.
Casein is precipitated when the solution is brought to 4-40. Give some examples of chromoproteins
isoelectric pH. Hemoglobin; Flavoproteins, Visuial purple.
4-30. What is the isoelectric pH of casein? 4-41. Give an example of a nutritionally rich protein (first
It is 4.6. class protein).
4-31 . What are the features of denaturation? Casein.
The secondary, tertiary and quaternary structures are 4-42. Why some proteins are nutriti,onally poor?
lost; but primary structure is preserved. The functional They lack in many essential annino acids and a diet
.. activity is lost. Denatured proteins are insoluble and based on these proteins will not even sustain the body
easily precipitated. weight.
4-32. What is heat coagulation? 4-43. Give an example of nutritionally poor protein.
,. When heated at isoelectric point, some proteins will Zein from corn lacks tryptophan and lysine.
denature irreversibly to produce thick floating conglo-
4-44. What is the advantage of biurnt method?
merates called coagulum. This is called heat coagulation.
The biuret method is simple onH step process, and is
4-33. Give examples of proteins that coagulate.
the most widely used method for plasma protein esti-
Albumin is easily coagulated , and globulins to a lesser
mations.
extent.
4-45. What is the disadvantage of b:iuret method ?
4-34. How are proteins classified?
The sensitivity of the method is less and is unsuitable
They may be classified (a) depending on the function
(b) based on the physicochemical characteristics or (c) for estimation of proteins in milligram or microgram
based on their nutritional value. quantities.
•
_ _ _ _ _Chapter 5
Enzymology:
General Concepts
and Enzyme Kinetics
Chapter at a Glance
l\,
l 0
Fischer's template theory
Koshland's induced fit theory
Michaelis constant, Km valu e, Vmax
0 Covalent modification
=JO lsoenzymes
Historical Perspectives
Berzelius in 1835 showed hydrolysis of starch by malt extract and put forward the theory of enzyme catalysis (see Table 1.1).
In 1878, Wilhelm Kuhne coined the word enzyme, which in Greek means "in yeast". Eduard Buchner (Nobel Prize 1907)
showed that cell-free extract of yeast could catalyze the fermentation of sucrose to ethanol. He named this active principle as
Zymase. Sir Arthur Harden in 1897 (Nobel Prize 1929) showed that m , a com lex mixtureo~, each catalyzing
a separate step in the degradation of sucrose. The rate of chemical reactions, chemical equi I num an alysis were studied
by Ostwald (Nobel Prize 1909). In 1926, James Sumner (Nobel Prize 1946) was the first to crystallize the en~ e yrea~e. In
1930, John Northrop (Nobel Prize, 1946) crystallized a number of proteolytic enzymes from gastrointestinal fr'act and proved
that they are all proteins.
Once upon a time there was a rich merchant. In his last catalysts. They enter into the reaction, but come out of
will and testament, he put aside his 17 white horses to the reaction without any chan~ Catalysts are substa-
his 3 sons to be shared thus; 1/2 for the 1st son, 1/3 for nces which ae;!!_erate the ra!9 tt
chemical reac-
the 2nd son and 1/9 for the 3rd son. After his death, the
sons started to quarrel , as the division could not produce.
tions, but do not change the equrfibriuJn. ~Ml-6t ..
whole number. Then their brother-in-law told them that AiiilinMHdioiiiiiljifi"
they should include his black horse also for the sharing
purpose. Thus now they had 17 + 1 = 18 horses, and so Life is possible due to the coordination of numerous
division was possible; 1st son got one-half or 9 horses; metabolic reactions inside the cells. Proteins can be
2nd son got 6 and 3rd son 2 horses. Now all the 17 white hydrolyzed with hydrochloric acid by boiling for a very
horses were correctly divided among the sons. The long time; bu_t
remaining black horse was taken back by the brother-in-
law. Catalysts are similar to this black horse. U D nzyme catalysis is very rapid; usually 1 mo e-
_, A chemical reaction, although theoretically prob- cule of an enzyme can act upon about 1000 molecules
able, becomes practically possible only with the help of of the substrate per minute. Lack of enzymes will lead to
Chapter 5: Enzymology: General Concepts and Enzyme Kinetics 53
a
Eduard Arthur James John Wilhelm
Buchner Harden Sumner Northrop Ostwald
NP 1907 NP 1929 NP 1946 NP 1946 NP 1909
1860-1 91 7 1865-1940 1887- 1955 1891- 1987 1853-1932
block in metabolic pathways causing inborn errors of First digit represents the class
metabolism. Second digit stands for the subclass
The substance upon which an enzyme acts, is called Third digit is the sub-sub class or subgroup
the substrate. The enzyme will convert the substrate Fourth digit gives the number of the particular
Q the product or products. enzyme in the list.
The enzymes are grouped into following six major
classes (Box 5.1).
i. Almost all enzymes are proteins. Enzymes follo lass 1: Ox1doreduc ses
the physical and chemical reactions of proteins.
This group of enzymes will c.aialyze o ~ of ~
ii. They are heat-labile~
~ cate with simultaneous re,duction of geethAc sub-
iii. They are water-soluble.
~ rate4 oenz9rne. This may be represented as
iv. They can ~ . I ecipitated by protein r recipitating
• reagents (a~ oft~§6Hlate or trich~f6ae ~<2~).
fl5 AH 2 +B - A+ BH2
For exam " ,.
Hexose-6-phosphate + ADP
The name of enzyme is Hexokinase.
j
IQBIIB SysJem of Classification C~2.~\\)
lnternation I Union of Biochemistry and Molecular Bi.,.,.. .........a s 3: Hidrolases
(D
logy (IU suggested the IUBMB system of nomen- This class~ / enzymes can hydrolyze er, er, pep--
w as first introduced in 1964 , tide or glyM sidic bonds by adding water and then break-
ing the bond.
Acetylcholine + Hp-----+ Choline + ace;.-ta
. t_e_ _ _
.....
enz me is Ace !choline esterase(AII diges;,
Uve enzymes are hy~
54 Section A: Chemical Basis of Life
BOX 5.1: Classification of enzymes BOX 5.2: Synthetase and synthase are ' . .
Cla. : Olridon!ductases-Transfer of hydrogen or addition of Synthetases are ATP-dependent enzymes cat alyzing biosyn-
oxygen; e.g. Lactate dehydrogenase (Coenzyme, NAO•); Glucose- thetic reactions; t hey belong to Ligases (class 6). Examples are
6-phosphate dehydrogenase (Coenzyme, NADP•); Succinate t;acbamoyl phosphate synthetase; ~ •succinate synthe-
dehydrogenase (Coenzyme, FAD); dioxygenases. tase; PRPP synthetase anrl 'ililTilWiPG§YOSbGTiil-Se,
a., ~ !es- Transfer of groups other than hydrogen. Synthases are enzymes catalyzing biosynthet ic reactions; but
Example, Aminotransferase. (Subclass: Kinase, transfer of phos- they do not require ATP directly; they belon g to classes o ther
phoryl group from ATP; e.g. Hexokinase). than Li gases. Examples are Glycogen synthase and ALA synthase.
c,-
Glyceraldehyde-3-phosphate Dilhydroxy acetone
phosphate
Enzyme is Triose phosphate isomorase.
~ - lntramolecular t ransfers. They include race-
mases and epimerases. Example, Triose phosphate isomerase.
~ ses-ATP dependent condensation of two molecules, lass&: Ligases
e.g. Acetyl-CoA carboxylase; Glutamine synthetase; PRPP syn-
thetase.
These enzymes link two substrates together, usually
with the siw':!Jtaneous bydcolysis at AIi~ (Latin, Ligare =
to bind). For example,
BOX 5.3: Salient features of coenzymes Acetyl-CoA + CO Malon I-CoA + ADP +Pi
1. The protejn part of the enzyme gives the necessary three- Enzyme i Acetyl-CoA carboxylas
dimensional infrastructure for chemical reaction; but the
A summary of classification is giiven in Box 5.1.
group is transferred from or accepted by t he coenzyme.
2. The coenzyme is essential for the biological activity of the The differences between synthetase and synthase are
enzym e. shown in Box 5.2.
3. Coenzyme is a low molecular weig ht organic substance. It is
heat-stable.
4. Generally, t he coenzymes combine loosely with the enzyme COENZYME § + J.t Ui,.+-~.~
+N u.f!\e.o'1o.-
<Pcfi-
molecules. The enzyme and coenzyme can be separated
easily by d ialysis.
Enzymes may be simple proteins, or complex enzymes,
5. Inside the body, when the reaction is completed, t he co- containing a non-protein part, called the prosthetic
enzyme is released from the apo-enzyme, and can bind to
group. The prosthetic group is called the coenzyme. It
another enzyme molecule. In the example shown in Figure
5.1, the reduced coenzyme, generated in the first reaction is heat stable. Salient featu res of coenzymes are shown
can take part in the second reaction. The coupling of these in Box 5.3. The protein part of the enzyme is then named
t wo reactions becomes essential in anaerobic glycolysis
the apoenzyme. It is heat labile. ThHse two portions
(Chapt er 1OJ for regeneration of NAO•.
6. One molecule of t he coenzyme is able to convert a large combined together are called the holocmzyme.
number of substrate molecules with the help of enzyme. Coenzymes may be divided into two groups:
7. Most of the coenzym es are derivatives of vitamin B complex
sub stances. a. Those taking part in reactions by oxido-
reductases by donating or acc ,epting hydrogen
atoms or electrons.
b. Those coenzymes taking part in reactions transfer-
These enzymes can remove groups from substrates @ ring groups other than hydrogen .
. or break bonds by mechanisms other than hydrolysis. First Group of Coenzymes
~ r example, J'l'\ll:70n ,•
ructose-1 , - In the fi rst group, the change occurring-¾
phate +dihydroxyacetone phosphate the substrate is counter-balanced by the
_ The enzyme is Aldolase (see Chapter 1O for details). coenzymes. Therefore, such coenzymes
may be considered as co-substrates or
Class 5: lsomerliil ,- s~ ry_ sub~ s. In the example
These enzymes can produ optical, geo tric or posi- shown in Figure 5.1, the substrate lactate Otto Heinrich
is oxidized, and simultaneously the COE!n- Warburg
tional isomers of substrate Racemase p imerases,
NP 1931
----------
C, is-trans isomerases are examples. zyme (co-substrate ) is reduced . When the 1883-1 970
Chapter 5: Enzymology: General Concepts and Enzyme Kinetics 55
Glyceraldehyde-3-phosphate
coo- Lactate
L
dehydrogenase dehydrogenase
Glyceraldehyde-3-P ( '\ 1-3 bisphosphoglycerate
CHOH
(') •
NAt NADH+ H+
CH3 NAO+ NADH+
Lactate · - )
Lactate dehydrogenase
Pyruvate
Lactate Pyruvate
F ig. 5.1: One coenzyme molecule can work with diff rent enzymes F ig. 5.2A: Reaction of lactate dehydrogenase
r-1 At>.,. \?Ut..':;) ~- C).CC.e.J)~
H
l ) ~ r \. N
Pyridoxal phosphate (PLP) Amino ~Jroup -N t'i"
Carbon dioxid e -CD
I 2H H+ I
R
Acyl groups -
R NAO• NAOH One carlbon groups
Fig . 5.28 : NAO• accepts hydride ion Adenosine triphosphate (ATP) Pho ·:11e
-
coenzymes and metal ions. Caeozyrn9 is an or:gaoic co- Histidine 12 acts as an acid and donates a proton. Histi-
factor. dine 119 accepts a pro ton and product is released.
Chapter 5: Enzymology: General Concepts and Enzyme Kinetics 57
A
I
B
I
C
______ 1_________ (A) Unfolded protein
with no activity ____ _
I Folding
process
(B) Folded protein
with enzyme activity
D
35Glu --+ I Asp 52
_,,µ.__ _ _ _ Amino acid side
E
chains from the
I active center of enzyme
F
Enzyme
Fig. 5.6: Active center of lysozyme. A, B, C, D, E, F are carbo- Fig. 5.7: Correct alignment of amino acids in the active center of
hydrate units (substrate). D = N-acetyl muramic acid; E= N-acetyl the enzyme
glucosamine. Bond is broken between D and E, with the help of Glu
and Asp residues in the enzyme, which are opposite to each other
attacks the substrate. This results in covalent bjndjna of
the substrate to the enztme.
••
complex on the enzyme; Enzyme- accept analog
substrate complex (ES) formed
•
-+ Fig. 5.9: Fischer's template theory
Active
site
•Substrate
Fig. 5.10: Enzyme and substrate are specific to each other. This
is similar to key and lock (Fischer's theory)
2 3 4
Alkaline phosphatase hydrolyzes a number of phos- with the substrate. At first, substrate binds to a specific
phate esters including glucose-6-phosphate. The active part of the enzyme. This leads to more secondary binding
cente·r of this enzyme contains a Serine residue, and the and conformational changes. The substrate induces
reaction is taking place in the following two steps: conformational changes in the enzyme, such that
a. E-Serine-OH+Glucose-6-P~E-Serine-O-P+Glucose precise orientation of catalytic groups is eff~ted (Fig.
b. E-Serine-O-P E-Serine-OH+Pi 5.7). A simplified explanation is that a glove is put on
Thus, the overall reaction is a hand. At first, the glove is in a partially folded position,
Glucose-6-P Glucose + Pi but hand can enter into it. When the hand is introduced,
In this reaction mixture, the enzyme substrate.c~m- the glove is further opened. Similarly, conformational
plex, E-Serine-0-f, has been isolated . changes occur in the enzyme when the substrate is
fixed (Fig. 5. 11 ).
When substrate analog is fixed to the enzyme, some
structural alteration may occur; but reaction does not
It states that the three-dimensional structur e active
take place due to lack of proper alignment (Figs. 5.7 and
site of the enzyme is complementary to the substrate.
5.9). Allosteric inhibition can also be explained by the
Thus enzyme and substrate fit each other. Substrate
hypothesis of Koshland.
fits on the enzyme, similar to lock and key. The lock
can be opened by its own key only (Figs. 5.9 and 5.10).
However, Fischer envisaged a rig id structure for enz-
ymes, which _could not explain the ·flexibility shown by
enzymes.
BOX 5.4: Active center of enzyme TABLE 5.4: Active center of enzymes
1. The region of the enzyme where substrate binding and Name of enzyme Imp ortant amino acid at the catalytic site
catalysis occurs is referred to as active site or active center Chymotrypsin His (57), Asp (102), Ser (195)
(Tables 5.3 and 5.4).
Trypsin Serine, Histidine
2. Although all parts are required for maintaining the exact
three-dimensional structure of the enzyme, the reaction is Thrombin Serine, Histidine
' taking place at the active site. The active site occupies only a
small portion of the whole enzyme.
Phosphoglucomutase Serine
Alkaline phosphatase Serine
3. Generali active site is situated in a crevice or cleft of the
Acetylcholinesterase Serine
enzyme molecule (Fig. 5.7). Tot e active site, the specific sub-
strate is bound. The binding of substrate to active site depends Carbonic anhydrase Cysteine
on the alignment of specific groups or atoms at active site. Hexokinase Histidine
4. During the binding, these groups may realign themselves to Carboxypeptidase Histidine, Arginine, Tyrosine
provide the u.!!i9ue conformatjonal orien!;ion so as to ~ -
Aldolase Lysine
mote exact fitfipg of substrate to the active site (Fig. 5.7).
5. The substrate binds to the enzyme at thea ctive site by non-
covalent bonds. These forces are hydrophobic in nature.
Urea > ammonia + CO2 + energy
The amino acids or groups that directly participate in making
or breaking..the. boods (present at the active site) are called At equilibrium of this reaction, the substrate will be
catalyticres1dues or catalytic groups. only 0.5% and product will be 99.5%. Such reactions are
J,,s;., The active site contai r i ~strate binding site and catalytic
generally irreversible.
site; sometimes these two may be separate.
lsothermic Reaction
When energy exchange is negligible, the reaction is
easily reversible, e.g.
Glycogen+ Glucose-1-phosphate
Catalysis occurs at the active center or active site. Sali-
At equilibrium of this reaction, 77% glycogen will be
ent features are shown in Box 5.4. See Tables 5.3, 5.4
unutilized and 23% glucose-1-phosphate will be formed.
and Figure 5.7 also.
Proteolytic enzymes having a serine residue at the
Endergonic or Endothermic Reaction
active center are called serine proteases, e.g. pan-
creatic proteases (Table 5.3), and coagulation factors. Energy is consumed and external energy is to be sup-
her examples are given in Table 5.4. plied for these reactions. In the body, this is usually
accomplished by coupling the endergonic reaction with
ERMODYNAMIC an exergonic reaction, e.g. Hexokinase catalyzes the
CONSIDERATIONS
-------
From the standpoint of energy, the enzymatic reactions
following reaction:
Glucose Glucose-6-Phosphate + ADP
BOX 5.5: Derivation of equ1l1bnum constant BOX 5.6: Derivation of Michaelis constant /Km)
Va. [Al [Bl Kl
At equilibrium, forward reaction and backward reaction are E+S Ki E-S E+ P
equal, so tha .____.,
"
If concentration of substrate is increased, the forward reaction Kl
is increased, and so K3 as well as total velocity is correspondingly
:J
enhanced. The three different constants may be made into one .t
1
• • • • I
Forward reacti
and backward reaction R2 = - - - --- -
At equilibrium, Rl -·
-----
_
--
Or, Kl [Al [Bl
Or, Kl = [Cl [DJ
K2 = [Al [Bl
K2 [C][D]
Keq or
Equilibrium constant.
- It . f rth h
aelis Constant
th
_,_
~_cm,.;;_.....
...... ---
l
l FACTORS INFLUENCING
'ENZYME ACTfV
;;..;..;;.;l:;.T..;..
.;;. Y_ __.---w~"'
The various factors which affect enzyme activity are
enumerated in Box 5.7. These are explained below.
.___ _ _ __ --'-
[E-'--1_ __ _ _ __ _ 4)Enzyme Concentration
Fig. 5.12: Effect of enzyme concentration
Rate of a reaction or velocity (V) is directly propor-
tional to the enzyme concentration, when sufficient
1. The equilibrium constant of the reaction is the
substrate is present. Velocity of reaction is increased
ratio of reaction rate constants of forward and back-
proportionately with the concentration of enzyme, pro-
ward reactions.
vided substrate concentration is unlimited (Fig. 5.12).
2. At equilibrium, forward and backward reactions are
Hence,Qhis property is made use of determining the
equal. Equilibrium is a dynamic state. Even though level of particular enzyme in plasma, serum or tissue9
n concentrations of substrate and ( Known volume of serum is incubated with substrate for a
fixed time, then reaction is stopped and product is quan- 1'
titated (endpoint method). Since the product formed
3. Numerical value of the constant can be calculated will be proportional to the enzyme concentration, the lat-
by finding the concentrations of substrates and ter could be assaye'
products.
4. If Keq is more than 1, the forward reaction is favo...&)Effect of Subs~ate c_oncentratio
red. In such instances, the reaction is spontaneous In a series of test tubes, equal volume of enzyme solu-
and exothermic. tion is taken, but increasing quantity of substrate is added
5. Concentration of enzyme does not affect the Keq. and the rate of reaction is assayed in each tube. The
Concentration of enzyme certainly increases the rate velocity (v) is expressed in micromoles of substrate con-
of reaction; but not the Keq or the ultimate state. In verted per minute.
Vmax Maximum velocity (Vmax)
½ Vmax
[SJ
Km
~~-\
value are shown in Box 5.8.
3
,o &,13~\ \'c.
-'r [ he lesser the numerical value of Km, the affinity
aeHs Constant 'm
of the enzyme for the substrate is more. To cite an ?
According to Michaelis theory, the ormation of enzyme- example, Km of glucokina&,e is 10 mrr,s:>1/L and that of
substrate complex is a reversible reaction, while the hexokinase is 0.05 mmol/LJ Thereforel_50% molecules
breakdown of the complex to enzyme + product is irre- of hexokinase are saturated even at a lower concentra-
versible. (See the derivation of the Michaelis constant in tion of glucos, {ln other words, hexokinase has more
Box 5.6). affinity for glucose than glucokinasy ]
62 Section A: Chemical Basis of Life
Km
Vmax
i
•• V
-
-1/Km 1/[SJ
i
V
down. So when product concentration is increased, the
reaction is slowed, stopped or even reversed. In inborn
venoJu.y-ol~ /. errors of metabolism, one enzyme of a~ etab lie ~athway
.___~..........,___,- ---~ ©
0 10 20 30 40 50 60
is blocked. For example, 1-l.,J Ci
Temperature in •c 2 E2
A~ B C - II ~ D
Fig. 5.17: Effect of temperature on veloci ty
CH2
II COOH
FADH2 C- H
I I
COOH COOH
P 2 4 6 't 8 10 12
iv. Coagulation factors are seen in blood as zymo-
Fig. 5.18: Effect of pH on enzyme velocity
gen form, their activation pathways are described
{ in Chapter 26.
1N These@_tivities are needed only occasional~ but
when needed, a large number of molecules are to be
produced instantaneously. Hence, the sascade systerp
of chemical amplification of such factors.
"
- 1/Km
Vmax
9Le
e Substrate
< >.
E-S complex
l7
'----+Prodoct
H 2N COOH
a Noncompetitive
inhibitor
Fig. 5.21 : Competitive inhibition
• Substrate
Vmax Without
inhibitor
With
fvmax 1nh1b1tor
=~-1
the heavy metal ions can react and thereby their enzyme. Inhibitor binds to enzyme-substrate complex;
poisonous effects are reduced. but not to the free enzyme. In such cases both Vmax
e. Acetylcholin esterase enzyme cleaves acetylcho- and ~m re decreased (Fig. 5~ 4 ). Inhibition of
1i line to form acetate and choline and therefore ter- alkaline tase Re.9art-:is'aloz¥me) by p a~-
minates the action of acetylcholine. Certain chemi- nine is an example of uncompetitive inhibition.
cals e.g. .diworogyl fluorophosphate (DFP) binds • • • d
to the active site, serine of acetylcholine esterase. t>.Suicide lnh1b1t1on ( M..e.c.Y'I OX")~'lo')in~,'tlE:'>'n)
As a result acetylcholine accumulates and over- It is a special type of irreversible inhibition of enzyme
stimulates autonomous nervous system including activity. It is also known as mechanism based inac-
heart, blood vessels and glands. This leads to vomi- tivation. The inhibitor makes use of the enzyme's
ting, salivation, sweating, and in worst cases even own reaction mechanism to inactivate it (mechanism
death. DFP forms an irreversible covalent bond based inactivation). In suicide inhibition, the structural
with acetylcholine esterase, and activity can be analog is converted to a more effective inhibitor with the
regained only if new enzyme is synthesized. help of the enzyme to be inhibited. The substrate-like
66 Section k Chemical Basis of Life
A
s
r -"7"'...::;,.- -.,,c--
With positive
modifier
AA ,___ ____ _ _ _ Sigmoid curve
\ _ _ y ! - Ain
½ Vmax
With negative
modifier
A= Active site; Allo = Allosteric s1le; S = Substrate;
AA= Allosteric activator; In = Inhibitor site;
Ain =Allosteric inhibitor. The enzyme has separate active site
and allosteric site. Figure 1 depicts that the activator is fixed at
the allosteric site, when active site has correct conformation,
and the substrate is correctly fixed. Figure 2 shows that the Fig. 5.26: Allosteric inhibition
inhibitor is fixed at the allosteric site when active site is deformed
and the substrate could not fix.
alloxanthine which is a more potent inhibitor of xanthine
Fig . 5.25: Action of allosteric enzymes
oxidase (see Chapter 38).
The anti-inflammatory action of Aspirin is also
BOX 5.10: Sahenl '" " -~. dllo~l"'"" regulation based on suicide inhibition. Arachidonic acid is converted
1. The inh ibitor is not a substrate analog. to prostaglandin by the enzyme Cyclo-oxygenase (see
2. It is partially reversible, when excess substrate is added Chapter 16). Aspirin acetylates a serine residue in the
3. Km is usually increased active center of cyclo-oxygenase, thus prostaglandin
4. Vmax is reduced synthesis is inhibited, and so infla
2...
5. The effect of allosteric modifier is maximum at or near sub-
strate concentration equivalent to Km (Fig. 5.26). When an f .Allosteric Regulatio
M
c;:~+me.chl
-+2,_eq ~\,I
~()XP>
Inhibitor binds to the allosteric site, the configuration of cata-
lytic site is modified such that substrate cannot bind properly Allosteric enzyme has one c ytic site where the sub-
6. Most allosteric enzymes possess quaternary structure. They strate binds and another separate allosterlc site where
are made up of subunits, e.g. Aspartate transcarbamoylase the modifier binds (a/lo = other) (Fig. 5.25). Allosteric
has 6 subunits and pyruvate kinase has 4 subunits. Examples
and substrate binding sites may or may not be physi-
of allosteric enzymes are shown in Table 5.7.
cally adjacent. The binding of the regulatory molecule
can either enhance the activity of the enzyme (allosteric
compound initially binds with the enzyme and the fi rst Cactivation), or inhibit the activity of the enzyme (allosteric
few steps of the pathway are catalyzed. This new product o)hibition). In the former case, the regulatory molecule is
irreversibly binds to the enzyme and inhibits further
known as the positive @>difier and in the latter case as
reactions.
the negativ difler. The binding of substrate to one
For example, ornithine decarboxylase (ODC) cata-
of the subuni s of the enzyme may enhance substrate
lyzes the conversion of ornithine to putrescine which is
binding by other sub-units. This effect is said to be posi-
necessary for p,Q!yamine synthesis (see Chapter 18).
tive@ operativity. If the binding of substrate to one of
When the ODC in trypanosoma is inhibited , multiplica-
the subunits decreases the avidi!)' of substrate binding by
tion of the parasite is arreste~. Ther~fore inhibitors of
other sites, the effect is called negative operatlvlty.
ODC enzyme such as difluordmethylornithine (DFMO)
' .
has been found to be effective against trypanosomla- In most cases, a combination is observed, resulting in
sis (sleeping sickness). DFMO is initially inert, but on a sigmoid shaped curve (Fig . 5.26). Salient features of
binding with the enzyme, forms irreversible covalent allosteric regulation are enumerated in Box 5.10.
complex with the coenzyme (pyridoxal phosphate) and f' K E
the amino acid residues of the enzyme. In mammalian ey nzyme
cells, the turnover rate of ODC is very high, and so-the· Body uses allosteric enzymes for regulating metabolic
inhibition by DFMO is only transient. So D'FMO kill s the pathways. Such a regulatory enzyme in a particu-
parasites with no side effects to the patient. lar pathway is called the key enzyme or rate limiting
A similar mechanism is observed in the case of enzyme. The flow of the whole pathway is constrained
Allopurinol that is oxidized by xanthine oxidase to as if there is a bottle neck at the level of the key enzyme.
Chapter 5: Enzymology: General Concepts and Enzyme Kinetics 67
A E1 B E2 C ~
---
Allosteric Allosteric
inhibitor activator Chapters In this pathway, if D inhibits E1 , it is called feedback
ATP, citrate AMP, F-2,6-P 10 inhibition.
Heme 22 One example is the inhibition of ALA synthase by
3. Aspartate trans- CTP ATP 38 heme. Other examples are AMP inhibiting the first step
carbamoylase
in purine synthesis (see Chapter 38). Usually such end
4. HMG CoA reductase Cholesterol 14
product inhibition is effected allosterically. Inhibition of
. Pyruvate carboxylase ADP Acetyl-CoA 10
aspartate transcarbamoylase by CTP is another example
. Acetyl-CoA carboxylase Acyl-CoA Citrate 13
for end product inhibition.
7. Citrate synthase ATP 20
8. Carbamoyl phosphate NAG 17 . Induction ~--~--e..-l-~--,J
synthetase I
9. Carbamoyl phosphate UTP 38 Induction is effected through the process of derepres-
synthetase II
sion. The inducer will relieve the repression on the oper-
ator site and will remove the block on the biosynthesis of
The allosteric inhibitor is most effective when substrate the enzyme molecules. Classical example is the induc-
concentration is low. This is metabolically very significant. tion of lactose-utilizing enzymes in the bacteria when
When more substrate molecules are available, there is the media contains lactose in the absence of glucose
less necessity for stringent regulation. (details in Chapter 42). There will be a minimal level of
A few examples are given in detail below: the enzyme inside the cell, but in presence of the indu-
a. Phosphofructokinase-1
cer, the level will go up to thousand or million times within
J
by insulin LA synthase is induced by barbiturates.
'1r __
an allosteric inhibitor (negative modifier) of PFK1.
When the cellular level of ATP rises, glycolysis can •
slow down and ATP binds to the allosteric site, thus!.Repression ~1-"'-.. _
inhibiting the reaction. A Wgb !eye! of AMP within Even though both inhibition and repression reduce the
the cell jndjcates a low level of ATP. Hence AMP enzyme velocity, the mechanisms are different. In the
acts as an allosteric activator (positive modifier) of case of inhibition, the inhibitor acts on the enzyme
the enzyme. directly; the inhibitory activity is noticed as soon as the
b. ALA synthase inhibitor is added; and the number of enzyme molecules
Succinyl-CoA + - - - delta- amino-levulinic is not changed by the inhibitor.
Glycine acid (ALA) On the contrary, repressor acts at the gene level;
the effect is noticeable only after a lag period of hours or
This is the @ p in Siiiii~im¥Dltbu~ The
days; and the number of enzyme molecules is reduced
end product, heme will allosterically inhibit the ALA
in the presence of repressor molecule. Details of repres-
synthase. This enzyme is the - .,-.u.'fme of heme
sion are given in Chapter 42. A summary of the mecha-
esi~ (see Chapter 22). Other such allosteric
nism of repression is given in Figure 5.27.
enzymes are listed in Table 5.7.
The key enzyme of heme synthesis, ALA synthase
is autoregulated by heme by means of repression. The
GJ. Feedback Inhibition structural gene is transcribed and later translated to pro-
The term feedback inhibition or end-producl inhibition duce the enzyme molecules. The transcription process
means that the activity of the enzyme is inhibited by the starts at the operator site when it is free. When heme
final product of the biosynthetic pathway. is not available, this operator site is open, and therefore
68 Section A: Chemical Basis of Life
-
ooc - Hc coo-
Regulator Operator Structural genes
II
c- coo-
l
CH-OH
! i.
H
I
A~orepressor } Holorepressor
t
ALA synthase
Fumaric acid (trans)
TABLE 5.8: Examples of covalent mod1ficat1on For example, heme synthesis (see Chapter 22), urea
Enzyme Phosphorylated enzyme cycle (see Chapter 17), gluconeogenesis (see Chapter 10).
Acetyl-CoA carboxylase Inact ive The intermediates have to be shuttled across the mito-
Glycogen synthase Inactive chondrial membrane for this purpose which provides a
Pyruvate dehydrogenase Inactive point where controls can be exerted.
HMG-CoA reductase Inactive
Pyruvate kinase Inactive
PFK2 Inactive
enzymes are proteins. Invariably, all rules will have 1. They may be products of different genes (more
exceptions. Ribozymes are RNA molecules with enzy_
matic activity, which catalyze cutting of in mRNA than one locus) in which case they are known as
:r::~ary trans~ t (see Chapter 40 ). true i"soenzymes. The genes may be located on
different chromosomes, e.g. salivary and pancrea-
tic amylase.
14 ody Metabolism is certain cases, all the different forms are presen
Controlled by Enzyme . . . _ ~ .,,· the same individual, e.g. lactate dehydrog
1
(1!1. a pipe, theoretically water can ltotlV'1be'l~ ~ lle":'"y)j::tt"""' nase (LOH) has 5 isoenzymes and all are seen i
¢ira>rficajjy me rjow 1s Ohldlfettlo(™. In the same way, all persons in the population (see Chapter 6).
although most of the individual enzyme activities are 3. The same locus of the gene may have diffetent
readily reversible, the pathway as a whole, tends to lead alleles alternate forms). Such alleleic isoenzymes
towards the final produ~ are called allozymes. In this case, only one form
/ n the reaction series, B C, as soon as C is will be present in one individual; but all the different
pro~ d, it is utilized for something else, so that the forms will be seen in total population. For example,
reactions as a whole, tend to go in the forward direction more than 400 distinct form of glucose-6-phos-
only. True equilibrium of chemical reactions in the cell phate dehydrogenase (GPD)~have been identi-
is achieved only when the cell is dead. In other words, fied; all of them are produced by the same locus
living cells avoid the state of chemical equilibrium. At oo the X chromosome. When isoenzymes due to
the same time, if we analyze a particular metabolite, the variation at a single locus occur with appr_eciable
concentration is more or less kept constant over_very frequency (more- than 1 % in population), it is said to
l_ong periods. This is called steady-state systerv For be
( example, QJ,Qnd .glucose lev.el is kept ithin a narrow 4. Molecular heterogeneity of enzymes may also be
\ n~. because the factors, which tend to increase the produced after the protein is synthesized -
level are at dynamic equilibrium with the factors that try These are called iso-
to decrease the leve} forms, e.g. sialic acid content in alkaline phospha-
Deficiency of the enzyme or its activit~ will lead to tase (ALP) isoenzymes. Different types of iso-forms
genetic defects, inborn errors of metabolism. may be seen in the same individual.
ISOENZVM
They are physica istinct forms same 1. In Agar gel or polyacrylamide gel electrophoresis
enzyme activity; Multiple molecular forms of an enzyme the isoenzymes have different mobility.
are described as iso-enzymes or isozymes. If One-rupee' n be separated by electro-
coins are examined carefully, there will be minor varia- phoresis.
tion~ of '}dges on the rims and number of dots below 2. Heat stability: One of the isoenzymes may be
i the year.\.ln ,tt,e market all these coins have the same easily denatured by heat, e.g.
face valuek ut to an experienced numismatist, these
variations will explain from which mint it was produced) 3. Inhibitors: One of the isoenzymes may be sensi-
In the same way, different molecular forms of the same tive to one inhibitor,
enzyme synthesized from various tissues are called iso- 4. Km value or substrate specificity may be different
enzymes. Hence, study of isoenzymes is very usefu,I to for isoenzymes,
understand diseases of different organsfuthe subunits
are all the same, the protein is a homomultime., repre- 5. Cofactor requirements may be different for iso-
sented by a single gene. If the subunits are different, enzymes.
protein is said to be a heteromultimer produced by t and the . ·:tl..'-1.:L.:
different gene1
70 Section A: Chemical Basis of Life
29. 11
by reversible protein phospho-
,-
30. are physically distinct forms of the
rylation or zymogen activation is a common mecha- same enzyme activity. They may be products of the
nism of short term regulation. Rate of synthesis of same gene or different genes.
enzyme proteins may be induced or repressed for
long term regulation.
-" 5-12.
5-13.
Michaelis Constant (Km).
Coenzymes.
5-17.
5-18.
Proenzymes.
Zymogens.
5-14. Metalloenzymes. 5-19. Effect of pH on enzyme activity.
5-15. Active site of enzyme. 5-20. Optimum temperature.
5-16. Kosh land's induced fit theory.
5-1 . An example of lyase is: 5-5. All the following statements are true with regard
A. Glutamine synthetase to the enzymes called synthases and synthetases,
B. Fumarase except:
C. Cholinesterase A. Synthetases and synthases are synonyms
D. Amylase B. Synthetases are enzymes belonging to ligases
5-2. The enzyme belonging to the class ligase is: C. Glutamine synthetase is belonging to class of
A. Glycogen synthase ligases
• B. Glutamine synthetase D. Glycogen synthase belongs to the class transfe-
C. Porphobilinogen deaminase rases
D. Histidine decarboxylase 5-6. The coenzyme:
5-3. Coenzymes are: A. Increases the affinity of apoenzyme to substrate
A. Dialyzable, nonprotein molecules B. Lowers the activation energy
B. Colloidal protein molecules C. Increases the number of active sites on the enzyme
C. Structural analogues of enzymes D. Accepts one of the products of the reaction
D. Different forms of the same enzyme 5-7. All the following metalloenzymes contain copper,
5-4. Digestive enzymes belong to the class of: except:
A. Hydrolases B. Ligases A. Superoxide dismutase
C. Lysases D. Oxidoreductases B. Tyrosinase
72 Section A: Chemical Basis of Life
C. Glutathione peroxidase C. Treatmen t of methanol poisoning by ethanol
D. Cytochrome oxidase D. Use of dicoumarol as an anticoagulant
5-8. Which one of the following enzymes is containing 5-17. In competitive inhibition:
molybdenum? A. Inhibitor has structural similarity to substrate
A. Cytochrome oxidase B. Km is decreased
B. Xanthine oxidase C. Vmax is decreased
C. Carbonic anhydrase D. Reaction rate is independent of substrate concen-
D. Phosphoglucomutase tration
5.9. The enzyme: 5-18. In a competitive inhibition:
A. Reduces the energy of activation A. Km is increased and Vmax is increased
B. Increases total energy of substrate
B. Km is decreased and Vmax is normal
C. Increases the equilibrium constant
C. Km is increased and Vmax is normal
D. Increases total energy of the product
D. Km is decreased and Vmax is increased
5.10. In enzyme kinetics, Vmax denotes:
5-19. The antimetabolite, methotrexate acts by:
A. The amount of an active enzyme
A. Noncompetitive inhibition
B. Substrate concentration
B. Formation of a covalent bond between enzyme
C. Half the substrate concentration
and inhibitor
D. Quantity of enzyme substrate complex
5-11. All the following statements are true with regard C. Increasing the Vmax of the reaction
to enzymes, except: D. Competitive inhibition
A. Enzymes lower activation energy 5-20. Ascaris worm is not digested by intestinal enzymes,
B. They alter equilibrium of the reaction because:
C. They accelerate the chemical reaction A. The worm produces a trypsin inhibitor
D. Most of the enzymes are proteins in nature B. Ascaris protein stimulates digestive enzymes
5-12. The Km value of an enzyme is: C. Ascaris has a tough cuticle
A. The substrate concentration at half maximal velocity D. Proteins of the worm is resistant to trypsin
B. Half the substrate concentration at maximal velocity 5-21 . The following are salient features of allosteric
C. Dissociation constant of enzyme-substrate complex enzymes, except:
D. The total enzyme concentration A. They are usually multiple subunit enzymes
5-13. Lineweaver-Burk (double reciprocal) graph is plot- B. They are involved in metabolic regulation
ted by: C. They do not show any change in Km and Vmax in
A. Substrate concentration against Vmax the presence of modifiers
B. Reciprocal of substrate concentration against D. On binding the inhibitor, usually Km is increased
reciprocal of velocity and Vmax is decreased
C . Reciprocal of initial velocity against substrate con- 5-22. Allosteric enzymes show all the following charac-
centration
teristics, except:
D. Km against initial velocity
A. Co-operative binding of the substrate
5-14. When substrate concentration is equal to Km value:
B. Sigmoid kinetics
A. Half of the enzyme molecules are bound to the
C. Binding between substrate and regulatory sites
substrate molecules and the other half are free
D. Substrate binding site and regulatory site are dif-
B. Maximum velocity is achieved
ferent
C. Maximum enzyme molecules are taking part in the
reaction
5-23. All the following enzymes are examples of cova-
lent modifications, except
•
D. The reaction is now at equilibrium
5-15. Enzymes which are synthesized in inactive form A. Chymotrypsinogen
are called: B. Pyruvate kinase
A. Coenzymes B. Apoenzymes C. Glycogen phosphorylase
C. Lysozymes D. Proenzymes D. Aspartate transcarbamoylase
5-16. All the following are examples of competitive inhi- 5-24. All are multienzyme complexes, except:
bition, except: A. Fatty acid synthase
A. Inhibition of enolase by fluoride ions B. Pyruvate dehydrogenase
B. Inhibition of dihydrofolate reductase by ametho- C . Alpha-ketoglutarate dehydrogenase
ptrine D. Glycogen synthase
Chapter 5: Enzymology: General Concepts and Enzyme Kinetics 73
5-45. The lock and key hypothesis explains all features C. Cannot be extrapolated from the double reciprocal
of enzyme action except: plot
A. Specificity of enzyme action D. A constant for all enzyme substrate pairs .,
B. Binding of substrate to active site 5-54. Enzyme inhibition is applicable in all except:
C. Three dimensional shape of active site A. Regulation of metabolic pathways
D. Effect of regulatory molecules B. Use of drugs as enzyme inhibitors l
5-46. Koshland's induced fit theory explains C. Enhance the action of toxins
A. Rigidity of active site D. To check excess effect of enzymes
B. Effect of modifiers on substrate binding 5-55. Reversible enzyme inhibition is:
C. Complementary shape of active site and substrate A . By a substrate analog
D. Presence of specific catalytic residues at active site B. Exhibited by toxins
5-47. Which of the following factors have NO effect on C. Characterized by a change in Vmax only
velocity of enzyme catalyzed reactions? D. Does not occur in biological systems
A. Concentration of substrate 5-56. The statement which is NOT correct regarding
B. pH of the medium competitive inhibition is:
C. Temperature of the medium A. Reversible
D. Equilibrium constant of the reaction B. Decreases Km
5-48. Velocity of enzyme action is dependent on all C. Decreases Vmax
except: D. Inhibitor binds to active site
A. Concentration of e nzyme molecules 5-57. The inhibitor increases the Km and its effect can
B. Turn over rate of enzyme molecules be reversed by increasing the substrate conce n-
C. Rate of synthesis of enzyme protein tration. Which of the following is an example of
D. Molecular weight of the enzyme protein competitive enzyme inhibition?
5-49. Maximum velocity is seen when: A. Effect of cyanide
A. All enzyme molecules are bound to substrate B. Inhibition of glycolysis by fl uoride
B. Substrate molecules are far in excess of enzyme
molecules
C. Arresting cell division by methotrexate
D. Toxic effect of arsenate •
C. Specificity of substrate binding is high 5-58. Which of the following is a folate antagonist in
D. Activators are present in the medium eukaryotes?
5-50. Km value indicates: A. Sulphanilamide
A. Affinity of enzyme for substrate B. Trimethoprim
B. Maximum velocity C. Amethopterine
C. Specificity of substrate D. Azaserine
D. Saturating concentration of substrate 5-59. All are mechanisms of enzyme regulation In bio-
5-51 . Michaelis-Menten substrate saturation curve is logical systems except:
obtained by plotting [S] against [V] Since Michae- A. Covalent modification
lis-Menten graph is a hyperbolic curve. B. End product inhibition
A. Binding properties of the enzyme are not clear C. Enzyme induction
B. Km cannot be accurately measured D. Enzyme denaturation
C. Velocity is not proportional at very low concen- 5-60. Allosteric enzymes can be regulated by the bind-
!ration of substrate ing of positive or negative modifiers to the allos-
D. Is not helpful to study effect of inhibitors teric site, thus affecting substrate binding to active i-
5-52. A double reciprocal plot, 1/[SJ Vs 1N is a straight catalytic site. Which of the following metabolic
line graph. All are true regarding the Lineweaver intermediates is an effective allosteric modifier?
Burke plot except: A. Pyruvate
A. It is a double reciprocal plot B. Lactate
B. Km can be accurately measured C. Citrate
C. X and Y intercepts directly indicate Km and Vmax D. Glutamate
D. Does not change in presence of inhibitors. 5-61 . Long term regulation of enzyme action is effected
5-53. Which of the following is true regarding Vmax? by:
A. Indicates the initial velocity at Km A. Altering the activity of enzyme proteins
B. Is measured as the number of moles of substrate B. Induction and repression of enzyme protein syn-
converted to product thesis
Chapter 5: Enzymology: General Concepts and Enzyme Kinetics 75
strate binds and a separate allosteric site where the Enzymes whose concentration in a cell is always the
modifier binds. same.
5-57. What are the features of allosteric inhibition? 5-64. What is repression?
(1) The inhibitor is not a substrate analogue. (2) It is Repression acts at the gene level; the number of enzyme
partially reversible, when excess substrate is added. molecules is reduced in the presence of repressor.
(3) Most allosteric enzymes are made up of subunits. 5-65. Give an example of repression .
5-58. Give examples for allosteric inhibition. ALA synthase is repressed by the heme.
ALA synthase, Aspartyl transcarbamoylase, HMG-CoA 5-66. What are the types of specificity?
reductase.
Absolute specificity, group specificity and streospeci-
5-59. What is covalent modification?
ficity.
Addition or removal of a group to the enzyme by a co-
5-67. Give an example for absolute specificity.
valent bond.
5-60. Give some examples of covalent modification. Urea is the only substrate for urease.
Glycogen phosphorylase is active when phosphory- 5-68. Give an example for group specificity.
lated and inactive when dephosphorylated. Trypsin can hydrolyse peptide bonds formed by ca r-
5-61 . What is meant by induction? boxyl groups of arginine or lysine residues.
Induction is effected at the level of DNA. The inducer 5-69. What are isoenzymes?
will relieve the repression on the operator site and will They are physically distinct forms of the same enzyme
-'
remove the block on the biosynthesis of the enzyme activity. They have identical catalytic properties, but
molecules. differ in structure.
_ _ _ _ _ Chapter
Clinical Enzymology
/
Chapter at a Glance
The learner will be able to answer questions on the following topics:
®
Plasma contains many functional enz;ymes, which are A "biomarker" is a t which is useful
actively secreted into plasma. For example, enzymes of in nctidn of an organ. Cardiac biomarkers
blood coagulation. are used to detect cardiac diseases, which may be:
On the other hand, there are a few non-functional a. Acute coronary syndrome resulting from myocardial
enzymes in plasma, which are coming out from cells ischemia
of various tissues due to normal wear and tear. Their
b. Congestive cardiac failure due to ventricular dys-
normal levels in blood are very low; but are drastically
function (Box 6.1). The different markers are used to:
increased during cell death (necrosis) or disease (Fig.
i. Detect myocardial ischemia at the eacliest
6.1 ). Therefore, assays of these enzymes are very
useful in diagnosis of diseases The reference values ii. Monitor the progression of the condition
of serum enzyme levels in normal people may vary iii. Predict the risk in cardiac dysfunction.
slightly depending on the method used. The normal Commonly used biomarkers for early detection of acut@
myocardial infarction are:
1. Cardiac troponins, Tnl and TnT
Unstable angina
Suspicious ECG i hanges
History suggestive of myocardial infarction
v. Following surgical coronary revascularization
vi. Patients with hypotension and s ne'
Chapter 6: Clinical Enzymology 79
0 2 3 4 5 6 7 8 9 10 11 12
Days after infarction
Mean percentage in
enzyme blood
MM(CK3)-,.._ s t_ ____;=-----==~+-
Le_a_ !'.'il
_O _ _ _ _--=-i
_% l
,,
Values_
SO .:t 4-0 K and Muscle Diseases
Normal serum value for CK is 15-100 U/L for maws an
10-80 U/L for females. ''0.i. 2 o U he level of CK in serum is very m~ h elevated in mus-
cular dystrophies (500-1500 IUILLJfu female carriers
Kand.Heart Attack of X-linked muscular dystrophy (heterozygous), CK is
CK value in serum is increased in myocardial infarction. moderately raised. £ K level is highly elevated in crush
The pattern of alteration in enzyme level with time is · · cerebrovascular accident~
shown in Figure 6.3 and Table 6.1. The CK level starts
to rise within 3-6 hours of infarction. Therefore, CK
estimation is very useful to detect early cases, where J
ECG changes may be ambiguous.
. The area under the ~ ~!!!l~A~C..!.TROPONINS (CTI/CTT)
peak and slope of initial rise are proportional to the size They are not enzymes. However, Troponins are now
of infarct. accepted as specific markers for myocardial infarction,
80 Section A: Chemical Basis of Life
-
Moderate (2-3 times) increa!jAin ALP level is saen in }
hepatic diseas~ such as i'l,.. n;aiye hepatitis, a1£'holic
ALANINE AMINOTRANSFERASE ~ patitis or hepatocellular carcinoma (see Chapter 24 ).
Very high levels of ALP (10-12 times of upper limit)
J may be noticed in extrahepatic obstruction (obstructive
In old literature, it was called as serum glutamate pyru- jaundice) caused by gallstones or by pressure on bile
vate transaminase SGPJ). The enzyme needs duct by carcinoma of head of pancreas. lntrahepatic
• ~l)_g§pha~e as coenzyme. Details of the reaction cholestasis may be caused by viral hepatitis or by drugs
are shown in [}Qu~i 3.19. Normal seru~ le~I of ALT (chlorpromazine). ALP is produced by w ithe!ialcells 'of
for male is 13-~?"u tL and for female is 1o~b U/L. Very biliary canaliculi and obstruction of biliary passage with
high values (300 to 1000 U/L) are seen in acute hepati- consequent irritation of epithelial cells leads to secretion
tis, either toxic or viral in origin (infective hepatitis). Both of ALP into serum.
ALT and AST levels are increased in liver disease, but Drastically high levels of ALP (10-25 times of
ALT > AST. Rise in ALT levels may be noticed several upper limit) are also seen in bone diseases where
days before clinical signs such as jaundice are mani- osteoblastic activity is enhanced such as I;egeW~
fested. Moderate increase (50 to 100 U/L) of ALT may be .........
(osteitis _JjeiQJ;mans), rickets, osteomalacia, osteo-
seen in chronic liver diseases such as..eifrhosis, .hepati- blastoma, metastatic carcinoma of bone and hyper-
tis C and oen-alcoholic steatohepatitis (NASH). parathyroidism.
82 Section A: Chemical Basis of Life
There a 6 oenzymes of alkaline phosphatase. is necessary for the liquefaction of seminal coagulum. In
Major fractio ound in serum are liver, bone and blood , it is bound to alpha-2-macroglobulin and alpha-1-
intestinal isoenzymes. Out of these, the Alpha-2 antitrypsin; a very small fraction is in the free form also.
variety is heat stable but is inhibited by phenylalao.ine.
It is of inte; tinal/placental origi~ . Heat stable placental
Normal value i.,.,..aa::w.~/ It is very specific for prostate
- - - ~ ~.g / ·.
isoenzyme is found in blood in normal pregnancy. A
--- - -
·- , .., . Values I
. It is named as
Regan isoenzyme (after the first patient in whom it was
detected) or carcinoplacental isoenzyme. Bone alkaline
C ollnesterase (ChE)
phosphatase (BAP), a h~ !~ fraction is used as a Acetylcholine esterase or true ChE can act mainly
marker of metabolic bone disease . ..Qlcio\L +o ~ t . . on acetyl choline. It is present in nerve endings and
~-
'Ii' .
Gamma-Glutamyltransferase (GGT)
in RBCs. Newly formed RBC will have high levels of
ChE which is slowly reduced according to the age of the
It can transfer gamma-glutamyl residues to substrate. cell. Therefore, ChE level in RBCs will be proportional
In the body it is used in ~ h nthesis of glutathione to the reticulocyte count.
(see Chapter 18). GGT h 11 oenzymes. It is seen irreversibly inhibit ChE
in liver, kidney, pancreas, i mal cells and prostate
gland. Normal serum value of GGT is 10-30 U/L. It is
moderately increased in i,tective hepatilis and ~msta~
.z.9pcec17.
*
GGT is clinically important because of its Pseudocholinesterase is nonspecific and can
sensitivity to detect alcohol abuse. GGT is increased hydrolyze acyl esters. It is produced mainly by liver i
in alcoholics even when other liver function tests are cells. Succinylcholine is a widely used muscle relaxant.
within normal limits. GGT level is rapidly decreased Succinylcholine is hydrolyzed by the liver ChE within r
within a few days when the person stops to take alcoho~ 2-4 minutes. But in certain persons the ChE activity may
be absent; this is a genetically transmitted condition. In
•
such individuals, when succinylcholine is given during
6, ~ Id Phosphatase (ACP) surgery, it may take hours to get the drug metabolized.
s . tma
It hydrolyzes phosphoric acid ester at pH between 4 and
6. Normal serum value for ACP is 2.5--12 U/L. ACP is
secreted by prostate cells, RBC, platelets and WBC.
The prostate isoenzyme is inactivated by tartaric acid.
Normal level of tartrate labile fraction of ACP is 1 U/L. ACP
total value is increased in prostate cancer and highly
It is a . It is an important
elevated in bone metastasis of prostate cancer. In these
enzyme in the hexose monophosphate shunt pathway
conditions, the tartrate labile isoenzyme is elevated. This
of glucose (see Chapter 10). It is mainly used for pro-
assay is very hel ful in follow-up of treatment of rostate
duction of NADPH. Hydrogen peroxide is continuously
cancers. So, (see
Chapter 48). Since blood cells contain excess quantity formed inside the RBC. Peroxide will destroy RBC cell
of ACP, care must be taken to prevent hemolysis while membrane. Glutathione and NADPH will prevent this
taking blood from the patient. Prostate massage may also process. Therefore, NADPH is very essential for pre-
increase the value. So blood may be collected for ACP serving the RBC integrity.
estimation before rectal examination of patient. Drug-induced hemolytic ~ i~P.}GPD is reduced in
drug induced hemolytic anemias. In the GPO deficient
PROSTATE SPECIFIC individuals, B<;c, lifespan ay ~ red d e
ANTIGEN (PSA) will b ~~I.SQ · I~ a~1AJ1-1e.,lil certain
drugs aspirin, mepacrine, primaquine, su a) re taken
It is produced from the secretory epithelium of prostate
by sueh · iduals, there will be sudden damage to
gland. It is normally secreted into seminal fluid, where it
RBCs. Primaquit ~t\~ 1 •roxide formation.
Chapter 6: Clinical Enzymology 83
Methemoglobinemia: NAOPH is also necessary for Marked increase in obstructive liver disease
- ~-;--
~-------·~~ --
- - - -
-
• •
- •
;-#
• ,;,~ - • A O 0
. -
Increased in obstructive and alcoholic liver
see Chapter 23). Hence in GPO deficient indi-
II. Myocardial Infarction
l als, methemoglobinemia may also be manifested.
-
Hydrolyzes triglyceride to beta-monoglyceride and fatty
Heat labile bone_isoenzyme (BAP) is elevated
V. Prostate cancer
acids. The enzyme is present in pancreatic secretion. The
level in blood is highly elevated in acute pancreatitis and Marker for prostate cancer.
Mild increase in benign prostate enlargeme
this •[lj§]fQt:I,- 14 days. Thus lipase remains elevated
..
M• • •• I f ,//1.. f !~•
longer than amylase. Moreover, lipase is not increased Marker for prostate cancer. Metastatic bone di se espe-
in mumps. Therefore, lipase estimation has advantage ciall from a rimar from rostate. Inhibited tartrate.
over amylase. It is moderately increased in carcinoma of
pa reas, biliary diseases and perforating peptic ulcers.
.
tand A value less than 20 mg/di is
pathognomonic of Wilson's hepatolenticular degenera-
clJer,
It is a glycolytic enzyme. Neuron-specific enolase (NSE) tion, in which per toxicity is manifested (see under
is an isoenzyme seen in neural tissues and Apudomas. Chapter 34 ).
NSE is a tumor marker (see Table 48.5) for cancers
associated with Adenosine Deaminase
Estimation of adenosine deaminase in serum and pleural
, etc. fluid is used to differentiate . . illlll;uu;:HJS pleural effusior\
Enzyme patterns in some important diseases are from other causes.
given in Box 6.4.
zymes irrother Body Fluids and Cells
1 eruloplasmin Enzyme levels in other body fluids are measured at time
It is otherwise called Ferroxidase and is described in for specific diagnostic purposes like amylase in urine
detail in Chapter 26. Normal serum level is - ~ u . utfl....l. LOH in CSF, pleural and ascitic fluids and leukocyte
Since it is an acute phase protein, it is increased in all alkt inL A os hatase in neutrophils.
84 Section A: Chemical Basis of Life
Related Topics
•• Clinical Case Study 6.2 Answer
Tumor markers are described in Chapter 48. Diagnosis: Gallstone pancreatitis.
Role of amylase: Enzyme for carbohydrate metabolism,
·• Clinical Case Study 6.1 used to digest glycogen and starch.
A patient presented with acute chest pain of half hour Acute pancreatitis is an inflammatory process
duration. The biochemical analysis reports are as follows: in which pancreatic enzymes are activated and cause
Blood Glucose - 350 mg%, Serum Cholesterol - 288 mg%, Alcohol use is the most
AST - 55 U/L, ALT - 15 U/L CPK and LOH were eleva- common cause, and episodes are often precipitated by
ted. Give your provisional diagnosis. What are the other C 4l . The next most common cause is ...,,_,
markers which can be estimated in this case? , usually passage of a gallstone into the
Chapter 6: Clinical Enzymology 85
-
mon cause, and that occurs when serum triglyceride lev- markers in hepatic diseases.
els are greater than 1000 mg/dl, as is seen in patients 3. &..e is also a useful marker in bone diseases.
with Abdominal pain 4. ~ r - . , t alcohol abuse and used as a
is the cardinal symptom of pancreatitis, and it is often marker o~lcoholic liver disease (ALD).
t 5. ACP and PSA,are used in t~ iagnosis of prostate
severe, typically in the upper abdomen with radiation
cancer.
to the back. The pain is often relieved by sitting up and
bending forward and is exacerbated by food. Pat:~:-:!~ 6. p~==.l. !.U:i!.!.sa..~ ~~~=:::
tate cancer.
also commonly have nausea and vomiting, also pre- 7. C olinesterase lChE)._is used as a marker in organo-
cipitated by oral intake. The treatment includes nothing phosphorus insecticide poisoning.
by mouth, intravenous hydration, pain control, and moni- 8. Pancreatic am lasels a marker of acute P,¥freatitis . . 4. ,.nu&
toring for complications. 9. Therap~,utically u~ fyl enzymes str~ioki~e . r<qro_J
or urdiarlase,~p~inaie1 hyall.lMnidase, all5fm1 - 1,: .. ,.~hc.
LEARNING POINTS, CHAPTER 6 antitrypsin, plf'l')Sin and t~in. e'j5
10. Some examples of enzymes used in diagnostic
-
1. Clinically useful biomarkers in diagnosis and pro-
gnosis of myocardial infarction are g<, CTI/CTT methods are peroxidase, glucose oxidase and alka-
and~T. line phosphatase.
6-1 . Write briefly about the enzymes that show variations in serum level in Myocardial infarction, and their clinical
i significance.
6-2. Name the enzymes that are used in the diagnosis of myocardial infarction. Explain the pattern of rise following
! an acute infarction.
6-3. Indicate the importance of enzyme studies in (a) Myocardial infarction; (b) Liver diseases; (c) Bone diseases;
(d) Malignancy.
6-4. Describe the importance of serum enzyme estimations in clinical diagnosis.
6-5. Indicate the use of enzyme studies in (a) Hepatobiliary diseases; (b) Bone diseases; (c) Muscle diseases; and
(d) Pancreatic diseases
• 6-1. When does creatine kinase (CK) level increased? carcinoma of lung, liver and gut. Regan isoenzyme is
Myocardial infarction; Muscular dystrophies. otherwise known as carcinoplacental isoenzyme.
6-2. What is the advantage of CK estimation? 6-8. Estimation of gamma-glutamyltransferase is use-
• CK level starts to rise within 3 hours of infarction. ful to detect which condition?
Therefore CK estimation is very useful to detect early Alcohol abuse.
cases. 6-9. Serum acid phosphatase level is increased in
6-3. What serum markers are helpful in the diagnosis which condition?
of myocardial infarction? Prostate carcinoma.
Creatine kinase (CK) CK-MB isoenzyme and Troponins. 6-10. What is the use of isoenzyme study of acid phos-
6-4. What are the isoenzymes of CK? phatase?
CK is a dimer; the subunits are called B for brain and M Tartrate labile isoenzyme is specific for prostate carci-
for muscle. Therefore three isoenzymes are possible. noma.
6-5. What is the advantage of cardiac troponin I? 6-11. What is advantage of prostate specific antigen?
Cardiac Troponin I is released into the blood within 4 PSA is very specific for prostate tissue.
hours after the onset of cardiac symptoms, peaks at 6-12. What are enzymes useful as tumor markers?
12-16 hours. Therefore, CTI is very useful as a marker (a) Regan isoenzyme of ALP for lung tumor; (b) Tartrate
at any time interval after the heart attack. labile isoenzyme of ACP; (c) Prostate specific antigen
6-6. Alkaline phosphatase level in serum is elevated in (PSA) for prostate carcinoma; and (d) Neuron specific
which conditions? enolase (NSE) for cancers of neuroendocrine origin.
Moderate increase is seen in hepatic diseases (infec- 6-13. Which enzyme deficiency is X-linked?
tive hepatitis); high levels in obstructive jaundice; and Glucose-6-phosphate-dehydrogenase.
very high levels in bone diseases such as Paget's 6-14. How the deficiency of GPO is manifested?
disease, rickets. metastatic carcinoma of bone. Drug induced hemolytic anemia.
6-7. What is Regan isoenzyme? 6-15. Acute pancreatitls can be diagnosed by estimating
It is the iso-enzyme of alkaline phosphatase. It is of which enzymes?
placental origin. It is elevated in about 15% cases of Amylase and lipase.
t
_ __ _ _Chapter 7
Chemistry of
Carbohydrates
Chapter at a Glance
The learner will be able to answer questions on the following topics:
Nomenclature and classification of sugars Amino sugars and deoxy sugars
Stereoisomers Pentoses
Glucose, mannose and galactose Sucrose, lactose and maltose
Fructose Starch, glycogen and cellu lose
Reactions of monosaccharides Heteroglycans, mucopolysaccharides
Glycosides
Gtf'h ~)_.I.,
The general molecular formu la ' of carbohydrate is 4. Glycoproteins and glycolipids are components of
C"(HPt For example, glucose has the molecular for- cell membranes and receptors.
p
mula C6 H 6 . Carbohydrates are polyhydroxy aide- · 5. Structural basis of many organisms: Celluloseaof
hydes or ketones or compounds which yield these on ~ s; exoskeleton of insects, c911 wall of micm-
®O
hydrolysis (Fig . 7.1 ). organisms, mucopolysaccharides as gro1,1nd sub-
-.~,..~
..--- 11-" .
stance in higher orga~isms. ; . · •
~·. :!'~=---·~..
-
. , - . - -
{ _1,___ .C.,_
ar~bohydrates are the main sources of energy in
Molecutes raving only, one actual or potential sugar
~' the body. Brain cells and RBCs are almost wholly
group are called monosaccharides (Greek, mono= one;
dependent on carbohydrates as the energy source.
l--~ production from carbohydrates will be
saccharide = sugar). They cannot be .further hydrolyzed
into smaller units. When two monosaccharides are com-
bined together with elimination of a water molecule, it is
2. Storage form of energy (starch and ~ gen). called a disaccharide (e.g. C12H2p 11 ). Trisaccharides
3. Excess carbohydrate is conv d t~ contain three sugar groups. Further addition of sugar
groups will correspondingly produce tetrasaccharides,
pentasaccharides and so on , commonly known as oligo-
saccharldes (Greek, oligo = a few). When more than
10 sugar units are combined, they are generally named
• as polysaccharides (Greek, poly= many). Polysaccha-
R R1 R H
rides having only one type of monosaccharide units are
Ketone Aldehyde
called homopolysaccharides and those having differ-
Fig. 7.1: Keto group and aldehyde group ent monosaccharide units are heteropolysaccharides.
Chapter 7: Chemistry of Carbohydrates 89
D-Mannose
gtw 12coteins - --
Constituent of ~ - rryucoproteins and
'
eins
CH2 0H
'
0-glucose 0-mannose D-galactose
uctose is a Ketohexose
In fructose, the keto group is on the 2nd carbon atom.
..
Thus secon~ om is the anomeric carbon atom.
Fructose has~~ - Each of them has D and L forms
with regard to 5th carbon atom. Fructose has the same
f Emil Fischer Walter Haworth Heyrovsky
NP 1902 NP 1937 NP 1959 molecular formula as glucose, but differs in structural
52-1919 1883-1950 1890-1967 formula. So glucose and fructose are function
(aldose-ketose) isomers.
three Representations of
o niy D variety is seen in biological systems. Fructose
ucose Structure remains predominantly as furanose ring structure
The 1st carbon, aldehyde group is condensed with the · . 7.8). Fructose is a major constituent of honey.
hydroxyl group of the 5th carbon to form a ring. Ring
structure represents• • • • •ill. which is the
an.aldehyde (or keto with a hydroxyl group.
, .
The • ~.::..._ ...... · ; - : - _ .~ _ ; • ' - ·., l ; I
c~oMl
ring structure of glucose were proposed by r erties will be seen together:
in 1883, and hence called Fischer's formula. Fischer • ...111111 I t: I • t ' ;'\_
H
.. H- C= O
6 CH2 0H 6CH 20H
t' 2
I
H- C-OH
I
H-t-OH I 5
0
HO - C-H
t, 3 HO-C-H 0
4
I
H-C-OH
I
I
H- i -OH
4
5 H-C-OH H- C
I I 3 2
6 CH2 -0H CH2 0 H
H
D-glucose. open chain a-D-glucose, closed a-D-glucopyranose, P-D-glucopyranose,
projection formula ·ng structure, Fischer formul Haworth formula Haworth formula
2
1
C=O
H20H 6
OH-H2C
1
CH2-0H
Sugar --+Enediol ~ u • •+--- CuS04
Oxidized Reduced
3 H0-6-H •
Sugar acid cu• - - 2Y
Cu(OH) - -cu20
ellow Red
ft,
4 H-6- oH '
5
I
H-C-OH
I
OH Fig. 7.9: Benedict's test, principle
'
6 CH,- OH I
D-fructose, open chair-it H
Needle-shaped crystals arranged
projection formula o.•D.fructo furanose, Haworth formula like a broom
Glucososazone
Fig. 7.8: Different representations of 0-fructose
H f y=o I HfITTOH
II
H-C-OH ,_----- C-OH ,_-----
I '-t Hedgehog or ' pincushion with
HO-C-H HO-C-H HO-C-H
,. pins" or flower of "touch-me-not" plant
I La<;tososazone
H-C-OH
I I
H-C-OH H-C-OH
I I I
H-C-OH H-C-OH H-C-OH
I I I
CH2-0H CH2-0H CH2-0H
Glucose 1,2-enediol form (intermediary) Fructose Sunflower-shaped or petal-shaped
-
,.
crystals of
Fig. 7.10: . - - - .-
_..,. - , ,
. .
~~-•Ill . _.
. - Maltosazone
l 100~ -C = O
I r
[ COOH Fig. 7 .11 : Shape of osazones under microscope
H-C-OH H-C - OH H-C-OH C
I I I
HO-C-H HO-C - H HO- C-H
I I I
H- C-OH H-C-OH H-C-OH
I I I
H-C- OH H-C- OH H- C-OH
I --i--.
CH2 -0H COOH 1 ! 600H
Gluconic acid Glucuronic acid lucosaccharic acid'
@ Be
Fig. 7 .12: Oxidation products of glucose
c'lCtn- ~~,ot. .
e let's Reaction t2,(1"
o/<'~ ~f">- ~e
to produce uronic acid. Thus glucose is oxidized to p[Oduces changes in tissues when they accumulate In
glucuronic acid, mannose to mannuronic acid and rma l amounts, e .g. cataract of lens.
galactose to galacturonic acid. The glucuronic 1-'.sV'~
D-,.."\l-
acid (Fig . 7.12) · ~''.Y~:tmpo~r-l.-
-.I iaL.u::_; •_• - • - -;i-- ;. - II :. When the hemiacetal group (hydroxyl group of the
(see Chapter 36) anomeric carbon) of a monosaccharide is condensed
and also for with an alcohol or phenol group, it is called a glycoside
c. Under strong oxidation conditions (nitric acid + heat), (Fig. 7 .14). The non-carbohydrate group is called agly-
the first and last carbon atoms are simultaneously cone. Glycosides do not reduce Benedict's reagent,
oxidized to form dicarboxylic acids, known as sac- because the sugar group is masked . They may be
charic acids (Fig. 7 .12). Glucose is thus oxidized to hydrolyzed by boiling with dilute acid, so that sugar is
lucosaccharic acid, mannose to mannaric acid and free and can then reduce copper,
The I . I f tif f 7
, while
Enzyme
hydro ysis thus affords a distinguishing
between the two forms. Some glycosides of medical
importance are given in Table 7.3. 1a~
r Monosaccharides when treated with concentrated sul- ant. forhtztrr ts usectto produce renal damage ip
furic acid undergo dehydration with the removal of 3
• molecules of water. Therefore hexoses give Jiydroxy-
ta nimals .
0
II
CH,OH
O
H CH, 0 H J
Glucose H H
xylose
H OH H 0~
Glucose indoxyl
CHO
I
HO- C- H
I
4 H- C- OH
I
H- C- OH
3 2 I
H NH-CO-CH3 HO - C- H
2-amino-D-glucose N-acetyl-glucosamine
or glucosamine or GluNac
5 t
OH-H2 CQ OH
H
4
H H OH
1 •
3 2
H0 H
-.,t.J.lJ~,-.•
~deoxy su!Jars with Schjffs reagent (Dye Fuch sine is
(%) Amjno Sugar!g decolorized by sulfurous acid).
O
H
-H,dG"
6 CH2OH
0
l
.I, 4 o/ H HO /H 20 H
f 3 4
HO H
2
OH Fructose component
Glucose component
Alpha-D-glucosyl-beta-0 -fructoside
t
6
O - -cH 2
Fructose Ketohexose
Disaccharides
Glucose + Galactose = Lactose (reducing)
4
Glucose + Glucose = Maltose (reducing)
Glucose + Fructose= Sucrose (non-reducir1g)
3 2
Two glucose residues in@iiiia,;1..6Jiok~g_e H
OH
units, but is highly branched with molecular weight
Fig. 7.22: lsomaltose more than 1 million. The branching points are made by
alpha-1,6 linkage (similar to isomaltos;e, Fig. 7.22).
on the configuration at the free anomeric carbon atom.
f\S\ It is a reducing di ccharide. It forms tal-sha ed drolysis of Starc/-1
~ _,s,Dls of malto eo zone (see Fig. 7.11 ). Starch will form a blue colored complex with iodine;
this color disappears on heating andl reappears when
laomaltose cooled. This is a sensitive test for starch. Starch is non-
It is also a reducing sugar. It contains 2 glucose units reducing because the free sugar groups are negligible in
combined in. alpha-1 ,6 linkage. Thus first carbon of one number. , smaller
glucose residue is attached to the sixth ca rbon of another and smaller fragments are produced. Thus hydrolysis for
glucose through a glycosidic linkage (Fig. 7.22). Partial a short time produces amylodextrin which gives violet ,.
hydrolysis of glycogen and starch produces isomaltose. color with iodine and is non-reducing. Further hydrolysis
The salient features of important sugars are shown produces erythrodextrin which gives red color with •
inBox.L iodine and mildly reduce the Benedict's solution. Later
ach·rodextrins (no color with iodine, but reducing) and
IDES {. Sf1"\ furthe'. on, maltose (no color _with iodine, b~t powerfully
These are polymerized products of many monosaccha- reducmw are fmmed op 9Pntmueg Jifrol SIS.
ride units. They may be: 4
1. Homoglycans are composed of single kind of mono- Action ofAmylases on Starct,r
saccharides, e.g. starch, glycogen and cellulose. a crea c a iefe are alpha-
2. Heteroglycans are composed of two or more diffe- amylases, which act at random on alpha-1,4 glycosidic
rent monosaccharides, e.g. hyaluronic ~ d, chon- bonds to split starch into smaller units (dextrins), and
L1. droi!i.(i_sulfa!e. • ,?t ,..,_,_ ~ 1...C mally to alpha-maltose. Beta-amyla1ses are of plant
n 0 M~Cf\NS. ,~~ _.,~~.,.....
rch \...-=>';· ), "'A..,..,..i;J-W origin (almond, germinating seeds, etc.) which split
l'J;>if"J i,'ff"lv starch to form beta-maltose. They act on amylase to split
Structure of Stare \.-: '6\0 maltose units consecutively. Thus the enzyme starts
. / It is the rate Sou~- its action from one end. When beta-amylase acts on
~ I ces are potatoes, tapioca, cereals (rice, wheat) and other
; food grains. Starch is composed of amylase and amylo-
amylopectin, maltose units are liberated from the ends of
the branches of amylopectin, until the action of enzyme '
:')"'· ~ ctin. When starch is treated with boiling water, 10-20% is blocked at the 1,6-glycosidic linkafJe. The ~ f
J ·•~ is solubilized; this part is called amylose. Amylase is beta-am lase st s at branchin oi ts, leaving a large
~v., made up of glucose units with alpha-1 ,4 glycosidic lin!<- molecule, calle llmlt-dextrin or residu " dextrin.
ages (Fig. 7.21) to form an unbranched long chain wi
a molecular weight 400,000 D or more. The insolubl
Jycogen t 3m) _, fm~Me,\ .a.l-n-f~
part absorbs water and forms paste like gel; this is called It is the reserve carbohydrate in animals. It is stored
amylopectin. Amylopectin is also made up of glucose in liver and muscle. About 5% of weight of liver is made
Chapter 7: Chemistry of Carbohydrates 97
-;~:,;tz;r
lnulin is a polysaccharide (carbohydrate) made up of fructose
units. It is used for renal function studies.
Insulin is a polypeptide (protein) hormone, with wide ranging
actions on carbohydrate and lipid metabolism.
H
0 H 0
1
0
0
•
l,,
0-
'1'
H OH H OH H NH-CO-CH3
Fig. 7 .24: Sulfated glucosamine-alpha-1 , 4-iduronic acid. Repeat- Fig. 7 .25: D-gluc uronic acid-be ta- 1, 3-N-acetylgalactosamine-4
ing units in heparin sulfate (units of chondroitin sulfate)
L :.. II
.
lir.:"'
•
., •
. e
.. · •" . - • -·4·. - - ,~ If-
,
(_~11')
It is present in ground substance of connective tissues
widely distributed in cartilage, bone, tendons, cornea
and skin. It is composed of repeating units of . . .
nd so on (Fig. 7.25).
carbohydrate content is less than 10%, it is generally Saccharin (Benzoic sulfimide) is an artificial sweet-
named as a glycoproteln. If the carbohydrate content ener. It is used to sweeten products such as drinks,
i is more than 10% it is a mucoprotein. (But some candies, medicines, and toothpaste. Although saccharin
authors use these words as synonyms). They are seen has no food energy, it can trigger the release of insulin in
in almost all tissues and cell membranes. About 5% of humans and rats, apparently as a result of its taste. It is
the weight of the cell membrane is carbohydrates. If the not banned anywhere in the world.
carbohydrate groups cover the entire surface of the cell
membrane, they are called glycocalyx. Glycoproteins Qletary Fiber
act as enzymes, hormones, transport proteins, struc- Dietary fiber is contributed by the unavailable carbo-
tural proteins and receptors. hydrates in the diet. Several different types of dietary
The oligosaccharide chains of glycoproteins are fiber have been found in different types of food items.
composed of varying numbers of the following carbo- They contri~ute the ~ k and assis~ normal bowel
hydrate residue: Glucose (Glu); mannose (Man); galac- movemenfsl)Cellulosef.Viemicellulos~ cti~ inates
tose (Gal); N-acetyl glucosamine (GluNAc); N-acetyl a urns are the usual glycans which form dietary fiber.
galactosamine (GalNAc); arabinose (Ara); Xylose (Xyl); Of these cellulose, a homoglycan made of glucose is
L-fucose (Fuc) (see Fig. 7.17) and N-acetyl neuraminic found in bran, flour and tubers. Pectins are mixtures of
acid (NANA). Glycoprotein metabolism is further elabo- homoglycans found in fruits like apples and berries.
rated in Chapter 10. Gums and alginates are found in legumes and oatmeal.
Carbohydrate group is attached to proteins either as
O-glycosidic linkages or as N-glycosidic linkages. The Bacterial Cell Wall
O-glycosidic linkage is GalNAc to serine or threonine Major constituents o roRaryotic (bacterial) cells are
residues of usual protein . However, galactose is added heteropolysaccharides, consisting of repeating units
to hydroxylysine residues of collagen. The N-glycosidic of N-acetylmuramic acid (NAM) and N-acetylglucosa-
linkages are made by addition of carbohydrate group to mine (NAG). This polysaccharide provides mechanical
nitrogen atom of asparagine or glutamine residues of strength. ~Synthesis of this complex polysaccharide is
proteins. blocked by penicillin. This inhibition is responsible for
F. l:'}i. ~ \ ) ~ ! the bactericidal action of penicillin. Penicillin was dis-
ommon Sugar Substitutes
covered by Sir Alexander Fleming in 1928. Later, Ernst
cesulfame-Potassium (Ace K) is made from aceto Chain isolated and purified penicillin. The first clinical
acetic acid. It is 2.90 times more sweet than sugar; but trial with penicillin was conducted by Howard Florey in
~rje content is negligible. It is present in artificial sweete- 1940. All the three were awarded Nobel prize in 1945.
ners, carbonated drinks and pharmaceutical products.
~ Aspartame is made from aspartic acid and phenyl
LEARNING POINTS, CHAPTER 7
~ . both are amino acids. It is 200 times sweeter
than sugar. Aspartame is not suitable for people with 1. Carbohydrates are polyhydroxy aldehydes or keto-
phenyl ketonuria. nes or compounds, which yield them on hydroly-
{f)saccharln is made from a.otbcaoilic acid. It is ~ sis. Simplest carbohydrates are monosaccharides
times sweeter than sugar. Calorie content is nil. which may be trioses (3C), tetroses (4C), pentoses
® Sucralose is made from sucrose or table sugar. (SC) and hexoses (6C).
2. Carbohydrates are classified into monosaccha-
Sucralose is approximately 600 times as sweet as
rides, disaccharides and polysaccharides, based
sucrose (table sugar), twice as sweet as sacc~arin, and
on the number of sugar/saccharide units they pos-
~ times as sweet as aspartame. Calorie content is nil.
sess. Disaccharides have 2 monosaccharide units,
'. ~ Cyclamate (Sodium cyclohexyl sulfamate) is ~ oligosaccharides around 10, and polysaccharides
ti~ sweeter thg_n sugar. Cyclamate: saccharin mixture more than 10. They could also be classified as
is found to increase the incidence of bladder cancer in aldoses and ketoses based on the functional group
rats. Its sale is banned in the United States. However, they possess.
Cyclamate is approved as a sweetener in over 55 3. Common examples of monosaccharides include
countries. Glucose, Fructose, Galactose, and Mannose.
100 Section A: Chemical Basis of Life
4. Common examples of disaccharides are Sucrose, 11. Optical isomerism is based on the rotation of plane
Lactose and Maltose. polarized light by a pure solution of the sugar. The
t,sofll~)
5. Monosaccharides exhibit stereoisomerism, optical
isomerism, anomerism and pyranose-furanose iso-
prefix 'd' or(+) and 'I' or(-) is used to indicate dextro
and levorotatory compounds respectively. •
'l,.
merism. 12. All reducing sugars form characteristic osazone
6. All carbohydrates are considered to be derived from crystals. Glucose and fructose form needle-shaped
glyceraldehyde by successive addition of carbons. crystals, maltose forms sunflower-shaped crystals
The penultimate carbon atom is thus the reference and lactose forms hedgehog-shaped crystals.
carbon atom for naming mirror images. 13. Amino sugars form important components of muco-
7. Stereoisomerism is the property of monosaccha- polysaccharides. For example, Galactosamine, Glu-
cosamine.
rides, due to the difference in orientation of H and
14. Sucrose is formed from glucose and fructose linked
OH around the reference carbon atom. The stere-
by 1, 2 glycosidic linkage.
oisomers are prefixed as 'D' or 'L'. D sugars are
15. Lactose is formed from galactose and glucose
naturally occurring and human body can metabo-
linked by beta 1,4 glycosidic linkage.
lize only D sugars. 16. Maltose is formed from two glucose molecules
8. A carbon atom bound by four different groups on all linked by alpha 1,4 glycosidic linkage.
its valencies is referred to as an asymmetric carbon. 17. Starch is made of two components; straight chain
When two sugars differ from each other in the con- amylose and branched amylopectin. The linkages
fig uration around one carbon atom (other than the are alpha-1,6 type at branch points while alpha-1,4
reference carbon), they are diastereoisomers. linkages form the straight chain.
9. A pair of monosaccharides which differ from each 18. Action of amylase on starch yields limit dextrins.
other in the configuration around a single carbon 19. Mucopolysaccharides or Glycosaminoglycans
atom are called epimers. Anomers of monosaccha- (GAGs) such as Hyaluronic acid, Chondroitin sul-
rides are produced by the spatial configuration with
reference to the first carbon atom in aldoses and
fate, Keratan sulfate, Dermatan sulfate are asso-
ciated with connective tissue.
,
the second carbon atom in ketoses. 20. Keratan sulfate is the only GAG that does not
10. Two anomers of glucose are alpha-O glucose and contain uronic acid .
beta-O glucose. Mutarotation is the result of anom- 21 . When the carbohydrate chains are attached to a
erism. polypeptide chain it is called a proteoglycan.
7-6. Name some important monosaccharides. The difference in glucose and fructose is dependent on
Glucose, fructose, mannose, galactose. the first and second carbon atoms; and this is masked
7-7. What are pentoses? by the osazone formation.
Monosaccharides with 5 carbon atoms. 7-25. On oxidation of glucose, what are produced?
7-8. Which is the reference carbon atom in sugars? Glucuronic acid; Gluconic acid and Glucosaccharic
Penultimate carbon atom. acid.
7-9. What is the difference between D and L sugars? 7-26. Name some important disaccharides.
They are mirror images with reference to penultimate Sucrose, lactose, maltose.
carbon atom. 7-27. What is the glycosidic linkage in lactose?
7-10. Which isomer is common in nature? Beta 1-4 linkage.
D variety of sugars are common in nature. 7-28. What is the glycosidic linkage in sucrose?
7-11. What is the difference between glucose and galac- 1-2 linkage.
tose? 7-29. Which disaccharide has no free aldehyde or ketone
Galactose is the 4th epimer of glucose. group?
7-12. Galactose is present in which food? Sucrose.
Lactose is present in milk. Lactose contains galactose 7-30. Sucrose (containing glucose and fructose) is a
and glucose. non-reducing sugar, why?
7-13. What is epimerism? Because the glycosidic linkage in sucrose involves 1st
When sugars are different from one another, only carbon of glucose and 2nd carbon of fructose; so both
in configuration with regard to a single carbon atom reducing groups are masked.
(other than the reference carbon atom), they are called 7-31 . Hydrolysis of maltose will give rise to what?
epimers. Two glucose units.
7-14. Give an example. 7-32. Which is the sugar found in milk?
Glucose and mannose are an epimeric pair which differ
only with respect to carbon atom 2. Similarly, galactose 7-33.
Lactose.
What are the component monosaccharides of lac- •
is the 4th epimer of glucose. tose?
7-15. Anomerism is produced with reference with which Galactose and glucose.
carbon atom? 7-34. Sucrose consists of what monosaccharides?
With reference to the first ca rbon atom in aldoses and Glucose + fructose.
second carbon atom in ketoses. 7-35. Name reducing disaccharides.
7-16. How alpha and beta sugars are produced? Lactose and Maltose.
These are anomers. 7-36. How polysaccharides are classified?
7-17. What is the basis of mutarotation? Homopolysaccharides (homoglycans) and hetero-
It is due to the anomeric carbon atom. polysaccharides (heteroglycans).
7-18. What is the difference between glucose and fruc- 7-37. What is a homoglycan?
tose? They are composed of single kind of monosaccharides.
Glucose is aldohexose; fructose is a ketohexose. 7-38. Give examples of homopolysaccharides.
7-19. What is the principle of Benedict's test? Starch, glycogen.
In alkaline medium, sugar reduces cupric ions, red 7-39. What are heteropolysaccharides?
precipitate. They are composed of two or more different monosac-
7-20. Benedict's test is commonly done for what? charides.
To detect the presence of glucose in urine. 7-40. What are the characteristics of glycogen?
7-21 . Name a few reducing sugars. It is composed of glucose units. It is the stored form of
l
-,
Glucose, fructose, mannose. carbohydrate in animal kingdom. It has a highly bran-
7-22. -Keto group is non-reducing; but fructose reduces ched structure.
Benedict's solution; what is the cause for this ano-
maly?
7-41 . Give examples of heteropolysaccharides.
Agar, hyaluronic acid, heparin, chondroitin sulfate.
~·
(
In alkaline medium, ketone group is converted to alde- 7-42. What are mucopolysaccharides?
hyde, through enediol formation. They contain uronic acid and amino sugars.
7-23. In the case of sugars, which of the properties go 7-43. Wh ich heteropolysaccharide does not contain
hand in hand? uronic acid?
Reducing property; osazone formation and Mutaro- Keratan sulfate.
tation. 7-44. Hyaluronic acid is seen in which tissues?
7-24. Glucose and fn,1ctose will form identical osazones, Connective tissue, Synovial fluid , tendons, vitreous
why? humor.
'
Chemistry of Lipids
-
Chapter at a Glance
The learner will be able to answer questions on t he following topics:
I
Classification of lipids ,______ { Phospholipids
Classification of fatty acids >,- D Phosphatidylcholine or lecithin
B Saturated and unsaturated fatty acids \ D Sphingomyelin
Neot,alf,ts o,t,;acylglymols l Non-phospho,ylated hp;d, ( Gt 1'.f u, \<pi d.()
(~L)
Lipids constitute a heterogeneous grou 1. Simple lipids: They are ~ s Qf~ acjds with
of biochemical importance. Li ids ma glycp,r.ol or other higher alcohols {Table 8.1 ).
compounds which are relativ :===:;:;=====:=~--=====-·
y insol le in wa er, but 2. nd lipids: They are fatty acids .esterified
freely soluble in non-polar organic olvents, such as with alcohol; but in addition they contain oj her
be~ne, ~ roform, ~ . hot alcohol, a~ e. etc. groups. Depending on these extra groups, they are
The functions of lipids are summari e in Box 8 .1. The subclassified in Table 8.1.
clinical applications are shown in B 8.2. ..JI a. Phospholipids, containing phosphoric acid.
"t' b. Nof'}.-phosphorylated lipids {Table 8.1)
ATIONOF 3. Derivetf ltpids, They are compounds, which are
d~ fr~ ~s of lipids, e.g.
fatty acids~ teroids. For details o{ cholesterol and
sterojds. see Chapter 14.
4 . Lipids complexed to other compounds.
1. Storage form of euer9ll (triacylglycerol) '1n"
2>,_ Structural components of biomembranes) W os,etwlipids an
(!)I cholesterol) lit')
Fatty acids, are included in the group of derived lipids.
3. Meta~ ic regulato~ st~ d hormones and p~staglandins)
4. Acta~m .Jllctal}_ts, Q&rgents and Q lsifying agents (amphi·
It is the most common component of lipids in the body.
pathic lipids) They are generally found in ester linkage in different
5. Act as el~ctric insulators io oemORs
6. Provide insulation against changes in external temperature BOX 8.2: Clinical appllcat1ons
(subcutaneous fat)
1. Excessive fat deposits cause obesity. Truncal obesity is a risk
7. Give sbjlpe and contour to t he body factor for heart attack.
8. Protect internal organs by providing a cushioning effect 2. Abnormality in cholesterol and lipoprotein metabolism leads to
(pads of fat) atherosclerosis and cardiovascular diseases (see Chapter 14).
9. Help in absorption of fat soluble vitamins {A, D, E and Kl 3. In diabetes rr.ellitus, the metabolisms of fatty acids and
10. Improve taste and palatability of food. lipoprotelns are deranged, leading to ketosis (see Chapter 13).
l_
TABLE 8.2 : Charactensllcs of common fatty acids
No. of
a. Triacylglycerol or Triglycerides or neutral fat
b. Waxes
Common name
carbon
atoms Chemical nature I
Occurrence
A. Even chain, saturated fatty acids
II. Compound lipids
Acetic 2 Saturated; small chain Vinegar
A. Phospholipids, containing phosphoric acid _
Butyric 4 do Butter
1. Nitrogen containing glycerophosphatides:
Caproic 6 do Butter
i. Lecithin (phosphatidylcholine)
Capric 10 Saturated medium Coconut oil
ii. Cephalin (phosphatidylethanolamine) chain
iii. Phosphat idylserine Laurie 12 do Coconut oil
2. Non-nitrogen glycerophosphatides Myristic 14 do
·-
Coconut oil
i. Phosphatidylinositol Palmitic 16 Sat urated; long cha in Body fat
ii. Phosphatidylglycerol Stearic 18 do do
iii. Diphosphat idylglycerol (cardiolipin) Arachidic 20 do Peanut oil
3. Plasmalogens, having long chain alcohol (Arachis oil)
G9
w6 famUz:. - Linoleic and Aracbidoni~ (Fig. 8.2)
2. Depending on length of hydrocarbon chain:
family - Oleic acid
a. Short chain with 2 to 6 carbon atoms
4. Arachidonic acid is the precursor of prostaglandins. Arachi-
b. Medium chain with 8 to 14 carbon atoms donic acid can be synthesized in the body, if the essential
c. Long chain with 16 and above, usually up to 24 carbon atom s fatty acids are supplied in the diet.
d. Very long chain fatty acids (more than 24 carbon). 5. Th® 'taenoiu c;u;14>resent in fish oils is of great nutritional
3. Depending on nature of hydrocarbon chain: importance ( w3 unsaturated fatty acid).
a. Saturated fatty acids (Table 8.2) 6. Eicosanoids (eicosa = twenty) are derived from 20 C ara-
b. Unsaturated fatty acids which may be subclassified into Mono- chidonic acid. They are polyenoic fatty acids. They are pros-
unsaturated (monoenoic) having single double bond or Poly- tanoids (prostaglandins, prostacyclins, thromboxanes) and
unsaturated (polyenoic) with 2 or more double bonds (Table 8.2) leukotrienes. See Chapter 16.
-----------------------------------------------------------------------------------
18 ro6 12 9 1
Linolenic (C A9. 12. 15 (three double bonds) (w3 family)
18)
CH3-CHz-CH = CH-CH2-CH = CH - CHcCH" = CH- (CH2h -COOH
18 Cil3 15 12 9 1
--
Safflower oil
(Kardi) by hard water and are used as detergents.
Sunflower oil 12 24 64
Butter 75 20 5 \ E er Formation
Ox(Tallow) 53 42 5
Both saturated and unsaturated fatty acids form esters
Pig (Lard) 42 46 12
with alcohols, especially with glycerol. Fatty acids can
Fish oi l 30 13 57
form mono-, di- or tri-esters with alcohol groups of
(*) t hese saturated fatty acids are medium chain fatty acids.
("") contains erucic acid, 22 C, 1 double bond.
glycerol. Triglycerides or triacylglycerols are also known
as neutral fat (Fig. 8.3).
Glycerol+ fatty acid -+ Monoacylglycerol (MAG)
@ ~ ation . .. Monoglyceride + fatty acid -+ Diacylglycerol (DAG)
When treated with halogens under mild condItIons, the Diglyceride + fatty acid -+ Triglyceride or
unsaturated fatty acids can take up two halogen atoms, triacylglyccerol (TAG)
at each double bond to form the halogenated derivativ~ . . .
of the fatty acid. For example, u, Oxidation at Fatty Acids
Oleic acid + 12 -+ Di-iodo oleic acid All fatty acids undergo oxidation in the body to give
The number of halogen atoms taken up will depend energy. Beta-oxidation is the major process by which
on the number· of double bonds and is an index of th~ fatty acids are oxidized (see Chapter 13). However, the
degree of unsaturati~m (see iodine number, under tn- unsaturated fatty acids can undergo auto-oxidation, due
acylglycerol). to the presence of the highly reactive double bonds and
;::;\ ariety 9f. r ducts r~ f3d.
~ ng Point ..~ :.>4+J '3.
The short and medium chain fatty acids are liquids, l!lttRAL F JS . ._,,-; ~..o.\tt>
whereas long chain fatty acids are solids at 25°C. The
Neutral fats are also called as triacylglycerols (TAG)
solubility in water decreases, while melting and boiling
or triglycerides (TG). These are e sters o7'the' trihydric
points increase with increase in chain length.
alcohol, glycerol with fatty acids (Fig. 8.3).
The unsaturated fatty acids have lower melting
point compared to saturated fatty acids with the sam omenclature of Carbon Atoms /
chain length. For example, stearic acid (C18 fatty acid,
As per International Union of Biochemistry (IUB) the
no double bond) has the melting point 69°C, oleic acid
correct designations are monoacylglycerol (MAG),
(C18, 1 double bond) has 13°C; linoleic acid (C1 8, 2
diacylglycerol (DAG) and triacylglycerol (TAG). But the
double bonds) has - 5°C and linolenic (C18, 3 double
old terminology of mono~ceride, digl~ ride and tri-
bonds) has -10°C.
gly~ ride are still popular, especially among clinical
- -
alkali.
•
CH3- COOH + NaOH -+ CH 3- COONa + Hp distinguish between 1,st3 od 3rd r.arbon atoms.
Chapter 8: Chemistry of Lipids 107
Lipase Lipase
(+) 3 NaOH
( \ • ( ' \ • Monoacyl- Triacytglycerol - - - - - - - Glycerol+3 R-COONa
oxidative rancidity. PUFA are more easily oxidized; so portion in their molecule (Figs. 8.7A and 8.8). The gly-
vegetable oils with a high content of PUFA are usually cerol along with the phosphoric acid and choline consti-
preserved with addition of antioxidants. tute the polar 'head' of a phospholipid molecule, whereas
Repeated heating of oils would lead to the formation the hydrocarbon chains of the fatty acids represent the
?,m);;'r
and polymerization of cyclic hydrocarbons. These will non-polar 'tail'.
impart an unpleasant taste and color to the oil. Coconu
oil having medium chain saturated fatty acids will with icellar Fon11ation (_
stand such polymerization. When phospholipids are distributed in water. their hydro-
@ Waxes
phobic parts keep away from water, forming molecular
aggregates called micelle (Fig. 8.78). These are involved
They fo rm the secretions of insects, leaves and fruits of in solubilization of lipids in aqueous media and help in
plants, e.g. Lanolin or wool fat, beeswax, whalesperm digestion and absorption of lipids.
oil, etc. They are esters of higher fatty acids with higher, ,
monohydroxy aliphatic alcohols and so have very Ion iposomes Ju.&~ 2,-)i .t;.oe.. no~ l.&
straight chains of 60-100 carbon atoms. They are used
as the base for the preparation of cosmetics, ointm~ . A lipid bilayer will close on itself under appropriQfcon-
polishes, lubricants and candles. (? ditions to form liposomes. Unilamellar or multilamellcy-
(3)
liposomes may be formed. They may be prepared by
PHOSPHOLIPIDS ( <; ~. . -tt sonication of mixtures of phospholipids and cholesterol
(Fig. 8.78). Liposomes are microscopic spherical vesicles.
© Phosphatidates When mixed in water under special conditions, the phos-
i. These are derivatives of pho~ pholipids arrange themselves to form a bilayer mem-
the simplest phospholipid brane which encloses some of the water in a phospholipid
ii. Phosphatidic acid is made up of one glycerol sp,.!:u;Le.. Drugs, proteins, enzymes, genes, etc. may be
to which two fatty acid residues are esterified to encapsulated by· the liposomes which could act as car-
carbon atoms 1 and 2. The 3rd hydroxyl group is r iers for these substances to target organs. Liposome-
esterified to a phosphoric acid (Fig. 8.6). entrapped drugs exhibit superior pharmacological
properties than those observed with conventional formu-
AmphiRathi~ Na ure la~ s. Liposom1:!~ ave important appl~ ions in can-
Phospholipids in general are amphipathic, particularly c~ hemoth~ p~ timicrobial thera~ ene therapy,
Lecithin. They have both hydrophobic and hydrophilic accines an~gnostic imaging.
.,~:~.~
·l
Heads orient
Hydrophilic toward water
polar head
water
QQQQOOOQ Phospho•
Hydrophobic
non-polar
ill::~,..
tail
Tails orient away from water
Fig. 8.7 A : Phospholipids form the bilayer Fig. 8.78 : Phospholipids form micelles and liposomes
Chapter 8: Chemistry of Lipids 109
R co1
H2C-o CO-R1 pholipids. Different phospholipases are involved in
o-?H o- L /CH3
2- the hyd rolysis of specific bonds in lecithin (Fig . 8.8).
c-01P-oic-c-N-CH
~2 [hA + "'CH:
Phospholipase A2 acts on an intact lecithin molecule
hydrolyzing the fatty acid esterified to the beta
(second) carbon atom. The products are Lysolecithin
3 4 and fatty acid . The fatty acid (often a PUFA) liberated
1 = Site of action of ob9sohgljpasc Al from ~ r boA is the precursor for the synthesis
2 = S11e of action of ,en§§g§gfiiiai\¢$2 of prostag andins Lysolecithin is a detergent and
3 = Site of action of J>hQ§PbPHPas., c hemo y~ The enzyme is present in the venom
4 = Site of action ofphospholipase D
of viper snakes. The hemolysis and consequent renal
Fig. 8.8: Lecithin R, and R2 are fatty acids. Red rectangle failure seen in viper poisoning cou ld be thus explained.
depicts glycerol group. The blue rectangle is choline which shows Actions of other phospholipases are shown in Figure
polar or hydrophilic property
8.8. The products formed in each case may be summa-
systems. They comprise the central solid nanocrystal- rized as follows:
line core coated with polyhydroxy oligomers onto which Phospholipase A2 L . h" f 'd
biochemically active molecules are adsorbed. The solid Lecithin yso1ecIt in+ atty acI
core provides the structural stability. The carbohydrate LecIt. h.In Phospholiphase A1 > A cyI gIycerop hosp horyI-
H2C-O-CO- R1 H C-0-CO-R
21 1
I
R2 - COO-CH OH R2-COO-CH OH
I I + I I
C-0-P- 0 - C-C-NH 3 C-0-P-O~H
OH
I II I I II
H2 O H. H. 2 0
HO
Fig. 8.9: Cephalin (Phosphatidylethanolamine) OH
OH
CHdCH2l12-CH=CH-CHOHiH-CH20H I
Fig. 8.11 : Ethanolamine plasmalogen
L _ _ _ __
biomembranes. This compound plays a vital role in the alcohol s hin osine (Fig. 8.12) which is attached
mejiaiioo at hacmopP...action on biomembranes and acts to a fatty acid in amide linkage to form a ceramide
as a second messenger (see Chapter 45). (Fig. 8.13). The fatty acid has a chain length varying
from C18 to C24.
@ Plasmilogens
I
t
These are phospholipids which have an aliphatic Ion hosph~phingqsides
chain a-~ unsaturated alcohol in ether linkage with the They contain phosphoric acid group. A common
first hydroxyl group of glycerol (Fig. 8.11 ). The second OH phosphosphingoside present abundantly in bi.ome.m.:..
group is esterified to a f~ . The phosphoric acid is branes, especially of the nervous system, is sphingo-
attached to ~ (Fig. 8.11 ). The alco- myelin. It contains choline (Ffg. 8.14).
hols have about C12 to C18 chain length. Plasmalogen
are found in biomembranes in brain and muscle. phlngomyelins (3m)-,(>)11'~
® Phosphatidy~lycerol l ~)
It is formed by esterification of phosphatidic acid to
Sphingomyelins are the only sphirw,2~ that
contain phosphate and have no sugar ffi81e . They
are found in large quantities in nervous system. Different
glycerol. When two molecules of phosphatidic acid sphingomyelins may be formed depending ~ the fatty
are linked with a molecule of glycerol, di9hosphaff~- acid attached. Common fatty acids found are~ ignoceric
g¥ cerol or cardlpfle!o is formed. It is the major lipid of (24 C),li'lervonic (24 C, one double bond) an~ c ·
mitochondrial membrane. Commercially, it is extracted (22 C, 6 double bonds) acids. Because of its amphipathic
from myocardium. Decreased cardiolipin level if£ds to nature sphingomyelin can act as an emulsifying agent
-
~dcial d ~nctioo, . 1s imp I d Ml-heart and detergent. The relative proportion of lecithin ·and
failuri,;iypothyr~ sm and som es yopath1'es.° sphingomyelin is important in biological fluids like yie,
a~ otic fluid, etc. Sphingomyelin combined wit~
Sphingolipids ( ... L) acid is called ceramide, which is a component of ~
The sphy,gosine pontaining lip.ids may~be of 3 types; spfungohp1dµ
Phosphdsphingosides,
' I
glycosphing ipids and sul-
J
- Clinical relevance of antiphospholipid antibody is
fatides. All sphingolipids have the long aliphatic amino
- described in Box. 8.4.
Chapter 8: Chemistry of Lipids 111
BOX 8.4: APL antibody syndrome 2. Compound lipids are Phospholipids, Sphingolipids,
Antiphospholipid antibodies have been found to be present S ulfolipids, etc. Compound lipids containing alcohol
in blood in patients with autoimmune diseases and in patients sphingosine and one or more carbohydrate resi-
with thr_ombotic episodes. In pregnancy, t.b£. presence ~ ese dues are called glycolipids.
ant 1b~ s can cause complications like ~ ~~rriage, edll ®Psia
and ~ erm labor. Here the antibodi!!s are developed against 3. Fatty acids are classified based on (i) Number of
me e phospfiolipid, cardlolipin. carbon atoms, (ii) Length of hydrocarbon chain, and
(iii) Nature of hydrocarbon chain. Depending on the
)
112 Section A: Chemical Basis of Life
20. Phospholipids are amphipathic in nature since they 24. Phosphosphingosides contain sphingosine as alco-
have a polar head and non-polar tail. Amphipathic hol. Sphingosine esterified to a fatty acid is called
nature is ideal for the role of phospholipids as com- ceramide.
ponents of biomembranes and for micelle formation. 25. Sphingomyelin is the only phosphosphingoside
21. Phosphatidic acid may combine with nitrogenous which contains choline.
base to form amino phospholipids like phosphatidyl 26. Sphingolipids have ceramide attached to carbohy-
choline, phosphatidyl ethanolamine and phosphati- drate residues to form glycolipids like cerebrosides
dyl serine. _ and lactosyl ceramid,z.
22. Phosphatidyl glycerol or cardiolipin is formed by the 27. When one molecule orNANA is attached to the
esterification of one molecule of glycerol simultane- ceramide oligosaccharide, it is called ganglioside.
ous! to 2 mole les of id. 28. Sulfatides are formed when sulfate is esterified to
23. Phospholipas A2 ydrolyses the ester bond bet- ceramide oligosaccharide.
ween the seco h droxyl group of glycerol and a 29. Cholesterol is an animal sterol which is a derived
PUFA . lipid. It is the precursor of all steroids in the body.
8-13. Sphingomyelin on hydrolysis yields all the follow- 8-17. Which one of the following is a dietary essential?
ing, except, A. Oleic acid B. Palmitic acid
A. Sphingosine B. Glucose C. Stearic acid D. Linolenic acid
C. Phosphate D. Choline 8-18. Which of the following lipids gives 2 fatty acids,
8-14. Cardiolipin: one molecule of glycerol and one molecule of
A. Contains Choline phosphoric acid on complete hydrolysis?
B. Is diphosphatidylglycerol A. Diacyl glycerol B. Phosphatidic acid
C. Is part of sphingolipid C. Lecithin D. Cephalin
D. Is found in endoplasmic reticulum 8-19. Which of the following fatty acids is present in
8-15. A ganglioside on hydrolysis gives all the following, surfactant?
except: A. Palmitic acid
A. Fatty acid B. Glycerol B. Stearic acid
C. Sphingosine D. N-acetylneuraminic acid C. Homogamma linolenic acid
8-16. Hydrolysis of fat by alkali is called: D. Arachidonic acid
A. Esterification 8-20. Which of the following is not an amphipathic mol-
B. Saponification ecule?
C. Mutarotation A. Sodium glycocholate B. Cholesterol ester
D. Alkylation C. Phosphatidylserine D. Potassium taurocholate
General Metabolism
Chapter at a Glance
The learner will be able to answer questions on the following topics:
i O Study on six levels of organizations '?> 0 Metabolic profile of organs
2 0 Metabolic pathways and control mechanisms Jt O Metabolic adaptations during starvation
PERIMENTAL STUDY OF To the perfusion fluid any cowpound may be added and
the flu.!£i emerging from the organ is a~fyzed for the
METABOLISM
metabolftes of the compound.
The study of metabolic sequences may be conducted
at six levels of organizations, each at deeper levels Level 3: Organ Slices
of cellular architecture, and ~ - The next lower level of study is using the slices of
s ectives to the s organs, abou( so micrometer tha :\_9tto Warburg (Nobel
Prize 1931 ) was the first scientisrlo study metabolic path-
Level 1: Intact Organism ways using organ slices. (Thµa.strument f ~y of
The essential nature of amino acids and vitamins, etc. tissue respiration is known a~ ~ urg apparatus)} The
was studied by feeding animals with dietc; lackin i advantage of this procedure is thaTihe cellular organelles
one of the ingredients of food.~ 1842 Friedrich Wohl were preserved inta Metabolic transformations of nutri-
showed that benzoic acid when injected Is excreted as ents could be studied in detail. If rat liver slices are incu-
hippuric acid (b~ ~); this was the starting bated with medium containing glucose, carbon dioxide is
point of metabolic study in animal~ adiolabeled iron evolvedj
( 59Fe) is given, and incorporation of the radioactivity in
)q CC'--9-bJ"_' IJ"6'v ,.
J..l kc• 0
Chapter 9: Overview of Metabolism 119
j_q "'\ g 'f::_C
TABLE 9. 1: Energy reserves of man BOX 9.1: Energy ut1ilzat1on of average person
Weight Energy equ,valent The energy consumption varies based on lifestyle in adults.
Stored fuel (in gram) (in kilo calories) Approximately 300 g of carbohydrates (1,200 kcal), 7 ~ f
Glycogen in liver 70 280 proteins (294 kcal) and 80 g of fats (720 kcal) are consumed by a
person with a sedentary lifestyle. Therefore about 60% calories
Glycogen in muscle 120 480
are derived from carbohydrates, 15% from proteins and rest from
Glucose in body fluids 20 80 fats. The energy reserves provide energy in between meals aod
Fat In adipose tissue 15,000 135,000 after over;;i ht-fa.stin I co enol sis and luconeo ene ·s).
.
.. - - ' : - • ..-'l", •
l
CO2 • The reducing eauiv,ajents are mainly gener- survive only a few minutes without blood supply. Occlu-
ated in the mitochondria by the final common oxi- sion of blood supply to brain causes unconsciousness
dative pathway, citric acid cycle. In this process, within 10 seconds. There is no stored fuel in the brain
NADH or FADHQare qruierated. This is called sec-
• Glucose can freely enter the brain cells.
ondary or intermediary metabolism.
iii. Then these reduced equivalents enter into the elec- The total consumption of glucose by brain is about
tron transport chain (ETC, or Respiratory chain), 120 g/day (480 kcal). Thus, about 60% of the total car-
where e'.!.e;Y is released . This is the tertiary bohydrate intake by the body is metabolized by the
120 Section 8 : General Metabolism
Liver Brain
Fed state -+ Glucose-+ Brain
Food
Glycogen
l
Nil F a t - -- - -'-- .. Fatty acid
i
VLDL- ---
Adipose tissue
= Sb\'. (:j)f>~
.Skeletal Musel P-f'I = ~oJtt-.\-hry
The skeletal muscle forms about 4~% of the total weight
of the body. About OJ,% muscle weight is due to glycogen
Adipose
tissue
t
Gluconeogenesis ..---Alanine
content. Following a meal, the muscle glycogen content
increases by about 1% of the total weight.
Fig. 9.3: Metabolism in fasting state
A . Muscle metabolism after a meal: The uptake and
brain. Moreover, about 25% of the oxygen consumed storage of glucose by the skeletal muscle is under the
influence of insulin. Following__a_meaJ? the level of the
by the adult body is due to glucose oxidation in brain. In ·r
children, this may be as high as 50%. gl~a..and insulin ar~ . So glycogen synthesis is
enhanced (Fig. 9.2). The resting muscle uses fatty acids
A Brain under conditions of anoxia: In anoxia the rate as a ~,;:iinr fif IPII (Aai;50011n ') .
of lactate production by glycoly~is rises to 5 or 8 times . . .
withih one minute. The(!Jisfeur eff½s) (see Chapter 1o)1?>. Muscle metabolism . dl.lfmg exerc1~e_ : Muscle us~s
is the brain's protection against conditions of anoxia . glycogen for short active spurts of. a~ §lyco_,.9en is
e le ~ ~ ~ a l . rapidly broken down to form 1~ 1':fhe lactate has to be
transported to liver to undergo gluconeogenesiS-(f,?ri's
B, Brain ~nd acetoacetate: The _brain is unable ~o utilize cycle in Chapter 1O). Mracle tlowever u es,fatty ado as
fatty acids as a source of fuel since the fatty acids com- f f b. • d d" t
. . ue I or ae o Ic exercIs an IOQ Is ance running.
plex:d to album,~ are un; le.~o t rave~j~e e
th
bz:°d
~r; nc .Muscle metabolis; ; during starvation: During star-
b~ . Bu~, brain can 1 . vation, maximum glucose is spared for the brain. The
This Is again a
free fatty acjd (l;.fA) mobilized from adipose tissue
s.
is the>-p referred fuel for muscle during starvation, FFA
C Brain and starvation: During starvation, a significant does ~qttif-e-t~in, and during fasti ·~sp lin level •
part (
is low {Tobie 9.3). uring prolonged starvation.1., muscle
met by 0 r
!(Fig. 9.1). protein breakdown occurs and alanine is relea~ d to
Under conditions of partial anoxia , the production of the bloodstream. It is transported to liver to provide
ammonia is increased. -rfils is immediately trapped as substrate for gluconeogenesis (glucose-alanine cycle in
glutamine. The NH2 group of glutamine and glutamate Fig. 10.27). The metabolic fuel during prolonged fasting
can be used for synthesis of oth er amino acids (s~e is ketone bodies. Branched chain amino a1\in.r......i;,re
Chapter 18). utilized by the skeletal muscle (Fig. 9.3 and Table 9.2).
Chapter 9: Overview of Metabolism 121
-
4. Purified enzymes and proteins may be used to
study i~ividual reactions and regulation _
5. Genomic studies are the main focus of current
- ~ ··
25/ Cardiac' my_scle..-al~ r . . . . a. c i ~
bodies as its preferred fuel under fed and fasting
research in medicine using a,en~knock out techni- state. N~ ~ ,OfheNJ
~es. 26. Cardiac muscle however has an <:,bligatory require-
6. Use of radioactive tracer is widely employed to ment of glucose which is continuously provided by
study the utilization and fate of compounds. the blood perfusing the heart.
7. Metabolic reactions generate chemical energy by 27. ~ iver is metabolically most active orqa~ Almost all
the degradation of compounds~..D metabolic pathways are active iin liver.
8. Precursors of cellular macromolecules are used for 28. During the fed state, liver stores glucose as glyco-
synthesis reactions gen. In the fed state, liver secrets TAG as VLDL
9. Amphibolic reactions 'arelti6'~e. where anabolic which can be taken up and utilized by peripheral
and catabolic reactions ar~i~ke'9 tissues (skeletal muscle and adiipose tissue).
10. All metabolic pathways are catalyzed by enzyme 29. After overnight fasting (8-10 hrs) the plasma
systems which are regulated by ~fferent Jffiha- glucose is maintained by glycogenolysis and gluco-
nisms. M~abolism has 3 phases;'primary, set'6nd- neogenesis.
ary an d
c.. ,ary.
.
30. 1:Jl' 24 houri of faiting. the glycogen stores are
11.(Erimary pb~ includes the digestion of macronutri- exhausted and fatty acids are mobilized from adi-
ents into small molecules which can be absorbed. pose tissue.
12.( § econdary pba~ includes all metabolic pathways 31. Muscle also produces alanine foir gluconeogenesis.
converging at citric acid cycle. 32. By third day of starvation, ketogenesis is active.
13C T.hjrd phasw ccurs at the mitochondrial electron Cardiac, skeletal muscle and aclipose tissue derive
transport chain where the reduced coenzymes are major fraction of energy from ke·tone bodies.
oxidized to generating energy as ATP. This is called 33. Substrate for gluconeogenesis is provided by gly-
oxidative hosphorylation. cerol part of fat and amino acid ireleased by muscle
14. Fed state represents a condition where sufficient protein breakdown.
energy is available. In this state, synthetic pathways 34.\ Ketone bodies start replacrn glucose as e ma r
predominate. source of energy for most tissues including brain.
15. ~ ~-oicarbohydrate is glycoS@J in liver 35. _However ac1;11m1rlation bodi9s in pro-
and~ longed starvation can lead ~s.,t~at[on ketosis.
_ _ _ _ _ Chapter 10
Metabolic
Pathways of Glucose
Chapter at a Glance
The learner wi ll be able to answer questions on the following topics:
)0 Digestion of carbohydrates O Glycogenesis; glycogen synthesis
lo Absorption of glucose and glucose transporters
0 Regulation of glycogen; Cyclic AMP
9, { D Glycolysis pathway and its regulation O Glycogen storage diseases
Energy yield from glycolysis D Hexose monophosphate shunt pathway
Cori's cycle
Glucose-6-phosphate dehydrogenase deficiency
D Pyruvate as a junction point
0 Glucuronic acid pathway
D Gluconeogenesis and malate shuttle
Essential pentosuria
0
D Glucose alanine cycle Polyol pathway
D Glycogenolysis; degradation of glycogen
,l,..a..9..~\t,°t".A
Historical Perspectives ~=~----
__~.,.n.., "::V , v_y "' F ' ''
Abbate Spallanzani (1768) showed that living tissues take up oxygen and give off carbon dioxide. In 18130, Louis Pastu ur
demonstrated the fermentation process o.f..glf.f,9_Se to alcohol by yeast. ,sck..(.1882) indicated that chemicail energy 1n mus le
is converted to contraction of muscle. lnlf~~ ustav G.eQ[ge..,Embden (1 874;1933) studied the lactic a1cid formation from
pyruvate. In 1919, Otto Fritz Meyerhof (1884-1951) enunciated most of the steps of the glycolytic pathway (Nobel Prize, 1922).
Hexokioase eozyro<> was first identified by v.2,n El.l.leL.Cllelpin in 1915 (Nobel Prize, 1929). Other enzym1es in the glycolyt~c
pathway were then identified rapidly; pyruvate decacbaxyla!'le by Neuberg in 1911 , pb.Qsphofructokinase by ~uu:iru:.der
in 1920 (Nobel Prize, 1929); phosphohexo:;e isomerase by L~hmann in 1933; p_yruvate kinase by P,amas..lJ:1-1934.;.enolase by
_l'{eyerhof jn 1!U,5; phosphoglucomutase by l.,tlloir in 1938 (Nobel Prize, 1970); ceraldeh de hosphate deh drogenas~ by
ti,!_~ arburg in 193.9~hosphog!yceromutase,.by 9.uthedand in 194~_(Nobel Prize, 1971 ). Between 1935 an 1H43, all enzymes of
~~'cl gTycolytic pathway were crystallized and characterized by Warburg. He was awarded Nobel Prize in 1931 for his earl ier work
i"l"~~ on cellu lar respiration. He was awarded Nobel Prize for a second time in 1944 for his contributiQ.{ls in glyccilysjs; but Hitler did
"'I( "\:, not permit him to receive it! P~ as was murdered by Stalinistic regime. '
tq\ --- --------·- -- --- acid will inhibit t~ action o f salivary amylase. In the
\.:1/ pancreatic juice another c1lpba-amx~ is available,
~liydr tes re presen S\-C~
In the d i e trb which will hydrolyze the alpha-1,4 gl1ycosidic linkages
~ari.des (s ch, ~ c gen)t.and to a minor extent, randoml~ so as to pr9duce smaller subunits like malt-
as"~ c and flk tose). They arewruo- ose, isomaltose, dexfrins and ~hed or..lL~<i~r-3-
~lO moOQ~ t'nm;units in the gastrointestinal olig&13'ccharides. The eel~ of rus ~ ~ s \ m e
_!Jr' ·tract. Cooking makes the digestion process easier. The contai~the enzymes, sJease, tase, iso~ltase
process of digestion starts in mouth by the salivary and laefase, They hydrolyze the corresponding disac-
/? ~ mylase. However, the time available for digestion charides into component monosacch,arides, which are
mouth is limited, because the gastric hydrochloric then absorbed. b
_,,,. rOll~
l.On$u.:n-,f1'l!W\ tr~~ f"Y\~ - S~ 'J'n~ nh1~ .,. .fibS?.o~f
Chapter 10: Metabolic Pathways of Glucose 127
b\a}O-C ·
Lazzaro Louis Arthur von Euler- Otto Warburg Karl Luis Le/oir
Spallanzani Pasteur Harden Chelpin NP 1931 Lohmann NP 1970
1729-1799 1822-1895 NP 1929 NP 1929 1883-1970 1898-1978 1906-1987
1865-1940 1873-1964
, ;
...
128 Section B:
Fig. 10.2: Glucose absorption (GluT2) Fig. 10.3: GluT4. Glucose transport in cells
r.~"
Another Uniport System C~ e , GLUCOSE METABOLISI\I
Releases Glucose into Blood tD £'GLUi)
Clinical Importance of Glucose
The same intestinal epithelial cells have a different
transport mechanism on the membrane facing capil- Glucose is the ~ e~ source of energy for most of the
laries (Fig. 10.2). lntestjnal cells__releas_e_gl.ucose.Jnto body tissues. Brain eels derive the energy mainly from
bloodstream by the carrier mechanism called Glucose glucose. When the glucose metabolism is deranged, life-
Transporter Type 2 (GluT2). This transporter is not threatening conditions may occur. A minimum amount
dependent on sodium. It is a uniport, facilitated of glucose is always required for normal functioning.
diffusion system (Fig. 10.2). GluT2 is involved in abs- Normal fasting plasma glucose level is 70 to 110 di.
orption of glucose from bloodstream to cells. GluT2 is
After a heavy carbohydrate meal, in a normal person,
present in intestinal epithelial cells, liver cells, beta
.____, . . -cells
-- this level i{ggow 150 mi@)
of pancreas and ~ Y- GluT2 e11ables the pancreas to
monitor the glucose level and adjust the rate of insuli._ ._ _
secretion (see Chapter 11). LYCOLYSIS (EMBDEN•
MEYERHOF.J>ARNAS PATHWAY)
Glucose Transporter 4
GluT4 is the major glucose transporter in skeleta efinition : In glycolytic pathway glucose is converted
muscle and adipose tissue (Fig. 10.3). GluT4 is under to pyruvate (aerobic condition) or lactate (anaerobic
the c~*cal ot jow)in. But otbec glllcase transporters are condition), along _wi~h pro~uction of a small quantity of
n un the trol of instffin. energy~ Glycol~s1s Is ~e:ved. f~om the Greek words,
Clinical applic~ :J.ns_u i promotes the translocation glykys - sweet, and lys1s - splitting.
of intracellular molecules to the cell surface an@ Site of reactions: All the reaction steps take place in:
thus increases gluc!,)se uptake. I~ Type 2 diabetes • the cytoplasm.
mell_itus (s~~ ~ a'8\rr 11 ~.EP~~ GluI4 is.reduced,
leading to insulin res ancelnn:iys.cl~s. In ignificance of Glycolysis Pathway
diabetes, entry of glucose into muscle is only half of
normal cells. GlyI5Js..thafructose.transporter. It is the only pathway that is t¾ing place in all the cells
of the body. Glycolysis is _the ~ ly source of energy in
Absorption t f other~ nosaccharides erythrocytes. In strenuous e~ercise, when muscle tissue
lacks enough oxygen, a~aero~ glycolysis forms the
9'. I \' Gluc are absorbed by the same
tl transporter, SGI It Is an energy dependent process, major source of energy for rn'Qscles.,.Glycolysis is the •
it,~1._, against a concentration gradient, and therefore absorp- preliminary step before complete oxida~'!n. The glycolytic
-y;:/ tion is almost complete from the intestine ...Q~ mono- . path"Y£ Y p~des carbon skeleti~ for:.sy~ thesis of~
.1- , .. [saccharides are absorbed by t arrier mediated facilitated esserftral ~ o a~ids as well as ~ 19cetii par1 of fat. Most
~ - transport. Therefore, absoro1LOD is not complete, and the of the reactions of the-gty~olytic pathway are reyersible,
~ cJremaining molecules in t,he intestine will be fermented which are also usec:NoFO-fcrconeogenesis. A summary of
,.,, \ by bacteria. ( Ot.L\."<~~ , the pathway is shown in Figure 10.4.
I.> \ \C, 0{\\1),.1
~ · w»tt- fo ~- ~om<-- kou.Ul
er
:p-.a.~ '"~ g chi£~.,.,_~ 0-\v "' u 'YE°9t . ~)\,_-tr
·• b.'~-2 lr(J~O'\¼ jn~uh'°f'. T ~ ttot''.~
tc'~ I..!. " L ve_q_
Chapter 10: Metabolic Pathways of Glucose 129
_-o Q
Glucose
e
(1) Hexok nase
•1-
c.,&A"r;;I
V- ATP
,o-r-o-c
757
II H2
\ ..~°:>~ Wit-ADP 4
Glucose-6-phosphate HH : HH
(2) Phosphohexose
H
t=.
isomerase 2 OH
Fructose-6-phosphate H OH H H
krn~e
e, I
J V r-·~
(3) Phosphofructo
.l, ""'
P,
.i(.C>''\
ROS
ADP
ATP
A'l l 0 Glucose
~ATP
\,____Mg+/
ADP
(4) ~ e
1
~~,. j1 PIP~pAl Step 1 of glycolysis; irreversible step
Occurrence
Km value
Phosphoenol pyruvate
Affinity to
substrate
Specificity Acts on qlucose, Acts only on glucose
tr.uc.lgse and
l~
Induction Not induced Induced by insulin and glucose
Function Even when blood Acts only when blood glucose
Fig . 10.4: Summary of glycolysis (Embden-Meyerhof - Parnas sugar level is low, le·1el is more than 100 mg/dl ;
pathway). Steps 1, 3 and 9 are key enzyems; these reactions are glucose is utilized then glucose is taken up by liver
irreversible. Steps 6 and 9 produce energy. Steps 5 and 10 are by body cells cells for glycogen synthesis
coupled for regeneration of NAO·
lucose Entry into Cells_ .. ~f'\ enz me. ~e kinase rep tio,il is · le. But this
.,,~ irreversibil" is c~ vented by another enzyme
Glucose transporter-4 (GluT · transports glucose from ,"•glucose-6 phosphatase (see gluconeogenesis).
the extracellular fluid to muscle cells and adipocytes (see Hexokinase and glucokinase may be considered
Table 10.1 ). This translocase is under the influence 9f as isoenzymes; their properties are compared in Table
i~ulir;i. In diabetes mellitus, insulin deficiency hinders the 10.2. Glue.okioase js under the influence of io.su~
entry of glucose into the peripheral cells. But Glu ·s the but hexokinase is not. Hexokinase is present in most
transporter in liver cells; it is not under the control of· sulin. tissues. Glucokinase is present in and ~ ells.
No1l .-~ ~Glucokinase is induced by insulin. The metabolic fates
tep 1 of Glycolysis of glucose-6-phosphate are shown in Figure 10.6. The
phosphorylation of glucose tra s it within the .c_eUs.
Glucose is phosphorylated to glucose-6-phosphate (Fig.
10.5). The enzyme is Hexoklnase (HK), which splits th i tep 2 of Glycolysis
ATP into ADP, and the Pi is added on to the glucose. Glucose-6-phosphate is isomerized to fructose-6-phos-
The energy released by the hydrolysis of ATP is utilized phate by phosphohexose isomera_Je. This is readily
for the forward reaction. He..xokioase is a ke lycol ic reversible. "
.::---
130 Section B: General Metabolism
Glycolysis to pyr~vate o
-/:Y-f-
E
Glucose ---+ Glucose-6-phosphate Glucose ( • ' ' - C
~ H2 0 CH2OH
Glycogen ".\,v l J'.l"l -o
'---- - -- - -- -- - - -- S
....hc...iup.
npt p°"a'-"
th+w+art-
y -.r-i.----J HH HO OH
Fig. 10.6 : Fate of glucose-6-phosphate
gr•
: ATP I ADP
BOX 10.1: D,phosphate and b,sphosphatc r1re different
When t~ phosph~te are I"
attached to a parent compound, it is called i
r and then
¥-- phosphate \ Mg++ __ t ....,~-Fructose-1 ,6-
Fructose-6- ---~......:::::- L.:::_
Phosphofructoklnase bisphosphate
adenosine-di-phosphate (see Fig. 5.3).
But wher("fil5ospbodc acid~ are present at ~~felf:nL
sites of the compound, it is named as bisphosphate,
- tructose-1,6-bisphos hate (Fig. 10.7).
CH 2OPO3
= H-C=O
I Triose ~ osphate
isomerase
I
C=O H-C-OH
Dihydroxyacetone phosphate
CH 2OPO 3 I I -
CH OPO -
)-r.~
I
: CH2-OH 2 3
CH20Po
®=
/ Site or cleavage I
C=O
~e.,•
t<"I'
Oil\ydroxyacetone phosphate Glyceraldehyde-3-phosphate
I
CH -0H
Fig. 10.9: Step 4-A: isomerization; reversible
/ 2
H- Cl·OH tep 4-A of G/yco/ysi
c~ H- C=O
H-C-OH Ptt~ \ Dihydroxyacetone phosphate is isomerized to glyceral-
I =
H- 6.0 H~
dehyde-3-phosphate by the enzyme ph~s~hcµriose
CH OPO
2 3 I -
CH2 OPO; isomerase. Thus net result is that glucose Is now
Fructose-1,6-bisphosphate Glyceraldehyde-3-P
cleaved int°'-2Tl]Olecules of glyceraldehyd~phate
Fig. 10.8: Step 4 of glycolysis; reversible (Fig. 10.9). The steps 4 and 4-A are together called the
SpJ itting Pbu i>. '("Cy°'-\
tep 3 of Glycolysis ( ~"mO~'(\j f~ Glycerol portion o th~ ral t can enter into
· l 1 glycolytic or gluconeogenic pathways at this point.
Fructose-6-phosphate is further phosphorylated to fruc-
tose1 ,6-bisphosphate (Fig. 10.7 and Box 10.1). Th tep 5 of Glycolysi$
.., enzyme is phosphofructoklnase (PFK). It is the e.Jim1- •1
Glyceraldehyde-3-phosphate is dehydrogenated and
~\q... mig enzyme of glycolys1s) t->FK is an all~feric, in~ ible,
simultaneously ribosphorylated to 1,3-bisphosphogly-
re¢atory enzyme. It is an important key enzyme of this
cerate (1,3-BPG) with the help of NAO• (Fig. 10.10).
pathway. This is again an activation process, and t!}e
The enzyme is glyceraldehyde-3-phosphate dehydro-
energy is derived from ATP. This reaction is an irreversi
genase. The product contains a high energy bond. This
ble step in glycolysis. However duri~ gluconeogenesis,
is a ~eversible ceac!ion. During this reaction, NAO• is
this difficulty is circumvented by fructose-1 ,6-bisphos- reduced to NADH.
phatase. The steps 1, 2 and 3 together are called as the
preparatory phase. 1° - tep 6 of Glycolysis'
Step 4 of G/ycolysis The energy of 1,3-BPG is trapped to synthesize one
V ,.. ATP molecule with the help of bisphosphogl cer te
One molecule of fructose-1 ,6-bisphosphate (6 carbons) Ki' e (Fig. "'I0'. 11 ). This is an example o level
is cleaved into two molecules of 3 carbon units; glyceral- iJ;;.iiihorylatloa(, where energy is trap()ed direct))l.fiom
dehyde-3-phosphate and dihydroxyacetone phosphate the substrate ~ he help of the c;'mplicated electron
(DHAP) (Fig. 10.8). Since the backward reaction is an transport chain reactions. When energy is trapped by
aldol condensation, the enzyme is called aldolase. This oxidation of reducing equivalents such as NADH, it is
reaction is reversible. called oxidative phosphorylation. Step 6 is reversible.
Chapter 10: Metabolic Pathways of Glucose 131
r- 1.
Fig. 10.10: Step 5 of glycolysis. Reversible step1 NADH gene- Fig. 10.11: Step 6 of glycolysis. Reversible reaction. ATP genera-
rating step I , -
1 tion step
(.,.C,, .
Phosphoglycero-
coo- mutase COO- Enolase coo-
l l
C=O
CH-OH l B-o-Po~ ~ •
I I
I -
CH OPO -
CH2.loHl
HO
2 CH 3
2 3 Phosphoenol pyruvate (PEP) Pyruvate
3-phosphoglycerate 2-phosphoglycerate Phosphoenol pyruvate
Fig. 10.12: Steps 7 and 8 of glycolysis Fig. 10.13: Step 9. ATP production (irreversible}
Fig. 10.14: Step 10; LOH reaction; reversbile two enzymes (pyruvate carboxylase and phosphoenol-
;. uvate carboxykinase) (see gluconeogenesis).
p 10 of Glycolysis H:! d
3-phosphoglycerate is isomerized to 2-phosphoglycerate
In anaerobic condition, ·
by shifting the phosphate group from 3rd to 2nd carbon
by tg te dehydro~ (LOH) (Fig. 10.14). (Greek; ·.
atom (Fig. 10.12). The enzyme is phosphoglyceromu-
an=not; aer=air; bios=life). LOH has 5 isoenzymes. A
tase This is a readily reversible reaction.
p summary of glycolysis is shown in Figure 10.4.
8 of Glycolysis In aerobic conditio , e pyruvate enters~ e~
..acid.cyc~ (or complete oxidation. The end product of ana-
2-phosphoglycerate is converted to phosphoenol pyru- erobic glycolysis i~ ~ Cori's cycl~
vate by the enzyme enolase. One water molecule is
removed (Fig . 10.12). A high energy phosphate bond ignificarice of Lactate Production "\
· duced. The reaction is reversible. En ·res
Steps 5 and 10 are Coupled i q Q_~Ll J
and by removing magnesium ion f uond ill
ersibly inhibit this enzyme. Thus, fluor stop )J.Jcose two mole-
the whole glycolysis. So whe~ collecting blood for sugar cules o '::: t{;A::;O!:.•:...a
:.::r:.:..e~re:.:::d:.:.u..:.
c:.:;
e~ = ~~ .1} The availability
estimation, fl~oride is added to blood. If no ,""glucose of co~ n mes inside a ce)J is limited. Therefore, this step . ,p-;
is metabolized by the blood cells, "so that lower blood becomes a bottleneck in the whole ~ e.
sugar values are obtained . This NADH is to be reconverted to NAO•. This can
\ be done by oxidative phosphorylation. However, during
p 9 of Glycolysis exercise, there is lack of oxygen. So, this reconversion
Phosphoenol pyruvate (PEP) is dephDsphorylated is not possible. Therefore, the cell has to couple some
to pyruvate, by pyruvate kinase. First PEP is made other reaction in which NAO• is regenerated in the
into a transient intermediary of enol pyruvate; which is cytoplasm itself. Hence pyruvate is reduced to lactate;
t' Cle,,..""vbZc 0J lJC!lhf;~ 1 "'7\vt2 -00 "'6" eo.ctt.un '"'hr t ~,z;tl.f
tlf''°"\'+-1-,• do.,\ve..0- 1)'°~ bo
cy c.\.e
fe,.. ,iurl:ow,\1-.oul
_-ti> NFi\J~, 1t'J-J.-1.
bv,r-.q
132 Section B: General Metabolism 1M~~ ~vi:~ N ~D
7
per molecule of glucose in the glycolytic pathway, under
Gly3PDH anaerobic conditions (Oxygen deficiency)
(Ste p 5)
NADH
!
No. ofATPs gained
1,3-bisphospho- Step Enzyme Source per glucose mo/
*
glycerate +
1 Hexokinase Minus 1
L
NADH NAO @
!
3 Phosphofructokinase Minus 1
6 1,3-bisphosphoglycerate kinase ATP 1x 2 = 2
\ ,LOH Lactate
Pyruvate
t 1
(S ep O) n \ : "" 9 Pyruvate ki nase ATP 1X2 = 2
" e~,en~""""tl'\ Total = 4 minus 2 = 2
Blocked in lack of oxygen 9\_ N I)
Acetyl CoA t) cle, BOX 10.2: Chicken 1s white. but duck 1s red 11
Fig. 10.15: Lactate formation is necessary for .~ f Actively contracting muscles that rapidly consume ATP can also
Nf .OH lo [)18.D:.d,_uring anaer®,iasis. · regenerate ATP entirely by anaerobic glycolysis. Fast twitch white
muscle fibers have very few mitochondria and predominate in
muscles capable of short bursts of activity (sprints). Slow twitch
red muscle fibers rich in mitochondria are found in muscles that
(1) contract slow ly and steadily (distance runners). Chicken mes
~---1--------+----=-'---_;;;:=.::..<---~---l only short bursts and has white fibers. Ducks on t he other hand
GK Insulin
are migratory and have red fibers suitable for sustained activity.
G) PK Insulin, Fl ,6-BP ) '------''----'---- - - - - - - - - - - - - - - = - -___,
(;;'l f---t-....:..._c_---+-'----'--'-'---"--.:........:........:....::L.::.:..;:____~~lR
PFK Insulin, AMP. F-6-P, Glucagon, ATP, citrate, low pH, cAMP
F-2,6-BP TABLE 10.S: Energy yield (number of ATP generated) per
Ji : U~e J e r\r-.r,1:-u<?.9-'~ rrr,1..,,~t'"""· • molecule of glucose in the glycolyttc pathway. under aerobic
G-) n ~rr,-6'"' ' " ' ~ . \ . (' I'\ :i:. -~ ,~, conditions (oxygen is available)
the NAO• thus generated is reutilized for uninterrupted!' J!!IIII No. ofATPs gained
operat~ the 5th step (Fig. 10.15). Step Enzyme Source per glucose mot
In~ . there are r:io mitochondria . Hence RBCs 1 Hexokinase Minus 1
P~ - =- 5 Glyceraldehyde-3- phosphate
dehydrogenase
NADH 2.5 x 2 = 5
TABLE 10.6: Energy yield (number of ATP generated) per molecule of glucose when it is completely oxidized through glycolysis plus
citric acid cycle, under aerobic conditions
No ofATPs gained
Method of ATP No.of ATPs as per old
Pathway Step Enzyme formation calculation
Glycolysis 1 Hexokinase Minus 1 Minus 1
Do 3 Phosphofructokinase Minus 1 Minus 1
Do 5 Glyceraldehyde•3·P DH NADH Respiratory chain 2.5 X 2 = 5 3 x 2=6
Do 6 1,3·BPG kinase ATP Substrate level 1 x2=2 l x2= 2
Do 9 Pyruvate kinase ATP Substrate level 1 x 2 =2 1X2=2
Pyruvate to acetyl•CoA . Pyruvate dehydrogenase NADH Respiratory chain 2.5 X 2 = 5 3x2=6
TCA cycle 3 lsocitrate DH NADH Respiratory chain 2.5 X 2 = 5 3 x 2=6
Do 4 alpha ketoglutarate DH NADH Respiratory chain 2.5 X 2 = 5 3X2=6
Do 5 Succinate thiokinase GTP Substrate level I x 2 =2 I X2=2
Do 6 Succinate DH FADH2 Respiratory chain 1.5 X 2 = 3 2x2 =4
Do 8 Malate DH NADH Respiratory chain 2.5 X 2 = 5 3 x 2=6
Net generation in glycolytic pathway 9 minus 2 = 7 10 minus 2 = 8
Generation in pyruvate dehydrogenation =S =6
Generation in citric acid cycle =20 =24
Net generation of ATP from one glucose mol =32 = 38
Nore: Previously, calculations were made assuming that in the electron transport chain, NADH produces 3 ATPs and FADH2 generates 2 ATPs.This will amount
net generation of 38 ATP per glucose molecule. Recent experiments show that these old values are wrong. Please also see Chapter 21 for details.
e
egulation of Glycolysis Glucose
-::1
The regulation is summarized in Figure 10.16. The '········· G6P
regulatory enzymes or key enzymes of glycolysis are:
1. ~ xokinase, step 1 (glucokinase, in liver)
2. Phosphofructokinase, step 3 (Table 10.3)
Glucocorti·
coides -
-
-
l
H
F6P _ . F2,6BP
+ ........ .J...... Insulin
........ .:
..............ADP
Pyruvate kinase, step 9. : FBP · ····················,
i t! ·············~ :
ctors Regulating Glycolysis I
:
G3P
u
+- DHAP i
:
: BPG :
Hexokinase having a hi_g_h affinity for glucose will act
even at low glucose concentrations. $0, brain and RBCs
~ . )lf
3PG
i:
can get necessary energy. Glucose-6-phosphate has a i: H
2PG
i
i
feedback inhibitory effect on the enzyme. G,.!Ygokinase
i H !
with a loi affinity a~ higll..Km-tor..g!1.1GGS0-is-;::>ffiSel:lt
o; ly irfTf, r and bet~ ells of pancreas. These are the $ , ···········•·····---·• _
PlEP
r . .. . ........ ADP
tissues where glucose is available in plenty. In the liver ''' ''
'
glucokinase phosphorylates glucose which can be used
for glycogen synthesis. l~
the glue · ·
eta cells of pancre~
·n to
::
'
''
:
\..
i
\
i
Pyruvate
-
is utilized by liver to produce glucose. Th~ cess.nee<:ls
ATP. The resultant increased oxygen consumption is the
explanation for the oxygen debt after vigorous exercise.
asteur Effect
Fig. 10.17: Cori cycle. Contracting muscle has lack of oxygen. Under aerobic conditions, glycolysis is inhibited. This
So pyruvate is reduced to lactate. This can be reconverted to
~ t o ~ effect~tgx ~ on qlycolysis is known as
r
glucose in liver by gluconeogenesis.(!.actate to pyruvate ratio Is
m:rrmaily approximately 25)> Pasteur effect.
\-\'f ft'<-O'>t\.PI \'Jll&
arburg Hypothesis CP.rNC£.R
6-bisphosphatase) are reciprocally regulated. The two
enzyme activities are present on the same polypeptide Otto Warburg showed that cancer cells utilize energy
chain , a tandem enzyme.Addition of a phosphate group to from qlycolysis and they require less ax~en than their
the tandem enzyme activates F-2,6-bisphosphatase and normal counterparts; this is called Warburg hypothesis
inactivates PFK2 leading to a fall in F-2,6-bisphosphate (1923). Inhibition of glycolysis severely depletes ATP in
and glycolysis slows down. Dephosphorylation has the can ells and leads to massive cell death. Because
opposite effect. the Warbur effect and hypoxia re frequently seen in
Pyruvate kinase is a regulatory enzyme of glyco- human cancers, these findings may have broad clinical
lysis. When energy is in plenty in the cell , glycolysis is
inhibited. Insulin increases its activity whereas glucago·,ft;".......-..= · .
inhibits. Insulin favors glycolysis by activating the ke apoport Luebermg Cyc:;.Je
~; glycolytic enzymes. Glucocorticoids inhibit glycolysis (BPG Shunti
and favors gluconeogenesis (Tables 10.3 and 10.7). Q in the erythrocytes, step 6 of glycolysis is bypassed.
Bisphosphoq!ycerate mutase.converts 1,3-bisphospho-
Cori Cycle or Lactic Acid Cycle glycerate (BPG) to 2,3-BPG. Then BPG-phosphatase
remo~e..pllos~ roup to form 3-phosphogly-
Q) oefinition: It is a process in which Qlµcose is converted..
to lactate inJba.muscle;.. and in the liver this lactate is
cerate (Fig. 10.18). i ,u;i-r-
.J
re:£9L1Yerted ·nt~ucese-_(Fig. 10.17). The muscl@ Significance of BPG
cramps, often associated with strenuous muscular exer-
cise, are thought to be due to lactate accumulation. Lac- 2,3-BPG binds to hemoglobin, and reduces the affi-
tic acid from muscle diffuses into the blood . Lactate then nity toward oxygen. So, in pr~ ce oL.2.3-BPG,
-\t ' Ra_p-Oll>O'i~ ~€$¼"3 d~ NO ~i? ~ ~ ~ & ' { ' ) . \ \
I -\'r-,0\J..<Jr\ ?, -¼ q~
Chapter 10: Metabolic Pathways of Glucose 135
II
CH3-CO-COO~
H Thiamine-PP ~ CH3- co-S-L- SH X HSCoA
"""'1!1111~
Pyruvate
11,Wulil · \!31
co
2 CH3-CHOH-TPP / L\ / L \ CH3-CO-SCoA
tat©
The compe_nsatory increase in 2,3-BPG in high altitudes C. Dihydrolipoyl dehydrogenase (Enzyme 3): The
favors oxygen dissociation. last step is the oxidation of lipoamide.
§r.culation;1n this shunt pathway~ TP is genera . A ,imilar enzyme complex brings about the oxidative
Please compare Figures 10.18 and 1o. . decarboxylation of alpha ketoglutarate to succinyl-CoA
in the TCA cycle (see Chapter 20).
METABOLIC FATE OF PYRUVATE t-l'C. l ~on: Alet: lost~'; i'1_\bi~ ;ruhe i r~ ucts
ace~l~~nd NA . ) "
Under aerobic conditions, pyruvate is comLedecL-to n, r) t
~J:Q.ol\ which enters the TCA cycle to be oxidized to Importance of Pyruva Dehydrogenase
CO 2• ATP is generated. .<~ Completely e}'l rs 'There is no.eafbwjY,
Glycolysis is taking place in cytoplasm. So ~_ume available in the body to cifE_umvent this,.step,,. Glucose
is gea,.e rate.d..ia..cyto_p!a~1:nsporte,d into mito- through this step is converted to acetyl-CoA from which
chondria by a pyruvate transporter. fa~cids.can.b.e~yntbesized. But the backward reaction
is not possible, and so there is no net synthesis of
Pyruvate Dehydrogenase Complex 9!!!.CAu.from,fat. HencQDH reaction ajhg romm;uaii)
.140~ . step toward com lete ox· o gly£Q_se .
Inside the ~ochon_d.cia, pyruvate is oxidatively decar- Diseases associated with glycolysis pathway are
boxylated to acetyl-CoA by pyruvate dehydrogenase described in Box 10.3.
(PDH). It is a multi-enzyme complex with ~ s
and~ enz~s. The coenzymes needed are: Pyruvate as a Junction Point
1. Thiamine gy_roph, hate (TPP)
2. (Cp.™vm.e.A.(.C~ \..c9'{(" , . ~>eJ.
3. FAD ,,- t
Z.Si+ 8C
WU
4 . NAO•
5. Lipoamide. The lipoic ~id, otherwise called .!!}i~ c =...;;...;.;;...;;._ ...:..;....:..;.~ ,ch is use eo enesis.
acid has two sulfur atoms and 8 carbon atoms. It These pathways are summarized in Figure 10.20.
can ac_seot or donate hydrogen ato~ . ~ON -..1-tO
6. The enzyme part of the PDH complex is made up of GLUCONEOGENESIS
three component enzymes (Fig. 10.19). - - --,- m
0
1-~-+-+-
A. Pyruvate dehydrogenase (Enzyme 1): Catalyze~ Deflnition \,,..\,n&.,ovo
C~
oxidative decarboxylation. TPP is required for this It is the process by which glucose molecules are produ-
step. So, Thiamine, a B complex group vitamin ced from non-carbohydrate precursors. These include
(_~)
136 Section B: General Metabolism
:i
for oxygen. On the other hand, a deficiency of e Cytoplasm Mitochondria
kinase leads t BP
Malate . = = ~- - ==== = ~ Malate
NAD+ +
MDH MDH
t NAD+ +H
NADH NADH
Oxaloacetate • Oxalo~
deficiency in alcoholism causes lactate accumulation in tissues. <" Glu , rrt,-\-
Inherited aldolase deficiency may lead to ·c.a sis. - ~''tt.-
Glu ~
AST
AST .,.,.-,i.
AKG AKG
Gfua.r !PYRUVATE
PK ½.
,:;:;;=,===- Alanine
Fig. 10.22:1Malate-aspartate shutt1J . MOH = Malate dehydro-
~ enase. AST = Aspartate aminotransferase. Glu = Glutamic acid.
AKG = Alpha ketoglutaric acid
PCl l PDH
Oxaloacetate Acetyl CoA - + Fatty acid Gluconeogenesis involves s es of gly-
coo- A
'co coo-
l '--- 2
l
C=O
I
CH2
PEPCK
4 r CO- P
II
CH
1----..
2
GTP GDP
( c~ (ITP) (IDP)
Glycerol
j
Oxaloacetate Phosphoenolpyruvate
Glyceraldehyde-3-phosphate - - - - DHAP
ate Carboxykinase
In the cytoplasm, PEPCK enzyme then converts oxalo- !i
1,3-Bisphosphoglycerate (1,3-BPG)
acetate to phos hoenol pyruvate bY. removing a molecule
~'\,e- l t , - .ADP
o~ 2
• GTP or ITP onates the phosphate (Fig. 10.23).
t---- ATP
The net effect of these two reactions is the
3-Phospr t ycerate
conversion of pyruvate to phosphoenol pyruvate. -
2-Phosphoglycerate
., t
Phosphoenol pyruvate
Partial Reversal of Glycolysis
The phosphoenol pyruvate undergoes further reactions ·f
Lactate
7
Glycine, Alanine,
catalyzed by the glyzilytic.....,filgy~ to form fW-~ Serine, Cysteine
1e-bise_hosphate (see glycolysis steps 8,7,6,5 and 4). fu<
All these reactions are fr~ JffilefS.ible. 1 Acetyl CoA
Fructose-1, 6-bisphosphatase \
Fructose-1 ,6-bisphosphate is then acted upon by fruc-
tose-1 ,6-bisphosphatase to form fructose-6-phosphate.
Asn
Asp
-----? Oxalo ~
acetate Citrate
j/E:~::
Malate \
glycolysis).
Fructose-1 ,6-bisphosphatase (enzyme) Phe,
r
Fructose-1 ,6-bisphosphate-,Fructose-6 phosphate+ Pi Tyr
\ Alpha keto
Asp,
Then fructose-6-phosphate is isomerized to glu- Asn - Fu~ rate /ta rate
cose-6-phosphate by the freely reversible reaction cata-
lyzed by hexose;_Qhospqate · mera (second step in "' / lie,
Propionate Succinyl CoA - - - - Met, Val
glycolysis)_. r r 'l Key gluconeogenlc enzymes:
1=Pyruvate carboxylase; 2=Phosphoenol pyruvate
carboxykinase; 3=Fructose-1 , 6-bisphosphatase;
Glucose-6~phosphatase Reactiorl 4:Glucose-6-phosphatase
Key glycolytic enzymes .
The glucose-6-phosphate is hydrolyzed to free glucose S=Hexokinase; 6=Phosphofructok1nase: 7:;Pyruvate kinase
by glucose-6-phosphatase. Substrates for gluconeogenesis are shown inside brown squares.
i
sent in and in,tgs't!nal muco~a to a lesser extent, Significance of Gluconeogenesis
but is absent in muscle. through gluco-
The detailed steps of gluconeogenesis are shown in neogenesis, because glucose-6-phosphatase is pre-
Figure 10.24. sent mainly in liver. So liver pJays the major role in
"- '>
lli...v-~\.--
Ne\
138 Section B: General Metabolism
CH3 NAO
( '\ NADH + H CH3 CH3 a-keto-
glutarate
Glutamate CH3
Z
hours of fasting. On prolo~ ) tarvation, the gluco-
neogenesis is speeded up ar\<U!'~ rr ca tabolism provi-
~des the substrates, namely glucogenic amino acids.
i LORt'"- C4t.le.
o/"1 (Qluconeogenesis by renal cortex increases:) ® Lactate - -- - - Lecta't'e - - - Lactate
1!:63@
erate one glucose molecule. Liver _
Blood Muscle
n,e>,I";..,
I",, ...
I' Fig . 10.27: Glucose-alanine cycle
Glycolysis
lucose , 'Lactate
Gluconeogenesi ·· the glycolytic pathway to form pyruvate, which in turn,
can be transaminated to alanine. Glucose-alanine cycle
Substrates for Gluconeogenesis is important in conditions of starvation (Fig. 10.27). Thus
Lactate and glucogenic amino acids are the most net transfer of amino acid (nitrogen) from muscle to liver
important substrates for gluconeogenesis. and corresponding transfer of glucose (energy) from liver
to muscle is affected. Alanine cycle is intimately related
Lactate "' } with Cori's cycle (compare Figs. 10.17 and 10.27).
Glycerol Glycerol-3-phosphate
kinase dehydrogenase Fructose-6-phosphate
(\ • Glycerol-3-
phosphate
( '\ Dihydroxy-
acetone
phosphate
(+) Citrate
ATP
Fructose-1,6-bisphosphate
Gluconeogenesis
Glucagon (+)
1l Glycolysis Enzyme
PC
Activation
Cortisol, Glucagon
Inhibition
Insulin, ADP
Glucocorticolds (+) f\f\\ p
Adrenalin, Acetyl-CoA
Insulin H Prt>t>
A'iP> G \:rols, (+) Insulin PEPCK (i,nO..~ Insulin
do
c..Am-P Pyruvate F-1,6-bisp hosphatase do F-1,6-BP,AMP,F-
2,6-BP
Hormonal regulation of gluco
G-6-phosphatase do Insuli n
• Pyruvate Carboxylase
It is an allosteric enzyme. Acetyl-CoA is an activator of BOX 10.4: Clinical significance of pyruvate
pyruvate carboxylase so that generation of oxaloacetate 1. Pyruvate carboxylase deficiency. It Is seen as an inb orn
is favored when acetyl-CoA level is sufficiently high error of metabolism, where mental retardation is manifested.
(Fig. 10.21). Its incidence Is 1 in 25,000 births. Lactic acidos1s is noticed.
2. Malignant hyperthermia. This may occur Yfil.en halothane
i given as an anesthetic to certain persons. The ryanodine
receptor, a calcium-release channel is defective, leading
to inappropriate release of calcium from sarcoplasmic
reticulum. This results in uncpntrolled heft ~ neration,
dama~ of muscle cells~TP depletion, lactic acidosis and
rhabddrnyolysis. CPK is markedly elevated. This defect is seen
in 1 per 50,000 population.
3. Ethanol (Ethyl alcohol). Inhibits gluconeogenesis. During
the metabolism of ethanol the level of cytoplasmic NADH
is raised. Thus, the Pyruvate Malate - > Oxaloacetate
Gluconeogenesis is enhanced by ATP. reactions are reversed. So, ingestion of alcohol
r~s in hye,oglycemia. Lactate also accumulates as NADH
Hormonal Regulation of Gluconeogenesis level is high (see Chapter 12).
Glycogen
phosphorylase
Glycogen Glycogen + Glucose-1-
G
with (n) + Pi with (n-1 ) phosphate
glucose (PLP) glucose
residues residues
l
sequentially
larger muscle mass. When blood glucose level lowers,
00000
0 0 0 0-0-0-0-0-00-- liver glycogen is broken down and helps to maintain
blOfil~Cose ~v~I (Figo.11 Q._3_\l-
Act1on of glycogen ( Transferase enzyme )
phosphorylase stops near ~ sfers a tn§pccharide 0..).'fter ":foobc..o~~o~r whi~h
the branching point unit to another.branch causes ~~63®r:amtasWiffn li~tA]_o'lR 5 h~ u e_r
taking food, the blood sugar tends tu.fall. But, glycogen
~ -- is lyzed to glucose so that the energy needs are met.
(,Oebranching enzyme)
µ
hydrolyses alpha-1 , 6 linkage
0-0-0-0-0-0-0-0--
Glucose unit 1s released:
branch point is removed
•
After about 18 hours fasting, most of the liver glycogen
is depleted, when depot fats are hydrolyzed and
energy reguireme~i~t ~ - The
function of muscle glycogen Is to act as reserve fuel for
G en phospho; las y
further acts
l
00
Glucose-1-phosphate units
are released sequentially
muscle contraction. All the enzymes related to glycogen
metabolism a r e ~.
......,....__
Carl Cori and Gerty Cori were awarded Nobel Prize ir:t, Glycogen phosphorylase removes gluco~e as glucose-
1947, for their work on glycogen degradatii:m. Based on 1-phosphate from glycogen (phosphorolysis) (Fig. 10.32).
his work on glycogen synthesis, Luis Leloir (Argentina) It contains pyridoxal phosphate (PLP) as a prosthetic
was awarded Nobel Prize in 1970. Earl Sutherland group. The alpha-1,4 linkages in the glycogen are
(Nobel Prize, 1971 ) studied the roje of cyclic 4,MP as ~ ea-f°e d. r-emoves glucose units one at a time. Enzyme
the second messenger in glycogenolysis. Glycogen ~ equentially hydrolyzes alpha-1,4 glycosidic linkages, till
is a @opolysaccha~ with glucose units linked in it reaches a glucose residue, 3-4 glucose units away
alpha-1 , 4 linkages (straight_!l,ne) and ajpha-1 , 6 linkages from a branch point (Fig. 10.33)(li cannot attack the 1,6
(branching oojnt). Branching makes the molecule more linkage at branch paint> If glycogen phosphorylase alone
globular and less space-consuming ..• For details of acts on a glycogen molecule, the final product is a highly
structure, see Figure 7.23. branched molecule; it is called limit dextrin.
L. , rp o CJ '1d .~)(r!f '-< cm tr1p1e 1 1)m 2\crti vo¥.'->r-- f')~ •
-: \_~ r"IQ.\ rn'-'.SrJ.e q r- C ?1'I'
.., Chapter 1O: Metabolic Pathways of Glucose 141
CH 2OH .
H2 GLYCOGEN SYNTHESIS
O
H H -o-:x- 0C- ~
OH (GLYCOGENESIS)
0
0 o-r-o-
II
OH H
The glycogen synthesis occurs by a pathway distinctly
different from the reversal of glycogen breakdown,
0 OH
H OH Cf which would prevent the operation of futile cycles. The
H OH
Glucose-6-phosphate steps are:
e reaction
-\ '\J> \)1 ()..'(-!.~
• •
Act1vat1on of Glucose
NO O
il
V-.
a.:
Deuranc h"mg by 1"() 0:-DI!.)
0 .1 er. •~ 1'1.A" UDP glucose is formed from gllJ...cose-1-phos.e,hate and
1 F V""'cu)s=- ~..., UTP (uridine triphosphate) by the enzyme UDP-glucose
QV.,
c,,c:'~,"o,,! ~s)e
(many cycles)
UDP
1 Glycogenin
alpha-1,4 linkages
phosphorylase kinase and protein phosphatases.
The intracellular concentration of cAMP therefore
decides the level of active PKA. The cAMP level depends
Branching enzyme
transfers
j on the activity of adenylate cyclase and phosphodi-
esterase. Cyclic AMP level is increased by glucagon and
decreased by insulin. P h~hO&.~!:
6 glucose residues
to form a new branch Adenylate cyclase ~ f!JI 0
Repeat action of
glycogen synthase and
branching enzyme
l Glycogen in
hosphory/ase Kinase Activation
The active protein kinase can now co Q_'ie.ct the phos-
•VT
~ 1 phosphorylation
1 of the active phosphorylase
'---- - - - - -- - -- - - - - - - - ~ pr~; e~~ phosphatase 1 (PP1) involves removal of
Fig. 10.35: Formation of branches in glycogen phosphate group from phosphorylase-a. When cAMP
level is high, PP1 is inhibited. The effeclol cycUe-AMFlis
but glycogen synthase becomes inactive on phos- OQL,o~~asiA9-loo..pbospl:lol)4atiGA-ef-em~ymes,
phorylation. The hormonal control by covalent modifi- but also by decreasing depbos.phol)'.iation.
cation and allosteric regulation are interrelated. These
hormones act through a second messenger, cyclic AM lycogen Phosphorylase in
(cAMP) (For structure see Fig 38.7). The mechanism is Liver and Muscle
shown in Figure 10.36.
Liver: The liver phosphorylase-b is the inactive form.
It becomes active on phosphorylation. The active
Generation of Cyclic AMP (cAMP)
enzyme is denoted as phosphorylase-a. The enzyme
Both liver and muscle phosphorylases are activated by a is inhibited by ATP and glucose-6-phosphate ~ee Fig.
c clic A mediated activation cascade triggered by 10.36).@lucose is an a"ikisteric inhibitor of phosphorylase
ttie ormonal signal. The hormones epinephrine and a. Insulin favors this effect by promoting the uptake and
glucagon can activate liver glycogen phosphorylase phosphorylation of glucose.
but glucagon has no effect on the muscle. When the Muscle: Skeletal muscle glycogen is degraded only
hormone binds to a specific receptor on the plasma when the demand for ATP is high. The regulation of
membrane, the enzyme adenyl cyclase is activated glycogenolysis in skeletal muscle is by epinephrine. ,
which converts ATP to cyclic AMP (cAMP). When level Glucagon has no effect on muscle glycogenolysis. AMP
of cyclic AMP rises, it will activate a protein kinase (PKA) formed by degradation of ATP during muscle contraction
by phosphorylation of a serine residue (Fig. 10.36). is an allosteric activator of phosphorylase-b.
6
Epinephrine/Glucagon attaches to receptor
', Glycogen synthase
'- - - (Active) (Dephosphorylated)
Cell membrane
1 Glycogen synthase
1915-1974
!
protein kinase (Glycogen synthesis inhibited)
(\
(inactive) (active)
(dephosphorylated) (phosphorylated)
ATP ADP
ATP
(\ (active) (phosphorylated)
+
ADP Glycogen ---'--+ Glycogen (n-1 units)+
(n residues) Glucose-1-phosphate
(Glycogen breakdown is favored)
Glycogen (n-1)
Glycogen Synthase
Glycogen synthesis
Glycogen synthase and phosphorylase activities are
Glycogen (n)
reciprocally regulated. The same protein kinase, which UDP-glucose
phosphorylates the phosphorylase kinase would also
[ Insulin (+) ycogen synthase
phosphorylate glycogen synthase. The activity of the
glycogen synthase is markedly decreased on phospho- UDP
Glycogen (n+1)
rylation. Insulin promotes glycogen synthesis by favor-
ing dephosphorylation . Glycogen synthase is active in Fig. 10.37: Effects of hormones on glycogen
the dephosphorylated state. Relative rates of glycogen
synthesis and breakdown are controlled by the action of stimulus of hypoglycemia. The key enzyme for glycogen
synthesis is glycogen synthase. Its activity is decreased
PKA, phosphorylase kinase and protein phosphatase.
by glucagon and epinephrine. Activity is enhanced by ins-
The regulation of glycogen phosphorylase and synthase
ulin, under the stimulus of hyperglycemia (Fig. 10.37).
is a typical example of multisite phosphorylation (pri-
Glycogen metabolism is regulated by coordinated
mary and secondary sites) for metabolic regulation .
regulation of glycogen synthase and glycogen phos-
phorylase. The regulatory mechanisms include allos-
Summary of Regulation teric control as well as hormonal control b covalent
The key enzyme for glycogenolysis is phosphorylase. rm-Pificati96)of enzymes. Th allosteric effect are
It is activated by glucagon and epinephrine, under the ATP, gluco'~-6-phosphate an AMP.
l-\~m on oJ 1 b\\v.t~oo.\ £.pi-neph~'l'\t...,
X.<-6u)'"' .
144 Section B: General Metabolism
limit dextrinosis Debranching enzyme ~ ( 9 -t Highly branched dextrin accumulates; Fasting hypoglycemia;
Cori's dise se (Liver and muscle) c;., hepatomegaly, myopathy
Amylopect inosis Branching enzyme f"lt.~_,<fJJ. Glycogen with few branches; h!_Patospleno~aly, irr~ t death
Anderson's disease (Liver and muscle) • · before age of 5
BOX 10.6. N/\D ,md NADP ,Ht' d1ffprenl Step 6, Transaldolase Reactio11
NADH ts used for reducing reactions in catabolic pathways, e.g.
The next group transfer reaction involves the transfer
and is g; e t . <
pyruvate to lactat . ADH enters the electron transport chain,
-:J of a 3 carbon unit, from sedoheptulose-7-phosphate to
glyceraldehyde-3-phosphate to form fr.JJctose-6-phos-
phate. Here also the donor is a ketose and acceptor is
an aldose (Step 6, Fig. 10.41 ).
CHO
i
OH
HO
OH
OH
CH2OPO3=
NADPH+H•
+OH
CH2 OPO 3=
Glucose-b-phosphate Ribulose-5-phosphatc
~e)~t~~)
Fig. 10.38: Oxidative phase of HMP shunt pathway; Steps 1, 2 and 3
i . 1)eh'-3~~~ohon 2.. - \.'~d•·nlhcm i.
cox~) s.1.,~'l",,I
I..:. dec:.a...-Ybo~ \.J \.O..,~
146 Section B: General Metabolism
r,ve.Y..e
.,.. kr6n ,. ,.
t::
Ribose-5-phosphate ceraldehyde-3-phosphate
CH2 0H
i lf
CHO
Transketolase
0 + OH
+oH
R
HO =c5H) CH20P03=
H
CH20P03=
OH H H
CH2 0 P0 3=
c~
CH2 0P03 = CH 20P03 =
HO
Xylulose-
5-phosphate
Phosphopentose
Eplme.uise
Ltf"t:,)
~==== ulose-
Phosphopentose
5-phosphate
l pf>\.J
=== :=, Ribose-
5-phosphate
H
Of CH2 0P03=
OH
OH
OH
CH20P03=
Xylulose-5-phosphate Sedoheptulose-7-phosphate
Fig. 10.39: Step 4 of HMP shunt pathway Fig. 10.40. Step 5; first transketolase reaction
5 x glucose-6-P
__:N'd:
. . :. =-.;..._-- -- -~ 6 x 3-keto-6-phosphogluconate
., t?>PG,if'\
2 x sedoheptulose-7-P
2,-t?- ~4+4-) 6
3-t~ 2 x erythrose-4-P 2 x fructose-6-P
2 x gr c: raldehyde-3-P 2 x fructre-6-P
1------ 1 x fructose-6-P J
1----- - 2 x fructose-6-P - - - -- -- -- --'-
- -- - - 2 x fructose-6-P - -- - - -- - -- - - -- - - -- - - - -
Enzymes·
1 = Glucose-6-phosphate dehydrogenase (GPO) 5 = Transketolase
2 = Gluconolactone hydrolase 6 = Transaldolase
3 = 6-phosphogluconate dehydrogenase 7 = Second transketolase
4A = lsomerase 8 = Reversal of glycolysis
48 = Eplmerase
Fig: 10.41 : Summary of shunt pathway; numbers show the steps referred to in the text
Chapter 10: Metabolic Pathways of Glucose 147
form fructose-6-phosphate and glyceraldehyde-3-phos- BOX 10.7: S1gnif1c;.mce of HMP shunt p;ithw;iy
phate (Step 7, Fig. 10.41). Metabolic importance:
1. To produce NADPH and pentose phosphates
.step 8, Regeneration of NADPH is required for
i. Reductive biosynthesis-fatty acids, cholesterol and ste-
Glucose-6-Phosphate roid hormones
Two molecules of glyceraldehyde-3-phosphate formed in ii. Free radical scavenging
step 7 are condensed to form one fructose-6-phosphate iii. Maintain RBC membrane integrity by keeping GSH in
reduced state
(reversal of step 4 of glycolysis). Fructose-6-phosphate
iv. Prevention of methemoglobin formation
is then converted to glucose-6-phosphate (reversal of v. Detoxification by hydroxylation
step 2 of glycolysis). A summary of the whole pathway is vi. Maintain the transparency of lens
depicted in Figure 10.41 . vii. Bactericidal activity of macrophages
2. Ribose-5-phosphate is required for nucleic acid synthesis
Regulation of HMP Shunt Pathway 3. Clinical importance
I. Glucose-6-phosphate dehydrogenase deficiency
ii. Drug-induced hemolytic anemia
iii. Methemoglobinemia
iv. Thiamine deficiency leads to reduced transketolase activity
athway is Significantly
Active in the Following Organs
i. Liver
ii . Adipose tissue
summarized as:
iii. Adrenal cortex
G-6-P + 12 NAOP• + 7Hp - - -- - iv. Mammary glands
6 CO2 + 12 NADPH + 12 W + Pi v. Testes and ovaries
vi. RBCs
Summary of Shunt Pathway vii. Lens of eye
A. The oxidative phase of the pathway is seen in
Suppose, 6 molecules of glucose (6 >< 6 = 36 carbons) the above organs, where NAOPH generation is
are entering in this pathway. The first carbon atoms of all required for lipid synthesis or steroid synthesis.
6 glucose molecules are removed as 6 molecules of CO2 • B. The non-oxidative phase is present in all tis-
(This is equivalent to complete oxidation of 1 mole- sues, and so synthesis of ribose is possible in
cule of glucose). In this process, 12 NAOPH are gene- all tissues of the body.
rated. The remaining 6 molecules of 5-carbon pentoses
(6 x 5 = 30C) are interchanged in such a way that Generation of Reducing Equivalents
molecules of glucose (5 >< 6 = 30C) are regenerated. The major metabolic role of the pathway is to provide
This is summarized in Figure 10.41 . cytoplasmic NADPH for reductive biosynthesis o ~
acids..cbalesteral and steroids (see Chapters 13 and 14).
Physiological Significance
of the Pathway ree Radical Scavenging
Free radicals (super oxide, hydrogen pemxide.},,ara-0en-
A. The oxidative phase of the pathway is seen in
ti~ ous1¥-Pmdu.ced in all caUs. These will ~ y__QW.
the above organs, where NADPH generation is
QLoteia.s~ds and. alL hiawolecuJ.es, and in turn
required for lipid synthesis or steroid synthesis. cjills..are-d cad. The fre · e inactivat
B. The non-oxidative phase is present in all tissues , by enzyme syste e superoxide · mutase (SOD),
and so synthesis of ribose is possible in all tissues peroxidase (POD) and glutathione reductase (.&?R). -(/;)
of the body. ~duced GR is regenerated with the help of NADE>H @
Please see summary in Box 10.7. (Fig. 10.42).
148 Section B: General Metabolism
1 l
o; +o; + 2H+ H202 + 0 2
0
POD ~·
•
( \
H202 2H 20
NADPH* 02
(,ed,o,d) 2 G ~ : ; - S G (o,;dOed) NADPH Oxidase
SOD MPO
--+ H20 2
I i
NADP+ o;- HCIO
+c1-
GPD • Bacteria
NADP+ NADPH Glucose
killed
Fig. 10.42: Free radical scavenging enzymes. SOD = superoxide Fig. 10.43: Generation of ROS in macrophages. MPO = myelope-
= =
dismutase; POD glutathione peroxidase; GSH glutathione; GR = roxidase
glutathione reductase; GPO = glucose-6-phosphate dehydrogenase
Detoxification of Drugs
Most of the drugs and other foreign substances are
,......a..;.;_..;;._,,.~~ .;.;.;::.::
olytic
detoxified by the liver microsomal P450 enzymes, with .:;.;,.,__i.,,. The eficiency is manifested only when exposed
the help of NADPH. to certain drugs or toxins, e.g. intake of ntimalarial
-
of lens. Similarly, in estion of toxic glycosjdes present in
f<lY,a-..beans ,may have simjlac,_ e~c avlsm). Sulfa
Macrophage Bactericidal Activity drug ~an . ad alg> QI:ecipitate thehemoly-
is will lead o jaundic fl"-5evere anemia.· The
NADPH is required for production of reactive oxygen
geographical distribution of~ deficienc corre-
species (R~) (superoxide anion radical) by macro-
lates well with the malarial f n~ lcity. The enzyme
phages to kill bacteria (Fig. 10.43).
deficiency offer re ·s o Plas odium infection
UP Availability of Ribose (see Fig. 23.10). The parasite requires reduced glu-
tathione for its survival, which will not be available in
Ribose ar.d deoxyribose are required for DNA and adequate amounts in deficiency of GPO. GPO defici-
RNA synthesis. Ribose is also necessary for nucleotide ency is reported from different parts of India.
Methemoglobinemia
GPO deficient persons will show i n c r e a s ~ -
globin in circulation, even though cyanosis may not 6e
!Jlenif~ -
C.
1amine Deficiency { 0 ,)
The transketolase reaction is measured in RBCs as
an index of the thiamine status of an individual. The
occurrence and manifestation of ermc e s orsa-
ko s s n rome encephalopath which is seen in
atcoboliC~JS1r:lG-thes& WI "1 ~wiile,_g_eficiencnis due to
a genetic defect in ttm enzymeftaAHift{'H( . .
GLUCURONIC ACID PATHWAY L
The pathway is shown in Figure 10.44.
I ~'"1
form of glucuronjc acid . It is used for the following
purposes:
1. Conjugation of bili~in
2. Conjugation of ster~s .... - r
3. Conjugation of various drugs which will make them
more w~ soluble and more e ~ excretable.
4. Synthesis of glycosamino glycans ~ )-
Effect of Drygs
A~ it£.p(U/II ~f"c,_ pa:...
Ba r b ~. anti'pyrine atld aminopyrine wilf increase
the .u~ id pathway, leading to availability of more
glucuroni}te for conjugation purpose.
E~ al Pentosuria
It is one of the_....rrwmbers of the (Qarrod's tetrj'a) The
incidence is 1 in 2,500 births. It is an inborn error of
metabolism. In the pathway (Fig . 10.44), b,-xx,lulose is
converted to ~ yllJ.Lose by ~ ~mes, xylitol dehy -
Glucose when converted to sorbitol, cannot diffuse
drogenase and xylulose reductase. Absence of any
of these enzymes leads to pentosuria. L-xylulose is out of the cell easily and gets trapped there. Sorbitol is
150 Section B: General Metabol ism
hyperlactatemia, metabolic acidosis, hyperlipidemia and 6. Energy generating steps of glycolysis are cata-
hyperuricemia. Clinical features include convulsions, lyzed by the enzymes glyceraldehyde-3-phosphate
enlargement of liver and kidneys, doll's face (rounded dehydrogenase: (NADH); 1,3-bisphosphoglycerate
cheeks due to fat deposition), growth retardation, normal kinase (ATP) and pyruvate kinase (ATP).
mental development, etc. Late complications include 7. Cori's cycle ensures efficient reutilization of lactate
renal stones, tubular defects, hypertension, changes in produced in the muscle.
skin and mucous membrane and altered platelet function 8. Energy yield per molecule of glucose in the glyco-
leading to bleeding . Renal conditions may necessitate lytic pathway under anaerobic conditions is 2 ATPs.
dialysis and transplantation. 9. Under anaerobic condition pyruvate is reduced to
No specific treatment is available. Primary goal of lactate by lactate dehydrogenase. Under aerobic
dietary therapy is to correct hypoglycemia and maintain conditions, it is oxidatively decarboxylated to acetyl-
normoglycemia by frequent small quantity of food. Intake CoA by the enzyme complex Pyruvate dehydro-
of fructose and galactose is to be limited, because they genase (PDH).
are converted to lactate, and not to glucose. Nasogastric 10. The PDH enzyme complex requires 5 cofactors for
glucose infusion, parenteral nutrition and oral admi- its activity viz. NAO•, FAD, TPP, Lipoamide and Co A.
nistration of raw cornstarch are useful options. 11 . The PDH reaction is a totally irreversible reaction.
Hence, there is no net synthesis of glucose from fat.
0 12. Key enzymes of gluconeogenesis are; pyruvate
-•• Clinical Case Study 10.3 Answer carboxylase, phosphoenolpyruvate carboxykinase,
GPO deficiency. Jaundice is hemolytic in type, kemicterus fructose-1,6-bisphosphatase and glucose-6-phos-
and anemia are present. Liver enzymes are normal and phatase.
lactate dehydrogenase levels are very high. Hemolytic 13. Major substrates for gluconeogenesis are lactate
anemic in GPO deficiency is triggered on many occasions and glucogenic amino acids.
by bacterial and viral infections as well as some antibiotics. 14. Glycogen is the storage polysaccharide of the body.
It is stored mainly in the liver and muscle.
0
8 Clinical Case Study 10.4 Answer; 15. Glycogen phosphorylase is activated by glucagon
and adrenaline, while glycogen synthase is acti-
Essential pentosuria. See chapter for biochemical basis. vated by insulin.
16. Glycogen storage diseases (GSD) are Inborn errors
LEARNING POINTS, CHAPTER 10 of metabolism. Type 1 is called van Gierke's disease.
17. The HMP shunt pathway, also known as pentose phos-
1. Deficiency of Lactase results in lactose intolerance.
phate pathway (PPP) generates NADPH required for
2. Insulin dependent GluT4 has been implicated in
reductive cytoplasmic biosynthesis of biomolecules
Type 2 diabetes mellitus.
3. Glycolysis occurs both in aerobic and anaerobic such as steroids, fatty acids and cholesterol.
conditions. Anaerobic glycolysis is the major source 18. NADPH generated as a result of HMP pathway is
of energy for muscles, when the muscle tissue lacks essential to maintain transparency of the eye lens,
oxygen. to prevent methemoglobinemia and to maintain
4. Phosphofructokinase (PFK) is the regulatory or rate erythrocyte membrane integrity.
limiting enzyme of glycolysis. It is an allosteric and 19. The pathway also provides pentose sugars (ribose
inducible enzyme. AMP is allosteric activator, while and deoxyribose) for nucleic acid synthesis.
citrate and ATP are allosteric inhibitors. 20. GPO deficiency is a common clinical condition,
5. The reaction catalysed by 1,3-bisphosphoglycerate transmitted as X linked recessive trait. Ingestion of
kinase and pyruvate kinase are examples of sub- fava beans (favism) and anti-malarials such as pri-
strate level phosphorylation. maquine precipitates the manifestations.
10-3. Describe the process of glycolysis. Explain how many molecules of ATP are formed in anaerobic and aerobic
conditions.
10-4. Assuming that oxygen is available in plenty, what are the steps in which emergy is trapped in glycolytic pathway?
Give the substrate, product, enzyme and coenzyme of each of these em!rgy-trapping steps.
10-5. How is glucose metabolized by the RBCs for producing energy? Indicate the energy yield from 1 molecule of
gl ucose in the RBCs.
10-6. In anaerobic glycolysis, lactic acid is generated. What is the reason for reduction of pyruvate to lactate?
10-7. What are the irreversible steps in glycolysls? How are these blocks circumvented?
10-8. Trace the pathway of gluconeogenesis starting from alanine. Mention thie key enzymes.
10-9. " Gluconeogenesis is not a simple reversal of glycolysis"; justify your ainswer by giving the reactions.
10-10. How is glycogen broken down in the body? Explain the hormonal regulation of the pathway.
10-11 . Outline the sequence of reactions involved in the breakdown of glyco1~en in the skeletal muscle. Explain the
purpose served by this process.
10-12. Write the reactions of the oxidative phase of the hexose monophosphate shunt pathway. Which tissues have
this pathway?
10-13. What is the significance of HMP shunt pathway?
C. There is net conversion of acetyl-CoA to glucose 10-40. The HMP shunt pathway is important for all the
during gluconeogenesis following, except
D. Glycerol part of fat forms a minor source for gluco- A. Generation of ATP
neogenesis B. Fatty acid biosynthesis
10-30. Pyruvate can be directly converted to all the follow- C. Synthesis of reduced glutathione
ing, except D. Synthesis of ribose
A. Alanine B. Lactate 10-41. All are true with regard to NADPH, except:
C. Oxaloacetate D. Phosphoenolpyruvate A. Generated by glucose-6-phosphate dehydroge-
10-31. Gluconeogenesis is taking place in : nase
A. Brain B. Liver B. Used for reductive biosynthesis of fatty acids
C. RBC D. Muscle C. Produces peroxides and free radicals in RBC
10-32. Both glycolysis and gluconeogenesis pathways D. Preserves integrity of RBC membrane
utilize all the following enzymes, except: 10-42. The hormone activating the enzyme phosphory-
A. Phosphofructokinase lase is:
B. Glyceraldehyde-3-phosphate dehydrogenase A. Epinephrine B. Insulin
C. 1,3-bisphosphoglycerate kinase C. Growth hormone D. Glucocorticoids
D. Phosphoglucomutase 10-43. Von Gierke's disease is characterized by the defi-
10-33. Lactic acidosis is seen in the following cases, except ciency of:
A. OXPHOS Diseases A. Glucose-6-phosphatase
B. Defective metabolism of pyruvate B. Glyceraldehyde-3-phosphate dehydrogenase
C. Excessive ingestion of alcohol C. Phosphofructokinase
D. Deficiency of glucose-6-phosphate dehydrogenase D. Phosphorylase
10-34. Which of the following metabolic intermediates is 10-44. McArdle's syndrome is due to the deficiency of:
not generated from glucose? A. Glucose-6-phosphatase
A. Dihydroxy acetone phosphate B. Glucose-6-phosphate dehydrogenase
B. 2,3-bisphosphoglycerate C. Muscle glycogen phosphorylase
C. Beta hydroxybutyrate D. Debranching enzyme
D. Glycerol-3-phosphate 10-45. Glycogen is:
10-35. Which reactions does not occur in the liver? A. Having unbranched or straight line structure
A. Acetoacetate to acetoacetyl-CoA B. Hydrolyzed to equal quantity of glucose and galac-
B. Hydroxylation of Cholecalciferol tose
C. Glucose to UDP glucose C. Hydrolysed by phosphorylase
D. Oxaloacetate to phosphoenol pyruvate D. Seen in brain
10-36. Transport of glucose from Intestinal lumen to 10-46. One glycosyl residue in glycogen is converted to 2
mucosal cell: lactic acid molecules, the net yield of ATP is:
A. Requires hydrolysis of ATP A. Two B. Three
B. Occurs by facilitated diffusion C. Four D. Thirty eight
10-47. Which enzyme is active in the phosphorylated state:
C. Is sodium dependent
D. Occurs by an antiport system A. Glycogen phosphorylase
10-37. Which enzyme generates NADPH? B. Pyruvate kinase (PK)
A. Pyruvate carboxylase C. PFK-2 (phosphofructokinase-2)
D. Glycogen synthase
B. Pyruvate dehydrogenase
10-48. Glycogen synthase is activated by:
C. Glucose-6-phosphate dehydrogenase
D. Lactate dehydrogenase A. Insulin B. Protein kinase
10-38. Transketolase activity is decreased in the defi- C. Adrenaline D. ATP
10-49. Wh ich enzyme uses NADP as coenzyme?
ciency of:
A. Pyruvate carboxylase
A. Thiamine pyrophosphate (TPP)
B. Pyruvate dehydrogenase
B. Nicotinamide adenine dinucleotide
C. Glucose-6-phosphate dehydrogenase
C. Flavin adenine dinucleotide
D. Lactate dehydrogenase
D. Pyridoxal phosphate
10-50. Glucose-6-phosphate dehydrogenase enzyme:
10-39. Deficiency of glucose-6-phosphate dehydrogenase
A. Is a member of glycolytic pathway
causes :
B. Is necessary for production of NADPH
A. Cataract B. Hemolytic anemia C. Requires the coenzyme ATP
C. Hypoglycemia D. Mental retardardation D. Is inhibited by insulin
Chapter 10: Metabolic Pathways of Glucose 155
10-51. Transketolase reaction ; 10-61. All are true regarding sorbitol, except:
A. Takes place between xylulose phosphate and A. Deficient production in diabetes mellitus
ribose phosphate B. Osmotically active
B. Occurs between glyceraldehyde phosphate and C. Used as a sweetner
fructose phosphate D. May be oxidsed to fructose
C. Needs pyridoxal phosphate as coenzyme 10-62. Alpha 1,4 linkages are hydrolysed by:
D. Occurs between glyceraldehyde phosphate and A. Lactase 8 . Sucrose
sedoheptulose phosphate C. Maltase D. lsomaltase
10-52. Hemolytic episode after administration of anti 10-63. Absorption of glucose from intestinal lumen to
malarial drug is due to the deficiency of enzyme: bloodstream involves all the following transport
A. 6-phosphogluconate dehydrogenase mechanisms, except
B. Glucose-6-phosphate dehydrogenase A. Cotransport
C. Glucose-6-phosphatase B. Active transport
D. Hexokinase C. Facilitated diffusion
10-53. Oxidative phase of HMP pathway produces all, D. Passive diffusion
except 10-64. Lactose intolerance is:
A. CO2 B. Pentose phosphates A. Seen only in newborns
C. ATP D. NADPH B. Due to relative deficiency of lactase
10-54. NAOPH is requ ired for all, except: C. Does not respond the lactose free diet
A. Phagocytic cells for respiratory burst D. Requires replacement of lactase
B. Drug hydroxylation 10-65. Which of the following tissues can derive energy
C. Substrate for electron transport chain only from glucose?
D. Detoxification of Reactive oxygen species A. Brain
10-55. Which of the following is not an intermediate of B. Cardiac muscle
HMP pathway? C. Erythrocytes
A. Fructose-6-phosphate D. Adipose tissue
B. Erythrose-4-phosphate 10-66. Insulin dependent uptake of glucose is seen in:
C. 2,3 bisphosphoglycerate A. Liver b. Brain
D. Ribose-5-phosphate C. Skeletal muscle D. Erythrocytes
10-56. Which of the following coenzymes is required for 10-67. Which of the intermediates is formed only in RBCs?
the HMP pathway? A. 1,3-BPG B. 2,3-BPG
A. PLP B. Biotin C. PEP D. 3-PG
C. FAD D. TPP 10-68. Which of the enzymes listed catalyzes a substrate
10-57. Which of the following is NOT true regarding the level phosphorylation?
HMP pathway? A. Phosphofructokinase
A. Glucose-6-phosphate is shunted through the HMP B. Hexokinase
pathway to glycolysis C. Pyruvate kinase
B. Used for energy production D. Glucokinase
C. Ribose-5-phosphate is produced for nucleotide 10-69. Lactate is the product of glycolysis in RBCs
synthesis because:
D. Pathway is essential for the erythrocytes. A. RBCs can convert lactate to glucose
10-58. HMP pathway is essential for all, except B. Formation of lactate produces more energy
A. Transparency of lens C. NAO' is regenerated in the cytoplasm
B. Integrity of RSC membrane D. Accumulation of lactate activates glycolysis.
C. Reduce methemoglobin level 10-70. Lactic acidosis can result from deficiency of:
D. Provide 2,3-BPG for RBCs A. Lactate dehydrogenase
10-59. Deficiency of HMP pathway can cause all, except 8. Pyruvate dehydrogenase
A. Hemolytic anemia C. Pyruvate kinase
B. Methemoglobinemia D. Phosphoenolpyruvate carboxykinase
C. Chronic granulomatous disease 10-71 . Which of the following cannot be converted back
D. Thromboembolic episodes to glucose?
10-60. Which of the following drugs can precipitate GPO A. Acetyl-CoA
deficiency? B. Lactate
A. Primaquin B. Aspirin C. Alanine
C. Statin D. Penicillin D. Propionyl-CoA
156 Section B: General Metabolism
10-72. Which of the following is not true regarding g luco - 10-73. Under conditions of starvation, the enzyme which
neogenesis? is inhibited is:
A. Requires ATP A. Glucose-6-phosphatase
B. Inhibited by a high NADH/NAD• ratio B. Alanine aminotransferase
C. Takes place in liver C. Glucokinase
D. Activated by insulin D. Pyruvate carboxylase
10-1 . Glucose is absorbed at the luminal side of gastro- 10-10. What is the difference between glucokinase and
intestinal cells by which mechanism? hexokinase?
Carrier mediated co-transport with sodium, named as Both add phosphate group to glucose. But glucokinase
Sodium dependent glucose transporter (SGluT). is present only in liver: acts specifically on glucose,
10-2. How glucose is released from intestinal cells into and is active when glucose level in blood is increased
the bloodstream? after food intake.
Glucose transporter type 2 {GluT2). 10-11. Which tissues prefer anaerobic glycolysis?
10-3. How glucose is taken up by cells? RBCs, exercising muscle, and cancer cells.
In tissues GluT2 helps in absorption of glucose from 10-1 2. During glycolysis, energy is produced during
blood. which steps?
10-4. What is the importance of GluT4? Step 5, Glyceraldehyde-3-phosphateto 1,3-bisphospho-
It is the glucose transporter present in muscle and glycerate; Step 6. 1,3-bisphosphoglycerate to 3-phos-
adipose tissue. phoglycerate; and Step 9, Phosphoenolpyruvale lo
10-5. What is its clinical significance? pyruvate.
Insulin induces these transporters. In diabetes mellitus, 10-13. Fluoride ions inhibit which enzyme?
entry of glucose into muscle is decreased, because Enolase (step 8).
GluT4 is reduced. 10-14. What is the clinical significance of the above inhi-
10-6. What is glycolysis? bition?
In this pathway, glucose is converted to pyruvate or Fluoride is used to prevent glycolysis , as preservative
lactate, along with production of a small quantity of for blood before glucose estimation.
energy. 10-15. In glycolysis, NAO• is reduced to NADH in wh ich
10-7. In which condition pyruvate is produced? reaction?
In aerobic condition pyruvate is produced. Glyceraldehyde-3-phosphate dehydrogenase reaction.
10-8. In which condition lactate is produced? 10-16. What are substrate level pt,osphorylation steps in
When oxygen is lacking, lactate is produced. glycolysis?
10-9. What is the significance of glycolysis? 1,3-bisphosphoglycerate kinase (step 6) and pyruvate
Anaerobic glycolysis forms the major source of energy kinase (step 9).
in actively contracting muscles. Moreover, glycolysis is 10-17. What is the purpose of lactic acid production under
the only source of energy in RBCs. anaerobic conditions?
Chapter 1O: Metabolic Pathways of Glucose 157
NADH generated in the 5th step has to be oxidized to Pyruvate carboxylase; Phosphoenolpyruvate carboxy
NAO'. This can be done by oxygen. But when oxygen kinase; Fructose-1 ,6-bisphosphatase and Glucose-6-
is lacking, the 5th step has to be coupled with the 10th phosphatase.
step for regeneration of NAO'. 10-34. Malate shuttle is used for what purpose?
10-18. What is Cori cycle (or lactic acid cycle)? Reactions of gluconeogenesis are taking place in
During exercise, lactate is produced in muscle. This cytosol. Hence the oxaloacetate has to be transported
lactate diffuses into the blood. Lactate then reaches from mitochondria to cytosol. This is done by malate
liver, where it is oxidized to pyruvate. It is converted shuttle.
to glucose. This glucose can enter into blood and then 10-35. Gluconeogenesis is seen in which tissue?
transported to muscle. Liver.
10-19. What is the purpose of Cori cycle? 10-36. How many ATP molecules are required to convert
By this means, the lactate is efficiently reutilized. 2 molecules of pyruvate into glucose?
10-20. What are the key glycolytic enzymes? Six.
Glucokinase; phosphofructokinase; pyruvate kinase 10-37. Blood glucose level can be raised by gluconeo-
10-21. What is the action of insulin on glycolysis? genesis only by liver, why?
Insulin stimulates glycolysis. Glucose-6-phosphatase is present only in liver.
10-22. What is the energy yield from glycolysis during 10-38. Muscle glycogen will not serve as a precursor of
anaerobic conditions? blood sugar, why?
2ATP. Glucose-6-phosphatase is absent in muscle.
10-23. In aerobic glycolysis, the net yield from one glu- 10-39. Which amino acids are both ketogenic and gluco-
genic?
cose molecule is how much?
Tyrosine and Tryptophan.
?ATP.
10-40. What hormone will inhibit gluconeogenesis?
10-24. During complete oxidation, what is the net yield of
Insulin.
ATP from one glucose molecule?
10-41. What hormones will stimulate gluconeogenesis?
32ATP
Glucagon and glucocorticoids.
10-25. How many ATPs are generated per one rotation of
10-42. What is the significance of gluconeogenesis?
the citric acid cycle?
Gluconeogenesis is necessary to maintain blood glu-
10ATP.
cose level, especially under conditions of starvation.
10-26. What is function of 2,3-bisphosphoglycerate?
10-43. What is the key enzyme of glycogenolysis?
It reduces the affinity of hemoglobin toward oxygen.
Glycogen phosphorylase.
10-27. What are the steps in which carbon dioxide is
10-44. Which hormones enhance glycogenolysis?
released from a glucose molecule?
Adrenaline and glucagon causes glycogenolysis
Pyruvate dehydrogenase; lsocitrate dehydrogenase;
10-45. What is the mechanism of action of adrenaline?
Alpha ketoglutarate dehydrogenase. Adrenaline increases cyclic AMP level which activates
10-28. What are the coenzymes necessary for oxidative glycogen phosphorylase
decarboxylation of pyruvate? 10-46. What will activate glycogen phosphorylase?
Thiamine pyrophosphate; NAD•; FAD; Lipoic acid; Epinephrine, Glucagon, Cyclic AMP.
CoenzymeA. 10-47. What is cyclic AMP (cAMP)?
10-29. There is no net synthesis of glucose from fatty It is a second messenger.
acids, why? 10-48. How cyclic AMP is produced?
Pyruvate to acetyl-CoA is a totally irreversible reaction. Adenyl cyclase enzyme acts on ATP to produce cAMP.
10-30. Which enzyme irreversibly channels glucose to 10-49. Which is the defective enzyme in von Gierke's
energy production? disease (glycogen storage disease type I)?
Pyruvate dehydrogenase is the irreversible reaction. Glucose-6-phosphatase.
10-31. What is gluconeogenesis? 10-50. What is the characteri stic clinica l features of von
Production of glucose from non-carbohydrate sources. Gierke's disease?
10-32. What are those non-carbohydrate sources? (What Fasting hypoglycemia, which does not respond to
are the substrates for gluconeogenesis?) adrenaline is very characteristic.
Glucogenic amino acids and lactate. 10-51. Which enzyme generates NADPH?
10-33. What are the key gluconeogenic enzymes? Glucose-6-phosphate dehydrogenase
158 Section 8: General Metabolism
10-52. Which is the key enzyme ofhexose monophosphate 10-60. What is the manifestation of glucose-6-phosphate
shunt pathway? dehydrogenase deficiency?
Glucose-6-phosphate dehydrogenase. Drug-induced hemolysis.
10-53. What are the tissues in which HMP shunt pathway 10-61. Acute hemolytic episode after administration of
is significant? antimalarial drug is due to what?
Liver, adipose tissue, RBC, adrenal cortex, ovary, Deficiency of glucose-6-phosphate dehydrogenase.
testis, mammary gland, lens. 10-62. What is the most common enzyme deficiency?
10-54. What hormone controls HMP shunt pathway? Glucose-6-phosphate dehydrogenase deficiency.
Insulin stimulates the pathway by activating the key 10-63. What is clinical significance of transketolase?
enzyme. The transketolase available in RBCs Is an index of the
10-55. What is the purpose of HMP shunt pathway? thiamine status of an individual.
It generates NADPH. 10-64. When is transketotase activity decreased?
10-56. What is the use of NADPH in biological systems? Deficiency of Thiamine pyrophosphate (TPP).
For reductive biosynthesis. 10-65. What is the purpose of uronic acid pathway?
10-57. Which synthetic pathways need NADPH? It is used for conjugation of bilirubin; steroids, synthesis
Fatty acid biosynthesis; synthesis of cholesterol, ste- of glucosaminoglycans.
raid hormones. 10-66. In lower animals, uronic acid pathway is used for
10-58. Apart from reductive synthesis, NADPH is used for what?
what purpose? For synthesis of ascorbic acid (vitamin C).
It is necessary to keep the integrity of RBC membrane; 10-67. What is essential pentosuria?
for keeping transparency of lens; and for superoxide Excretion of pentose (L-xylulose) in urine.
production inside macrophages. 10-68. What is it due to?
10-59. Is NADPH used for ATP generation? Due to the deficiency of xylulose reductase or xylulose
No. NADPH is not used for ATP generation. reductase.
_ _ _ _ _ Chapter 11
Regulation of Blood Glucose,
Insulin and Diabetes Mellitus
Chapter at a Glance
Historical Perspectives
The word "insulin" is derived from Latin, insula, meaning island (islet). In 1869, Langerhans identified the alpha and beta cells
in islets of pancreas. In 1889, von Mering and Minkowski produced experimental diabetes by pancreatectomy. In 1922, Banting
and Best extracted insulin from pancreas. Insulin was the first hormone to be isolated in a pure form. They injected the extract
to a diabetic dog, Marjorie, who was kept alive by regular insulin injections. For this work Banting was awarded Nobel Prize in
1923. But Best was deleted in the list. As a compensation, Banting declared that half his share of the prize will go to Best. In
1954 , Sanger studied the amino acid sequence of insulin. For this work Sanger got Nobel Prize in 1958.
The term "diabetes" is derived from the Greek words dia {= through), bainein (= to go) and diabetes literally means pass
through. The disease causes loss of weight as if the body mass is passed through the urine. The Greek word, mellitus, means
sweet, as it is known to early workers, that the urine of the patient contains sugar. Diabetes mellitus is a disease known from
very ancient times. Charaka in his treatise (circa 400 BC) gives a very elaborate clinical description of madhumeha (= sweet
urine). He had the vision that carbohydrate and fat metabolisms are altered in this disease. In Western literature, Thomas Willis
in 1670 noticed the sweet taste of diabetic urine. In 1838, Bouchardt and Peligot proved that the sugar of diabetic urine is the
same as that present in grape sugar. A crude test for urine sugar was first developed by Trommer in 1841. Qualitative test for
urine sugar was perfected by Hermann Fehling (1848) and semi-quantitative test by Stanley Benedict (1908). Folin in 1919
identified a method for quantitative determination of sugar in blood .
i
BOX 11 .1 : F.ic-" r" rn2,~t,i1r·111,1 l,lo,)cJ sugar High blood
1. The plasma glucose level at an instant depends on the balance
between glucose entering and leaving the extracellular fluid
Lowblood
gl,rose I!!! glucose
~'""''":J ~ @i;"
Alpha cells Beta cells
a. Absorption from intestines
b. Glycogenolysis (breakdown of glycogen)
c. Gluconeogenesis
d. Hyperglycemic hormones (glucagon, steroids)
4. Factors leading to depletion of glucose in blood are:
Glycogenolys~·s Peripheral tissue
a. Utilization by tissues for energy in liver; cells take
b. Glycogen synthesis glucose glucose from
released to blood blood
c. Conversion of glucose into fat (lipogenesis)
d. Hypoglycemic hormone (insulin) Nonna! blood glucose level attained
i
Liver Liver Glycogen synthesis
Muscle
Gluconeogenesis
;;~~~:':'°'
Glycogenoly~ r
Glucose Glucot i
FFA
Fig. 11.2A: Blood glucose regulation during fasting state (high Fig. 11.28: Blood glucose regulation during postprandial state
Glucagon). In fasting state, blood glucose level is maintained (high insulin). In postprandial state, glucose level is high; then
by glycogenolysis and gluconeogenesis; further, adipose tissue blood glucose level is lowered by tissue utilization, glycogen syn-
releases free fatty acids as alternate source of energy. Red arrows thesis and lipogenesis. Red arrows indicate activation; blue arrow
indicate activation; blue arrow indicates inhibition indicates inhibition
stimulates the secretion of insulin by the beta ce lls of that supplies the glucose for maintaining blood gluco se
islets of Langerhans of pancreas. The uptake of g lucose level (Fig. 11 .1 ). Hormones like glucagon, epinephrine,
by extrahepatic tissues, except brain is dependent on glucocorticoids, growth hormone, ACTH and thy roxine
insulin . Moreover, insulin helps in the storage of g lucose will tend to inc rease the blood glucose level. They a re
as glycogen or its conversion to fat (Fig . 11 .2A) referred to as anti-insulin hormones or hyperglycemic
hormones. An overview of the regu latory mechanism is
Regulation in Fasting State s hown in F igure 11.3. Effects of hormones are shown in
Normally, 2 to 2½ hours after a mea l, the blood glu- Box 11 .2.
cose level falls to near fasting levels. It may go down
further; but this is prevented by processes that contri-
Determination of Glucose in Body Fluids
bute glucose to the blood. Fo r another 3 hours , hepatic Estimation of glucose is the most common analysis
glycogenolysis will take care of the blood sugar level. done b clinical laboratories. The blood is collected using
Thereafte r, gluconeogenesis will take c harge of the an anticoagulant (potassium oxalate) and an inhib ito r of
situation (Figs. 11.2A and B). Liver is the major o rgan glycolysis (sodium fluoride).
Chapter 11 : Regulation of Blood Glucose, Insulin and Diabetes Mellitus 161
Gluconeogenesis in liver /
Hypoglycemic Hormone Lipogenesis (synthesis of
(Amino acids to glucose)
Insulin fatty acid and fat deposit)
BOX 11 .2: Effects of hormones on glucose level in blood (glucose oxidase peroxidase) method is most commonly
A. Effect of insulin (hypoglycemic hormone) used to assess the blood glucose level. The reaction
1. Lowers blood glucose generates a color, which is read in a photometer. The
2. Favors glycogen synthesis newer automated systems use hexokinase method.
3. Promotes glycolysis The above GOD reaction mixture is immobilized on
4. Inhibits gluconeogenesis a plastic film (dry analysis). The intensity of the color is
B. Glucagon (hyperglycemic hormone)
measured by reflectance photometry. The instrument is
1. Increases blood glucose
named as glucometer. It is useful for patients to have
2. Promotes glycogenolysis
3. Enhances gluconeogenesis self-analysis at home. But the instrument is less accurate.
4. Depresses glycogen synthesis
5. Inhibits glycolysis (Details given below) Commonly Employed Terms
C. Cortisol (hyperglycemic hormone) Regarding Glucose
1. Increases blood sugar level
1. Blood sugar analyzed at any time of the day, without
2. Increases gluconeogenesis
3. Releases amino acids from the muscle
any prior preparations, is called random blood sugar.
D. Epinephri1 ..: or Adrenaline (hyperglycc:-:iic) 2. Glucose estimated in the early morning, before tak-
1. Increases blood sugar level ing any breakfast is called fasting blood glucose.
2. Promotes glycogenolysis Fasting state means, glucose is estimated after an
3. Increases gluconeogenesis overnight fast (12 hours after the food) (postab-
4. Favors uptake of amino acids
sorptive state).
E. Growth hormone (hyperglycemic)
3. The test done about 2 hours after a good meal is
1. Increases blood sugar level
2. Decreases glycolysis
called postprandial blood glucose (Latin = after
3. Mobilizes fatty acids from adipose tissue food).
4. When blood glucose level is within normal limits, it
Fluoride inhibits the enzyme, enolase, and so glyco- is referred to as normoglycemia. When values are
lysis on the whole is inhibited . If fluoride is not added, above the normal range, it is known as hypergly-
cells will utilize glucose and fa lse low value may be cemia. When values are below the normal range,
obtained. Capillary blood from finger tips may also be it is called hypoglycemia. (Greek, hyper= above;
used for glucose estimation by strip method. hypo = below).
5. When the blood glucose is below 50 mg/di, it is a
Enzymatic Method very serious condition. Hyperglycemia is harmful in
This is highly specific, giving 'true glucose' values (fasting the long run; while hypoglycemia even for a short
70-110 mg/dl). In the medical laboratory, the GOD-POD while is dangerous, and may even be fatal.
162 Section B: General Metabolism
TABLE 11.1: The plasma sugar levels in OGTT In normal BOX 11 .3: D1agnost1c cntena for diabetes melhtus
persons and In d1abet1c patients
1. If the fasting plasma sugar is more than 126 mg/dl, on more
Criteria for Criteria for than one occasion (Table 11 .1)
Normal persons diabetes diagnosing /GT 2. Or, if 2-hour post-glucose load value of OGTT is more than
Fasting < 110 mg/dl > 126mg/dL 110 to 200 mg / dl (even at one occasion)
126 mg/ dl 3. Or, if both fasting and 2-hour values are above these levels,
1 hr (peak) < 160 mg/dL Not prescribed Not prescribed on the same occasion
after glucose 4. If the random plasma sugar level is more than 200 mg/dl, on
2 hr after < 140 mg/dL > 200mg/dL 140to more than one occasion. Diagnosis should not be based on a
glucose 199 mg/ dl sing le random test alone; it should be repeated
5. Glycated hemoglobin (Glyco-Hb) or HbA 1c level more
than 6.5% at any occasion. As per the recommendations of
6. The ability of a person to metabolize a given load of American Association of Clinical Chemistry and American
glucose is referred to as glucose tolerance. Diabetes Association, HbA 1c level is the preferred method
for initial diagnosis of diabetes mellitus.
Conducting the Glucos e Tolerance Test
At about 8 am, a sample of blood is collected in the fast- Classical Oral Glucose
ing state. Urine sample is also obtained. This is denoted Tolerance Test (OGTT)
as the "O" hour sample.
Glucose tolerance test is artificial, because in day-to-day
Glucose load dose: The dose is 75 g anhydrous
life, such a large quantity of glucose does not enter into
glucose (82.5 g of glucose monohydrate) in 250-300ml
blood. However, the GTT is a well-standardized test,
of water. This dose is fixed for an adult, irrespective of
and is highly useful to diagnose diabetes mellitus in
body weight. (When the test is done in children , the
doubtful cases.
glucose dose is adjusted as 1.75 g/kg body weight).
In order to prevent vomiting, patient is asked to drink it
Indications for OGTT
slowly (within about 5 minutes). Flavoring of the solution
will also reduce the tendency to vomit. 1. Patient has symptoms suggestive of diabetes
Sample collection: As per current WHO recommenda- mellitus; but fasting blood sugar value is inconclusive
tions, 2 samples are collected, one at fasting ("O" hour (between 100 and 126 mg/dl).
sample) and 2-hour post-glucose load. Urine samples 2. During pregnancy, excessive weight gaining is
may also be collected along with these blood samples. noticed, with a past history of big baby (more than
This is sufficient to get a correct assessment of the 4 kg) or a past history of miscarriage.
patient. 3. To rule out benign renal glucosuria.
4. GTT has no role in follow-up of diabetes. It is indi-
Normal Values and Interpretations cated only for the initial diagnosis.
As per WHO recommendation, In a normal person,
fasting plasma glucose is 70-110 mg/dl. The present Preparation of the Patient
day tendency is to view values above 100 mg/ml as
The patient is instructed to have good carbohydrate
suspicious. Value more than 100 mg/dl is one of the
criteria for the metabolic syndrome . diet for 3 days prior to the test. Patient should not take
Following the glucose load, in normal persons, the food after 8 PM the previous night. Should not take
level rises and reaches a peak within 1 hour and then any breakfast. This is to ensure 12 hours fasting.Th e
comes down to normal fasting levels by 2 to 2½ hours. patients are advised to remain in the hospital during the
This is due to the secretion of insulin in response to the waiting period of two hours without any active exercise.
elevation in blood glucose. None of the urine sample Figure 11.4 represents the graph, when plasma glucose
shows any evidence of glucose. Diagnostic criteria for values are plotted on the vertical axis against the time
diabetes mellitus are given in Table 11.1 and Box 11 .3. of collection on the horizontal axis.
Chapter 11: Regulation of Blood Glucose, Insulin and Diabetes Mellitus 163
Alimentary Glucosuria
Here the fasting and 2-hour values are normal; but an
exaggerated rise in blood glucose following the inges-
tion of glucose is seen. This is due to an increased rate
of absorption of glucose from the intestine.This is seen
50
in patients after a gastrectomy or in hyperthyroidism.
Renal Glucosuria
0 2 Hours Normal renal threshold for glucose is 175-180 mg/dl.
Fig. 11.4: Oral glucose tolerance test (OGTT) If blood sugar rises above this, glucose starts to appear
in urine.
Causes for Abnormal GTT Curve Generally, the increased blood sugar level is reflec-
ted in urine. But when renal threshold is lowered,
Impaired Glucose Tolerance (JG T) glucose is excreted in urine. In these cases, the blood
sugar levels are within normal limits. This is called renal
It is otherwise called as Impaired Glucose Regulation
glycosuria.
(IGR). Here blood sugar values are above the normal Renal threshold is lowered physiologically in preg-
level, but below the diabetic levels (see Table 11.1 ). nancy. Renal glucosuria is associated with renal dise-
In IGT, the fasting plasma glucose level is between ases with renal tubular transport defects; e.g. Fanconi's
110 and 126 mg/dl and 2-hour post-glucose value is syndrome. In these cases glucosuria is seen along with
between 140 and 200 mg/dl (Fig. 11.4). amino aciduria and phosphaturia.
Such persons need careful follow-up because IGT In some cases, renal threshold may be increased
progresses to frank diabetes at the rate of 2% patients when glucose will not appear in urine, even though
blood sugar is elevated. Here GFR is decreased with
per year.
minimal or no impairment of tubular reabsorption. This
is seen in old age (arteriosclerosis) and in Kimmelsteil-
Impaired Fasting Glycemia (/FG)
Wilson Syndrome (diabetic nephrosclerosis).
In this condition, fasting plasma sugar is above normal
(between 110 and 126 mg/dl); but the 2-hour post- Factors Affecting GTT
glucose value is within normal limits (less than 140mg/dl). In acute infections, cortisol is secreted, and so curve is
These persons need no immediate treatment; but are to elevated and prolonged. In hyperthyroidism there will
be kept under constant check up. be steep rise in curve. A flat curve is seen in hypo-
thyroidism.
Gestational Diabetes Mellitus (GDM)
REDUCING SUBSTANCES IN URINE
This term is used when carbohydrate intolerance is
Normally glucose is not excreted in urine. But if blood
noticed, for the first time, during a pregnancy. A known
sugar is more than 180 mg/dl, urine contains glucose.
diabetic patient, who becomes pregnant, is not The blood level of glucose above which glucose is
included in this category. excreted is called renal threshold.
Women with GDM are at increased risk for subse- The excretion of reducing substances in urine is
quent development of frank diabetes. GDM is associ- detected by a positive Benedict's test. (see Chapter
ated with an increased incidence of neonatal mortality. 7). About 0.5 ml of urine is boiled with 5 ml Benedict's
Maternal hyperglycemia causes the fetus to secrete reagent for 2 minutes (or kept for 2 minutes in water bath
more insulin, causing stimulation of fetal growth and which is already boiling). The formation of a precipitate
164 Section B: General Metabolism
Glucose ent~ Ca++entry When insulin binds with alpha unit, the beta unit transmits
K -ATP channel signal to cytoplasm
GLUT2
Glucose/
more;
ATP more
@
/ "'"~"@
@
Insulin in granules @
l
Carbohydrate Translocase Stimulation Glycolysis
Glucokinase Stimulation favored
Phosphofructokinase Stimulation
Pyruvate kinase Stimulation
Pyruvate carboxylase Inhibit ion Gluconeogenesis Hypoglycemia
PEPCK
Fructose-1,6-bisphosphatase
Glucose-6-phosphatase
Glycogen synthase
Glycogen phosphorylase
Inhibit ion
Inhibition
Inhibition
Activation ]
Inactivation
l depressed
Glycogen deposition -
GPD Stimulation
1 Generation of NADPH l
Lipid Acetyl-CoA carboxylase
Glycerol kinase
Stimulation
Stimulation
j Lipogenesis favored J Glucose is used for lipogenesis;
glucose lowered; decreased
Hormone sensitive lipase Inhibition Lipolysis inhibited ketogenesis
HMG-CoA reductase Stimulation Cholesterol synthesis
lj
Protein Transam inases Inhibition Catabolism inhibited
Ornithine transcarbamoylase Inhibition
RNA polymerase Protein synthesis General anabolism
and ribosome assembly Favored favored
Chapter 11 : Regulation of Blood Glucose, Insulin and Diabetes Mellitus 167
low blood
Other General Effects glucose
i. Insulin depresses HMG-CoA synthase and so keto-
Glucose Glucagon
genesis is decreased. Insulin also favors fatty acid uptake/storage glucocorticoids
synthesis from acetyl-CoA. All these factors reduce
Fig. 11 .8: Combined action of insulin and glucagon will keep the
the availability of acetyl-CoA, so that production of blood sugar level within normal limits. High blood sugar stimulates
ketone bodies reduced . insulin secretion. Low blood sugar causes glucagon secretion
168 Section B: General Metabolism
synthesis. Gluconeogenesis is favored by glucagon by suffer from this disease. It is a major cause for morbidity
inducing enzymes like PEPCK, glucose-6-phosphatase and mortality. Insulin deficiency leads to increased blood
and fructose-1 ,6-bisphosphatase. Glucagon increases glucose level. In spite of this high blood glucose, the entry
plasma free fatty acid level. In adipose tissue gluca- of glucose into the cell is inefficient. Hence all cells are
gon favors beta-oxidation, as it activates carnitine starved for glucose.
acyl transferase. The mitochondrial acetyl-CoA level Criteria for diagnosis of diabetes mellitus are shown
increases. Ketogenesis is favored. in Table 11 .1, under glucose tolerance test. The disease
may be classified as follows (WHO recommendation,
Mechanism of Action 1999):
Glucagon combines with a membrane bound receptor.
This activates G protein and adenylate cyclase (see Type 1 Diabetes Mellitus
Chapter 45). Thus ATP is converted to cAMP. Cyclic
(Formerly known as Insulin-dependent diabetes mel-
AMP activates glycogen phosphorylase, and inactivates
litus; IDDM). About 5% of total diabetic patients are of
glycogen synthase.
type 1. Here circulating insulin level is deficient. It is sub-
Anti-insulin Hormones classified as:
a. Immune mediated and
Regulation of carbohydrate metabolism in general de-
b. Idiopathic.
pends on the balance between insulin and anti-insulin
hormones. A summary is given in Box 11.2. See also
Table 11 .5. Glucocorticoids act mainly by stimulating Type 2 Diabetes Mellitus
gluconeogenesis. But growth hormone antagonizes (Formerly known as noninsulin dependent diabetes
insulin in ma ny key metabolic reactions (Table 11.5). mellitus; NI DDM). Most of the patients belong to this
Bernado Houssey demonstrated that in pancreatecto-
type. Here circulating insulin level is normal or mildly
mized animals, the requirement of insulin was about 100
elevated or slightly decreased, depending on the stage
units per day. When anterior pituitary was also ablated
of the disease. This type is further classified as:
in such animals, the requirement of insulin came down to
10 units or so. This shows that growth hormone antago- a. Obese
nizes insulin. Houssay was awarded Nobel Prize in 1947. b. Nonobese
Secondary to Other Known Causes BOX 11. 4 : Cntem1 for d1agnos1s of metabolic syndrome
i. Elevated waist circumference: (For men >90 cm and for
a. Endocrinopathies (Cushing's disease, thyrotoxico- women, >80 cm).
sis, acromegaly); ii. Elevated triglycerides: > 150 mg/ dl
b. Drug induced (steroids, beta blockers, etc.); iii. Reduced HDL ("good") cholesterol: For men, <40 mg/ dl; for
c. Pancreatic diseases (chronic pancreatitis, fibrocal- women, < 50 mg/dl
culus pancreatitis, hemochromatosis, cystic fibrosis) iv. Elevated blood pressure: > 130/ 85 mm Hg
d. Anti-insulin receptor autoantibodies (Type B insulin v. Elevated fasting glucose: > 100 mg/dl
vi. Insulin resistance (hyperinsulinemia)
resistance)
vii. Additional parameteri include coagulation abnormalities,
e. Mutations in the insulin gene or insulin receptor
hyperuricemia, microalbuminuria, non-alcoholic steatohepa-
gene (acanthosis nigricans) titis (NASH) and increased CRP
f. MODY (Maturity Onset Diabetes of Young). viii. Diagnosis is made, if the first criterion and any two of other
MODY was previously considered to be a third criteria are present.
form of type 2 diabetes. However, with the discovery of
specific mutations leading to MODY, it is now classified where it is re-esterified. The consequent increase in
under secondary diabetes. MODY is characterized by diacylglycerol (DAG), a second messenger, leads to
onset prior to age 25, impaired beta cell function and reduced signal transduction by insulin leading to insulin
insulin resistance. Mutations of about 10 different genes resistance.
have been correlated with the development of MODY. A high-caloric diet coupled with a sedentary lifestyle
are the major contributing factors in the development
Type 1 Diabetes Mellitus (T1 DM) of the insulin resistance. A major susceptibility locus
It is due to decreased insulin production. Circulating for type 2 diabetes, named as NIDDM 1, is located on
insulin level is very low. These patients are dependent chromosome 2. Lipoprotein (a) or Lp(a) (see Chapter
on insulin injections. Onset is usually below 30 years 15) is associated inversely with risk of type 2 diabetes.
of age, most commonly during adolescence. They are
more prone to develop ketosis. Metabolic Syndrome (MetS)
An autoimmune basis is attributed to most of these It is a combination of abdominal obesity, atherogenic
cases. Circulating antibodies against insulin is seen in dyslipidemia (hypertriglyceridemia and low HDL cho-
50% cases. Type 1 diabetes mellitus is an autoimmune
lesterol), elevated blood pressure and elevated plasma
disease in which pathologic, autoreactive T cells of the
glucose. The characteristic features are abdominal
immune system attack the insulin-secreting pancreatic
obesity, insulin resistance and decreased glucose tole-
islets of Langerhans. There is excessive secretion of
rance (Box 11.4). The body cannot properly use glucose
glucagon in IDDM patients.
even in presence of normal insulin level. In other words,
body cannot use insulin efficiently. Therefore, the meta-
Type 2 Diabetes Mellitus (T2DM) bolic syndrome is also called the insulin resistance
95% of the patients belong to this type. The disease syndrome. People with Mets are at increased risk of
is due to the decreased biological response to insulin, coronary heart disease and type 2 diabetes. The Mets
otherwise called insulin resistance. So, there is a has become increasingly common in the developing
relative insulin deficiency. Type 2 disease is commonly countries. Diagnostic criteria are shown in Box 11 .4.
seen in individuals above 40 years. These patients are Abdominal obesity is the most prevalent manifesta-
less prone to develop ketosis. About 60% of patients tion of metabolic syndrome. Obesity and adipocytokines
are obese. These patients have insulin resistance and are discussed in Chapter 35.
high/normal plasma insulin levels.
Insulin resistance develops as a consequence of
Metabolic Syndrome (MetS) and
excess accumulation of fat in liver and skeletal muscle. Polycystic Ovary Syndrome (PCOS)
The free fatty acid level increases, exceeds the capacity They have overlapping features. The common factors
of mitochondrial oxidation and spills over to cytoplasm are insulin resistance and obesity. In PCOS there is
170 Section B: General Metabolism
Skeletal muscle
l
Glucose - - - - - - - - - - - - - - Hyperglycemia
1
l\
Fal1)' acid
l
Gluconeogenesls - - - - - -- - -- Oxaloacetate + Cit,{
Acetyl-CoA - - - - - - Ketone bodies
~ sterol l
Increase
Decrease
TCAcycle
l
Atheroscleros,s and
cardiovascular diseases
Ketonuna
Alpha ketoglutarate
Glycolysis is inhibited; gluconeogenesis is favored. Fat is broken down; FFA is increased; Acetyl-CoA is in plenty. This could not be fully
utilized in TCA cycle, because availability of oxaloacetate is reduced. So acetyl-CoA is shunted to ketone body formation.
The best index of long-term control of blood glucose Disadvantage of HbA 1c estimation
level is measurement of glycated hemoglobin or glyco- Any type of anemia, (e.g., abnormal hemoglobins,
hemoglobin. Enzymatic addition of any sugar to a protein hemoglobinopathies) where RBC life span is reduced,
is called "glycosylatlon", while nonenzymatic process is will reflect in lowered HbA 1c value, because the time
termed "glycation". When once attached, glucose is averaged value is less.
not removed from hemoglobin. Therefore, it remains
inside the erythrocyte, throughout the lifespan of RBCs How Frequently the Estimation to be Done
(120 days) (Fig . 11.1 0). The glycated hemoglobins are HbA1c estimation indicates the average blood glucose
together called HbA 1 fraction. Out of this 80% molecules concentration for the past 120 days (life span of RBCs).
are HbA1c, where glucose is attached to the N-terminal However, the value is weighted towards the younger
valine of beta chain of hemoglobin. RBCs. About half of the value is contributed by the
RBCs of the age of 1 month or less. Therefore, it is ideal
Interpretation of to repeat the test every month. This will give an idea to
Glycohemoglobin Values the physician that the treatment is effective or not.
The determination of glycated hemoglobin is not for dia-
gnosis of diabetes mellitus; but only for monitoring the Fructosamines
response to treatment. The Hb A 1c level reveals the Along with other proteins, albumin is also glycated in
mean glucose level over the previous 10-12 weeks. diabetes mellitus. Glycated albumin is more correctly
It is unaffected by recent food intake or recent changes called as fructosamine albumin. As half-life of albumin is
in blood sugar levels. The estimation should be done at about 20 days, glucoalbumin concentration reflects the
least every 3 months in all diabetic patients; however, it glucose control over a recent past, for a period of last
Chapter 11 : Regulation of Blood Glucose, Insulin and Diabetes Mellitus 173
7
0 •headache • palpitation drugs are not sufficient. The availability of human insulin
iii • confusion • tremor
• slurred speech • sweating prepared by recombinant DNA technology has markedly
7
40 • seizures improved the response of patients.
• coma
• death Prevention of c:omplications
B. When the diagnosis is confirmed and treatment interval, 4.0-5.5.0%). What are the various types of
started, how will you monitor the patient? methods used for measuring HbA1c? How do Hb
C. What are the possible complications that can be variants interfere with each of these HbA 1c methods?
avoided by proper monitoring of the patient? What actions should be taken when a spurious HbA1c
e result is present?
• Clinical Case Study 11.2 e
A person is brought to the Emergency Department in a • Clinical Case Study 11 .7
comatose state. The following test results were obtained A 40-year-old male was brought to the emergency room
Blood sugar - 400 mg%, Benedict's test (Urine) - Red complaining of dizziness and weakness. History revealed
precipitate, Rothera's test (Urine) - Positive, Serum that he had skipped breakfast. Random blood sugar
Bicarbonate - 12 mEq/L, Plasma pH - 7.14. What is value was 40 mg%. What is the probable diagnosis?
your probable diagnosis?
e
e
a Clinical Case Study 11.3
• Clinical Case Study 11 8
A40-year-old man presented with complaints of frequent
A person is brought to the Emergency Department episodes of dizziness and numbness in legs. On exami-
in a comatose state. The following test results were nation, he is obese, leads a sedentary lifestyle, has a BP
obtained - Blood sugar-40 mg%, Benedict's test (Urine) of 200/120 mm Hg, has fasting hyperglycemia, hyperin-
- Negative, Rothera's test (Urine) - Positive, Serum sulinemia, dyslipidemia and glucose intolerance. What
Bicarbonate - 12 mEq/L, Plasma pH - 7.14. What is is the diagnosis? What is the pathogenesis involved?
ae
your probable diagnosis?
•
1?. Clinical Case Study 11 .4
Cllnlcal Case Study 11 .9.
A 30-year old woman during her second pregnancy had
A 19-year-old with 4 year history of juvenile diabetes a glucose tolerance test and the results are:
mellitus was brought to the Emergency Department in Fasting glucose level: 125 mg/dl
state of coma. The following laboratory results were 1 hour glucose level: 210 mg/dl
obtained - Blood sugar - 1300 mg%, Plasma pH - 7.1 , 2 hour value: 170 mg/dl.
pCO2 - 13 mm Hg, Pulse rate - 120/min, Respiratory A. Plot a GTT graph with these results.
rate - 28/min. What is your probable diagnosis? What is B. Comment on the GTT results.
the pathophysiology of the above condition? C. What will be the result of Benedict's test with the urine
e sample collected along with each blood sample?
• Cllnlcal Case Study 11 .5 D. How will you follow-up the patient?
E. What is the importance of assessing the glucose
A 55-year-old man with long standing diabetes melli-
tolerance in a pregnant lady?
tus presented with fever, pruritis, delirium and low urine
F. How do you rule out lactosuria in this case?
output. His blood urea level was 135 mg% and urea clea-
rance was 35 ml/min . What is the most likely diagnosis? e
• Clinical Case Study 11 .10
e
a Clinical Case Study 11 .6
An apparently healthy man, on a routine checkup, was
found to have fasting blood sugar of 80 mg/dl, and
A 52-year-old woman with a medical history of hepatitis B, urine showed no abnormal constituents. After a heavy
hyperlipidemia, hypertension and anemia, presented to breakfast of one-and-half hours, his blood sugar was.
the medicine department for a routine visit. Laboratory 140 mg/dl and urine sample at that time was positive
tests 3 months previously had revealed an impaired for Benedict's test.
fasting glucose concentration of 118 mg/dl) [reference A. What is the diagnosis?
interval, 70-110 mg/dl]. Therefore, a hemoglobin HbA1c B. How do you further investigate?
analysis was performed. The initial HbA1c evaluation C. What is the line of treatment?
by HPLC showed an HbA1c value of 12.8% (reference D. What is the course of this disease?
Chapter 11: Regulation of Blood Glucose, Insulin and Diabetes Mellitus 175
The activated IRS in tum will activate other enzyme 27. Glycolysis and glycogen synthesis are stimulated,
system and cascades causing metabol c effects. thus decreasing glucose level in plasma.
25. Insulin has its effects at the level of gen es by induc- 28. Insulin also favours lipogenesis where by excess
tion and repression of enzymes as well as covalent glucose is converted to fat for storage.
modification of enzymes. 29. Gluconeogenesis and glycogenolysis are inhibited
26. Insulin recruits GluT4 in cells to the membrane by insulin, favouring fall in plasma glucose level.
enhancing glucose uptake.
11-1 . The normal fasting plasma glucose level is: A. Untreated diabetes mellitus with ketosis
A. 40-60 mg /100 ml B. Diabetes mellitus treated with overdose of insulin
B. 70-110 mg/100 ml C. Renal glycosuria
C. 120-150 mg/100 ml D. Thyrotoxicosis
D. 60-180 mg/100 ml 11-5. Activities of all the following enzymes are enhan-
11-2. Insulin activates:
ced in starvation, except:
A. Lipolysis B. Ketogenesis
A. Phosphoenolpyruvate carboxykinase
C. Gluconeogenesis D. Glycolysis
11..J. When a standard oral glucose tole1ance test is
B. Carnitine acyl transferase
done, the blood glucose levels of the patient were C. Pyruvate ca rboxyiase
found to be as follows: 0 min (fasting)= 120 mg/ D. Acetyl-CoA carboxylase
dl; 60 min= 170 mg/dl ; 120 min= 150 mg/dl. The 11-6. Insulin increases activity of all the following enzy-
patient has: mes, except
A. Normal glucose tolerance A. Acetyl-CoA carboxylase
B. Impaired glucose tolerance 8. Hormone sensitive lipase
C. Mild diabetes mellitus C. Glycogen synthase
D. Severe diabetes mellitus
D. Glucose-6-phosphate dehydrogenase
11-4. A comatose patient with tremors ad nitted to the
11-7. The following are llpolytic hormones, except
hospital has a blood glucose value >f 300 mg/dl
A. Glucagon B. Cortisol
and his urine was positive for both F.othera's test
and Benedict's test. He is suffering from: C. Epinephrine D. Insulin
178 Section B: General Metabolism
11-8. Growth hormone causes: C. Diabetes mellitus treated with overdose of insulin
A. Decreased peripheral utilization of glucose D. Renal glucosuria
B. Decreased hepatic production of glucose by glu- 11-17. Benedict's reagent will be reduced by all the fol-
coneogenesis lowing substances seen in urine, except:
C. Increased glycolysis in muscle A. Fructose B. Galactose
D. Decreased lipolysis C. Pentoses D. Sucrose
11-9. All are true with regard to Glucagon, except: 11-18. All affect glucose tolerance test, except:
A. Increases lipolysis A. Liver diseases
B. Stimulates glycogenolysis B. Acute infections
C. Favours gluconeogenesis C. Vitamin B12 deficiency
D. Produces glycogen synthesis D. Hyperthyroidism
11-10. All are true with regard to Glycohemoglobin, except: 11-19. Which statement is true with regard to insulin?
A. Seen as high levels in sickle cell anemia A. Secretion is from the beta cells of pancreas
B. Otherwise known as HbA1c B. Insulin favours gluconeogenesis
C. Useful index of long-term control of blood glucose C. Insulin molecule has two chains connected by
level disulphide bridges
D. About 3-5% of hemoglobin in normal cases D. Insulin level in blood is regulated by the growth
11-11. A lactating mother's urine was found to be positive hormone level
for Benedict's test. Her fasting blood sugar was 80 11-20. All the following cellular activities are decreased
mg/dl and postprandial blood sugar after one and under the influence of insulin, except:
half hours after a breakfast was 140 mg/dl. She A. Protein breakdown
may be a case of: B. Ketogenesis
A. Untreated diabetes mellitus C. Glycogen synthesis
B. Diabetes mellitus treated with inadequate dose of D. Lipolysis
insulin 11-21. Insulin increases activity of all the following enzy-
C. Lactosuria mes, except:
D. Renal glucosuria A. Acetyl-CoA carboxylase
11-12. Diabetes mellitus is caused by: B. Hormone-sensitive lipase
A. Anti insulin antibodies C. Glycogen synthase
B. Low levels of glucocorticoids D. Glucose-6-phosphate dehydrogenase
C. Receptor abnormalities on target tissues 11-22. Insulin secretion is mainly controlled by:
D. Reduced secretion of growth hormone A. Blood lipid level
11-13. Which tissue is most insulin sensitive? B. Hypothalamic mechanism
A. Brain B. Liver C. HCI in stomach
C. Adipose tissue D. Cardiac muscle D. Glucose level in blood
11-14. Which laboratory finding is abnormal in a pregnant 11-23. All are true with Insulin receptor, except:
woman at 28 weeks gestation: A. It is a glycoprotein
A. Serum alkaline phosphatase level of 25 KAU/dl B. Alpha subunit has tyrosine kinase activity
B. Plasma 1hour postprandial glucose of 200 mg/d l C. It has 4 subunits
C. Alpha fetoprotein level of 120 ng/ml D. Alpha subunits are located on the exterior of cell
D. Plasma triglyceride level of 150 mg/dl membrane
11-15. In the enzymatic method of glucose estimation, 11-24. The following are lipolytic hormones, except:
the following reactants are used, except: A. Glucagon B. Cortisol
A. Glucose oxidase B. NAD+ C. Epinephrine D. Insulin
C. Peroxidase D. Hydrogen peroxide 11-25. When glucagon binds to its receptors on the liver
11-16. An apparently healthy man, on a routine check up, cells, the following changes take place, except:
was found to have fasting blood sugar of 80 mg/dl, A. Activation of adenylate cyclase
and urine showed no abnormal constituents. One B. Increase in concentration of cAMP
and half hours after a heavy breakfast, his blood C. Inactivation of glycogen phosphorylase
sugar was 120 mg/dL and urine sample at that time D. Activation of protein kinase
was positive for Benedict's test. He is suffering from : 11-26. Growth hormone causes:
A . Untreated diabetes mellitus A. Decreased peripheral utilization of glucose
B. Diabetes mellitus treated with inadequate dose of B. Decreased hepatic production of glucose by gluco-
insulin neogenesis
Chapter 11: Regulation of Blood Glucose, Insulin and Diabetes Mellitus 179
11-1 . What is the normal level of fasting blood sugar? 11-3. Which hormone is hypoglycemic?
70-110 mg/dl. Insulin.
11-2. What are the sources of blood glucose? 11-4. What are the major actions of Insulin?
(a) Absorption from intestine; (b) hepatic gluconeo- Insulin decreases blood sugar; it stimulates glycolysis;
inhibits gluconeogenesis; enhances glycogen synthe-
genesis; and (c) hepatic glycogenolysis.
sis and inhibits lipolysis.
180 Section 8 : General Metabolism
11-5. What are the hyperglycemic hormones? 11 -21 . What are the pathways stimulated by insulin?
Glucagon, adrenaline, corticosteroids, growth hormone. Glycolysis, Glycogen synthesis, HMP shunt pathway,
11 -6. W hat are the major actions of glucagon? lipogenesis.
Promotes glycogenolysis; enhances gluconeogenesis, 11-22. What are the enzymes stimulated by Insulin?
depresses glycogen synthesis, inhibits glycolysis. Phosphofructokinase; Glycogen synthase, Glucose-6-
11-7. What is the major indication for doing an oral phosphate dehydrogenase; Acetyl-CoA carboxylase.
glucose tolerance test (OGTT)? 11 -23. What are the pathways inhibited by insulin?
Patient has symptoms suggestive of diabetes mellitus; Gluconeogenesis. Glycogenolysis, lipolysis, Ketoge-
but fasting blood sugar value is inconclusive (between nesis.
110 and 126 mg/dl). 11-24. What are the enzymes inhibited by insulin?
11-8. What is the criteria to diagnose diabetes mellitus,
Glucose-6-phosphatase, Glycogen phosphorylase,
with regard to fasting blood glucose level? Hormone-sensitive lipase.
Fasting blood glucose level is more than 126 mg/dl, 11-25. How is insulin secretion controlled?
on more than one occasion.
Glucose is the major stimulant of insulin secretion.
11-9. What is impai~ed glucose tolerance (IGT)?
11-26. How glucose stimulates insulin secretion?
When fasting plasma glucose level is between 110 and
GluT2 receptors act as sensor mechanism for glucose
126 mg/dl or 2-hour post-glucose value is between
level. Cyclic AMP along with calcium causes the insulin
140 and 200.
secretion.
11-10. What is Gestational Diabetes Mellitus (GDM)?
11-27. Which tissue utilises glucose maximally?
This term is used when carbohydrate intolerance is
At basal rates, brain utilises 60% of sugar oxidized.
noticed, for the first time, during a pregnancy.
11-28. What are the cardinal symptoms of diabetes mel-
11-11 . What is the clinical significance of GDM?
litus?
Women with GDM are at increased risk for subsequent
Polyuria, polydipsia, polyphagia, weight loss.
development of frank diabetes. GDM is associated with
11-29. What is the reason for polyuria in diabetes?
increased birth weight of child and increased incidence
of neonatal mortality. When the blood glucose level exceeds the renal
11-12. What is renal glucosuria? threshold glucose is excreted in urine. Due to osmotic
effect, more water accompanies the glucose.
Glucose is excreted in urine due to a lowering of renal
11-30. What is the reason for polydipsia in diabetes?
threshold. The blood sugar levels are within normal limits.
11-13. What is normal renal threshold for glucose? To compensate for this loss of water, thirst centre is
180 mg/100 ml. activated, and more water is taken (polydipsia).
11-14. What are reducing substances seen in urine? 11-31. What is the reason for weight loss in diabetes?
Glucose, fructose, lactose, galactose, pentoses, ascor- The loss and ineffective utilisation of glucose leads to
bic acid, glucuronides. breakdown of fat and protein. This would lead to loss
11-15. Fructosuria is due to what? of weight.
Due to the deficiency of fructokinase or aldolase B. 11-32. What is the reason for polyphagia in diabetes?
11-16. What is lactosuria? To compensate the loss of glucose and protein, patient
It is observed in the urine of normal women during 3rd takes more food.
trimester of pregnancy and during lactation. 11 -33. What is microalbuminuria?
11 -17. What is the clinical importance of lactosuria? Albumin 30 to 300 mg/day in urine. It is a predictor
The condition is harmless. But it is important to dis- of progressive renal damage, atherosclerotic diseases
tinguish lactosuria from glucosuria. and cardiovascular mortality.
11-18. What is the test for reducing sugars in urine? 11-34. What is the difference between glycosylation and
Benedict's test. glycation?
11-19. Where ls insulin synthesized? ·-:, Enzymatic addition of any sugar to a protein is called
Beta cells of islets of Langerhans of Pancreas. "glycosylation", while nonenzymatic process is termed
11-20. What is pro-insulin? "glycation".
Insulin is synthesised as a large single polypeptide. 11-35. What is significance of glycated hemoglobin?
Middle part of it is then removed, to form the A and B An elevated glycohemoglobin indicates poor control of
chains of insulin. diabetes mellitus.
Metabolic Pathways of
Other Carbohydrates
Chapter at a Glance
The learner will be able to answer questions on the following topics:
D Fructose metabolism
D Glycoproteins
D Galactose metabolism
D Blood group antigens
D Metabolism of alcohol
D Mucopolysaccharidosis
D Amino sugars
Dihydroxyacetone phosphate
T ©
I
C=O C=O C-0
I I CHO
r
I
'\~
HO-C-H HO-C-H CH2OH Trlose kinase I
I ATP ADP I Absent in H-C-OH
+
H-C-OH H-y-OH Hereditary CHO
I I tHztjP03=1
H-y-OH H-y--OH Fructose ATP ADP
Absent in Intolerance H-y-OH
CH2OH CH2OH Glyceraldehyde-
Fructosuria CH2OH 3-phosphate
Fructose-1-
Fructose
phosphate Glyceraldehyde 3P~f\
Fig. 12.1: Fructose entering glycolysis
182 Section B: General Metabolism
Galactosemia
;;;,,__ _ _ _ _ Mental retardation
There is deficiency of enzyme galactose-1-phosphate
:---.,,- - - - - Congenital cataract uridyl transferase. It is an inborn error of metabolism.
- - - - Jaundice The incidence is 1 in 35,000 births. Herman Kalckar
Hepatomegaly described it in 1958. Due to the block in this enzyme,
Kidney damage galactose-1-phosphate will accumulate in liver. This will
inhibit galactokinase as well as glycogen phosphorylase.
Hypoglycemia is the result (see Box 12.1 ).
Bilirubin uptake is less and bilirubin conjugation is
reduced; so unconjugated bilirubin level is increased in
blood (for bilirubin, see Chapter 22). There is enlargement
of liver, jaundice and severe mental retardation. Free
Galactose and galactose accumulates, leading to galactosemia. It is
Amino acids in Herman Kalckar
urine partly excreted in urine (galactosuria). Galactose is
reduced to dulcitol (see Chapter 7). The accumulation of
Fig. 12.3: Clinical features of galactosemia
dulcitol in the lens results in cataract due to its osmotic
effect. This is called congenital cataract and is a very
UDP galactose + glucose Lactose synthase
characteristic feature of galactosemia. Galactose-1-
Lactose
phosphate may get deposited in renal tubules, producing
Lactose synthesis is seen in lactating mammary glands tubular damage leading to generalized aminoaciduria.
(see Chapter 29). These salient clinical manifestations are summarized in
ii. Synthesis of glycosaminoglycans Figure 12.3.
iii. Synthesis of cerebrosides
Diagnosis: Clinical manifestation including congenital
iv. Synthesis of glycolipids
cataract and presence of galactose in urine as well as
v. Synthesis of glycoproteins.
elevated blood galactose levels will help in the diagnosis.
Galactose Utilizing Steps Collection of fetal cells by amniocentesis may be useful
in prenatal diagnosis. Heterozygous parents could be
1. Galactokinase reaction: Galactose is first phos- detected by elevated galactose level in blood after a
phorylated by galactokinase to galactose-1-phos-
galactose load.
phate (Step 1, Fig .12.2).
Treatment: If lactose is withdrawn from the diet, most
2. Galactose-1-phosphate uridyl transferase (GALT):
of the symptoms recede. But mental retardation, when
This is the rate-limiting enzyme in galactose meta-
established, will not improve. Hence early detection is
bolism (Step 2, Fig.12.2). UDP-galactose may be
most important. For affected infant lactose-free diet
used as such for synthesis of compounds contain-
is given. Such special diets may be withdrawn after
ing galactose (e.g. lactose).
4 years, when galactose-1-phosphate pyrophosphorylase
3. Epimerase reaction : By this reaction, galactose is
(Step 4, see Fig. 12.2) becomes active.
channeled to the metabolism of glucose (Step 3,
Fig. 12.2). Since the reaction is freely reversible,
even if the dietary supply of galactose is deficient,
Galactokinase Deficiency
UDP-glucose can be epimerized to UDP-galactose. A variant of the disease occurs due to the deficiency of
4. Alternate pathway: The galactose-1-phosphate galactokinase. But here the symptoms are milder. This
pyrophosphorylase in liver becomes active only is because galactose-1-phosphate is not formed and
after 4 or 5 years of life. The enzyme will produce hence no toxic effects of this compound are manifested.
UDP-galactose directly, which can be epimerized to However, cataract is seen. Galactokinase deficiency is
UDP-glucose (Step 4 , Fig. 12.2). reported to be 1 in 40,000 births.
184 Section B: General Metabolism
NAO+ NAOH + H
+
CH3 - COOH
Epimerase Glu-6-P
N-acetyl-glucosamine N-acetyl-
mannosamine i
F-6-P - - - - - . GluN-1 -P - -- • UDP-GluN
i i
l
~ ATP
GluN-6-P - - - GluNAc- 1-P
, N-acetyl- +-- -- -- --
Mutase
~ ADP
i i
N-acetyl- GluNAc-6-P ---+ GluNAc Glycoproteins
i
!
mannosamine- mannosamine- and GAG
-6-phosphate -1-phosphate
ManNAc-6-P
+ Phosphoenol pyruvate i GalNAc /
J
Aldolase enzyme NeuraNAc-9-P
l
N-acetyl-neuramlnic acid -9-phosphate
Fig. 12.6: Interrelations of amino sugars
P = phosphate; Glu = glucose; F = fructose; GluN = g lucos-
amine; Neura = neuraminic; NAc = N-acetyl; UDP = uridine
Fig. 12.5: Synthesis of N-acetyl neuraminic acid (NANA) diphosphate; GAG = glycosamino glycans
Immunity lmmunoglobulins, Blood group antigens Keratin sulfate N-Acetyl g lucosamine I} 1,4 Cornea,
Ty pes I and II and Galactose car tilage
Lubricant Mucin
Heparan N-Acetyl glucosamine a -1 ,4 Skin
M essage transfer Receptor proteins on cell surfaces
Cell adhesion Selectins and integrins
The H locus codes for fucosyl transferase. In a per-
Glycoproteins are widely distributed in tissues. All son belonging to blood group A, N-acetyl-galactosami-
plasma proteins are glycoproteins. nyl transferase is present. In group B person, galactosyl
In glycoproteins, the carbohydrate groups are atta- transferase is seen. Lack of both leads to blood group
ched to the polypeptide chain by the following types of 0 , while AB persons have both enzymes.
linkages:
a. Through the amide group of asparagine to N-acetyl Proteog lycans
glucosamine (N-glycosidic linkages). These are widely distributed complex molecules having
b. Through hydroxyl group of serine, threonine, hydroxy- glycosaminoglycans (GAG) and proteins (Table 12.2).
lysine and hydroxyproline to N-acetyl glucosamine The GAGs containing repeating disaccharide units are
or galactose or xylose (beta-O-glycosidic linka- covalently bound to the peptide chain to form proteogly-
ges). cans. Structures of the repeating sugar units are shown
There can be many (usually 20-30) oligosaccharide in the end of Chapter 7. The different GAGs found in
chains attached to a single protein. Each sugar different tissues are shown in Table 12.3.
chain may range from 1 to 20 in length.
Enzymatic addition of specific carbohydrate unit on Biosynthesis of Glycoproteins
the protein is called glycosylation, while nonenzymatic
The biosynthesis of GAG is taking place in endoplas-
spontaneous addition is called glycation.
mic reticulum and Golgi bodies, with the help of specific
Blood Group Substances (Antigens) glycosyl transferases.
The glycoproteins having mannose-6-P as the end
The RBC membrane contains several antigenic sub-
residue will be directed to lysosomes. Mannose phos-
stances, based on which persons are classified into dif-
phate acts as the tag to deliver such glycoproteins into
ferent blood groups. More than 160 different antigens
lysosomes. Defect in this correct tagging will lead to
are known. Of these, ABO system and Lewis system
defective targeting. See I-cell disease, described in
are known to involve glycoproteins. ABO system is asso-
Chapter 41 .
ciated with 3 blood group substances on RBCs desig-
nated A, B and H antigens. The H antigen is the basic
Congenital Disorders of
structure. It has the following structure:
Glycosylation (COG)
Fucose-Gal- GalNAc- Protein
These are autosomal recessive disorders. They are
The RBCs carrying such H antigen are denoted as
multisystem disorders, mostly affecting CNS, with men-
blood group 0 . A and B antigens differ from this in hav-
tal retardation .
ing additional sugar residues.
A antigen: - Fucose-Gal-GalNAc-Protein
Paroxysmal Nocturnal
I
GalNAc Hemoglobinuria (PNH)
B antigen: - Fucose-Gal-GalNAc-Protein There is somatic mutations in PIG (phosphatidyl inositol
I glycan) gene in hematopoietic cells. During sleep, there
Gal is a slight drop in pH of blood, causing mild RBC lysis by
Chapter 12: Metabolic Pathways of Other Carbohydrates 187
complement factors, which leads to excretion of hemo- ground substances. In general, defective degrada-
globin in urine. Normal persons can withstand this lysis. tion of heparan sulfate leads to mental retardation
predominantly whereas accumulation of other GAGs
Rheumatoid Arthritis leads to mesenchymal abnormalities. A characteristic
There is reduced glycosylation of circulating immuno- finding is the excessive excretion of any one of the
globulin G, these are aglycosyl lgG molecules. These will GAGs in urine. All these diseases are inherited as auto-
activate complement system, causing chronic inflamma- somal recessive traits, except Hunter's disease, which is
tion of synovial membranes of joint cavities. X-linked (see Table 12.1).
The inborn errors associated with carbohydrate
Viruses
metabolism, are shown in Table 12.4.
"
Many viruses will attach on human cells through spe-
cific glycoproteins. HIV virus (causing AI DS), attaches
on lymphocytes by r:ieans of a surface glycoprotein
•i i• Clinical Case Study 12.1
(gp120) of the virus (see Chapter 47). Influenza virus A 2-year-old child presented with liver enlargement.
attaches by hemagglutinin (H). So neuraminidase inhibi- Investigations showed the following results: Blood sugar
tors are used as antiviral drugs against H1 N1 . - 50 mg%, Uric acid - 10 mg%, Lactic acid - 15 mmol/L,
Plasma cholesterol - 300 mg% and ketone bodies were
M ucopolysaccharidoses present.
These are a group of inborn errors of metabolism char- What is the likel y diagnosis?
acterized by excessive intralysosomal accumulation What is the biochemical basis of the disorder and its
of GAG in various tissues. They are progressive disor- treatment?
ders. The clinical manifestations include coarse facial
features, thick skin and corneal opacity due to accumu-
lation of GAG.
a0 Clinical Case Study 12.2
Mental retardation, growth deficiency and skeletal A 3-year-old boy is brought to the emergency depart-
dysplasia are also seen due to defective formation of ment after several episodes of vomiting and lethargy.
188 Section 8 : General Metabolism
His pediatrician has been concerned about his failure Biochemical basis of disorder: Because of a genetic
to thrive and possible hepatic failure along with recur- disorder, the hepatic aldolase B enzyme is defective,
rent episodes of the vomiting and lethargy. After a care- and functions normally in glycolysis but not in fructose
ful history is taken, it is found that these episodes occur metabolism. Glucose production is inhibited by eleva-
after ingestion of certain types of food, especially high ted fructose-1-phosphate. When fructose is ingested,
in fructose. His blood sugar was checked in the emer- severe hypoglycemia results.
gency department and was extremely low. Treatment Avoid dietary fructose.
1. What is the most likely diagnosis? Clinical correlation: Deficiency of aldolase B is an auto-
2. What is the biochemical basis for the clinical symp- somal recessive disease, leading to fructose intolerance.
toms? It does not cause difficulty as long as the patient does
3. What is the treatment of the disorder? not consume any foods with fructose or sucrose. Fre-
a0
quently, children with fructose intolerance avoid candy
Clinical Case Study 12.3 and fruit. However, if chronically exposed to fructose-
containing foods. infants and small children may have
A 10-year-old child was brought to the OP Department
poor weight gain and abdominal cramping or vomiting.
with complaints of constant dribbling of thick mucus from
mouth and not responding to surroundings. Clinical his- 0
tory revealed delayed developmental milestones since •ti• Clinical Case Study 12.3 Answer
2 years of age. The child has coarse facial features and
Diagnosis is Hurler's syndrome, a type of mucopoly-
thick mucus and skeletal deformities. Urinalysis revealed
presence of heparan sulfate and dermatan sulfate. saccharidoses (MPS-I). There is deficiency of a-L-
ldurionidase. This leads to defective degradation of der-
What is the probable diagnosis?
matan sulfate and heparan sulfate. They accumulate in
What is the biochemical basis of this disorder?
tissues and are also excreted in urine. Clinical features
12-1 . Describe the process by which galactose is converted into glucose. Indicate the metabolic errors associated
with this pathway.
12-1. All are true with regard to fructose intolerance, 12-3. Features of galactosemia include the following,
except except
A. Defective enzyme is aldolase B A. Cataract B. Hepatosplenomegaly
B. Fructose-1-phosphate accumulates C. Mental retardation D. Hemolytic anaemia
C. Glycogen phosphorylase is inhibited 12-4. Which inborn error will result in mental retardation?
D. Urine is positive for Rothera's test A. Fructosuria
12-2. All are correct with regard to fructose metabolism, B. Essential pentosuria
except C. Galactosemia
A. Fructose is metabolized in liver D. Glucose-6-phosphate dehydrogenase deficiency
B. Free fructose is seen in seminal plasma 12-5. Excess intake of alcohol may produce lactic aci-
C. Block in seminal vessels is indicated by the abs- dosis, because:
ence of fructose in semen A. Alcohol is oxidized to lactic acid
D. Compared to glucose, fructose is sluggishly meta- B. Alcohol is oxidized to acetaldehyde and then to
bolized lactic acid
190 Section B: General Metabolism
C . NADH is g enerated, which converts pyruvate to 12-9. Fructokinase catalyses which reaction?
lactate A . Fructose ---> fructose-6-phosphate
D . Ethanol induces Cori cycle, by wh ich lactic acid B. Fructose---> fructose-1-phosphate
produced from muscle is taken to liver C. Fructose ---> fructose-1 ,6-bisphosphate
12-6. N-acetyl neuraminic acid (NANA) (sialic acid) is D. Fructose-> fructose-2,6-bisphosphate
synthesized by: 12-10. Which is true with regard to fructosuria?
A . N-acetyl glucosamine + glyceraldehyde-3-phos- A . It is due to the absence of Aldolase-B
12-1. What is fructose intolerance? 12-7. Why five years? Why not life-long?
Due defective aldolase B. So, fructose-1 -phosphate By five years, the a lternate pathway (galatose-1-phos-
accumulates. phate pyrophosphorylase) becomes active.
12-2. What is the clinical application of fructose esti- 12-8. Why intake of alcohol produce lactic acidosis?
mation in semen? During alcohol oxidation, NADH is generated, which
Fructose is secreted by seminal vesicles. A block in converts pyruvate to lactate.
seminal vessels in indicated by the absence of fructose 12-9. Why excess intake of alcohol produce hypoglyce-
in semen. mia?
12-3. Neonatal hypoglycemia is seen in which conditions? Because ethanol inhibits gluconeogenesis.
Gly_!'.:ogen storage disease, type I; Galactosemia; Fruc- 12-10. What are the features of chronic alcoholism?
tose intolerance. Polyneuropathy; Fatty liver; Cirrhosis, encephalopathy.
12-4. Congenital cataractis seen in which condition? 12-11 . In glycoproteins, carbohydrate residues are atta-
Galactosemia. ched to which group of the polypeptide chain?
12-5. Galactosemia is due the absence of which enzyme? Hydroxyl group of serine or threonine.
Galactose-1-phosphate uridyl transferase. 12-12. Increased glucosaminoglycans in urine is seen in
12-6. What is the treatment for galactosemia? which condition?
Lactose free diet is given for first five years of life. Mucopolysaccharidosis.
.,______ _ __ Chapter 13
Metabolism of
Fatty Acids
Chapter at a Glance
The learner will be able to answer questions on the following topics:
D Digestion of Lipids D Omega Oxidation
D Absorption of Lipids D De Novo Synthesis of Fatty Acids
D Chylomicrons D Synthesis of Triacylglycerols
D Beta-Oxidation of Fatty Acids D Metabolism of Adipose Tissue
D Oxidation of Odd Chain Fatty Acids D Fatty Liver and Lipotropic Factors
D Alpha Oxidation D Ketogenesis and Ketolysis
e D1gest1on in
/ii,
Stomach -,
'
for Digestion of Lipids
The bile salts present in the bile (sodium glycocholate
The lingua(i)pase from the mouth ent~ ~ ,§.\qlJlfch and sodium taurocholate) lo~r surface _tension~They
i:r-.
along with the food. It h-;;;s an optimum p H ofIT~ 5~The emulsify the fat droplets. The emulsification increases
enzyme therefore continues to be active in the stomach. the sliliace area of the particles for enhanced activity of
It acts on sh_ort chain triglycerides (SCT). SCTs are pre- enzymes (Fig. 13.1 ).
sent in milk, butter and hee. The action of lingual lipase - O -=========~
0
is observed to be more significant in the newborn infants.t)Lipolyti c Enzymes in Intestines
~'}Afric lipase is acid stable, with an optimum pH about 1 _ Pancreatic lipase with co-lipase ( R\.-)
_ 4. It is secreted by Chief cells, the secretion is stimu- 2 _ Cholesterol esterase C. (.E.)
lated by Gastcin. Up to'@_0% digestion__,,of triglycerides 3 _ Phospholipase ~- c~ C.f:l~ c. 1
occurs in stomach. The bile (pH 7.7) , entering the duodenum serves to
neutralize the acid chyme from the stomach and provides
@ Digestion in Intestines _, a pH favorable for the action of pfil!_creatic _enz~mes.
Emulsification is a pre-re_guisiter for digestion of lipids. A list of physiologically important lipases is shown in
The lipids are dispersed into smaller droplets; $.!Ifface Table 13.1.
~rnw..(, "\j co}\on e ~u< d.-~~~'f/2)~
rt t_.
,,.. , .. TABLE 13.1 : Phys1olog1cally important llpases
'
.._ /\
i
I
,,,'
, Lipase
Lingual/acid-
Site ofaction Preferred substrate Product(s)
..,. ' -I
Fat Mouth, TAGs with short and FFA+ DAG
-,,,,,,, stable lipase stomach medium chain FAs
'
, I'
- "if .. ___,. I
,,I Pancreat ic
lipase+
Small
intest ine
TAGs with long-
chain FAs
FFA +
2MAG
,. ,, ,,.
I l
.. ,. ,
, co-lipase
I Intestinal lipase Small TAGs with medium 3 FFA +
,,' I
with bile acids
Phospholipase
intestine
Small
chain FAs
Pls with unsat. FA
glycerol
Unsat FFA
A + bile acids intestine on position 2 Lysolecithin
Fig. 13. Acti Ie The hydrophobic portions of bile
Capillary TAGs in chylomicron FFA+
salts intercalate into the large aggregated lipid, with the hydr.
walls orVLDL glycerol
philic domains remaining at the surface. This leads to breakd
of large aggregates into smaller and smaller droplets. Thus t Adipocytes TAG stored in FFA + DAG
surface area for action of lipase is increased adipose tissue
~ .
CH2- HO + RrCOOH
Lipase I Lipase
CH-0- CO- R2
I
CH - 0-CO-R
+ H20
2 3
.
Triacylglycerol 2,3-diacylglycerol
Fig. Complete hydrolysis of triglyceride. In the intestines, general y fats are only partially hydrolyzed
f ,_cv C\ t,;.
'i)()t"'"U.'-" \-o "f("'\-e. CV bi
cf) Digestion of Triacylglycerols
Pancreatic lipase can easily hydrolyze the fatty acids
esterifie™ a~ d~ s of glycerol
I 1-.· forming - on l'iefd and two molecules of fatty
'. P ,,rc---..I·acid (Fig. 13.2). Then ari isomerase shifts the ester bond
\~ _ from position 2 to 1. The bond in the 1st position is then
-V' ,w .drolyzed by the lipase to form free glycerol and fatty Bergstrom
~-€ acid (Fig. 13.2). The major end products of the diges- NP 1982
\ e.~~tion ofTAG are 2-MAG (78%), 1-MAG (6%), glycerol and 1916-2004
J fatty acids (14%). Thus digestion ofTAG is partial (incom-
A~ 4pl~). Cholesterol ester may be hydrolyzed to free cho- Long chain fatty ac,d
OH OH
I
R~ ~y~,o~>:f \~ ,1 ~ f
c'tfolesterol, f)h cisbholipids and ljio~hospholipids into
ABSORPTION OF LIPIDS -:J f(' 0 ~ f 'molecular aggregates to form__mixed micelle (Fig. 13.3).
~ mely 2 '- 0io~°Jglycerols:~'9P~~~rin fatty acids,
Chylomicrons
carrying
Adipose - ----1+-- -<,, TAG
tissue with
stored fat -,,..=-------l--f-- Albumin
carrying
Muscle and- - -~,a.i.+- free fatty
peripheral acids
tissues
G_ uj-u-o\ ®
~.11e.:l
10,~ w<&U--- 1-;,,,.J. c.!o-e'.:.
194 Section B: General Metabolism ,,.,. K_o. mo\:,
'
BOX 13.2: Absence of d1gest1ve Juices L BETA-OXIDATION OF FATTY ACIDS
1. In pancreatic deficiency: Steatorrhea; unsplit fat is present
This) process ii./<nown as beta-oxidation, because the
in stools
oxm:ation and splitting of tw.9-carboOJW.l!s occur at the
2. When bile is not available: Absorption is defective; split fat
l'r 6eta.carb..on_.atom. The oxidation of the hydrocarbon
is present in stools; defective absorption of vitamin K leads to
Rrolon ed rothrombin time. chain occurs by a sequential cleavage of two-carbon
atoms (Fray Knoop, 1904).
can directly eriterjDto blood vessefs. then top-9rtal v.e.in , Preparative Steps for Beta-Oxidation
finally !o___!tter where they are immeeiately utilized for
energf fig. 13.4). Their absorption is rapid. They are The c9,~nzyme A is a~mplex molecule containing B
better absorbed than long chain fatty acids. Peculiarities com1t1lf~ min Q.9n~ ffienic_gg_d and a molecule of
in digestion and absorption of medium chain fatty acids bi ~ tmer.capto ethaooJarnine; this SH group forms thio-
8
are given Table 16.1.
0
ester bond in acyl-CoA (Fig. 13.6). To emphasize the
function of the SH group, the CoA is sometimes written
repara
- --3·
~t . St
1ve
ll
ep .
'
2 2
Beta-mercapto
I a. ,:J
Chapter 13: Metabolism of Fatty Acids 195
r, J JJ)
X
BOX 13.3: Acetyl and Acyl groups are different Mitochondrial
membrane--tt-·
1. Acetyl-CoA is the combination of acetate or acetic acid
(2 carbon unit) with Coenzyme A.
Ac11l-
2. Acyl-CoA means acyl group (any fatty acid, C4 to C26 in
Carnitine co:a.
length) combined with Coenzyme A.
®
Preparative Step 4: Translocase
Acyl
carnitine
CoA Co.~
A P_!:.~tejn tran_slo~ will gm:yJh,.e_ac_ylcamitine..across
the me brane to the matrix of mitochondria. On the Cytosol Mitochondri;1
matrix side of the ~ e another enzyme, carnitine
acyl transferase-lll{S1T-!!),-'will transfer the acyl group Fig. 13.7: Role of camitine in transport of acyl groups
( = Carnitine acyl transferase
back to co-enzyme A ~cule (Fig. 13.7). Carnitine is
.....,
returned to the cytosolic side by the translocase.
Beta-Oxidation Steps
Clinical Applications The next 4 reactions are sequentially repeated for com-
MC.f'F'I, .SC ff!\ plete oxidation of fatty acids. After one round of four
• 1. Medium chain and short chain fatty acids do @ metabolic steps, one acetyl-CoA unit is split off and acyl-
req uire carnitine for transport across the inner mito- CoA with 2 carbon atoms less is generated. This would
chondrial membrane. So, medium chain and short undergo the same series of reactions again until the
chain f~ acids re easily oxidized. fatty acid is completely oxidized.
2. Carnitme dl _
1 1e~5;~repo~ed in. P.rn.nts,
in wh . dtattv'aciB°ox~~s no ice . So Step 1: FAD Linked Dehydrogenase ,. IJ~ ~o.tt,-t\)
~ r ~ ~ se,Js0 utilized , resulting in episodes of The fatty acyl-CoA is dehydrogenated to a transenoyl-
hypoglycemia. CoA with FAD accepting the hydrogen atoms (Step 1,
.. , _ ,. __,., :. Fig. 13.8). FADH 2 when oxidised in electron transp'.lrt
Carmtme -=> NI n n G.rui €. } ~ain will produce 1.5 ATP molecules.
Carnitine is s nthesized in the @ c and ~ s "om Step 2: Hydration \--rtJ :::h·'l ri..ro~'\
I sirre and met ·onin . During ro or pregnanc the I
requirement o migbt exceed.its natural produc- This is catalyzed by an enoyl-CoA hydratase (step 2,
tion. Human genetic disorders, affecting different steps Fig. 13.8). This step forms a beta-hydroxy fatty acyl-
of carnitine metabolism will cause deficiency of fatty acid CoA. The L isomer alone is formed during the hydration
of the trans double bond.
oxidation.
During the aging process, camitine concentration in
I' t I~ b
cells diminishes. B e are particularly affected adver-
Step 3: NAO+ Dependent Dehydrogenase~
sely, leading t~ opo in elderly subjects. Admin- The beta-hydro~y fatty acyl-CoA is again oxidized to fo1m
istration of carnitine 1s capable of improving the clinical beta-keto fatty acyl-CoA. (Step 3, Fig. 13.8). This dehydro-
condition. .iLl ~J ~!oh~ genase acts only on L isomer. The NADH when oxidiz13d
v The class~ pifi~entati~ frprffuary carnitin~~fi- in electron transport chain will generate 2.5 ATPs.
ciencBs hep~ megaly, ele~ ted transaminases, and
hyper'a~monemia. T~ causes for seJttl[1dary carnitine Step 4: Cleavage r \"\Ar\ q fl c. t" l. ~ '
deficiency are organ~ cidurias, rd dfi!'g -induced (val- The beta-keto fatty acyl-CoA now undergoe~ thiolytic
proic acid,~gyudine). \URlNm~eet\ cleavage, splitting off a molecule of acetyl-CoA and
;lf <!- WXj wid:lii ~t
196 Section B: General Metabolism
ll
ll-hydroxy-t NAO+ BOX 13.4: Summary of beta-ox1dat1on
f.itty acyl-CoA-
dehydrogenase + When one molecule of palmitate undergoes beta-oxidation, the
(Step 3 NADH+H- 2.5ATP net reaction is:
R- CH2- CO - CHr CO-SCoA
®
l
f.l -keto fatty acyl-CoA Palmitoyl-CoA 8 Acetyl-CoA
+ 7 FAD + 7 FADH2
!3-k~· Thiolase + CoA-SH + 7 NAO' - -- + 7 NADH
(Step 4)
+ 7 H2O + 7 H"
R- CH 2 CO-SCoA + CH 3 CO-SCoA
+ 7 HSCoA
Fatty acyl-CoA + Acetyl-CoA
I ( - l,C..) I
(Steps 1,2,3,4)
...._;_;.__ ___;_ _ TCAcycle (10ATP)
(further cycles) gives 10 molecules of ATP. Each molecule of FADH2
Fig. 13.8: Beta-oxidation of fatty acids
produces 1.5 molecules of ATP and each NADH gene-
Important to remember that the first step is FAD dependent rates 2.5 molecules of ATP, when oxidized in the elec-
and the third step is NAO• dependent. tron transport chain . Hence, the energy yield from one
molecule of palmitate may be calculated as:
leaving behind a fatty acid with 2 carbon atoms less 8 acetyl-CoA x 10 = 80 ATP
(step 4, Fig . 13.8). 7 FADH2 x 1.5 = 10.5 ATP
7 NADH x 2.5 = 17.5 ATP
Further Cycles Gross total = 108 ATP
The newly formed fatty acyl-CoA will sequentially under- Net yield = 108 minus 2 =106 ATP
go further cycles of step s 1, 2 , 3 and 4 of beta-oxidation (In the initial activation reaction, the equivalents of 2
until the fatty acid i(s_ofiip_Ieteiy__sor1vertei!_@ acetyl-C0) high energy b s a e utilized). The efficiency of beta-
(Fig. 13.9). A summary is shown in Box 13.4. oxidation is bou 33°0 The differences in oxidation of
unsaturated fa y I s are shown in Chapter 16.
Energetics of Beta-Oxidation A few years back, calculations were made assum-
(ATP Yield) ing that NADH produces 3 ATPs and FADH generates
2 ATPs. This will amount to a net generation of 129 ATP
Palmitic acid (16 C) needs 7 cycles of beta-oxidation . per palmitate molecule. Recent experiments show that
So, it gives rise to 8 molecules of acetyl-CoA. Every these old values are wrong, and net generation is only
molecule of acetyl-CoA when oxidized in the TCA cycle 106ATPs.
Methylmalonyl-CoA '\ Ketoacidos1s, hypotonia, 1- 1')
mutase or B12 yiJ:,/ hypoglyce'mia, hyper- ,::,.
coenzyme ammonemrci, hyperuricemi : '
Ketoacidosis, hypotonia, 7-
vomilng, lethargy
Acidosis, hyperammone,rnia;
hypoglycemia, fatty liver
Long chain acyl-CoA Nonketotic hypoglycemia,
dehydrogenase low carnitine, increased acyl '
carnitine
Glutaryl-CoA Ketoacidosis, convulsionf
dehydrogenase progressive neurological '
defects, cerebral palsy '
Regulation of Beta-Oxidation
i. The availability of free fatty acid (FFA) regulates the Fig. 13.1 O: Metabolism of propionyl-CoA
CO2, and hence as a general rule, fatty acids cannot Infantile Refsum's Qis.aase
be used for gluconeogenesis. However, propionate is
It is a(tSer o Xl.J·soS~ . , similar to Zellweger synd-
entering into the citric acid cycle at a point after the CO2 rome and adr e'!li e ~o_Eh-y (see Chapter 41 ).
elimination step~ so propionate can be channelled to
= -
gluconeogenesis. Thus 3 carbon units from odd car-
Hence phyta ic acid accumulates along with VLCFA.
Children do not survive long. 'f.oll'5
*bon fatty acids are gluconeogenic. Cow's milk con- &o,.'Y\if,
tains significant quantity of odd chain fatty acids. Lc@GA OXIDATION :;,,le] ~Mil J
CGio.-u.u.1ev Rarrde,'J . . th t k' I
It Is a minor pa way a 1ng p ace . .
1
J
1crosomes, wI·th
th,,.,r- '
Inborn Errors of Propionate Metabol ism the help of hydroxylase enzymes involving NADPH and
1. Propionyl-CoA carboxylase deficiency. It is chara- cytochrome P-450. The CH3 group is converted to CHpH
§._..-1- cterized by propionic acidemia, ketoacidosis, and and subsequently oxidized with the help of NAO· to a
developmental abnormalities. COOH group to produce@1carboxylic acJ~. w-oxidation
2. Methyl malonic aciduria. Some patients respond becomes important when 13-oxidation is defective and
to treatment with pharmacological doses of B12 . This dicarboxylic acids (6C and BC acids} are excreted in
group had deficiency in the formation of adenosyl urine causing dicarboxylic aciduria. Peroxisomal oxi-
B12 with deficient mutase activity. The second type dation is described in Chapter 16, under VLCFA.
did not respond to cyanocobalamin and had defi-
ciency of the enzyme racemase or mutase. Methyl- Inherited Disorders
malonate affects tht£'®abo~ of brQin leading to Inherited defects in the 1'.nzymes of b~-oxidatioJ;Hlnd
meQ,tal retardati~ ·n these cases. r _, ketog~esis also lead to'Wonket~~~oglycemia, ~ ma,
=~ v-iti (~ n
1
and fatty liver. Defects are known in 3-hydroxyacyl-CoA
1 ALPHA-ox1DAT10N _,,.;;; ..c "irCl--r' dehydrogenase, 3-ketoacyl-CoA thiolase and HMG-CoA
It is a process by which fatty acids are oxidized by lyase deficiency. Dicarboxylic aciduria is characterized
re.rD,Q',illl carbon-atoms, o,oe~ a.t~ om the car OET by the excretion of dicarboxylic acids and by nonketotic
end. The process is important io brain. The fatty acid hypoglycemia. It is caused by a lack of mitochondrial
does not need activation. Hydroxylation occurs at the medium-chain acyl-CoA dehydrogenase.
alpha-carbon atom. It is then oxidized to alpha-keto More than 25 enzymes havebeenidenti~ for fatty
acid. The keto acid then undergoes decarboxylation acid metabolism in humans, out of which at>;Q.ub 5 are
yielding a molecule of CO2 and a fatty acid with one car- known to be associated with metabolic disorders.
bon atom less. This process occurs in the endoplasmic
reticulum, does not require CoA, but does not gene- DE NOVO SYNTHESIS
rate energy. Some fatty acids undergo alpha oxidation OF FATTY ACIDS f \-~
in peroxisomes also. -----
The process of fatty acid synthesis was studied by
Alpha-oxidation is mainly used for fatty acids that
Feodor Lynen, w h o ~ ize in 1964. The pathway
-
have a methyl group at the beta-carbon, which blocks
is referred to as L nen s s ira It is not a reversal of
beta-oxidation. A major dietary methylated fatty acid is
oxidation . Important di nces in synthesis and break-
phytanic acid. It is derived from phytol present in chlo-
down of fatty acids are given in Table 13.3.
rophyll, milk and animal fats.
This pathway operates in the cytoplasm. So it is
referred to as extramitochondrial las ·c fatty
Refsum's Disease acid synthase system.
It is a metabolic error due to lack of alpha-hydroxylase The major fatty acid synth~sized de novo is palmitic
(phytanic acid oxidase) so that alpha oxidation does not acid, the 16C aturated fatty acid. The process occurs
occur and phytanic acid accumulat~ in the tissues. The in b"·adipose tissue, kidney, ~ n, and m mmary
patie,m presents wit~evere nearologic~ symptoms, glands.
polyne_1Kopathy, retinitis pigmentosa, nerve~afness and
..tet> Q ~ " ' j 1,... ~~N>•r·
ti; I,. ,..,., CoT:f .....
cerebe'llar ataxia. Regression of symptoms is observed ransport of /1+.. etyl-CoA to Cytoplasm
with restricted dietary intake of phytanic acid. Milk is a Acet I-CoA is formedinside the mYt'~ Ro~ l from pyru-
good source of phytanic acid, which may be avoided. vate. The inner membrane is not freely permeable to
....
.. . . .
TABLE 13.3: Difference 1n the twc pathways Fatty acid
Site
Intermediates
Beta-oxidation
Mitochondria
Present as Ce>A
Gllft!llllMS
Fatty acid synthesis
Cytoplasm
Covalent lyilliikicl to
~of/0
l MITOCHONDRIA
beta-oxidation
Acetyl-CoA
Malaw
acetyl-CoA. Hence the,acetY.1-CoA units are ~ d
to the cytoplasm citra Fi9i 13.11 ). In the crtoplg2~1 NADP+ .j
citrate is cleaved to o~aloa~e ~nd ace~ CoA' in th"e1
cytoplasm. The enzyme is ATP citrafe lyase. The oxalo-
acetate can return to the mitochodtia as malat or
NADPH --1
pyruvate (Fig. 1 1. < e.,N
4~ · ~ ~ ~ .~ - - - ~~ -- - ~
l Y'> . Fig. 13.11: Transfer of acetyl-CoA from mitochondria to
Fatty'Acid Synthase (FAS) Complex cytoplasm by malate-oxaloacetate shuttle
= = =
1 citrate synthetase; 2 ATP-citrate lyase; 3 malate
This system exists as a multi-enzyme complex. The dehydro-genase; 4 = malic enzyme
enzymes form a dimer with identical subunits. Each
subunit of the complex is organized into 3 domains with Third Domain or Releasing Unit l I )
7 enzymes (Fig. 13.12).
It is involved in the release of the pa Imitate synthesized.
Advantages of Multi-enzyme Complex It contains th jo-esterase JbE~ or de-acylase (Fig. 13.12).
lo. Th~ \N>.. ~ ' t ~ -
in_teraGt Step 1: Carboxylation of Acety/-CoA 3oo~
The first step in the~ \.synthesis is the•.wroo_xx- rod!t
b. of ~ yl-CoA to form~ . Acetyl-CoA
carboxylase is not a part of the multi-enzyme complex.
C. But it is the rate-limiting enzyme. a member
t): ' CJIL of B complex _vi tamios, is necess.ary for this reaction
First Domain or Condensing Unit 0
u.1step 1 in Fig. 13. 13). --
It is the initial substrate bin\~~ site. The en= invl The enz~me i~ aJ!,R_~eric~_lly regulated1) ~~- i1?.!o .d-1
ved are b £ ~ ~ ~ a s e or condensin.g_enz.~ effectors being g trate (pos1t1ve) and palm, oyl-Co J
(CE);o.~ etyl transferase'(AI) and malo ny) transacylase (negative). The reaction is similar to carboxylation of
(MT) (Fig. 13. 12). - ~ M . ~ '\:.&.~ pyruvate to form oxaloacetate.
roi c..~1 The a
Second Domain or Reduction Unit the 2-carbon units am
( 4) added as 3-carbon, malonyl units. The whole reaction
It contains the dehydratase (DH); enoyl r.edu.ctase (ER); sequence occurs while the intermediates ace bouru:Uo
beta-keto acyl ~ as.e (KR) and a~ ~e.r.. prote~ ACP (acyl carrier protein). _. A 't·\;..~-' , a,.~
(AC~ _Fig. 13.12). The acyl carrier protein ha~ - =-
~ tottieine group, to which the acyl groups are atta- Step 2: Three C and Two C Units are Added
ched in thioester linkage. So ACP acts like the CoA A. Acetyl transacylase (AT in Fig. 13.12) catalyze!;
carrying fatty acyl groups. the transfer of the acetyl group (2 carbons) to the
0.'
'( <;,),...."f". Condens ing unit , , Reduction unit
@
\
CE - AT - MT----''--.,.----- DH - ER - KR - ACP- - - - -- .
',,,,,
yys ', Translocation
SH
SH '
I ', I
SH ',
',
' ',
TE - - - - - - - - - -ACP- KR - ER - DH - - - - - ' - , - MT- AT - CE
R9lualng 111111 Reduction unit ' Condensing unit
~~:J
lil!d .. ,.
Fig. 13.12: Fatty acid synthase complex
Upper and lower units are two monomers of the complex. Dotted line represents functional division.
f'Qrf'> 1~ L'tllte ~oo~__,,c,..\t\
• ~l V t
cyst~inyl SH group of the condensing enzyme Cycling of Reactions
(CE) of the other monomer of the fatty acid sm--
thase complex (step 2A in Fig. 13. 13). The butyryl group (4C) is now transferred to the SH
B. One molecule of acetyl-CoA (2 carbon ) and one group of the condensing enzyme on the other mono-
molecule of malon I-CoA 3 carbon) bind to the mer and a 2nd malonyl-CoA molecule binds to the
.~ ti-enzyme comp~ . Malonyl transacylase (MT fili(~mlQ.:Pantot; ! sH rou ~~ he sequ~J'.\ce of reac-
(.t'I°<'~ :~v-fn~ e r s the malonyl group to the SH tior~ , namely con · ;}tion, reduc ion, de dr ion and
~.,~ group of the ACP (step 28 in Fig. 13.13). reo'at~n (steps 3,4,5,6) are repeated. The cycles are
~ ,- repeated a total of seven times, till the 16-Carbon pal-
/ Step 3: Condensation mitic acid is formed. rr co , .,,..
M.e..-r N\a.llLL "- CP-" \\
~ C ) a~ inafo-,,yr (3g units are condensed to
form aceto acetr AcP (4C). During this process one Step 7: Palmitic acid is Released
carbon is lost ast;02 (step 3 in Fig. 13.13). The enzyme
Th io-esterase or deacylase activity (TE) releases
is called condensing enzyme (CE in Figs.13.12). palmitate from the multi-enzyme complex (step 7, Fig.
13.13).
Step 4: Reduction The end point is Palmitic acid 16 C) in liver and
Acetoacetyl ACP is reduced by NADPJ:L.d.epeodent adipose tissue. Bu in rnar:nma land, the
reductase (KR in Fig. 13.12) to form ~ tty end products are Ca ric 10 C) and Laurie 12_C) acids.
a~ P (step 4 in Fig. 13.13). r
-OH A Ad') Mother's milk contains these me rum-c arn fatty acids.
Cow's milk contains odd numbered fatty acids.
i~~~~ tep 5: Dehydration
-~ It is then dehydrated by a ehydratase (D j) to form Summary of De Novo Synthesis
e- gru:zyl ACP otherwise known as (unsaturated acyl ACP) The net reaction of de nova synthesis of fatty acid may
(step 5 Fig. 13.13). be sum
'°"V(('
-+
Step 6: Second Reduction
The enoyl ACP is again reduced by enoyl reductase Fatty acid synthesis is not an exact reversa of beta-
(ER) utilizing a 2nd molecule of NADPH to forfflbu~ ox dation. A comparison of these pathways is given in
ACP (step 6 in Fig. 13.1 3). '-r- Taole 13.3.
~,4,..'.> Cl.Sl~
Chapter 13: Metabolism of Fatty Acids 201
l
(Step 2A) active HMP shunt pa ay also.
Acetyl transacylase (AT)
Acetyl-CoA + (CE)-SH-+ Acetyl S- (CE) + CoA
(Step 28)
Regulation of Fatty Acid C thesis '1"
Malonyl transacylase (MT) Availability of Substrates o.e (l) eJ
Malonyl-CoA + ACP-S~ Malonyl-S-ACP + CoA
Fatty acid synthesis oc9J1!.is when carbohydratiLJs abun-
(CE)- S- CO- CH 3 + ACP- S- CO- CH2 COOH
dant and the level off~ s .is low. The availability of
Acetyl- S- (CE) MalonylACP
l~C) ( a c.) q.tcate io the cytoplasm is the fll.O,SWIJlPOrtsot rea.ulaj_Ory
(Step 3) CO2 factor producing a short-term effect.
Condensing enzyme
or keto acyl synthase (CE)-SH
(rO
ACP- S-CO- CH2- CO- CH3 e
At-./P
cetoacetyl ACP ~ )
(Step 4 ) beta keto acyl ACP
Keto acyl reductase NADPH + fatty acid synthesis is enhanced when eoei:mw:bar9e- is
bi9bCEafty..acid syaihe~is de~1eases when glucose level
NADP+
a'f!)rner~ arge is low. The enzyme is inhibited by pal-
"'.a\-.
ACP- s - co- CH2- CHOH-CH3
Beta-hydroxy butyryl ACP e
mitoyl-CoA, the end product.
;cu?; (.ll!!'9)
11
v:---1
,•
Dehydratase~
Insulin Fa vors Lipogenesis ~'d)...o J~
(Step 5)
H2 0 Insulin enhances the u tal,<e oL ll,ico e by/adipocytes
ACP-S- CO - CH==CH-CH3 (Enoyl ACP) and increases the activity of py,a,~ ~aatl¥d~~ase,
1 Pl>l-1
= ~""'"""...._,,.,._t0xytase and 91¥-C,eu..,i-+u.1
Enoyl reductaser : NADPH + H+ transferase (see Table 11.4 ). Insulin also depresses (G 11
(Step 6}
hormone-sensitive lipase (Fig . 13.14 ). J
NADP+
__
(Steps 3,4,5,6)
,__......;___;_ _;_ __l Butyryl ACP (4 carbons)
Membrane
ATP •
\ ·•--.:t,
=···
Cyclic AMP
,__ P_h_
os_p_hod_ie_s_te_ra_se
_ _ }AM~ ~ &A
... ..
ch~~
T\
Inactive Active
hormone-sensitive hormone-sensitive
lipase lipase
(no..Qh9sphate group) ATP ADP • (phosphorylated)
-
Acyl transferase
- r=
cerol (TAG) synthesis. T TIX synthesis in adipose HSCoA
tissue is for storage of energy. But TAG is secreted ~yl dihydroxy acetone phosphate
as VLDL and is transported. The TAG is synthesized NADPH+H•
by esterification of fatty acyl-CoA with either glycerol- Reductase
NADP+
3-phosphate or dihydroxy acetone phosphate (DHAP)
(Fig. 13.15). The glycerol part of the fat is derived from Lysophosphatidic acid { 1-.P'P.l)
the metabolism of glucose. DHAP is an intermediate Fatty acyl-CoA
of glycolysis. Glycerol-3-phosphate may be formed by Acyl transferase
r=
phosphorylation of glycerol or by reduction of dihydroxy HSCoA
~ ETABOLISM OF ADIPOSE TISSUE tissue are not inert. They undergo a daily turnover with
The adipose tissue serves as a storage site for excess new triacylglycerol molecules being synthesized and a
calories ingested. The triglycerides stored in the adipose definite fraction being broken down.
Chapter 13: Metabolism of Fatty Acids 203
'
FATTY LIVER AND the capacity to synthesize VLDL is affected leading
to fatty infiltration of liver (step no. 3 in Fig. 13.17).
( LIPOTROPIC FACTORS
ii. In protei calorie malnutrition, amino acids req-
Fatty liver~efers ;Jth)dffosltio~of ek!ss tfigtycerides ire to synt esise apoproteins may be lacking.
in the liver cells. The balance between the factors caus- iii. Hepatitis B virus infection reduces the function of
ing fat deposition in liver versus factors causing removal hepatic cells.
of fat from liver, determines the outcome.
Alcoholism
Causes of Fatty Liver It is the most common cause of fatty liver and cirrhosis in
A. Causes of fat deposition in liver India. The metabolism of alcohol is described in Chapter
1. Mobilization of fatty acids (FFA or NEFA) from 12. Alcohol is oxidized to acetaldehyde. This reaction
adipose tissue . produces increased quantities of NADH, which converts
• -.@ More synthesis of fatty acid from glucose. oxaloacetate to malate. As the availa bility of oxaloace-
.' B. Reduced removal of fat from liver tate is reduced, the oxidation of acetyl-CoA through citric
, 3. ·n· to liver. Secretion of VLDL needs acid cycle is reduced (block in step 4 of Fig. 13.17). So
'•, ,,,....-:\ synth · Bf :too and apo.C. fatty acid accumulates leading to TAG deposits in liver.
·- \V Qijci'easeo oxidation of fat by hepatic cells. An
increase in factors (1 ) and (2) or a decrease in Nonalcoholic Fatty Liver Disease
factors (3) and (4) will cause excessive accumu- (NAFLD) and Nonalcoholic
lation, leading to fatty liver. These pathways are Steatohepatitis NASH
summarized in Figure 13.17. Hi h fat diet and unco
most common causes
Excessive Mobilization of Fat
cytes. As it progresse
The capacity of liver to take up the fatty acids from blood which is t
far exceeds its capacity for excretion as VLDL. So fatty (NASH).
Chapter 13: Metabolism of Fatty Acids 205
E
oetyl-CoA
cess fibrous tissue ·sJaid..dow!l, caus· fibrosis of liver,
Step 2 H2 0
otherwise known as cirrhosis. ...,,, ction tests (see
Chapter 24) will show abnormal v CoA
COOH-CH2- C(CH3 ) (OH)- CH2 - CO-SCoA
Betahydroxy beta methyl glutaryl-CoA (HMG-CoA)
Lipotropic Factors
They are required for the normal mobilization of fat Step 31
from liver. Therefore deficiency of these factors may
CH3 - CO- CH2- COOH + CH3- CO-SCoA
result in fatty liver. They can afford protection against
the development of fatty liver. Acetoaceta~te + : c:: ~ : :
1. Choline
Step 4
2. Lecithin and methionine: They help in synthesis
NAO•
of apoprotein and choline formation. The deficiency
of methyl groups for carnitine synthesis may also CH3- CO- CH 3 CH 3 - CHOH- CH2 - COOH
Acetone Beta-hydroxy butyrate
hinder fatty acid oxidation.
3. Vitamin E and selenium give protection due to Step 1 = Aci?t : CoA synthase;
their antioxidant effect. Step 2 = HM A synthase;
4. 0 ega-3 fatty acids present in marine oils have a Step 3 = H • oA lyase;
protective effect against fatty liver. Step 4 = Dehydrogenase; (.D t-r)
Step 5 is nonenzymatic and spontaneous .
l
Ketone bodies Ketone bodies
J~= l
gluconeogenesis. The increased rate of lipolysis is to
provide alternate source of fuel. The excess acetyl-
CoA is converted to ketone bodies. The high glucagon
favors ketogenesis. The brain derives 75% of energy
Citric acid cycle
Acetone
in lungs
Ketone
bodies in urine
i
CO2
from ketone bodies under conditions of fasting. Hyper-
emesis (vomiting) in early pregnancy may also lead to
starvation-like condition and may lead to ketosis.
Fig. 13.19: Formation, utilization and excretion of ketone bodies
Explanation for Ketogenesis
Ketolysis
i. During starvation and diabetes mellitus, the blood
The ketone bodies are formed in the liver; but they are level of glucagon is increased . Glucagon (see
utilized by extrahepatic tissues. The heart muscle and Chapter 11) inhibits glycolysis, activates gluconeo-
renal cortex prefer the ketone bodies to glucose as fuel. genesis, activates lipolysis, and stimulates keto-
Tissues like skeletal muscle and brain can also utilize genesis. High glucagon-insulin ratio is potentially
the ketone bodies as alternate sources of energy, if glu- ketogenic.
cose is not available. Acetoacetate is activated to ace- ii. Insulin (see Chapter 11) has the opposite effect; it
toacetyl-CoA by thiophorase enzyme. favors glycolysis, inhibits gluconeogenesis, depres-
Almost all tissues and cell types can use ketone ses lipolysis, and decreases ketogenesis. The
bodies as fuel, with the exception of liver and RBC. ketone body formation is regulated at the following
Thiophorase 3 levels:
Aceto acetate Acetoacetyl-CoA
+ Succinyl-CoA Succinate Level 1: Lipolysis
Then acetoacetyl-CoA enters the beta-oxidation
Precursors of ketone bodies are free fatty acids. So
pathway to produce energy. Summary of ketone body
mobilization of fatty acid from adipose tissue will influ-
metabolism is shown in Figure 13.19.
ence ketogenesis (Fig. 13.20). Insulin inhibits lipolysis,
Normally the rate of synthesis of ketone bodies by the Level 2: Entry of Fatty Acid to Mitochondria
liver is minimal. So they can be easily metabolized by the
The mobilized fatty acid then enters mitochondria for
extrahepatic tissues. Hence, the blood level of ketone
beta-oxidation. Carnitine acyl transferase I (CAT-I) regu-
bodies is less than 1 mg/dl. Ketone bodies are not
detected in urine. But when the rate of synthesis exce- lates this entry (see Fig. 13.7). Malonyl-CoA is the major
eds the ability of extrahepatic tissues to utilize them, regulator of CAT-I activity. In diabetes and starvation,
there will be accumulation of ketone bodies in blood. glucagon is increased, which decreases malonyl-CoA
This leads to ketonemia, excretion in urine (ketonuria) (Fig. 13.20).
and smell of acetone in breath. All these three together
constitute the condition known as ketosis. Level 3: Oxidation of Acetyl-CoA
When the above two steps are increased , more acetyl-
Causes for Ketosis
CoA is produced. Normally, acetyl-CoA is completely
Diabetes mellitus: Untreated diabetes mellitus is the oxidized in the citric acid cycle. In both diabetes mel-
most common cause for ketosis. The deficiency of litus and starvation, the oxaloacetate is channelled to
Chapter 13: Metabolism of Fatty Acids 207
Starvation/Diab ties M ' ill.IS Supportive evidence may be derived from estimation of
(Increased Glucagon/Decreased Insulin) serum electrolytes, acid-base parameters, glucose ana
---+ Level 2:
l
Increased CAT-I
The urine of a patient with diabetic ketoacidosis will
give positive Benedict's test as well as Rothera's test.
Decreased malonyl-CoA
But in starvation ketosis, Benedict's test is negative, but
i
Increased beta oxidaion
Rothera's test will be positive.
- - - . Level 3: Increased gluconeogenesis
Decreased oxaloacetate Management of Ketoacidosis
i. Treatment is to give insulin and glucose. When
Ketogenes1s -+- Increased generation of acetyl-CoA glucose and insulin are given intravenously, potas-
and decreased utilization of acetyl-CoA
sium is trapped within the cells. Hence, the clinician
Fig. 13.20: Summary of ketosis should always monitor the electrolytes.
ii. Administration of bicarbonate, and maintenanc1:i
gluconeogenesis; so the availability of oxaloacetate is of electrolyte and fluid balance are very important
decreased. Hence acetyl-CoA cannot be fully oxidised aspects.
in the TCA cycle. When oxaloacetate is diverted for
gluconeogenesis; citric acid cycle cannot function opti- • · Clinical Case Study 13.1
mally. Thus, on the one hand, acetyl-CoA is generated in
A teenage girl was brought to the hospital with com-
excess, on the other hand, its utilization is reduced. This
plaints that she gets too tired and has muscle pains. .A.
excess acetyl-CoA is channeled into ketogenic pathway.
consulting neurologist found muscle weakness in arms
(See Fig. 11.9 also).
and legs. Biochemical investigations revealed elevated
amounts oftriacylglycerols esterified with long chain fatly
Salient Features of Ketosis
acids. Muscle biopsy report showed significant number
1. Metabolic acidosis. Acetoacetate and beta-hy- of lipid vacuoles. What is the probable diagnosis? What
droxy butyrate are acids. When they accumulate, is the cause of these symptoms?
metabolic acidosis results. (see Chapter 27).
2. Reduced buffers. The plasma bicarbonate is used 8 Clinical Case Study 13.2
up for buffering of these acids.
A 22-year-old primigravid female at 36 weeks of gesta-
3. Kussmaul's respiration. Patients will have typical
tion presented with nausea, vomiting, and malaise ov1:ir
acidotic breathing due to compensatory hyperventi-
the last several days. On examination she had high
lation.
blood pressure (190/110 mm Hg) and yellowish discol-
4. Smell of acetone in patient's breath .
oration of sclera. Laboratory results revealed protei 1-
5. Osmotic diuresis induced by ketonuria may lead
uria, impaired liver function tests, prolonged clotting
to dehydration.
time, hyperbilirubinemia , hypoglycemia and hypofibrino-
6. Sodium loss. The ketone bodies are excreted in
genemia. The condition was diagnosed as acute fa1ty
urine as their sodium salt, leading to loss of cations
liver of pregnancy. An emergency cesarean section was
from the body.
done, but hypoglycemia worsened and the patient went
7. Dehydration. The sodium loss further aggravates
into coagulopathy, renal failure and hepatic coma. What
the dehydration.
is the cause of acute fatty liver of pregnancy? What is
8. Coma. Dehydration and acidosis are contributing
the cause for hypoglycemia?
for the lethal effect of ketosis.
4. Defective absorption of lipids occurs in celiac dis- 16. Acetoacetate is the primary ketone body. Beta
ease, Crohn's disease. hydroxy butyric acid and acetone are secondary
5. Mammalian tissues oxidize fatty acids primarily ketone bodies.
by the beta-oxidation pathway which occurs in the 17. Ketosis is seen in diabetes mellitus and starvation.
mitochondria. 18. Rothera's test is commonly used to detect pres-
6. Transport of fatty acids {long chain acyl-CoA) ence of ketone bodies in urine.
through the inner mitochondrial membrane is facili- 19. Acetyl-CoA formed from fatty acids is further oxi-
tated by carnitine acyl transferase and translocase. dized in TCA cycle to generate energy, when avail-
7. Net yield of ATP from one molecule of palmitic acid ability of oxaloacetate is sufficient.
is 106ATP. 20. Under conditions of fasting and starvation , the oxa-
8. Oxidation of odd chain fatty acids produces propio- loacetate is channelled to gluconeogenesis. Excess
nyl-CoA, which may be further metabolized by the acetyl-CoA is then used for ketogenesis by liver.
TCAcycle. 21. A similar situation is seen in uncontrolled diabetes
9. Alpha oxidation and omega oxidation are two other mellitus where gluconeogenesis and lipolysis are
modes of fatty acid oxidation. both enhanced.
10. De novo synthesis of fatty acids occurs in the cyto- 22. The excess acetyl-CoA is converted to ketone
plasm with the help of a dimeric multi-enzyme com- bodies in hepatic mitochondria .
plex termed Fatty acid synthase. 23. The HMG-CoA formed is cleaved by liver enzyme
11 . Synthesis of fatty acid requires NADPH, while lyase to the primary ketone body acetoacetate.
degradation requires NAO and FAD. 24. Acetoacetate may be reduced to beta-hydroxy buty-
12. Insulin favors fatty acid synthesis. rate or spontaneously decarboxylated to acetone.
13. The white adipose tissue is concerned with energy 25. Ketone bodies are synthesized by liver and meta-
storage and the brown adipose tissue is concerned bolized by extrahepatic tissues, mainly cardiac
with thermogenesis. muscle and skeletal muscle.
14. Obesity is the result of an increase in the fat content 26. Under conditions of starvation, brain starts metabo-
of the adipose tissue. It is associated with insulin lizing ketone bodies for energy needs.
resistance. 27. Since ketone bodies are acids, metabolic acidosis
15. Fatty liver refers to deposition of excess triglyceri- occurs. Excessive accumulation of ketone bodies
des in the liver cells. It is facilitated by lipotropic can be dangerous since it can result in acidosis,
factors such as methionine, choline and lecithin . dehydration and coma.
13-16. In the biosynthesis of triacylglycerol from glyce- 13-28. All the em:ymes are required for de novo synthesis
rol phosphate, all the following are intermediates, of fatty acid, except:
except: A. Acetyl ..CoA carboxylase
A. Lysophosphatidic acid B. Glucose-6-phosphate dehydrogenase
B. Phosphatidic acid C. Thiolase
C. 1,2-diacylglycerol D. Malonyl transacylase
D. Monoacylglycerol 13-29. The action of caffeine include all the following, except
13-17. In adipose tissue, activity of hormone-sensitive A. Inhibition of phospho diesterase
lipase is increased by all the following hormones, B. Prolongation of action of cyclic AMP
except C. Increase in activity of hormone-sensitive lipase
A. Adrenalin B. Growth hormone D. Inhibition of synthesis of prostaglandins
C. Corticosteroids D. Insulin 13-30. Ketosis is often seen in:
13-18. Chain elongation of fatty acids takes place in which A. Nephritis
of the following subcellular fraction? B. Coronary artery disease
A. Cytoplasm B. Nucleus C. Muscle degeneration
C. Microsomes D. Golgi complex D. Diabetes mellitus
13-19. HMG-CoA is a precursor of all the following, except: 13-31 . The pathway which takes place in the mitochon-
A. Arachidonic acid B. Cholesterol dria. is:
C. Acetone D. Beta hydroxy butyrate A. KetogEmesis
13-20. HMG-CoA is directly converted to all the following, B. Glycolysis
except: C. HMP shunt pathway
A. Acetoacetyl-CoA B. Mevalonate D. Cholesterol synthesis
C. Acetoacetate D. Acetyl-CoA 13-32. Utilizationi of ketone bodies by peripheral tissues
13-21. Fatty liver may be prevented by the following, except needs:
A. Ethanol B. Choline A. NADPH dependent dehydrogenase
C. Methionine D. Lecithin B. Biotin dependent carbon dioxide fixation reaction
13-22. Ketone bodies are produced mainly in : C. Carbon dioxide elimination reaction
A. Brain B. Liver D. Succinyl-CoA dependent thiophorase reaction
C. Erythrocytes D. Skeletal muscles 13-33. All are trw! with regard to ketone bodies except
13-23. Ketosis is due to increased: A. Acetoacetate, acetone and betahydroxy butyrate
A. Hepatic glucose liberation B. Seen in urine in uncontrolled diabetes mellitus
B. Carbohydrate utilisation C. Mainly produced in adipose tissue
C. Gluconeogenesis D. Seen in urine during prolonged starvation
D. Fatty acid oxidation 13-34. Defects ini oxidation of fatty acids can produce all
13-24. Which of the following tissues are capable of oxi- except
dizing ketone bodies? A. Hypoglycemia B. Ketonemia
A. Liver B. Heart C. Myopa1thy D. Hyperammonemia
C. RBCs D. Plasma 13-35. Defective digestion and absorption of fats can lead
13-25. Ketone bodies in urine are identified by: to the foll,c,wing, except
A. Heat and acetic acid test A. Steatorrhoea
B. Rothera's test B. Hypocalcemia
C. Benzidine test C. Defective coagulation
D. Benedict's test D. Difficullty in dim light
13-26. Beta-oxidation of fatty acids yields all the follow- 13-36. Which of the following tissues cannot derive
ing, except energy trc,m fatty acids?
A. NADH B. Acetyl-CoA A. Brain B. Cardiac Muscle
C. FADH2 D. Malonyl-CoA C. Skeletal Muscle D. Erythrocytes
13-27. Regarding propionic acid metabolism 13-37. Wh ich of the following processes does not occur
A. Propionyl-CoA carboxylase is biotin dependent in adiposi! tissue?
B. Methyl-malonyl-CoA racemase is vitamin 812 A. Formation of triose phosphates
dependent B. Generation of NADPH
C. Succinyl-CoA is converted to propionyl-CoA C. Hydrolysis of Triacyl glycerol
D. Leucine is catabolized to produce propionyl-CoA D. Phosphorylation of glycerol
212 Section B: General Metabolism
13-38. The enzyme which is active only in the liver is 13-39. Fatty acidl synthesis differs from beta oxidation in
A. HMG-CoA reductase all, except
B. Carnitine acyl transferase A. Uses NAD PH as reducing power
C. Lecithin cholesterol acyl transferase B. Requires coenzyme A
C. Catalysed by multienzyme complex
D. HMG-CoA lyase
D. Activated by insulin
13-18. What is the net generation of ATP, when one mole- From dihydroxy acetone phosphate, derived from glu-
cule of palmitic acid (16 carbon) is oxidised com- cose.
pletely? 13-34. White adipose tissue is concernt;!d with what?
106. Energy storage.
13-19. What is the product of beta-oxidation of odd chain 13-35. Brown adlipose tissue is involved in what?
fatty acids? Thermoge1nesis.
Propionyl-CoA. 13-36. What enzyme is involved in lipolysis?
13-20. What is the metabolism of propionyl-CoA? By hormone-sensitive lipase.
Propionyl-CoA is first carboxylated to methyl malonyl-
13-37. What is its action?
CoA then to form succinyl-CoA. The succinyl-CoA then
It hydrolyses triglyceride into fatty-acid.
enters TCA cycle.
13-38. In blood, fatty acids are transported as what?
13-21. What are the coenzymes required for the conver-
Albumin is the carrier of free fatty-acid.
sion of propionyl-CoA to succinyl-CoA?
13-39. Which hormones activate hormone-sensitive lip-
Biotin, ATP, Vitamin B1 2.
13-22. Succinyl-CoA is generated from which substances? ase? or, what are the lipolytic hormones?
Odd chain fatty acids, Propionic acid, Valine, lsoleu- Growth hormone; Corticosteroids; ACTH; Adrenalin;
cine, Threonine Glucagon
13-23. Succinyl-CoA is utilised for what purposes? 13-40. In diabet«?s mellitus, non-esterified fatty acid level
Porphyrin biosynthesis; Activation of acetoacetate; in blood is increased; why?
Oxidation in TCA cycle. Insulin inhibits hormone-sensitive lipase; in diabetes,
13-24. Where is alpha oxidation taking place? this inhibition is removed, so, more lipolysis is taking
In endoplasmic reticulum (microsomes). place.
13-25. Refsum's disease is due to what? 13-41. What are the sources of acetyl-CoA?
Accumulation of phytanic acid , due to defective alpha
Pyruvate; fatty acids, acetoacetyl-CoA; Leucine.
oxidation.
13-42. What substances will prevent fatty liver?
13-26. What is the rate limiting enzyme of de novo syn-
Choline; Methionine; Lecithin.
thesis of fatty acid?
13-43. What are ketone bodies?
Acetyl-CoA carboxylase.
13-27. What is the reaction? Acetoacetate; Beta hydroxy butyric acid; and Acetone.
Acetyl-CoA + CO2 to produce Malonyl-CoA 13-44. Ketone bodies are formed in which tissue?
13-28. What are co-enzymes required for the reaction? Liver.
Biotin and ATP 13-45. KetogenEisis is taking place in which subcellular
13-29. How NADPH is made available? organelle1?
In the HMP shunt pathway, glucose-6-phosphate de- Mitochondria.
hydrogenase reaction produces NADPH. 13-46. What is the rate limiting step in ketone body forma-
13-30. Acetyl-CoA is used for what purposes? tion?
Fatty acid synthesis; Oxidation in citric acid cycle for
HMG-CoJ~ synthase.
generation of energy; Cholesterol synthesis and Ke-
13-47. Ketone body utilization is taking place in which
tone body formation
organs?
13-31 . How is fatty acid synthesis regulated?
Ketolysis is taking place in extra hepatic tissues. (All
Key enzyme, acetyl-CoA carboxylase, is stimulated by
Citrate and inhibited by palmitoyl-CoA. other tissues, except liver).
13-32. Which hormone enhances the synthesis of triacyl 13-48. Utilization of ketone bodies by peripheral tissue
glycerol? needs which enzyme?
Insulin. Succinyl-CoA dependent thiophorase.
13-33. In adipose tissue, what is the source of glycerol 13-49. What test is used to identify ketone bodies?
phosphate for triglyceride formation? Rothera's test.
_ _ _ _ _ Chapter 14
Cholesterol and
Lipoproteins
Chapter at a Glance
The learner will be able to answer questions on the following topics:
F' Struct ure of cholesterol Low density lipoproteins
,e. Biosynthesis of cho lesterol High density lipoproteins
Plasma lipids Free fatty acid
Chylomicrons Formation of bile acids and bile salts
Very low d~nsity lipoproteins
CoA
70-S-Co.A, yH20H
Tota/no. No. ofcarbon
Name ofsteroid
of carbon atoms inside
atoms chain Importance
fH2 rH3
HO- T-CH3 HO- T-CH3
Cholesterol 27 8 Most important
animal sterol CH2 2NADPH+2H• 2NADP CH2
Bile acids 24 5 Emulsifying agents I I
COOH COOH
Glucocorticoids 21 2 Influences HMG-CoA Mevalonate
and metabolism
Mineralocortioids as well as fluid and Fig.14.2: Step 3 of cholesterol synthesis
electrolyte balance
Testosterone 19 Male sex hormones
Estrogens 18 Female sex hormones
Step 1 : Conidensation
The acetyl-CoA is provided by the ATP-citrate lyase reac-
tion as in the ca:se of fatty acid synthesis. Two molecules
of acetyl-CoA condense to form aoetoacetyl-CoA cata-
lyzed by cytoplasmic acetoacetyl-CoA synthase (see
Chapter 13, fatty acid synthesis).
, .:--_ ...._,
'(-.; ~le~\ \ A.cm- c.'r-.o'lu \~~\ ~ -\: e.Q
216 Section B: General Metabolism ooi.. ~ , ~~\-, \ c\r\o\atero\
0 0
II II Step 1
H2C- O- P- O- P- OH
Acetoacetyl-CoA synthase
1 I I
CH2 OH OH Acetyl CoA + Acetyl CoA "'-: Acetoacetyl-CoA
I \ + Acetyl-CoA
~c ep 2
H2C "cH3 CoA
MG-CoA synthase
t:
Fig. 14.3: lsopentenyl pyrophosphate; 5 carbon unit CoA
Beta hydroxy beta methyl glutaryl-CoA (HMG-CoA)
'i
lsopentenyl pyrophosphate (5 Carbons)
B. Then the double bond migrates from 8-9 position
to 5-6 position, when desmosterol is formed. Des-
mosterol is present in fetal brain. It is absent in adult
Step 5-A; lsomerase
l
Dimethyl allyl pyrophosphate (5 C)
brain and reappears in gliomas (brain tumor). ~'Y( . + Dimethyl allyl
Step 5-B, Transferase l pyrophosphate
C. Finally, the double bond in the side chain (between Geranyl pyrophosphate (10 C)
carbon 24-25) is reduced by NADPH when choles- -+< lsopentenyl
terol is formed. A summary of the whole pathway of Step 5-C ; Transferase pyrophosphate
PPi
cholesterol synthesis is given in Figure 14.4.
Farnesyl pyrophosphate (15 C)
NADPH J+ Farnesyl
Regulation of Cholesterol Synthesis
&
l
NADPH
is HMG-CoA reductase. Long-term regulation involves Step 6-A: Epoxidase
regulation of transcription of the gene for HMG-CoA 02
Squalene epoxide
reductase. When sufficient cholesterol is present in the
cell, transcription of the gene for HMG-CoA reductase
is suppressed, and cellular synthesis of cholesterol is
1 Step 6-B: Cyclase
Lanosterol (30 C)
decreased. When c_!1olesterol jn djet i ~ , synthesis is
J.ocreased (Fig. 14.5). Cholesterol regulates the expres-
1 Step 7-A
Zymosterol (27 C) d.
sion of tiMG-CoA reductase gene and LDLR (LDL
r~ ce toI) g ~ --- l step7-B /
Translation
-~ lucago~ f'.) -
!
Enzyme ~ Insulin
ort,sone HMG-CoA Thyroxin
·statins· reductase
' ->.! ic~ r)
HMG-CoA reductase - - - - - . Cholesterol
Fig. 14.5 : Regulation of cholesterol synthesis
60%
90%
2% -r·a :• · -...::
'11111111111111 , 20%
3%
Chylomlcron (CM) Very low-density lipoprotein (VLDL) Low-density lipoprotein (LDL) High-density
llpoprotein (HDL}
D Triacylglycerol Protein Ill Phospholipids D Cholesterol
, -- ~ ~ ~ ~:;::== =~AAip~o~-si-:44EB~
- =;;;n~lo't'~o~)-_:::
J __ produce the 8-48 protein (Fig. 14.11). 8-48 is so
0-,-e. ro, r-.o r amed because it is only 40% of the size of 8 -100.
74. Apo-C-11. ActivatesJipoprot!'tl!:! lipase')
t 5. Apo-E. It is present in chylom icrons, LDL and
\ VLDL. Apo-E has I, 11, Il l and IV isoforms. A po-E-IV
\."ii. isoform is implicated in the development of senile
~ , ..
Cholesterol
Phospholipid outer cover dementia and Alzheimer's disease.
Phospholipld
B-48e
Triglyceride
Cholesterol
c-u•
B-48e
l e A
B-48e e A Fig. 14.1 1: Low density lipoprotein (LDL)
,')..' Adipose c-11• E
Nascent chylo tissue
Chylo remnantl
onlyTG Mainly Ch (YERY LOW DENSITY LIPOPROTEINS
Synthesis of VLDL
Triacylglycerol synthesized in liver is incorporated into
Apo-E-receptor VLDL along with hepatic cholesterol. Apo-B-100 is the
major lipoprotein present in VLDL when it is secreted.
, /LDLwith
Clathrin- ~ ¥" cholesterol
coated pits ~ - B- 100
Cytosol
1
~! o
Recycling
Liver - VLDL- LDL
Coated
vesicle
@
HDL - -----
Lysosome
-
The endosome vesicle thus formed fuses with lyso-
somes. The LDL particle, along with apoproteins and arterial walls, and are taken up by macrophages. This
cholesterol ester are hydrolyzed to form fr~olesterol. is the starting event of a heroscierosi leading to myo-
-"-- ___,,A.
The free receptors can now return to the membrane cardial infarction (see coronary artery diseases in Chap-
surface to bind further LDL molecules (Fig. 14.12). For ter 15). When macrophages are filled with cholesterol,
their work on LDL receptors, Michael Brown and Joseph foam cells are formed. They get deposited in the sub-
Goldstein were awarded Nobel Prize in 1985. endothelial space and leads to the formation of athe.!9-
The free cholesterol is either incorporated into plasma matous plague (see Fig. 15.3). Th~ results in increased
membranes or esterified (by ACAT) and stored within chances of thro<,Wbosis and corb~ary artery disease
the cell. The excess cholesterol tends to be deposited (see Fig. 15.4).
within the arteries, leading to atherosclerosis. Since LDL-cholesterol is thus deposited in tissues, the
Function of LDL
LDL (low density lipoprotein) variety is calle~~
->-
Ji~
terol" in common parlance (Fig.14.13 and Box 14.2).
About 75% of the plasma cholesterol is incorporated into lns.ulio...aru:1Jri-iodothyronine (T3} increase the bind-
-4= --- --
the LDL particles. LDL transports cholesterol from liver ing of LDL to liver cells. This explains the ~ er ~s-
to the peripheral tissues. The cholesterol thus liber- tec.oJecmaseBriTrLaiatfetes an oth ro1d1sl'n.
ated in the cell has three major fates: Defects in LDL receptor synthesis leads to fami-
i. It is used for the synthesis of other steroids like lial hypercholesterolemia, which is further described in
steroid hormones. Chapter 15.
222 Section B: General Metabolism
Lp(a) has homology BOX 14.3: Lp(a) and apo-A are different
with plasminogen
Apo-A is a constituent of HDL. This"A" is always written in capital
l
Plasminogen
Lp(a) apo-B-100 letters. It is seen in all persons. It Is antiatherogenic.
l
Activation to plasmin Lp(a) binds to fibrin
Lp(a) is seen in high levels only in some persons. When present,
it is associated with LDL This •a• is always written in small let-
!
and Inhibits plasmi- ters. It is highly atherogenic and connected with heart attacks in
nogen activation younger age group.
Liver Intestine
PL, A1, C, E
Pl.
Discoid
l Heart and
blood vessels
c;,i Nascent HDL
! LCAT Ch
PL
Excretion of A1 cE
Ch+ ChA .,S~ .~ Mature HDL
PL= phospholipid, HDL = high densitr,lp~ro\~~~ cholesterol, ChE = cholesterol ester.
ChA = cholic acid, LCAT = lecithin cholesterol acyl transferase
Fig. 14.15: HDL metabolism
I•••
Cholesterol Cholesterol ester
Fig. 14.16: Lecithin cholesterol acyl transferase (LCAT)
Chyle
•
HDL 11
ee •;
remnants
coronary artery diseases. A summary of the lipoprotein
•••
1
Blood
metabolism is shown in Figure 14.17.
l •
Chylomicrons vessel
HDL-specific Receptors
\
Blood
In liver, specific recepto , R-8 bjnds apo-A-1 present vessel
o..Q..!:!Q..L. Then~ les erol is internali .
1'-Prnpkmate
rl•I
Chenodeoxycholic acid (24C)
HS- CoA
Chenodeoxycholyl-CoA
V Taurine
~ CoA
Glycocholic acid Taurochenodeoxycholic acid
alpha-oriented (projecting below the plane of ring) Fig. 14.19: Formation of bile salts
hydroxyl group at position 7. The r~ctioos foe synth96is
of bile acjos are summarized below:
1. Cholesterol hydroxylated at ¥!.f.12 QOS~ S
2. Removal of 3-carbon unit, to make it 24 C
3. Conjugation with glycine
4. Secretion into intestinal canal
5. In the intestine, deconjugation and removal of Adolph Adolf Otto Heinrich Otto
hydroxyl groups. Strecker Windaus Wieland
1822- 1871
* Hydroxylation Reactions
NP 1928 NP 1927
1876-1959 1877-1957
14-1. Enumerate the major steps of synthesis of cholesterol. Name the rate-limiting step of cholesterol synth esis.
Describe the role of cholesterol in atherosclerosis.
14-2. Classify lipoproteins. Explain their biological significance.
14-3. What is the normal cholesterol level in plasma? Explain how the cholesterol is transported from liver to periphe-
ral tissues and back.
14-4. How are dietary triglycerides absorbed and transported in plasma? Explain briefly the transport of dietary TAG
from intestine to liver.
14-5. How are endogenously produced triglycerides transported in plasma? Explain the transport of them from liver.
Indicate the abnormalities associated with this process.
14-6. Biologically important compounds derived from 14-10. Give the normal blood level of cholesterol.
cholesterol. 14-11. Apo-B-100.
14-7. HMG-CoA reductase. 14-12. Chylomicrons.
14-8. Regulation of cholesterol synthesis. 14-13. Lipoprotein lipase.
14-9. Key enzymes of cholesterol biosynthesis.
226 Section B: General Metabolism
14-1. All are useful substances produced from choleste- C. Cholesterol from peripheral tissues to liver
rol, except D. Free fatty acids (NEFA) from adipose tissue
A. Vitamin D B. Bile salts 14-12. Function of LDL Is the transport of:
C. Bile pigments D. Cortisol A. Triglycerides from intestine to adipose tissue
14-2. The ring system of cholesterol is called: B. Cholesterol from liver to peripheral tissues
A. Cholanthrene ring C. Cholesterol from peripheral tissues to liver
B. Cyclopentano phenanthrene D. Free fatty acids (NEFA) from adipose tissue
C. Naphthoquinone 14-13. The cyclopentano phenanthrene ring structure is
D. Corrin ring seen in all the following compounds, except
14-3. During cholesterol blosynthesis, the first sterol A. Testosterone B. Adrenocorticotropin
ring synthesizes is: C. Glycocholic acid D. Vitamin D
A. Squalene B. Zymosterol 14-14. All are intermediates in the biosynthesis of choles-
C. Lanosterol D. Desmosterol terol, except
14-4. The key enzyme In the pathway of cholesterol blo- A. Acetyl-CoA B. Cholyl-CoA
synthesis is: C. Acetoacetyl-CoA D. HMG-CoA
14-15. All are true with respect to plasma lipoproteins, except
A. HMG-CoA synthase
B. HMG-CoA lyase A. Lipoprotein lipase is activated by apo-C-11
C. HMG-CoA reductase B. LCAT is activated by Apo-A-I
C. Apo-E is involved in receptor mediated uptake of
D. Mevalonate kinase
chylomicron remnants
14-5. Cholesterol contains how many carbon atoms?
D. Apo-A-I is atherogenic
A. 6 B. 12
14-16. Select the correct statement regarding HDL:
C. 27 D. 30
A. Normal HDL cholesterol in serum is 150- 250 mg%
14-6. The serum lipoprotein with the highest cholesterol
B. It contains apoprotein 8-48
content Is:
C. It is associated with enzymatic conversion of free
A. Alpha lipoprotein (HDL)
cholesterol to cholesterol ester
B. Beta lipoprotein (LDL)
D. Mature HDL has discoid shape
C. Prebeta lipoprotein (VLDL)
14-17. Function of albumin is the transport of:
D. Chylomicrons
A . Triglycerides from intestine to adipose tissue
14-7. The main apoprotein present in the low density
B. Cholesterol from liver to peripheral tissues
lipoprotein (LDL) is:
C. Cholesterol from peripheral tissues to liver
A. A and D B. B-1 00
D. Free fatty acids (NEFA) from adipose tissue to
C. E2 and C D. B-48
peripheral tissues
14-8. The main apoprotein present in chylomicron:
14-18. All are true in familial hyperlipoproteinemia Type
A. D B. B100 II-A, except
C. E2 D. B48 A. TAG level in blood is increased
14-9. The main apoproteln present In HDL Is: B. LDL level in blood is increased
A. A1 B. B-100 C. LDL receptor defect is seen
C. E2 and C D. B-48 D. Cholesterol level in blood is increased
14-10. Function of chylomicron is the transport of: 14-19. The plasma sample of a 35 year old man after over-
A. Triglycerides from intestine to adipose tissue night refrigeration showed a creamy layer on top
B. Cholesterol from liver to peripheral tissues and opalescence below. The condition which is
C. Cholesterol from peripheral tissues to liver excluded is
D. Free fatty acids (NEFA) from adipose tissue A. Diabetes mellitus
14-11. Function of HDL is the transport of B. Alcoholism
A. Triglycerides from intestine to adipose tissue C. Hypothyroidism
B. Cholesterol from liver to peripheral tissues D. Familial hypercholesterolemia
14-1. What is the ring structure present in cholesterol? 14-16. What is lipoprotein(a) or Lp(a)?
Perhydro cyclopentano phenanthrene ring. It has homology with plasminogen. So it interferes with
14-2. What are substances derived from cholesterol? plasminogen activation and impairs fibrinolysis. This
Glucocorticoids, mineralocorticoids, testosterone, estro- leads to unopposed intravascular thrombosis and pos-
gen, bile acids. sible myocardial infarction.
14-3. What is the rate limiting step in the cholesterol bio- 14-17. What is the significance of Lp(a)?
synthesis? Lp(a) is associated with heart attacks at the age of 30
HMG-CoA reductase. or 40 years.
14-4. How cholesterol is excreted? 14-18. What is the function of HDL?
Through bile, partly as cholesterol itself, and partly as Transport of cholesterol from peripheral tissues to liver.
bile salts. 14-19. What is "good cholesterol"?
14-5. What is the normal level of total cholesterol? HDL cholesterol.
140-200 mg/dl. 14-20. Why is it called so?
14-6. Maximum cholesterol content is in which lipo- HDL transports cholesterol from peripheral tissues to
protein? liver, and so helps in excretion of cholesterol from the
LDL (Low density lipoprotein). body. So HDL is antiatherogenic.
14-7. What is the function of chylomicron? 14-21 . What is the main apoprotein present in HDL?
Transport of triglycerides from intestine to adipose Apo-A-I; it is the ligand for HDL receptor.
tissue. 14-22. What is LCAT?
14-8. What is the main apoprotein present in chylo- Lecithin cholesterol acyl transferase.
micron? 14-23. Where is it present?
Apoprotein, B-48. LCAT present in plasma is activated by apo-A-I, when
14-9. Highest content of triglycerides is seen in which LCAT binds to HDL disk.
lipoprotein? 14-24. What is its importance?
Chylomicrons. The free cholesterol is esterified by LCAT; the esteri-
14-10. Which carries endogenous triglycerides? fied cholesterol is then incorporated into HDL. So for
VLDL (Very low densinty lipoprotein). excretion of cholesterol, LCAT is necessary.
14-11. What is the main apoprotein present in LDL? 14-25. What is the importance of PUFA in cholesterol
B-100; it is the ligand for LDL receptor. metabolism?
14-12. How LDL is taken up by the cells? PUFA present in lecithin is transferred to cholesterol
When the apo-B-100 binds to the receptor, the recep- by the enzyme LCAT. The esterified cholesterol is then
tor-LDL complex is internalised by endocytosis. taken by HDL, and finally excreted through liver. So,
14-13. What is the function of LDL? for excretion of cholesterol PUFA is required. Thus,
Transport of cholesterol from liver to peripheral tis- PUFA will lower the blood level of cholesterol.
sues. 14-26. Free fatty acids of plasma are bound to what?
14-14. What is "bad cholesterol"? Bound to serum albumin.
LDL cholesterol. 14-27. Triglycerides present in adipose tissue are hydro-
14-15. Why is it called so? lyzed by what enzyme?
LDL transports cholesterol from liver to peripheral Hormone sensitive lipase.
tissues, where it is deposited, and causes athero- 14-28. Bile acids are derived from what substance?
sclerosis. From cholesterol.
_ _ _ __Chapter 15
Hyperlipidemias and
Cardiovascular Diseases
Chapter at a Glance
The learner wi ll be able t o answer questions on the following topics:
0 Hyperlipidemias O Prevention of Atherosclerosis
0 Atherosclerosis and Coronary Artery Disease O Hypolipoproteinemias
\
0 Risk Factors for Coronary Artery Disease
I CLINICAL SIGNIFICANCE
OF CHOLESTEROL
coronary artery disease (CAD). It is estimated that by
the year 2020, it will account for 33% of all deaths.
Students should be familiar with cholesterol and lipo- Atherosclerosis and LDL \flc.e..
proteins described in detail in Chapter 14. A summary Stage I: Formation of Foam Cells
of lipoproteins is given in Table 15.1 and their metabolic
relati nships are shown in_Eigure---4 5:1 . is said to Increased levels of cholesterol for prolonged periods
be "b~ tmles erol an HD is ,,._"goo cholesterol (Fig. will favor deposits in the ~ ntimal reg.ion of a@ries.
15.2). High cholesterol leve s are associated with athero- Aorta, coronary arteries and cerebral vessels are pre-
sclerosis. Abnormality of cholesterol metabolism may dominantly affected by the atherosclerotic process. The
lead to cardiovascular accidents and heart attacks. LDL cholesterol, especially oxidized LDL particles are
deposited in the walls of arteries. Plasma LDL is mainly
catab · d via apo-B-LDL receptor pathway. But a small
p~a~rt~O!!.JJ~Rfil!~~~~l!a!l~.Clt.-~~~~~ake
Greek word, scleros· b:t.., macrapbaqFjls. Fr idative
obstructjon and m')(aimdia.Liofa[;~ damage of LDL will accelerate this process. Later, the
macrophages become overloaded with cholesterol, and
Intestine Liver
l•••Chylomicron
remnants
••• Blood e •
••
t
LDL
HDL lt • Liver -
"Good"
VLDL- LDL
l
cholesterol
C hylomicrons vessel • • • {Excretion)
\ HDL .,__- - - - -
,,
Blood
vessel
Heart
2.
Cigarette smoking
High total cholesterol I RISK FACTORS FOR
ATHEROSCLEROSIS
@Y
--
3. High LDL cholesterol Risk factors for atherosclerosis and future myocardial
4. Low HDL cholesterol
infarction (Ml) am shown Box 15.3. Out of these, the total
5. High fat/cholesterol diet
cholesterol, HDL. and LDL levels are the most impor-
Jr.' Left ventricular hypertrophy (LVH)
..;r. Thrombogenic factors , tant indices (Box 15.4, item A). Cardiac markers, which
Class 2: Modifiable risk factors; Interventions are likely to indicate the presence of acute myocardial infarction
lower CAD risk are listed in Box 15.4, item B.
....a. Upoprotein (a) or Lp(a)
$. Diabetes mellitus
.,ffi. Hypertension
Serum Choliesterol Level
-.)1 . Physical inactivity
Framingham epidemiological study demonstrated that
_.)-2. Obesity
increase in serum cholesterol level is associated with
13. High triglycerides C) , as c..reasecLcislWlL..deatb-fr om-GH . For every 10%
14. High homocysteine ./1 C. -
15. Increased high-sensitivity-CRP (hs-CRP) 'yn lowering of cholester ( c!HD mortality).,as reduced by
J8'. Stress 13%_.,•~-in healthy persons, cholesterol level varies from
Class 3: Nonmodlflable risk factors 150 to 200 mg/dl (Table 15.2). If other risk factors are
17. Age pre e o st,~rol level should b~. t eferably
18. Male gender b,l_o w 180 ..._ml)/~µ. Values a~i'.? 22ij m will have
19. Family history of CAD m o ~ n d valu 40 m dl will need
active treatmen~;
sterol which affoirds protection against atherosclerosis.
~ LASMA LIPID PROFILE Plasma cholesterol levels would tend to slowly rise after
The sample of serum should be taken after 12 hours of the 4th decade of life in men and postmenopausal women.
fasting . A complete lipid profile is assessed by estimat-
1. Total cholesterol
2. HDL-cholesterol
t
ing the following parameters in plasma/serum.
(- ,,.,\-n CX-- ~
1·r ·· -, -
Ir,, tz1"4-.\
LDL-Choles1:erol Level
National Cholesterol Education Program (NC~ P) identi-
fied elevated LDL--C as a primary risk factorforCH D. Blood
3. LDL-cholesterol levels less than ·130 mg/dl are desirable (Table 15.2).
4. Triglycerides } <. Levels between 130 and 159 are borderline; while above
5. Apo-B level 1 160 mg/dl carry definite risk. Hence LDL is "bad" chole-
6. Apo-A-I level f 6c:;...(;.va..... 'o~ sterol (Fig. 15.2). Ox~ edD)Uinitiates fatty streaks,
7. Lp(a) level ' K.i.H .. dJ.~c:_ \"\f ·· which is the starting p~
-
eroma formation.
---::::::....;.
*' ®\ ~gU<P
1 V.
•,r0j,c.o..~'vP\...., A c..U.'ce.. :e. "6'ful·
Chapter 15: Hyper/ipidemias and Cardio ascular Oise ses 231
accidents.
Serum Triglyceride
Lp(a) Normal level is 50-150 mg/dl. Blood level more than
Lp{~ h~ rinolysis. Levels more than 30 mg/dL 150 mg/dl is injurious to health. ( AOO- J.t·OO ':'5 /d.L
increase the risk 3 times; and when increased Lp(a)
is a~ with jncre.asecU.DL, the risk is increased
Diabetes Mellitus J"i ~g') bcm:la-U~)
6 tim s. (S~ Lp(a) in Ch.apter 14). Nicotinic acid will Cardiovascular disease is respon~ for 80% of total
reauce serum Lp(a) level. mortality in diabetes. It is associated with an increase
wrct& ~¥;. 1M, ~ -
232 Section B: General Metabolism li~d.,y ,;.;g --'> A~\. t,O~ Cho\-. ~ o}h
in LDL, high TAG and low HDL levels. In the absence BOX 1S.6 : Treatment policy in high risk patients. Targets to be
,.of insulin, hormone-sensitive lipase is activated, more achieved
ee fatt ac· are formed, which are catabolized to 1. Reduce total cholesterol below 180 mg/ dl
produce ce - o These cannot be read.il,! utilized, as 2. Decrease LDL-cholesterol below 130 mg/ dl; In persons with
.£Pi. the a~ailability of oxaloacetate isceducect and citric acid documented CHD, the goal of therapy is to reduce LDL to
1
from the cells. reg_l:!lc!~ xercise, balarfced die es~ ion of smoking,
Smoking __., )',vl\tJ.n. m;int~ ~ roper weight, cdhlr:o1 o.Lh.yQfilte.o_sion,
di ~ ~ dysliQjg,emia. The aim is to reduce total
Cigarette smoking is the most important modifiable risk cholesterol below 180 mg/dl ; to decrease LDL-cho-
factor for CAD Box 15.3). Risk from smoking is dose- lesterol below 130 mg/di and to keep HDL-cholesterol
dependant; ~e on t a~at which the person above 35 mg/dl (Box 15.6).
started smoking an umber4 of cigarettes smoked
Reduce Dietary Cholesterol
--
per day. Smoking enhances oxidation of LDL, reduces
HDL, increases CRP and augments a regation and
Cholesterol in the diet should be kept less than 200 mg
per day. ~ s and ~ at contain high cholesterol. One
egg yolk contains about 500 mg of cholesterol (Fig.
15.5A). One double omelet increases the blood choles-
terol § m~ th.~n the original level.
Hypertension f'M=--
Vegetable Oils a~ PUFA B
Systolic blood pressure more than 160 further increa-
ses the risk of CAD. An-.kl~asaJo 10 mm oL will Vegetable oils (e.g. stJOfiower oil) and fi ails contain
rfil!LJ.C..e l.ife~ oectanc'l by 10. y~ s. n~ ea.s.e..oc1f-5 mm Hg polyunsaturated fatty acids (PUFA). They are required
of diastolic pressure is associated with 34% increase in for the esterification and final excretion of cholesterol.
stroke risk. - ' -;r...<t:. So PUFA is helpful to reduce cholesterol level in blood
(Fig. 15.5B)~ mega-3 fatty acid;;from fish oils reduce
Obesity and Sedentary LDL and decrease the risk of CAD. i""tc_;_:--:,,"Tj-r":·r- ded
The classical description of Pickwick (in Pickwick papers) intake of omega-3 fa acid fish oils) is PA
by Charles Dickens reminds of a person with high risk and DHA combined). Clinical studie ave suggested
for CAD. People with "apple type" of obesity or truncal tha~ct.,ocosahexenoic aci ) P
obesity are more prone to get myocardial infarction. Aper- enoi c iicici'ffower · · , w e ii
son is obese when BMI exceeds 27.8 kg/m 2 in men and r~ u e s ; as well as reduce the risk..Qf.h_eart
-,.. - l '~ attack.
27.3 kg/m2 in.. women. ~ ~ifUauses gl~ e ilJ!2Ie-
rance, insulin-~ sistance, ~ pertJ-rlsion and d'/Sfioidemia.
Adipose tissue releases a large number of bioactive Moderation in Fat Intake
mediators that influence insulin resistance leading to The accepted standard is t h a t ~ oUotal calwies
endothelial dysfunction and atherosclerosis. A summary may be obtained from fat, out of which about one-third
of adipose tissue function is given in Chapter 35.
;i.01- --,
c
from saturate-a, another one-third from monounsaturated
v3, _., ~
Y; --
'b•
\..._., v 3 - Pv P~
Chapter 15: Hyperlipicfemias and Cardiovascular Diseases 233
Figs. 15.5A and B: (A) Reduce dietary cholesterol by avoiding Fig. 15.6: Green leafy vege- Fig. 15.7: Avoid cigarettes
egg omelet; (B) Sunflower oil and other vegetable oils contain tables are very 901Jd for heart.
PUFA
of oils and about_J-3 g_gf PUFA per day for a normal Plant derived fiber: Reduces serum cholesterol
adult. - '5 1. no..p:rn Legumes: Reduces cholesterol even on high fat diet
'
Onion and garlic: Beduces serum cholesterol and triacylglycerol
Embelia Ribes (Vidanga): Dried berries alone or along with amla
Green Leafy Vegetables has hypolipidemiic effect _,
Due to their high fiber content, leafy vegetables will Cammiphora Muku/ (Guggulu): Hypolipidemic and cardiopro-
tective
increase the motility of bowels and reduce reabsorption
Cyperus Rotundus (Musto): Hypolipidemic; improves metabolic
of bile salts Fi . 1~ tables also contain plant activity
sterols @tostero which decrease the absorption of Spices, flavanoids, red wine: Natural antioxidants prevent oxida-
cholesterol. A out 400 B'day of fruit and vegetables are tive modification of LDL
desirable. R.<;.
( Avoid Sucrose and Cigarette cholesterol will be converted to bile acids, thus
reducing the cholesterol level.
Cigarett~ oking Fig. 15.7) is t s1ngle most im iv. is widely used to ~ ')t!Gs,Jo.uDa-
tant modifiable risk factor for CAD (Box 15. c os tion, because of its anti-platelet activity (see Chap-
will rais£ plasma TAGs. High ca ohydrate diet, e'sr/e- ter 16).
cially sucrose, should be avoided by patients with 1
v. Anti-oxidanits such as • vitamin E · wilLroini.rn.Lz.e
hypercholesterolemia. oxi~ ~ and so, atheroscl;rosis may be
reduced.
Exercise vi. Plant deriv,ad·products having cholesterol-lowering
Regular moderate ex~~ @ min per day) will lower action are enumerated in Box 15. 7. The guggul
LDL (b~ lesterol~ raise Hpl (gqg(cholesterol) (resins) from the Mukul myrrh tree (Commiphora
levels in blood. It will also reduce obesity. Individuals Mukul) has cholesterol lowering action.
spending more than 2000 kcal/week in exercise are at
a lower risk. Avoid Trans Fatty Acids (TFA) ·
H • •d • D Trans fatty acids (with double bonds having trans con-
y po11P1; emic rugs \tt,j7 J figuration) are formed during the partial hydrogenation
i. HMG ~ A re9ucta~hibitors ("statins"): Ator- of vegetable oils. They are widely used in food industry
vastatin and ~ stalin are popular drugs in this because of their long shelf-life. Trans fatty acids (TFA)
group. They are effective in reducing the cholesterol are found to be, more atherogenic than saturated fattv
level and decreasing the incidence ?~AD. acids. It alters se& etion and composition of apo-8100
ii. Bi~ a}cid binding resins (Chol~fyramine and containing lipo~!f:Rteins (LD~ nd VLDL). It increases
Co · ipol) decrease the reabs.9.!B!ion of bile acids. catabolism of ~ -A-1, decr~ es HDL and increases
iii. Probuc I increases LDL catabolism and prevents u ® ev~ Reclucing the intake of TFA to 2- 7 g/day is
accumulatio ;of ~
. in arterial walls. So more now strongly advised .
( cm~o.6 , ome.~ o.. ,1
234 Section B: General Metabolism
-, • I I t .. It t t • I , creased)
Lipoprotein
fraction Cholesterol Appearance of
Type elevated level TAG/eve/ plasma after 24 hr Metabolic defect Features Management
Type I Chylomicrons N ii Creamy layer over Lipoprotein Eruptive Restriction of fat intake.
clear plasma lipase deficiency xanthoma; Supplementat ion w ith
hepatomegaly; medium chain triglycerides
Pain abdomen.
Type IIA LDL ii N Clear LDL Receptor Atherosclerosis, Low cholest erol diet.
defect; Apo-8 i coronary artery Decreased int ake o f
disease, Tuberous saturated fat. Give PUFA
xanthoma and drugs like statins.
Type 118 LDL andVLDL ii i Slightly cloudy Apo-8 t Corneal arcus Do
Apo-CII
Type Ill Broad beta- it i Cloudy Abnormal apo-E; Palmar xanthoma. Reduction of weight,
VLDL and Apo-CII"' High incidence of restrictio n of fat and chol.
Chylo microns vascular disease Give PUFA and drugs
Type IV VLDL t it Cloudy or m ilky Over-pro duction Associated with Reduction of body weight.
ofVLDL; Apo - diabetes, heart Restrict carbohydrate and
CII t disease, obesity. cholest erol
Type V VLDL N tt Creamy layer over Secondary t o Chronic High PUFA intake,
Chylomicrons milky plasma other causes pancreatitis hypocholipidemlc drug
TABLE 1s.s: Secondary hyperl1p1dem1as BOX 15.8: Clinical class1f1cat1on of hyperllp1derrnas (for treat-
Serum cholesterol Serum triglyceride ment purpose)
Diabetes Increased Increased Classification Type Salient features
Nephrotlc syndrome Increased Increased Hypercholesterolemia Type Ila Increased LDL
Hypothyroidism Increased Increased Hypertriglyceridemia Type I, Type IV and Increased VLDL
Biliary obstruction Increased Normal TypeV
Pregnancy Normal Increased Increased chylomicrons,
Alcoholism Normal Increased Combined hyperlipidemia Type lib and Type Ill Increased VLDL,
Oral contraceptives Normal Increased Increased IDL
!t
¥ Clinical Case Study 15.1
likely diagnosis? What biochemical shuttle may be active
to produce more adenosine triphosphate (ATP) per
glucose molecule?
A 48-year-old male presents to the clinic because of
concerns about heart disease.The patient reports chest 0
pain occasionally with ambulation around his house
• · Clinical Case Study 15.3
and is not able to climb stairs without significant chest A48-year-old male presented to OP with chest pain. Family
pain and shortness of breath. The physical examina- history shows that his father died of a heart attack at the
tion is normal, and the physician orders an electrocar- age of 46, and his elder brother also had a heart attack at
diogram (ECG), exercise stress test, and blood work. the same age. The patient reports that he gets chest pain
The patient's cholesterol result comes back as 350 mg/ occasionally with ambulation and is not able to climb stairs
dl (normal 200). The physician prescribes medication, without significant chest pain and shortness of breath. His
which he states is directed at the rate limiting step of plasma cholesterol level was 450 mg%. What is the pos-
cholesterol biosynthesis. sible diagnosis?
What is the rate-limiting step of cholesterol metabo-
lism? What is the class of medication prescribed? 0
• • Clinical Case Study 15.1 Answer
0
• · Clinical Case Study 15.2 Diagnosis: Hypercholesterolemia.
Rate-limiting step: The enzyme hydroxymethylglutaryl-
A 51-year-old male presents to the emergency center with
CoA reductase (HMG-CoA reductase) catalyzes an
chest pain. He states that he has had chest discomfort
early rate-limiting step in cholesterol biosynthesis.
or pressure intermittently over the last year especially
Likely medication: HMG-CoA reductase inhibitor,
with increased activity. He describes the chest pain as a
otherwise known as "statinn medications.
pressure behind his breastbone that spreads to the left
Clinical correlation: Hyperlipidemia is one of the most
side of his neck. Unlike previous episodes, he was lying
treatable risk factors of atherosclerotic vascular disease.
down, watching television. The chest pain lasted approxi-
In particular, the level of the low-density lipoprotein
mately 15 minutes then subsided on its own. He also noti- (LDL) correlates with the pathogenesis of atherosclero-
ced that he was nauseated and sweating during the pain sis. Exercise, dietary adjustments, and weight loss are
episode. He has no medical problems that he is aware the initial therapy of hyperlipidemia. If these are not suffi-
of and has not been to a physician for several years. On cient, then pharmacologic therapy is required. The exact
examination, he is in no acute distress with normal vital LDL targets depend on the patient's risk of cardiovascular
signs. His lungs were clear to auscultation bilaterally, and disease. For example, if an individual has had a car-
his heart had a regular rate and rhythm with no murmurs. diovascular event previously (heart attack or stroke),
An electrocardiogram (ECG) revealed ST segment eleva- the LDL target is 100 mg/dl ; 1 to 2 risk factors with-
tion and peaked T waves in leads II, Ill, and aVF. Serum out prior events = 130 mg/dl ; and no risk factors =
troponin I and T levels are elevated. What is the most 160 mg/dl.
Chapter 15: Hyperlipidemias and Cardiovascular Diseases 237
13. Vegetable oils supply mainly omega-6 fatty acids, 18. Tangier disease due defective efflux of cholesterol
where as fish is rich in atheroprotective omega-3 from cells occurs when ABC-A1 protein is deficient,
fatty acids. The ideal ratio of omega-6 to omega-3 with cholesterol ester accumulating in tissues, e.g.
in the diet is 4:1. Orange tonsil.
14. When diet control and exercise do not bring the 19. Frederickson's classification is based on the type
cholesterol to desired levels, use of hypolipidemic of lipoprotein elevated. Six types have been descri-
drugs may be considered. bed. Most common is Type IIA, which is due to defec-
15. "Statins" that inhibit HMG-CoA reductase is the tive LDL receptor, leading to decreased uptake of LDL
most common drug prescribed to lower cholesterol 20. For purposes of management, a clinical classifica-
levels. In addition, antiplatelet agents like aspirin tion to 3 typ,es is used, (a) hypercholesterolemia,
are also given. In persons with significant hypertri- (b) hypertrigilyceridemia and (c) combined hyper-
glyceridemia, fibrates may be given. lipidemia.
16. Abetalipoproteinemia is due to a deficiency of TAG 21. Hypertriglyceridemia is seen in Type I where lipo-
transfer protein which interferes with the incorpo- protein lipase activity is deficient. In type IV and
ration of apo-B-100 and apo-B-48 into VLDL and Type V diseases, the TAG pool is increased .
chylomicrons. 22. Combined hyperiipidemia is seen in Type IIB, where
17. Hypoalphalipoproteinemia is due to low HDL level apo-B levels are high. In Type Ill disease, the apo-E
with high risk of CAD. deficiency causes defective hepatic uptake of IDL.
15-1. HDL cholesterol is said to be "good" cholesterol, A. TAG level in blood is increased
because B. LDL level in blood is increased
A. HDL contains contains enzymes to break down C. LDL receptor defect is seen
cholesterol D. Cholesterol level in blood is increased
B. HDL carries cholesterol from liver to tissues where 15-4. Normal blc,od cholesterol level is:
it is broken down A. 40-60 rng/ 100 ml
C. HDL carries cholesterol from tissues to liver from B. 70-110 mg/100 ml
where cholesterol is excreted C. 120-150 mg/100 ml
D. HDL inhibits cholesterol synthesis D. 150-200 mg/100 ml
15-2. All the following statements are true with regard to 15-5. Hyperchol,esterolemia is seen in the following con-
type IIA hyperlipoproteinemia, except ditions, except:
A. Premature artherosclerosis A. DiabetEi s mellitus
B. Elevated plasma LDL cholesterol B. Thyrotoxicosis
C. Creamy layer on top of the serum C. Nephroitic syndrome
D. Xanthomata D. Obstructive jaundice
15-3. All are true in familial hyperlipoprotenemia Type IIA, 15-6. The characteristic finding in hypobeta-lipoprote-
except: inemia is:
Chapter 15: Hyperlipid'emias and Cardiovascular Diseases 239
15-1 . What are the salient features of hyperlipoproteine- 15-5. What are other risk factors associated with coro-
mia Type IIA? nary artery diseases?
Premature atherosclerosis; Elevated plasma LDL cho-
Cigarette smoking, hypertension, diabetes mellitus,
lesterol; Prominent beta band on electrophoresis.
serum trig1lyceride level above 200 mg/di; homocyst-
15-2. What is it due to?
Defect in LDL receptor. eine level; sedentary life style, obesity.
15-3. Hypercholesterolemia is seen in what conditions? 15-6. What advise you will give to a person with
Diabetes mellitus; Nephrotic syndrome; Obstructive increased! cholesterol level?
jaundice; Hypothyroidism. Reduce cholesterol content of food; include PUFA and
15-4. What are the important risk factors of coronary
omega-3 fatty acids in diet; reduction of total fat intake;
artery diseases?
increase gIreen leafy vegetables; exercise.
Serum cholesterol level above 200 mg/di; LDL-cho-
lesterol level above 160 mg/di; HDL-cholesterol level
below 35 mg/di; Lp(a) above 30 mg/di.
_ _ _ _ _Chapter 16
MCFA, PUFA, Prostaglandins
and Compound !Lipids
Chapter at a Glance
The learner will be able to answer questions on the following topics:
0 Medium chain fatty acids (MCFA) 0 Leukotrienes
0 Monounsaturated fatty acids (MUFA) 0 Very long chain fatty acids (VLCFA)
0 Polyunsaturated fatty acids (PUFA) 0 Glycerophosphatides
0 Saturation and desaturation of fatty acids 0 Phosphatidylcholine synthesis
0 Essential fatty acids 0 Sphingolipid .and sphingomyelin synthesis
0 Eicosanoids 0 Lipid storage diseases
0 Prostaglandins
Fatty acids having carbon atoms 4 to 6 are called small catalyzes the complete hydrolysis of SCT/MCT into
chain fatty acids (SCFA); those with 8 to 14 carbon atoms glycerol and small/medium chain fatty acids. These free
are known as medium chain fatty acids (MCFA); those MCFAs diffuse directly into portal circulation. SCFA
with 16 to 18 carbon atoms are long chain fatty acids and MCFA are preferentially oxidized by peripheral
(LCFA); and those carrying 20 or more carbon atoms cells, and so they are not deposited in adipose tissues.
are named as very long chain fatty acids (VLCFA) A comparison is uiven in Table 16.1 .
(see Table 8.3).
The SCFAs, butyric acid (4C) and caproic acid (6C) Very Long Chain Fatty Acids (VLCFA)
are seen in butter and ghee; the name butyric is derived
Fatty acids having 20 or more carbon atoms are called
from butter. MCFAs, capric acid (10C), lauric acid (12C)
very long chain fatty acids (VLCFA). Eicosapentaenoic
and myristic acid (14C) are present in coconut oil and
acid (EPA) (C-20, 5 double bonds) and docosahexaenoic
human milk. Normal skin has a very thin layer of sebum
acid (DHA) (C-2,!, 6 double bonds) are good examples
secretion with MCT (medium chain triglyceride) contain-
of VLCFA. DHA is synthesized in liver from linolenic acid
ing lauric acid, which prevents bacterial entry into the
(Omega-3, C-18, 3 double bond}. DHA is available in
body.
large quantities in fish oils. DHA is especially required for
the development of brain and retina. Low level of DHA
STION OF MEDIUM
in blood is seen in patients with retinitis pigmentosa.
N FATTY ACIDS- - - - In human beings, DHA accumulates in brain before
Digestion and metabolism of SCFAs and MCFAs are birth and for up to, 12 weeks afterwards. Outer segments
entirely different from those of LCFAs. Triglycerides con- of retinal rods contain high concentrations of DHA, which
taining small and medium chain fatty acids (SCT and MCT) gives high fluidity to the membranes. This is required
do not require prolonged digestion. Pancreatic lipase for the lateral anid rotational movement of rhodopsin
and bile salts are not required. MCT-specific lipase within the membrane during photoactivation.
Chapter 16: MCFA, PUFA, Prostaglandins and Compound Lipids 241
Examples Butyric acid (C4) in butter and !auric acid (Cl 2) in Palnnitic acid (C16) and stearic acid (C18) in
coconut oil veg,etable oils and animal fats
Di estion in stomach Hydrolyzed Not hydrolyzed
Pancreatic lipase Not necessary Esse:ntial
Bile salts Not necessary Abs,olutely essential
Inside intestinal cells TAG is hydro I ed to form fatty acids Free• fatty acids are re-esterified to form TAG
Absorbed Directly to blood To li1mphatics, then to thoracic duct
Absorbed as Free fatty acid carried by albumin TAG, carried by chylomicrons
Immediate fate Oxidized by peripheral cells Deposited in the adipose tissue
Carnitine Not required for oxidation Required for oxidation
Clinical application No effect on atherosclerosis Hypercholesterolemia and atherosclerosis
VLCFAs (20C and above) are partly oxidized in (1st step in Fig.13.9) is not needed. Thus in the case of
peroxisomes to smaller (18C) fatty acids, which then unsaturated fatty acids, the energy yield is less by 1.5
leave peroxisomes to enter mitochondria. This peroxi- ATP molecules per double bond.
somal oxidation differs from beta-oxidation in that the
electrons from FADH 2 (step 1 of beta-oxidation) are directly I POLYUNSATURATED
donated to oxygen to form hydrogen peroxide. So this
step does not produce ATP.
lEMJy ACIDS (PUFA}_ _
Deficient oxidation of VLCFA by peroxisomal The important poly unsaturated fatty acids are:
enzyme systems leads to adrenoleukodystrophy (see 1. Linoleic acid (18C, 2 double bonds)
Chapter 2), where VLCFAs are accumulated and myelin 2. Linolenic acid (18C, 3 double bonds)
sheaths are destroyed. It is an X-linked condition. The 3. Arachidonic acid (20C, 4 double bonds)
child usually dies during the first decade of life. They are pn:!sent in significant quantities in veget-
able oils such as sunflower oil (see Fig. 8.2 and Table 8.4 ).
I MONOUNSATURATED They are used to esterify cholesterol, whereby the latter
lEAI:rY
ACIDS (MUFA
can be excreted. So, PUFA in general are antiathero-
genic (see Chapters 14 and 15). Functions of PUFA are
Palmitoleic (C16, 1 double bond) and oleic (C18, 1 double shown in Box 16.1. Clinical manifestations of PUFA/ EFA
bond) acids are present in human body fat, as well as deficiency are shown in Box 16.2.
many vegetable oils. Erucic acid (C22, 1 double bond) is Fish oils contain PUFAs with 5 or 6 double bonds.
a constituent of mustard oil and rapeseed oil. Nervonic They are important for development of human brain.
acid (C24, 1 double bond) is present in substantial
quantities in brain. Lipid Peroxiidation
In vitro, peroxidation would lead to rancidity of fats and
Modified Beta-Oxidation of MUFA oils. In vivo the membrane lipids are more liable to attack
The oxidation of unsaturated fatty acids proceeds as in by free radicals ,and produce damage to the integrity of
the case of saturated fatty acids, till the double bond is the membrane (see Chapter 30). In naturally occurring
reached . Thus palmitoleic acid (16 C monounsaturated) lipids antioxidanits prevent lipid peroxidation. Vitamin E
undergoes 3 cycles of beta-oxidation to yield t.3-cis or tocopherol is an important antioxidant in the human
enoyl-CoA with 10 carbon atoms. Here the double body.
bond is cis type; the dehydrogenase cannot act on that
Elongation iof Fatty Acids
bond. Therefore, an isomerase changes the cis 63
double bond to 62-trans double bond. The double bond The Microsomal system (microsomal fatty acid
between 3rd and 4th carbon atoms is shifted to between elongase system) elongates saturated or unsaturated
2nd and 3rd carbon atoms. It will then undergo 2nd, 3rd fatty acyl-CoA by successive addition of two-carbon units.
and 4th step reactions of beta-oxidation (see Fig. 13.9). Malonyl-CoA is the donor of two carbon acetyl groups.
So in this cycle the FAD dependent dehydrogenation NADPH is required for the reaction. This system can
242 Section B: General Metabolism
BOX 16.1: S1gnif1c,mce of PUFA BOX 16.2: Clln1cal s1gnif1cance of PUFA and EFA
1. PUFAs are seen in vegetable oils. 1. Persons with normal diet w ill not have any deficiency; but
2. Linoleic and linolenic acids are nutritionally essential; and are t hose who are on parenteral nutrition for long periods will have
called Essential Fatty Acids (EFA). deficiency.
3. Prostaglandins, thromboxanes and leukotrlenes are produced 2. PUFAs are used for esterification and excretion of cholesterol.
from arachidonic acid. PUFA will reduce serum cholesterol level (see Chapter 15)
4. PUFAs form integral part of mitochondrial membranes. In defi- 3. Deficiency of EFA causes acanthocytosis, hyperkeratosis,
ciency of PUFA, the efficiency of biological oxidation is reduced. acrodermatitis and hypercholesterolemia.
5. They are components of membranes. Arachidonic acid is 4. EFA deficiency is connected with acrodermatitis enteropathica,
10-15% of the fatty acids of membranes. hepatorenal syndrome and CNS manifestations.
6 . As double bonds are in cis configuration; the PUFA molecules 5. Elevated PUFA levels are seen in Zellweger's syndrome.
cannot be closely packed. So PUFAs will increase the fluidity 6. DHA levels in blood are low in patients with retinitis pigmentosa.
of the membrane. 7. Trans fatty acids will compete with EFAs, and may increase the
7. As PUFAs are easily liable to undergo peroxidation, the EFA deficiency and decrease fluidity of membranes.
membranes containing PUFAs are more prone for damage by 8. Trans fatty acids decrease HDL-cholesterol and may cause
free radicals. atherosclerosis.
8. The production of DHA (docosahexaenoic acid) is synthesized
to a limited extent from alpha linolenic acid. DHA is present in
fish oils. DHA is present in high concentrations in retina, cerebral Essential Fatty Acids (EFA)
cortex and sperm s.
Normal dietary allowance of PUFA is 2-3% of total
calories. Linoleic acid and linolenic acid are the only
NADH + H+ NAO+
y-linolenoyl-CoA
fatty acids which cannot be synthesized in the body.
Linoleoyl-CoA
- (18C , A6, 9, 12) Their structures are shown in Figure 8.2. They have to be
(18C, 9 • 12 ) 6 6 desaturase (Linolenic)
(Linole,c) provided in the food; hence they are essential fatty acids.
02 2Hp Arachidonic acid can be formed, if the dietary supply of
t
NADPH NADP+ linoleic acid is sufficient. For deficiency manifestations
Homogamma- of EFA, (see Box 16.2). The relation of PUFAwith chole-
y-linolenoyl-CoA - linolenoyl-CoA
Elongase (20C , 6 8, 11, 14) sterol is described in Chapters 14 and 15.
H
___ __ ___ Malonyl-CoA ___ CO2 ________ _
(A)
,,c:,9
11
7
13
5 3
20
OH
John R Vane
NP 1982
1927-2004
Ulf von Euler
NP 1970
1905-1983
Bengt
Samue/sson
NP 1982
b. 1934
(B) h 20
OH OH
Most of the omega-6 PUFAs consumed in the diet
9
are from vegetable oils such as soybean oil, corn oil,
(6
J:::
etc. Linoleic acid is converted to arachidonic acid (see (C)
Fig. 16.1 ). When gamma-linolenic acid (GLA) is available 20
'12
in food, GLA is converted to arachidonic acid.
Omega-3 and omega-6 fatty acids are incorporated
into cell membranes. These membrane lipids serve Figs. 16.2A to C: (A) Prostanoic acid; (B) Prostanglandin-F2;
(C) Thromboxane A2
as precursors for the synthesis of important signaling
molecules involved in cell growth and inflammation.
The most important omega-6 PUFA is arachidonic awarded to Beni~t Samuelsson (biosynthesis of PGs)
acid. On stimulation of the cell, arachidonic acid is and Sir John Vane (effect of aspirin on PGs). PGs were
released from cell membranes through the action of phos- originally isolated from prostate tissue and hence the
pholipase A 2 (PLAJ The released arachidonate then name. But they are present in almost all tissues. They
serves as the precursor for the synthesis of the prosta- are the most potent biologically active substances;
glandins (PGs), thromboxanes (TXs), and leukotrienes. as low as one n!~/ml of PG will cause smooth muscle
(l Ts). These eicosanoids will cause platelet and leuko- contraction. The diverse physiological roles of prosta-
cyte activation , signaling of pain, induction of broncho- glandins confer on them the status of local hormones.
constriction, and regulation of gastric secretions. These
activities are targets of nonsteroidal anti-inflammatory Chemical Stiructure
drugs (NSAIDs), COX-2 inhibitors, and leukotriene All prostaglandins are considered to be derived from
antagonists.
the 20C cyclic saturated fatty acid, prostanoic acid (Fig.
Dietary omega-3 PUFAs compete with the pharma-
16.2A). The five carbon ring is saturated. All naturally
cological activities of omega-6 PUFAs because they
occurring PGs have an alpha-oriented OH group at
displace arachidonic acid from cell membranes. Increas-
C15.
ing dietary consumption of omega-3 PUFAs will reduce
the activity of leukocytes and platelets.
Classification of Prostaglandins
I EICOSANOIDS According to thei attachment of different substituent
groups to the rin!g, PGs are named with capital letters
They are 20C compounds (Greek, eikosi = twenty),
such as A, B, E and F (Table 16.2). PGF is designated as
derived from arachidonic acid . Their names are:
alpha to denote the projection of the OH group in natu-
1. Prostanoids, containing:
rally occurring prostaglandins.
a. Prostaglandins (PGs);
In the same series, depending on number of double
b. Prostacyclins (PGls);
bonds on the side chains they are denoted by a sub-
c. Thromboxanes (TXs)
script after the capital letter, e.g. PGE 1, PGE2 , PGE3 , etc.
2. Leukotrienes (LTs)
(Table 16.2).
l Aspirin H
PGD OH group at C9; keto group at Cl 1 Cyclo- ~
PGE Keto group at C9; OH group at Cl 1 oxygenase lndomethacin H
PGF OH groups at C9 and Cl 1 Prostaglandin G2
IPeroxidase
i
PGG Two oxygen atoms, interconnected to each other, and
bonded at C9 and Cl 1; hydroperoxide group at Cl 5 Prostacyclin Thromboxane
PGH Same ring as PGG; but C15 has OH group
syntha,se PG]; : y; hase T><Ai
PGI Double ring. Oxygen attached to C6 and C9, to form
another 5-membered ring. Hence called prostacyclin. o ,..a.. o'>-'
,-.Y(!I 't>;
7 PGD, -~
".;\ &,<
Reductase & '
Structure of PGF2 is shown in Figure 16.28. Throm-
boxanes have 6 membered oxane ring (Fig.16.2C).
PG\+--Pr --+ looci"'• PGs
Biosynthesis of Prostaglandins Adenyl cyclase
Prostaglandins are derived from the following PUFA. ATP - - - - - • Cyclic AMP - + Biological effects I I
1 series (1 double bond)-from Linoleic acid Fig. 16.3: Synthesis and action of prostaglandins
2 series (2 double bonds)-from Arachidonic acid
3 series (3 double bonds)-Eicosapentaenoic acid
BOX 16.3: Mi>chan1sm of c1ct1on of ilsp1r111
Naturally occurring PGs belong to the 2 series. Aspirin irreversibly acetylates and inhibits cyclo-oxygenase.
i. PGs are not stored as such; the precursor fatty Platelets cannot regenerate cyclo-oxygenase and so thromboxane A2
acids are present in membrane as phospholipids. is not formed in platelets. Hence, there is decreased platelet aggre-
gation. Therefore, aspirin is useful in prevention of heart attacks.
The arachidonic acid is released by the action of
By inhibiting cyclo-oxygenase, aspirin also reduces PGl2; but endo-
phospholipase A2 on phospholipids (Fig.16.3). thelial cells after a few hours will resynthesize cyclo-oxygenase. So
ii. Synthesis is catalyzed by prostaglandin H synthase aspirin completely blocks TXA2, but only partially inhibits PGl2. Other
(PGHS) . It contains two separate enzyme activities, anti-inflammatory drugs (indomethacin and ibuprofen) also cause
irreversible inhibition of enzyme. Paracetamol is a reversible Inhibitor.
cyclo-oxygenase and peroxidase.
iii. PGG 2 and PGH2 are formed as intermediates during
the synthesis of other PGs. Specific enzymes functions. The inducible form mediates the inflammatory
convert PGH2 to other prostaglandins (Fig. 16.3). response. Prostaglandins have only very short half-life,
of about 30 seconds.
Regulation of Synthesis
Mechanism of Action
The phospholipase (PL) is activated by epinephrine,
thrombin, angiotensin II, bradykinin and vasopressin. Prostaglandins are local hormones. They function
Steroids inhibit PL and prevent release of arachidonic through G-protein coupled receptors (see Chapter 45).
acid from membranes (Fig.16.3). Cyclo-oxygenase is In most tissues, PGE increases cAMP (cyclic AMP)
activated by catecholamines and inhibited by nonsteroid level. But in adipose tissue and in renal tubular cells,
anti-inflammatory drugs (NSAIDs). Aspirin acetylates PGE lowers cAMP level. PGI activates adenyl cyclase
serine at the active site and irreversibly inhibits the and TXA inhibits it (Fig.16.3).
cyclo-oxygenase (Box 16.3).
Cyclo-oxygenase is a "suicide" enzyme, self cataly- Biological Actions and
zed destruction rapidly inactivates the enzyme (see Clinical Applications
suicide inhibition in Chapter 5). This would prevent exces-
Effects on CVS
sive production of PGs. Cyclo-oxygenase exists in two
different forms. COX- 1, the constitutive form produces Prostacyclin or PGl 2 is synthesized by the vascular
prostaglandins, that mediate gastric, renal and platelet endothelium. Major effect is vasodilatation. It also
Chapter 16: MCFA, PUFA, Prostaglandins and Compound Lipids 245
Structure
Site of formation
PG/,
Cyclopentane ring
Endothelium
TXA?
Oxane ring
Platelets
1'.~r,....,......
Cyclic AMP level Increased Decreased 5-hydro-peroxy-eicosa-tetra-enoic acid (5 HPETE)
l
Platelet aggregation
Blood vessel
Inhibited
Vasodilatation
Enhanced
Constriction
l''"' · " ' ' " ' \ ~
- H 2O
LTA synthase
Peroxidase
HETE
l•
Bronchioles Relaxation Constriction + Glutathione
\,, L •
f:'. oleic acid
1-palmitoyl lysolecithin Phosphatidylcholine Elhanolamine
l
LLAT (1-palmitoyl, phosphotransferase
2-arachidonyl lecithin) 1, 2-diacylglycerol - - - - - - + Phosphatidylcholine
+ CDP-choline + CMP
1-palmitoyl lysolecithin ( \ • f"hosphatidylcholine
Ethanolamine
Arachidonyl- kinase
1
CoA HS-CoA Ethanolam ine + Phospho-ethanolamine + AD
1
1-palmitoyl
CoA HS-CoA
-----7""""'?""""""'""-+• Dipalmitoyl
1, 2-diacylglycerol +
CDP-ethanolamin,e
Phosphotransferase
-----+ Phosphati~yl-
ethanolamme + CMP
lysolecithin ( • lecithin
1-palmitoyl Glycerophosphoryl Phosphalidyl - - - - - - - + Phosphatidyl serine
lysolecithin choline ethanolamine + Serine + ethanolamine
1. Gaucher's disease Glucocerebro side 3 types- adult, infant ile, juvenile hepatosplenomegaly, erosion
of bone, moderate anemia
3. Metach romatic Arylsulfatase A Sulfogalactocerebroside Accumulates in most tissues. Neurological deficit, difficulty in
leukodyst rophy speech and opt ic atrophy. Demyelination is also seen
4. Tay-Sachs disease Hexosaminidase A Ganglioside (GM2) Incidence 1 in 6,000 births. M ental retardation. Cherry red spot in
the macula. Progressive deterioration. Death by 3- 4 years
5. Generalized ~-galactosidase Ganglioside (GM I ) Mental retardation, hepatomegaly, skeletal deformit ies. Foam
gangliosidoses cells in bone m.arrow. Cherry red spot in the retina
248 Section B: General Metabolism
0
Ceramide- Glu- Gal + CMP-NANA •i i• Clinical Case Study 16.1 Answer
Diagnosis: Gastric ulcer due to NSAID
~ CMP
Biochemical etiology: NSAID inhibits gastric
Ceramide- Glu - Gal- NANA ----------------------------• GM3
enzyme (COX-1 ) required for synthesis of prostaglandins
UDP-N-Acetyl-Galactosamine that have a protective effect on the gastric mucosa.
those with 8 to 14 carbon atoms are called medium 13. The arachidlonic acid is converted to prostaglandins
chain fatty acids (MCFA) (e.g. Laurie acid). Fatty by cyclo-oxygenase pathway.
acids having more than 20 carbon atoms are called 14. Thromboxanes are the major prostaglandins pro-
Very Long Chain Fatty Acids (VLCFA). duced by platelets causing aggregation and vaso-
2. Monounsaturated fatty acids (MUFA) have a single constriction .
double bond like oleic acid (C18) or palmitoleic acid 15. Prostacyclins synthesized in endothelial cells inhibit
(C16). Poly unsaturated fatty acids (PUFA) have 2 platelet aggIregation and cause vasodilatation.
or more double bonds. 16. PGF2 stimulates uterine contraction. Most impor-
3. MCFA digestion does not require pancreatic lipase tant therapeutic uses are in inducing labor, termi-
and bile salts. They diffuse directly into the portal nation of pregnancy and controlling postpartum
circulation. hemorrhage. Leukotrienes LTC4 , LTD4 and LTE4
4. Linoleic acid (C18, 2 double bonds) and linolenic constitute tlhe slow reacting substance of anaphy-
acid (C18, 3 double bonds) are essential fatty acids laxis (SRS-.A).
to be supplied in the diet. Arachidonic acid (C20, 4 17. Cyclo-oxygenase is an example of a 'suicide en-
double bonds) and Eicosapentaenoic acid (C20, 5 zyme'. Aspirin irreversibly inhibits the enzyme. Inhibi-
double bonds) are also biologically important PUFA. tors of cyclo-oxygenase are used as anti-inflamma-
5. Unsaturated fatty acids require isomerization of tory agents (NSAID) and antiplatelet drugs (Aspirin).
double bonds at specific carbon atoms before beta- 18. Cortisol inhibits phospholipase A2 thus reducing
oxidation can continue. PG synthesis.
6. VLCFA undergo initial shortening in peroxisomes 19. Compound lipids contain molecules other than
before they can undergo beta-oxidation. fatty acid and alcohol. Sphingomyelins are the only
7. Defective peroxisomal oxidation can lead to Zell- sphingolipicls, which contain phosphate and does
weger's syndrome (empty peroxisomes) and not contain a sugar residue.
Adrenoleukodystrophy. 20. Cardiolipin or diphosphatidyl glycerol is formed by
8. MUFA can be formed in the body by desaturation of reaction between CDP-diacylglycerol and glycerol-
saturated fatty acids. Thus oleic acid can be formed 3-phosphate.
by desaturation of stearic acid. The microsomal 21 . Sphingosin,e is the alcohol present in glycolipids
desaturase system requires NADH, molecular oxy- and sphingomyelin.
gen and cytochrome B5. 22. Sulfatides are formed by addition of active sulfate
9. Nutritionally essential linoleic and linolenic acid can from PAPS ..
be further elongated and desaturated to form Homo- 23. Gangliosidei synthesis requires one or more sialic
gamma linolenic acid (GLA) and arachidonic acid . acid (NANP1) residues.
10. Omega-3 fatty acids are found mainly in fish liver 24. Defective catabolism of these compound lipids can
oils and have atheroprotective effect. lead to a group of lysosomal lipid storage disorders.
11. Arachidonic acid is the precursor of prostaglandins, (Gaucher's, Neimann-Pick's, Tay-Sachs)
thromboxanes and leukotrienes. 25. All these conditions are characterized by accumu-
12. Prostaglandins are local hormones synthesized lation of a single compound lipid, severe mental
from arachidonic acid. retardation and neurological deficit.
•
Chapter 16: MCFA, PUFA, Prostaglandins and Compound Lipids 251
MCT containing triglycerides are digested by MCT- Prostaglandins are derived from the PUFA. The Series 2
specific lipase. Pancreatic lipase and bile salts are not (with 2 double bonds) are derived from. Arachidonic
required. acid. All naturally occurring PGs belong to the 2 series.
16-5. What is the difference in absorption of MCFA from 16-14. What are the enzymes necessary for prostaglandin
that of LCFA? synthesis?
MC FA is absorbed directly to blood (not to lacteals, as The arachidonic acid is released by the action of phos-
in the case of LCFA). A fter absorption, MCFA is carried pholipase A2. Prostaglandin synthesis is catalyzed
by albumin in blood, whereas LCFA are absorbed as by Prostaglandin H synthase (PGHS). It contains two
triglycerides and carried by chylomicrons. separate enzyme activities, cyclo-oxygenase and
16-6. When unsaturated fatty acids are oxidised, how peroxidase.
many ATP is formed? 16-15. How prostaglandin synthesis is regulated?
The energy yield is less by 1.5 ATP molecules per The phospholipase is activated by epinephrine. Steroids
double bond, when compared to the corresponding inhibit PL and prevent release of arachidonic acid from
chain length saturated fatty acid. Because, the FAD membranes.
dependent dehydrogenation (step 1 of beta-oxidation) 16-16. What is the importance of cyclo-oxygenase?
does not occur at the double bond. Cyclo-oxygenase is activated by catecholamines and
16-7. Name polyunsaturated fatty acids. inhibited by nonsteroid anti-inflammatory drugs (NSAIDS).
Linoleic, linolenic and arachidonic acids. Cyclo-oxygenase is a •suicide" enzyme.
16-8. What are essential fatty acids? 16-17. What is the mechanism of action of aspirin?
Those cannot be synthesized by the body. So they are Aspirin acetylates serine in the active site and irrever-
to be provided in the diet. sibly inhibits the cyclo-oxygenase.
16-9. Name the essential fatty acids. 16-18. What is the mechanism of action of prostaglandins?
Linoleic and linolenic acids are the only fatty acids Prostaglandins are local hormones. and function
which cannot be synthesized in the body. through G-protein coupled receptors.
16-10. Where is desaturation of fatty acid takes place? 16-19. What is the importance of DHA?
In the endoplasmic reticulum. DHA is synthesized linolenic acid. DHA can be obtained
16-11 . What are the substances derived from PUFA? from fish oils or from milk. DHA is especially required for
Prostaglandins, Prostacycline, Thromboxanes, Leuko- the development of brain and retina. Low levels of DHA
trienes. in blood is seen in patients with retinitis pigmentosa.
16-12. What are the functions of PUFA? 16-20. Niemann-Pick disease is due to deficiency of?
Synthesis of prostaglandins, synthesis of phospholipids Sphingomyelinase.
and esterification of cholesterol. 16-21 . What is accumulated in Niemann-Pick disease?
16-13. Prostaglandins are derived from what? Sphingomyelin.
_ _ _ _ _Chapter 17
General Amino Acid
Metabolism (Urea Cycle antt
One-Carbon Metabolism)
Chapter at a Glance
The learner will be able to answer questions on the following topics:
l Digestion of proteins -:r Formation of ammonia
Absorption of amino acids Urea cycle
3 Meister cycle ) ' .9 Urea cycle disorders
)t Cathepsins, proteasomes ,1' 0
10 Urea level in blood
lnterorgan transport of amino acids O It One carbon metabolism
{. Transamination and transdeamination .4ovJ Hyperammonemia
--
CO>\.,~ P1f'(1te\ ·C. ~ l (_L-1 -- 1/
V u
The main role of amino acids is in the ~ ntbe~ ,if l DIGESTION OF PROTEINS El.
strycturill and functional proteins. A 70 kg man has
an average protein turnavec rate of @oo g per da) (same The dietary proteins are denatured on coo~ and
amount synthesized and same amount broken down). therefore mo~ i asily digested. All these e!)_z~ s
: The(nonessenii)I amino acids are either ~~m, are hydrol~ s tciat s 3 enzt > ~~ natu r e ~ i c
the diet or synthesized in the body. The>'f!~aJ e ~ are secrete&f.~t~;ctive 2_~':Jns which are
amino acids are obtained from the diet. Even if one ~rted to their active "#6r'i! in the i estinal lumen.
amino acid is deficient, protein synthesis cannot take This would prevent autodigestion of the secretory acinr.
' place. The body amino acid pool is always in a dynamic The protEiolytic enzymes include:
steady state. In an adult, the rate of synthesis of proteins 1. @n~ot'eptidase~ They act on peptide bonds ioside
balances the rate of degradation, so that ~ n the protein molecule, so that the protein becomes
-\ b~ is maintained proteins are generaUy not US!;1d successiv~ small1S\and s~~er units. Thi~ roup
\, for ruoviding energy (Fig. 17 .1 ). includes P~ sin, Try~fn, Chy~ trypsin and El~ tase.
~"' T, EJoJt (~he.) c;p,, == Pf T
tf (Y'tL '\ -~ cb:-. + Ammonia g/day)
)' Dietary proteins- - - -- - Body _____.. Keto acid _
(50-75 g/day) amino ,I ~ x ldatlon
l:P 1'
l
acid pool
(J).,N, (100 g) l>eo..tofl'I otl,
luconeogenesis
Breakdo~ n of _ _ _ _ _ _ . / a£i-et J.rivl"j
body proteins onessential amino acids
(300-400 g/day)
Syntl;tuia of body pto~ns f300-400 gtday) isvntbub_of.&o!cla~iud ~ .1:-1cts (heme, purine,, pyrimidines, cr~tjne;- , · -:,
______ . , a ; : - ~onzymei;, _
.t,onnones, etc.)• - ,:_ ;_ ·.:_· -•.:.; -;_.::-
0
• _ - - ::·
bond located at the ends of the polypeptide chain. Ala, Gly, Ser
This group includes: 1~ ~ -----+-_:__:_:__ _ ___:::..!..,__:J_.:...__ _----1
a. Carboxypeptidase, which acts only on the
( -terminal basic amino acid
peptide bond at the(sarboxy term]bal>end of the
chain.
b. Aminopeptidase, which acts only on the peptide the peetide hormones: Chol~ Jdnin an ancreo-
i
dlfbf1iin
l PX)
nM ~,S~"M
zymin. ·
M,on W ~Jhe chi iQ
pro\ em is e#efiifai y eitr~i1t :ff • Pancreatic ju}'jf) contains ~ importan~ dopepti-
A. Stomach das~ namely Tl)Tpsin, Chyrirotrypsin, E'fas'tase and
B. Carboxypeptidase.
C. These enzymes are also secreted as zymogens
/'Jv, Pn~ • (trypsinogen, chymotrypsinogen and proelastase), so that
Gas fie Digestion of Proteins
-C...~ P i ! c - thei nf.;a~~io~eus are not aotolyzedj\ll the three
In the stomach{hy~ ~nc'acid IS secreted (see Chapter areeC ase: i.e. the active centers of these
24 ). It rnakes e~pH optimum for the ~ction of epsin t . . .d
, - enzymes con am serine res, ues.
and also acJivates pepsin. The acid als ~PP. '-· "·
proteins. 1fu=-.&,•,,( t ~ ....,iua ~ ) 9-'2. -i""G =fJ ~~~noui
(U,'r:~ oJ OJTT ~ lJSltJJ\... ' ) E:~-
Renn'm ® CJ,1 ~moon L Cm) Trypsinogen is a~ a~ terokjnaspl (enteropep-
tidase) present on thd'.iritestioal mjcroyjUuslnembranes.
Rennin otherwise called Chymosin, is active in infants
Once activated, the trypsin activates other enzyme mole-
and is involved in the curdling of milk. (Box 17.1 ). It is . . .
absent in adults. Milk p rotein, casein is converted to cules. Trypsin is activated by the(remaval af a bexaReP
paracasein by the action o(Turinfu)This denatured protein tide from N-terminal end . Trypsin catalyzes hydrolysis
is ; asilyd igested further by pepsin. . of the bonds formed by carboxyl groups of Arg and Lys.
C'o.&"L11' :W.DD'f) c . f>~ Acute pancreatitis: P~ atll(e activa1iool>f trypsi-
Pepsin ( P) r J (;"Q
1
~ i~ c-, nogen inside the pancreas itself, will result in the autg-
- "eferej,i, ~fi~ digestion..oLpaocreatic c.eJls, The result is acute pancrea-
lt is ~ecreted by the ecsc . s as inacti~e titis. It is a life-threatening condition.
pepsmogeri. The conversion of ~eJ>smogen to pepsin
is brought about b rem ova1 or ~4 ~~1cids from@;hymotrypsin l C,)
tha. tl,l-ter0lioat.evd, by the hydrochloric acid. The opti-
mum pH for activity of pepsi~ s arourtd 2. epsin is an I.JlYRSin wHiacvt:,n chymotrypsinogen, in such a manner
endope tidase, (Table-tt'. 1). Pepsin catalyzes~ dr~- that &,_B a,11d C peoti,®~ f9rmed. These 3 segments
- are approximated, so that the"active site is formed. Thus
sie\of th~ onds formed by carboxyl groups of e yr, . - . . {{)
~=
\&.I . . x- ) ~ I' ( selective proteolys_w produces the catalytic sit
Trp and ~ Y the act1~ f pepsin, proteins are bro-
ken into pr~ses and peptt>nes. @Jarboxypeptidases (C p)
Pa~ atic Diges~ Trypsin and chymotrypsin degrade the proteins into
small peptides; these are fucth~ hydral¥3ed into dipep-
The optimum pH for the activit of Pt ncreatic enzy~ep tjges aod trjpeptide.s by carbox tidasesl pre ·
(p~ by the lkahne · a n d ~ c the pancreatic juice. The procarboxypeptidase is
juice. The secretion ~ 1eatic juice is stimulated by
PZ CC.\<
II O'OC
254 Section 8 : General Metabolism
y
~ Miester Cycle (Gamma-Glutamyl Cycle) loss of proteins t u h th astrointestinal tract.
p. 1?, (.,
intestines, kidney tubules and brain, the absorption Intracellular Protein Degradation
~uT _ of neutral amino acids is effected b the gamma-gluta-
All proteins in the body are constantly being degraded.
1?JO{td,myl cv,sLe;.l llEtripeptide glutathione H) (gamma-
glutarh~ in I;ll~ i~) reacts with [lino acid to
form gamma-glu ~ i n o aci . hat is then cleaved
- -==--
to give the free amino acid. The -----~ - __.
- - - -4
~--
- ... ' -
__ ..........._..,,_~ ~-
• • •
I ~G
Valine? Glutam,ne
Alanine
l
Muscle
lanine - -- - - -+
Muscle
*
~e
Fig. 17.2: lnterorgan transport of amino acids during fasting con- Fig. 17.3: lnterorgan transport of amino acids after taking food
ditions (postprandial condition) ~ -
2 ? I• '.
In the Fed State " Ol '- ,, l rr ) CJ-, ' •
Diet - - - - ·~ ---+ Ammonia -+ Urea (57%)
Amino acids absorbed from the diet are taken up by dif-
---+ Plasma proteins (7%) ferent tissues (Fig. 17.3). Both muscle and brain take up
Amino acid
pool branched chain amino acids, and release glutamine and
---+ Body proteins (14%)
Body- - + alanine. The glutamine is delivered to kidneys to aid in
protein
catabolism ---+ Circulation (22%) regulation of acid-base balance, while alanine is taken
up by liver.
Fig. 17.4: Amino acid pool
Gener~ Metabolism of Amino Acids
because it is io all cells_abundantly. It is a small
1. The ahabolic reactions where proteins are syn-
protein with 7§,c~ idu~ (molecular weight, 8.5 kDa). thesized.
Ubiquitin is attached to proteins. 2. Synthesis of specialized products such as heme,
(D ' cr~e, purines and pyrimidines.
Proteasomes -- Pzro,ient ")'r\ 3. T~ tabolic reactions where dietary proteins
Ubiquitin tagged proteins are immediately broken down ~ d body proteins are broken down to amino acids.
~ ransamination: Amino group is removed to pro-
-=
inside tbe proteasomes of the cells. Ciechanover, Hershko
and Rose were awarded Nobel Prize in 2004 for their
duce the carbon skeleton (keto acid). The amino
group is excret~ds ure1 f- ·- · - - ·
discovery of ubiguitin-mediated protein degradation. li'\The carbon sk ton is us d for synthesis of non-
\J_)ssential amin a ~
lnterorgan Transport of Amino Acids 6. It is also used for 'gruconeogenesis or for com-
plete oxidation.
Breakdown of muscle protein is the source gf _amino
7. Amino acids are used for minor metabolic functions
acids for tissues while liver is the site of disposal (Figs. like conjugation, methylation, amidation, etc. Figure
17.2 and 17.3). 17.1 gives a summary of amino acid metabolism. The
,(~ c:9.>~' amino acid pool in the body is shown in Figure 17.4.
In Fasting State J,.e.e.
The musde releases mainly alanine and glutamine of which FORMATION OF AMMONIA
by liver and glutamine by kidneys The sources and fate of ammonia are shown in Figure
es the amino rou and converts 17.5. The first step in the catabolism of amino acids is to
it to ure ~~msJ!t r on skeleto is used for gluco- remove the amino group;; ammonia. This
neog;.:;ti. S de s should also see glucose-alanine source of ammonia. However, ~ s of ammo-
cycle, under gl cbfili'agth~sft)~}ig.
10.27). The brain nia may also be formed from c.51abolism of purine and
predominantly takes up branched chain amino acids. pyrimidine bases.
'8"" ~\<. A'-~
j 0£4\\-a. ha.{
rvce¥ °-p (';06~,,l)Of) 0J
256 Section B: General Metabolism )SY/>
. fo X 'fC
'-" V
~,I
(;) First Step of Catabolism r~""2r,l\ g Aspartate amino transferase (AST) and Alanine amino
In this first step, ammonia is removed, and the carbon transferase (ALT) are induced by glucocorticoids, which
sk eIeton of the ammo· ac,·d lPJW:,
t S in t h a r,c path way.
· to cad'.3 favor gluconeogenesis. AST is increased in myocardlc;il
A 'I'f'~~ \..~")
I.JVSynthesis of Non-essenr,a/ Amino Acids
'--> ~4B~'& R,9""-t
infarction and .i.\
r
· live dise~s~_lhe~-
im(?Ortance is given in Chapter 6. V\t.J,Y(.
B ransdeamination 1· ~\o...
t1 11....
Bymeansoftransamination, allnonessentialaminoacid (Of\,(.Lll)'lrxm
l"\~\f,> can be synthesized by the body from keto acids avail- .._ J
1'" _.IJ, able from other sources. For example, pyruvate can be 1. The amino woup of most of tn-e amino acids is
!~, transaminated to synthesize alanine. Similarly oxalo- released by a coupled reaction, transdeamination,
_J•~ acetate produces aspartic acid. Alpha ketoglutarate that is transamination followed by oxidative
µ,"' is transaminated to form glutamic acid. Those amino deamin'1ltlori.
; / acids, which n t be s nthesized in this manner, are 2. Transamination takes place in the cyfc$Pasm of all
therefore s enti ; they shou e made available in the the cells of the body; the amino group is transported
food (See Box 3.1 for essential amino acids). to liver as glutamic acid.,_w.hlG~ finally oxidatively
. .<kak.0.9
n, nterconversion of Amino Acids <aeaminated in the roitnchondr.ia_6f hepatocytes.
3. Thus, the two, components of the reaction are physi-
If amino acid no.1 is high and no. 2 is low; the amino
group from no.1 may be transferred to a $ cally far away, but physiologically they are coupled.
Hence, the term transdeamination (Fig. 17 .7).
ru,,i • Q}a,.2.
0..0.,1 ~o.. 0..0-2..
f'\a> bh- toi l Y'A:l~iljDO'OJ ~ f ' \fl.i( k-0 :l •lf9-9"'
~ . , ~fubi p i ~16.a.k,o,. y,ga.A/JO-{ (G~ pt"~~f\ doli!~ \
enera/ Amino Acid Metabolism (Urea Cycle and One-Carbon Metabolism) 257
r -----:-- - - - --iw-~- ..::,..,;;;i.....:__.s;.,~~~::!...!_T V ab
Any amino acid
Corresponding Glutamate
ketoacid :)
I ti • f\11 .Jt~~• I,,),> ff;"). ti~• \~, •~t f toJiZl-. J._(J'6 j-~f
faction 'in the gastrointestinal tract.
DISPOSAUDETOXIFICATION I)
of all other amino acids is funneled into glutamate. Hence, OF AMMONIA ~1oo,.dlL «9\-~+~
t~e glut~mate dehydrogena~e reaction is the fi~al re~c- First Line of Defense /'> ¼ ~Oril-
111
tion, which removes the amino group of all amino acids (Trapping of Ammonia) ~ , w
(Fi~.17.7). ltneedsNAD•as en~ . . ½cd\e.q,~ ,,tu)
enzyme; it is activate Dg.,and inhibited b GTP. Being highly toxic, ammonia 'Shouldoe eliminated or
The hydrolysis of glutamine also yields NH but this detoxified , as and when it is formed. Even very minute
3
occurs mainly in the kidney where the NH; excretion is quantity of ammonia may produce toxicity in central
d for acid-base regulation. £em nervous system. But, ammonia is always produced by
r 'L"' \);h \4-:.,{J almost all cells, including neurons. The intracellular
r Pathways of De, minati ~IJ· ,':}.f ammonia is immediately trapped by glutamic acid to
1. L-amino acid oxidase can ~i ct on all amino acids form glutamine. especially in brain cells (Fig. 17.9). The
-
except hydroxy.A~J..1'18 acids and dicarboxylic a23i.!JO glutamine is then t.@_nsported to lj~ r. where the reaction
acids. It uses'l=~ cl£benzyme. The peroxfde is reversed by the enzyme glutaminase (Fig. 17.9). The
formed in this reaction is decomposed by catalase ammonia thus generated is immediately detoxified into
in the peroxisomes (Fig. 17.8). urea.' Aspartic acid may also undergo similar reaction to
2.(®amino ci xidase can oxidiz~ and form asparagine (see Chapter 18).
a mino · that ay be form~cterial r-ho-N.p~r
me a olis . ses a t!oenzyme.
Transportation of Ammonia ~ o ,
3. Small quantities of ammonia may be formed in Inside the cells of almost all tissues, the transamination
the body through minor reactions like o~ of amino acids produce glutamic acid. However, gluta-
' 1 2 ~ (mono amine oxidase) (see mate dehydrogenase is available only in the liver. There-
Chapter 19, under Tyrosine metabolism). fore, the final deamination and production of ammonia
"-4"'~ I.~
I
Argininosuccinate
Argi~
Fig. 17.9: Ammonia trapping as glutamine
Fig. 17.11 : U rea cycle and its relation w'ith citric acid cycle
\
Step 2: Formation of Citrulline (Figs. 17 .10 and 17 .11, Step 4 ). The e..!)ZWejS inhibited
QY-1.uroarate.. But this is a'iQided by the c toRlasmic
The second reaction is also mitochondrial. The carba-
lo_p aliza!iQn._o.Lthe eo__zy__m.e. The fumarate formed may be
moyl group is transferred to the NH2 group of ornithine funnelled jnto TCA.cycle to be converted to malate and
by ornithine transcarbamoylase (OTC) (Figs. 17.10 then to oxaloacetate to be transaminated to aspartate
and 17.11 , Step 2). The citrulline leaves the mitochon- (Fig. 17.11 ). Thus the urea cycle is linked to TCA cycle
dria and further reactions are taking place in cytoplasm. through fumarate. The 3rd and 4th steps taken together
proteins 1JCL.U1 may be summarize as:
. d , " _,
..
' c.. ·r
"',.
01
1
Citrulline + aspai ate -l Arginine + fu1Ilj3rate
A similar reaction of donation of amino group by
Step 3: Formation of Argininosuccinate aspartate takes place in purine nucleotide synthesis also
One molecule of aspartic acid adds to citrulline form- (see Chapter 38).
ing c(larbon to ni@ gen bong),vhich provides the 2nd
nitrogen atom of urea. Argininosuccinate synthetase Step 5: Formation of Urea
catalyzes the reaction (Figs. 17.10 and 17.11 , Step 3). The final reaction of the cycle is the hydrolysis of arginine
This needs hydrolysis of ATP to AMP level , so two high to urea and ornithine by arginase (Figs. 17.10 and 17.11 ,
energy phosphate bonds are utilized. Step 5). ThelorrJitbine returns to.the_mito_choodcia to react
with another molecule of carbamoyl phosphate so that
Step 4: Formation of Arginine the cycle is repeated. Thus ornithine may be considered
Argininosuccinate is cleaved by argininosucclnate as a catalyst which enters the reaction and is regen_e-
lyase (argininosuccinase) to arginine and fumarate rated.
OTC ~iOd\C-Lj :: -*Hnke.0. ~o~cl£sl
~ : lbl'b'& <9'v o}'nm,u..J-~ hee~
Hyperammonemia type I Very high NH3 levels in blood. Autosomal recessive. Mental etardation. Incidence is 1 in
100,000
Hyperam monemia type II (OTC) Ornithine Ammonia level high in blood. Increased g lutamine in blood, CSF and urine. Orotic aciduria
t ranscarbamoylase due to channeling of carbamoyl phosphate into Pyrimidine synthesis. X-linked 1
Failure to import ornithine fro m cytoplasm t o mitochondria. Defect in ORNT1 g ne.
transporter protein yperornithinemia, hyperammonemia and homocitrullinuria is seen (HJiH syndrome).
~c,. Decreased urea in blood. Autosomal recessive condition t<...O~
I,;\
~.,,. l~
Arg ininosuccinate Autosomal recessive inheritance. High blood levels of synthetase ammo nia and cit rulline.
\.9'
I----------+-=------+----_:....,------------':,__-'--
Citrullinuria (Citrulline excreted in urine 1-2 g / day).
O, Arg ininosuccinic aciduria Argininosuccinate Argininosuccinate in blood and urine. Friable brittle t ufted hair (Trichorrhexis nodosa).
- -- - - -------;
\!,' f - ---'- - - - - - + -:.t..
ly..:..
as:..:
e_ _ _ _-+-.:.:.
ln:..:
c:..:
id.:.:
en___c:..:
e:..:
2___in_ 2
::..:0:..:
0.:.:,00..:..0
:....::..:
bi.:.:. .:.:.._ _ _ _ _JI!.·-="'m.:.:.o.._\.,,L.c-_f'-.-st:~~--1::A~_;_:,mai=,Fr,:..._--,
rt.:..:.
hs
ti' Hyper argininemia Arginase Argini ne increased In blood and CSF. Instead of arg inine, cysteine and lysine are lost in
urine. Incidence 1 in 100,000
L-----------'--------'---------,~---'-------------------~
t)m))"f1"9 ~L,orr-h
Energetics of Urea Cycle isorders of Urea Cycle
The overall reaction may be summarized as: ~ ~Deficiency of an of the urea cycle enzymes would result
NH3 + CO2 + Aspartate -+ Urea + Fumarate 1" in perammonemi When the block is in one of the
In the urea cycle 2 ATPs are used in the first reac- eru:lier..ste~ q g ition is [1.0@..S.evereofiQ~ ~~~,"1::11
tion. Another ATP is converted to AMP an~ ~ nia itself accumulates. Deficiencies of late~ i met~-1.
equivalent to @ .The urea cycle consumes 4 high result in the accumulation of other intermediates, which
energy phosphate bonds. However, fumarate formed are l~oxic and hence symptoms are less. As a general
in the 4th step may be converted to malate. Malate when ffl~iption, disorders of urea cycle are characterized
'J \
oxidized to oxaloacetate produces 1 NADH equivalent to Y yperammon!,_T ip, ~rnc~ppalopathy agd resp:ratory
2.5 ATP. So, net energy expenditure is only 1.5 high alkc1losis. Clinical sy~ toms include '@Olllio , ircila6iti~
energy phosphates. The urea cycle T c cle are lethargy and severe mental retardation. Infan ts a~ear
interlinked, and so, it is called as "urea bicy~le°y uarmal at birth, but within days progra"ssive leth~
main lines of treatment are t . ~ ~in diet. 2. Bowel meter for the assessment of a variety of ge~ti£ and
disinfection using antibiotics and clearance by _lactulose. acquired conditions. The most important gen€tic causes
"'00")
3. Avoid hepatotoxjg dru~ . 4 . Maintenance of electro- of hyperammonemia are urea cycle disorders and organic
lyte and acid base balan 'NU 'k~~, acidurias (Fig. 17.12). Organic acidurias usually pre-
. ~e.ch-o.l ~ .e..t-e...~ , sent with life-threatening metabolic acidosis and hyper-
Urea Level m Blood 0n\,:,l:>- ~ ~ Q kO , ~ ammonemia; w hereas urea cycle disorders often have
In clinical practice, blood urea level § ?a~n as an indi- metabolic alkalosis along with hyperammonemia. Ammo-
cator of renal function . The normal urea level in plasma nia levels may be elevated in hepatic coma (hepatic
is from 20-40 mg/di. Blood urea level is increased encephalopathy). In this condition liver function tests will
where renal function is inadequate. Details of causes of also often be grossly abnormal. Other conditions where
uremia are given in Chapter 25. When protein intake is ammonia is elevated include cor pulmonale, pulmonary
high, the urea level in blood will be slightly increased, emphysema and renal failure. Treatment policy is shown
but within the upper limit of the normal values.
Prmm1:1'0, <Ji ~ - ,
L.:+.:Y~,:-hepatic coma.
-Fd\on
\ ) ~ Q_ - L l~
262 Section 8 : General Metabolism
ll
17 • Hydroxymethyl -CH 2OH N10 - hydroxymethyl- THFA
~Formyl
G $ Methylene - CH2- N5,N10 -methylene-THFA N Methiony1 RNA
ll
l '.
Choline ~ Hydroxy-+ Methylene- + Glycine
Serine Methyl THFA Se ne
N THFA dU P-+ dTMP
Reductase
Melhy1THFA
E
T nsmethylation
S rlne _,. Choline
---• Creatin
Fig. 17.13: Tetrahydrofolic acid (THFA). Methylene group is atta-
Epinephrine
ched to N 5 and N 10 methionine
-
2. Glycine cleavage system also produces~Tethyl~ methyl-THF ccur. THFA is not regenerated;
groups (see Fig. 18.3). this is called folate trap (See Folic acid in Chapter 33).
Chapter 17: General Amino Acid Metabolism (Urea Cycle and One-Carbon Metabolism) 263
Utilization of One-Carbon Groups hyperargininemia, which is the least severe). NAG syn-
thase (NAGS) dleficiency mimics CPS-I deficiency.
A summary of the generation and utilization of one- Clinical symptoms of urea cycle disorders are leth-
carbon groups is shown in Figure 17.14. The one-carbon argy, anorexia, hyperventilation or hypoventilation, hypo-
units are used for synthesis of the following compounds: thermia, seizures and coma. Hyperammonemia may be
1. C2 of purine { ~m@ j triggered by illness or stress, which along with a normal
2. Formylation of methionyl tRNA anion gap, is tt1e classical hallmark of urea cycle dis-
3. CS of purine £S ,e,} order. Definitive: diagnosis depends on enzyme assay
4. Gly~ine (see Fig. 18.1)Jl p ~\ from liver biopsir specimen and genetic analysis.
5. Senne J · 1 Treatment includes dialysis to reduce ammonia
6. Choline (see Chapter 18) (F-) levels, intravenous arginine chloride and nitrogen scaven-
7. Deoxy TMP... '"''11),&J ger drugs (phenyl acetate, benzoate) to activate altera-
8. Transmethylation reactions including creatine, tive pathways for ammonia excretion, and protein
choline and epinephrine synthesis restricted diet. Chronic therapy includes protein restricted
9. Excreted as carbon dioxide. diet, phenyl bultyrate, arginine, citrulline supplements,
0 and if necessary, liver transplantation. Genetic coun-
•• Clinical Case Study 17.1 seling and prenatal diagnosis are advised.
17-1. What is the action of pepsin? 17-17. What are the physiological significance of trans-
It hydrolyses peptide bonds formed by carboxyl groups aminatiori1?
of Phenylalanine, Tyrosine, Tryptophan and Methionine. It synthesises nonessential amino acids; it is the first
17-2. What are zymogens? step of amino acid break down pathways.
They are proenzymes; inactive at the time of secretion, 17-18. What is the clinical significance of transamination?
but will be activated in the gastro intestinal tract. Transaminases are increased in blood in liver and car-
17-3. What is its biological significance? diac diseases.
Zymogens prevent autodigestion of the cells. 17-19. What is transdeamination?
17-4. What is its clinical significance? Transamination takes place in all the cells of the body;
Acute pancreatitis results when trypsinogen is activated
the amino group is transported to liver as glutamic acid
prematurely.
which is finally oxidatively deaminated in liver. Thus
17-5. What are the enzymes in pancreatic juice?
the two components of the reaction are physically far
Pancreatic juice contains the endopeptidases trypsin,
away, but physiologically they are coupled. Hence the
chymotrypsin and elastase.
term transdeamination
17-6. How trypsinogen is activated ?
17-20. Which amino acid is oxidatively deaminated in liver?
Removal of a hexapeptide from N-terminal end by
enterokinase (enteropeptidase) Glutamic acid.
17-7. What is the action of trypsin ? 17-21 . Nitrogen atoms in the urea is derived from what
It hydrolyses peptide bonds formed by carboxyl groups precurso1rs?
of arginine and lysine. One from ammonia and another from aspartic acid.
17-8. How chymotrypsinogen is activated? 17-22. Ammonia1is trapped in brain by what?
It is activated by trypsin. Glutamine! synthetase.
17-9. What is an endopeptidase? 17-23. What is the key enzyme of urea synthesis?
It acts on peptide bonds inside the protein molecule, Carbamoyl phosphate synthetase.
so that the protein becomes successively smaller and 17-24. What are t he two carbamoyl-hosphate synthetases?
smaller units. CPS-I is involved in urea synthesis; CPS-II is required
17-10. Give some examples of an endopeptidase. for pyrimidine synthesis. CPS-I is seen in mitochon-
Trypsin, pepsin. dria, while, CPS-II is in cytosol.
17-11 . What are exopeptidases? 17-25. What is the normal blood urea level?
They act at one end of the protein molecule, liberating
20-40 mg/di.
amino acids sequentially, one at a time.
17-26. Blood unea level is markedly increased in which
17-12. Give an example.
condition?
Carboxypeptidase.
Renal diseases.
17-13. Carboxypeptidase contains which metal?
17-27. Tetrahydrofolic acid is used for what purpose?
It is a metalloenzyme containing zinc.
It is the ca1rrier of one carbon compounds.
17-14. In fasting state, nitrogen is transported from mus-
cle as what form? 17-28. Name on1~-carbon compounds?
In fasting state, the muscle releases alanine and gluta- Form,:I, formimino, methenyl, hydroxymethyl, methy-
mine of which alanine is taken up by liver and gluta- lene, and methyl.
mine by kidneys. 17-29. Which an~ the donors to one carbon pool?
17-15. Transamination of glutamic acid produces what? Serine, choline, glycine, tryptophan, histidine.
Alpha ketoglutaric acid 17-30. One carbon units are used for what?
17-16. What is the coenzyme for transamination reaction? C2 of purine, CB of purine, serine, choline, creatine,
Pyridoxal phosphate epinephrine.
Aliphatic Amino Acids
Chapter at a Glance
(~~N'I~)
Serine hyaroxymethyl transferase coo-
COO- Glycine
l
~-t~ _
+ I +
Serine Glycine CH- NH Threonine
3 CH2- NH3
____;ca_ld_ol-'-
as'""e_ _
coo- coo- +
l
CH-NH 3
+ l +
CH2-NH 3 I @) CHO
CH20H
CH3 I Acetaldehyde
Threonine CH 3
Fig. 18.1: Formation of glycine from serine Fig. 18.2: Formation of glycine from threonine
Proteins Proteins
Glycine
i l~ r-
Glycine ~eavage system
Serine - G ......,____-.......,.
ammonia -+ Urea
~~]' ...,_ 1 carbon unit
-""(:,.\ul!lic.~"lolt- I Serine -+ Pyruvate -+ Glucose
Amino methyl group "'J Cysteine
, Choline
+ CO2 + Glyoxanc acid -+ Oxalate
THFA ( t·,,o
) amlde * NADH+H NH.c3 +} Creatin~-+ CP -+ Creatinine
@ 0 0
Reduced +
1
c;.'c.;#
5 7 of purine ring C l.~g
·Tie!>
Melhylene lipoamide NAO ALA -+ Heme
v THFA tA~~~ ,. :r •
) ')-"" f Cl> Glutathione
. NH3 H11- .., . ._ L ~(.,J.J}>
...< \,v
'<~ -t::-
t
r....eS ·~ • ,..,., ~t>l.,) N\'f:
) Bile salt
v~• A= Glycine decarb<?~ase B = Aminomethyl transferase· ( liI> ) ..__ _ _ _ __ ___c_o_n_iu_ga_ti_
·o_n,_e_.g_.h
_i_
pp_u_ric_ ac_id_ ___,
C = Meth lenelf'Al..?t, nt as D = Lipoamide dehydrogenase; Fig. 18.4: Overview of glycine metabolism
THFA = Tetrahydrofolic acid; PLP = Pyndoxal phosphate
Fig. 18.3: Glycine cleavage system. Glycine is completely deg- total muscle weight. It is synthesized from 3 amino acidsJ
raded to CO2 , ammonia and one-carbon unit methylene THFA. ~ e. argioia.e and methionine. ('; r r
The reactions a.qi~ · reversible, when the enzymes are A. First step (Guanidoacetic acid); The amidino group
1
together calle~d~G~1~ · ~ ~ ~I"'
of arginine is transferred to glycine to form guanido-
acetic acid, catalyzed by arajdo transfetase·(Step 1,
unit methylene THFA (Fig. 18.3). This pathway is the
Fig.18.5). It is seen in mitochondria of kidney and
major catabolic route cine. The glycine cleavage
pancreas, but not in liver.
system is ulti-enzyme co It needs the,.w -
B. Second step (Creatine): Guanido acetic acid is
enzymes, 'A , lipoamide, tetra ydrofolic acid and p?ri-
m;!9Ylated lby S-adenosyl methionine (SAM) by
doxal hos hate. . ,.,_ u- re-"" w,\J.,t) _ o ~;_
met~mJ§:~e to form creatine. This methyla-
t::':)..
,." ,_ .. --,
, -¥'
b> • J \:-o ·1 tion reaction takes place in liver (Step 2, Fig.18.5).
Vl!l2.Gluc;og'fmi9c ath~ay s lAP- ..Q..."'p C. Third step (Creatine phosphate): Creatine is phos-
Glycine is~ airfty7hatm~ci into the glucogenic pathway phorylated to creatine phosphate (Step 3, Fig.18.5).
by getting first converted to serine. This is the reversal Th~ enzy~j ere~ ine kinase (CK) is present in
of serine hydroxy methyl transferase reaction (Fig.1 8.1 ). muscle, brtfin ana Iver. The reac . n .eds hydro-
The serine is then converted to pyruvate by serine dehy- lysis of ATP . The stored~ atine ho h e in the
dr~tase (Fig.18.9). b ~'rm'""' ,S S. ') u > f?
muscle serv«:?s as an immediate s
4,,~ he muscle. During muscle contraction, the energ•y
·
Special Metabolic Functions of Glycine ,tv.c,uc<.. is first derived from ATP hydrolysis. Thereafter, the
ATP is regenerated by the hydrolysis of creatine
Glycine may be used for the biosynthesis of the follow-
phosphate (Fig.18.6). This is called the Lohman n's
ing compound§ (Fig.18.4 ):
reaction.
i. Creatine, creatine phosphate and creatinine D. Fourth step, (Creatinine): The creatine phosphate
ii. Heme µ~ 9.1.,.o.9- .
may be conv,erted to i s anhy_g_ri9e, creatinine (Step 4,
iii. Purine nucleotides rrein --~~ " ~e. Fig.18.5). It is a~ -enzymatic. spontaneous reac-
iv. Glutathione ha.n..,,-cc...."', tion. Creatinine is excreted in urine. The blood level
v. Conjugating agent
vi . Neurotransmitter. lt--o..e. J.o G.w.. I ½ of creatine and creatinine, and urinary excretion
of creatinine are more or less constant. But creati-
l
nine level is dependent on the total muscle mass.
CREATINE AND
CREATINE PHOSPHATE Clinical Applications
The word creati e is derived from the Greek term, kreas, ~ ; serum creatinine level is 0..Z..toJA..mgl.dl and
which means flesh. Creatine constitutes. about 0.5% of serum creatine level is Q2=0_4 mg/di. Urine contains
-OM--:
0 L.,, m'- .
C,-'ll"tat\'Af. f" .3l' 'lt'11\ C..~0$ln;-r-a,
,, 1s7rg
rc')Qn
,
NH 2
ATP g-rs(»-'O' purine ring (C4, C5 and N7 ) (s~~apter 38).
, A-1f l
"\r-t 9 ~~R
Synthesis of Glutathione .._
J.D\luJ\OM
- ~ OC
coo A DP CC~ 9 I Glutathione is ;a ! ( i ~ formed from glutamic acid,
~H -4 '--i C~~H2 cystei_ne a~d @~ The functions of glutathione are
2
I Creatine Creati- ' N-CH3 described In thei section on metabolism of cysteine.
N-CH3 phosphate_ nin\ I
I ('~ C=NH Glycine as a Conjugating Agent
C=NH m:M) -~ Pi+H20 I
, - svJl(v-, , N A. Bile acids . Glycine is used to conjugate bile acids,
NH-Po; \,l}V~ H
to pr~duce: pile salts. ~yeocbalic acid and EtC't>~
_s.be~ ~ acid are the main conjugated
bile a ids (see Chapter 14).
B. Benzoic acid. It is used as Q!eservative in foods.
4 = Spontaneous (non-enzymaticl,
SAM = S-adenosyl methionine
Glycine is iused for detoxification of benzoic acid to
- SAH = S-adenosyl homocysteine form hippuric acid (~ Chapter 36).
Glycine: + Benzoyl-CoA -+
Fig. 18.5: Creatine metabolism •
>v(l..(l.0 + NC\Av-.e-. Benzoyl glycine (hippuric acid) + CoA
ne ~e amounts of creatine in normal males. But in T~ a tion occurs in liver and so it is an index ~a\)
m ~r dystrophies, the ~od c~atine and urinary of~ r functic~. Hippuric acid was first isolated from
~~::!!~jg_.c.reased. Creatinine level in blood 1s a~ ~ e ~ j hence the name.
indicator of renal function (see Chapter 25). The enzyme~
CK is clinically important as it is elevated in myocardial Glycine as a Neurotransmitter
infarction (see Chapter 6).
The spontaneous loss of creatine and of phospho-
creatine to creatinine ~uires that creatine be continu-
ously replaced ; this occurs by a combination of diet
and efl.dogenous synth_!!sis . ~tiaos 9btairi.-aJJ:oost
no d~ reatine synthesis makes major Glycine as a Constituent of Protein
demands on the metabolism of glycine, arginine, and
methionine. Children with inborn errors of creatine In collagen, ev~~ 3rd :_Q}ino acid i€. i : ?
-~~-
\Jn..A.. 6
l ~ _., rr...,.q~
Cn.a}\n,l\ e. Re.J'\O.}~
... __\•,
\~
. , C K.
270 Section B: General Metabolism
Dehydrogenase
3-phosphog.occerato ( \ • 3-phosphohydroxypyruvate
r\
Serine decarboxylase
\
Serine lmino acid Pyruvate CH-NH3
\
I I
coo- COCJ coo- CH2-OH
(PLP) CH2-OH
I -+ H2O I H2O NH3 I
Serine
CO2
CH-NH3 C = NH C=O Et hanolamlne
I I I
CH 2-OH CH3 CH3 Fig. 18.10: Decarboxylalion of serine
l®
Glycine Serine Ethanolamine Pyruvate -+ Glucose
Glycine /
Ethanolamine -+ Choline
+CH, Choline-+ Acetylcholine
Phosphatidylserine-+
Sarcoslne or / Methyl other phospholipids
methyl glycine ethanolamine
·
tD CH3
CH3 +CH 3
1 "'CH3
®
is added by protein kinases which are se~/threonine
kinases. In gJYJcoproteins, the C1:f ~ y ~ o up
are usually attached to the hJ2!oW rou s of selioe
or threonine residues of the protein. Serine forms the
Betaine Choline or active catalytic residue of many enzymes (serine pro-
or trlmethyl glycine trimethyl ethanolamine
teases), e.g. trypsin and coagulation factors.
An overview of serine metabolism is given in Figure
18.12.
/
SelenocystE!ine (SeCys) OOC- C - C -S,eH
, 'H
Fig . 18.11 : Glycine~( e-cholin (21st amino acid)
~e.,~dono Selenocysteine i s abbreviated as SeCys. It is seen at
(Fig.18.10). Figure 18.11 -s ows the addition of 3 the active site of the following eri.,zymes: a) Thioredoxin ' ~~-~J
methyl groups to ethanol amine to form choline. ''~'..,-- v -
reductase; b) Gl~ thione peroxidase, which scaven-
B. Choline is used for acetylcholine synthesis, which ges peroxides; c) De-iodinase that removes iodine from
is an important neurotransmitter. thyroxine to malke triiodothyronine and d) p ele.nopco.:
C. From choline, 3 one-carbon groups (-CH3) can be tei n~ . a glycoprotein seen in mammalian blood. Their
removed. So, choline is an important one-carbon concentration fallls in selenium deficiency.
donor (Fig. 18.11 ).
-
Its structure is COOH-CHNH 2- CH,-SeH. Biosyn-
thesis of selenocysteine is by two steps: • I
Me~\,e:::;
.
phate groups , e.g. casein. Glycogen phospj,orylase is SeCys is inserted as ~uc durini p,i:otein _biosyn-
activated by phosphorylation, while pyruv5ttkinase and thesis. Its genetic code is,__Y~A. The tRN~~~c rs first
r i
phosphofructok inase-2 are activated by aep ospho~ ctiarged with smi~ then it is-'c2nverted into SeC_y..s.
lation. This covalent modification serves as a mecha- This is then i~~~dJ_nto the correct ~ ition, when pro-
nism of regulation of enzyme activity. The phosphate group tein is synthesiz,ad.
272 Section 8: General Metabolism
Beta alanine
+ CH3 (1-C unitj
Vitamin B 12
Cysteine synthesis
!·"~
Here the amino group is attached to the beta carbon ~~~Of")'l'tC- F - , U . ~ ~ + ~'ISIN.
atom. It is fo rmed during the ~at~ of th.s).QWroidioe
=
1 methionine adenosy~ ~ ~e (MAT); 2 methyltransferase; =
3 =adenosine homocysteinase; 4 =homocysteine
bases, cytosine and ~ (see Chapter 38). It is mainly
melhyltransferase, with vitamin B12 as coenzyme
used for the synthesis of Coenzyme A (see Chapter 33).
Fig. 18.13: Formation of active methionine
l!_HREONINE THR) (~T)_
It is an es~tial amiDQ_ acid. It is 9!llcogenic. Threo- 2. Methyl transfer. In methionine, the thio-ether link-
nine has 2 asymmetric carbon atoms, hence it has 4 age (~ C) is very stable. In SAM, due to the
diastereoi~ hreonine. L-threonine, L- presenceoT a high energy bond, the methyl group
">KalJ.otbreooine and D-allothreoni e Tq~ ~do s not is labile, and may be transferred easily to other
r directly undergo transamination, ~ \'tl..l?i'a'erc +- -~ 00 acceptors (Step 2, Fig.18.13; and Table 18.1).
A nation forming alpha ketobutyric acid reaction similar to 3. Homocysteine. From the S-adenosyl homocys-
'N~ LW~ rine, Fig. 18.9). The enzyme is threonine dehydratase. teine (SAH), the adenosyl group is removed to form
The H gro f threonine residue in protein serves homocysteine, which is the hjaher homolog~ of
-a si ·on (as in the case of c~steine (St13p 3, Fig.18.13).
is OH group also serves for combining car- 4. ~ nine synthesis. Homocysteine can be con-
\oJ:~@
C
1..i.tJu.,....u..a.Lw..1.......,u.w=,.!.l,l..l,,W,!,,IS;,!lai!;> , ~-te ~ glycopro- verted to me!thionine by addition of a methyl group.
teins. /.::7 (r\e,rvv.J v
'J ..... rv!. This methyl group is donated from one-carbon
·· J .,gool with the help,.ofvita~ Step4,F lg.18.1 3).
IMETHIONINE (MET) (M)
r·
__ 5. Homocysteine degradation: Homocysteine con-
It is sulfur-containing, e ~ial, glu~ eni~ amino ~enses with1 serine _to form ~st~thionine:)This
acid. Degradation of methionine results in the synthesis . is cata_lyz~dl by pyndoxal phpsp hate _dependent
e; of cysteine. The sparing action of cysteine on methio- (l~ c stath1ornrn:i-beta synthase (No.5, Fig. 18.14).
nine is thus explained . Absence of this enzyme leads to homocystinuria.
~ ~,...... 1. Activation of methionine to SAM : In the major 6. Cysteine sy·nthesis: In the next step cystathionine
_/
'""1~ 9,.~i°"IC. \:'ru.Q_. ' N\eln~O()) N2_
*
· coO- CH - CH2- CH2-CH2-CH-COO-
I I I
NH; SH NH;
Ethanolamine C. hhd!! Cystathlonine
Carnosine
Acetyl serotonin
adz~
GA.l!h.~ J::..g.p) 6 (+)H20 ©
l:'t~oo-
°'-:f
-OOC-i:tr-@
Serine ~ omoserine Cysteine
Histidine
Lysine Methyl lysine
tRNA Methylated t RNA
.2)_,
i ~NH3
7. Final oxid~ton: Homoserine is dWminated and Alpha keto hydroxy butyrate -OOC-C-CH2-CH 2-0H
......_
THFA . 18.16: Pyruvate formation from cysteine
l!
Homocysteine Methylated j
Box 18.1: Summary of MeUCys metabolism 2 ATP+ SO4- ---+ PAPS+ ADP+ PPi
•
Methionine 0 0
S-adenosyl Met--... Transmethylation
II
Ho- s -
II
o - P- O-H C Adenine
Creatine
Methyl :::.-+ Epinephrine UV\Cfc1 bH , ~
~ Choline (.I
Melatonin
S-adenosyl homocysteine
•
Serinex Homocysteine
O-Po;
Homoserine ~Cysteine
\Pyrtvate Glucose
CoA
a. inorganic sulfates,
b. organic or ethereal sulfates, and
c. neutral sulfur.
rr,- ,~.
Me olism of Sulfur etc.
The ~ p o n e n t o~oteins
ill!.::W.Jllli1~1Y--i~LLIU~jds) or as pai't"\)f swfatides
1cvsTINURIA -1
...,cans (GAG). n~anlc sul
~---~~c: is Cystinuria is one of the inborn errors of metabolism
derived from the sulfur-containing amino~acids. Th S included in the\Garrod's tetra.ii) It is an autosomal rece-
derived from c s ine--may be oxidized to sulfites ssive condition. The disorder is attributed to the
thiosulfat and further oxidized to sulfateB c,iency in Jransport,nf a~ n acjds (Table 18.2). Signs
tory forms of sulfur in urine are: and symptoms include:
~ ; 1..r "' ~ zr ,r-a ~CL ~ ~v.9.i°'
C4.tu&ne- G'f~ W - \ ~ ~
276 Section B: General MetabolisrH O.N.~a-~ N \11<0 ~ '& ~ t'
Fig. 18.21 : Cystine crystals in urine Fig. 18.22: Cystine stone in the bladder
i. Abnormal excrz:tion of cystine and to a Box 18.2. Ho111ocyste1r1e dnd t1ec1rt ,1tt:icks
extent lysine, orWi?hine and arginine. Hence the
An increase of 5 micromol/L of homocysteine in serum elevates
condition is also called Cystine-lysinuria. the risk of coronary artery disease by as much as cholesterol inc-
ii. ~ @ g _ caU~uh fbrrt!a~,1. In acidic pH , cys- rease of 20 mg/di. Homocysteine interacts with lysyl residues of
tine crystals are formed in urine (Fig. 18.21 ). collagen and bind to fibrillin producing endothelial dysfunction.
iii. Obstructive urogruhy, which may lead to renal insu- Providing adequate quantity of pyridoxine, vitamin B12 and folic
fficiency (Fig. 18.22). acid will keep homocysteine in blood at normal levels.
-
by®kalanizatjon of urini}y giving sodium bicarbo-
nate.
@ Cyanide-Nitroprusside Test
romol/L. In diseases, it may be increased~o@:0 to 1Q£
oderate .increase .Is seen .In agg(dt!__Persons,
\.,,
tamin B42 ?tr B6 deficiency, tot:>a co smokers, alee olics
1
.
vi-
Glutamic acid
COO °o NH3 c.l COO COO
j (PLP)
I
CH-NH +
3 f i lCH-NH+3
2 I
CH-NH+
3
I I .., I CO2 / ! 1 ,:,. ...._{.
CH2 CH2 CH2
Gamma-amino butyric
6H2 ATP ADP+Pi 6H2 l NH
3
6H2
; c ~ABA)
I I I
COO CO-NH2 COO 2
Succlnic semialdehyde
Fig. 18.23:~ glutamine synthetasei 2 = lutaminase
NAO+~
3 i)
Gf0\ J'P.- · - incy of pyridoxal phosphate would lead to convul- NADH + H+
11 }'q ,lt«1 Sodium valproate which~ nhibits GABA oxidase is Succinate -+ TCA cycle
used in the treatment of epilepsy.
= =
1 glutamate decarboxylase; 2 GABA oxidase;
Congenital deficiencies of GABA aminotransferase 3 = succinate semialdehyde dehydrogenase I
and succinic semialdehyde dehydrogenase (leading
-~ - --
Fig. 18.24: GABA metabolism
to~~~tyric acidurla} are reported, but are very
rare.
Glutamate Transporters
IGLUTAMINE (GLN~) ~<Q~ - - ,..L -Glutamate (~
'--'-- l.
is the major excitato~eurotranwiit-
-
Glutamine was isolated as free amino acidffi.om beeti:ooi) ter. Different transportations are available in the brain.
by Schulze and Bosshard in 18 33 _Glutamine was iden- Dysfunction of transportation mechanism of gultamate
tified as a member of all proteins by Charles Chibnall cause neurodegenerative conditions such as Alzheimer's
and Manayath Damodaran in 1932. disease.
Histidine - - + Glutamic
Arginine - - + Alpha ketoglutarate -+ TCA cycle-+ Glucose
acid (E) ,\;,party!
Praline - - +
trans-
Fig. 18.26: summary metabolisms of aspartic acid, asparagine, glutamic acid, and glutamine
280 Section B: General Metabolism
l
Creat!ne - Creatine phosphate
~ Proteins
Urea
1·
-t- Ornithine --+ Putrescine --+ Spermidine
!
Glutamate - - - Glucose
Nitric oxide
Fig. 18.27: Metabolism of arginine and ornilhine
.:..~~ .:.I
important signal molecule in the body.
Pre1vents platelet
~ ITRIC OXIDE (No · aggregation
'
It is a toxic pollutant of air and automobile exhausts.
But now it is shown to possess more potential biological
functions than any other known molecule. In 1977, Ferid Functions as a
.·.··1:.\~.~
neurotransmitter
Murad showed that the vasodilatory effect of nitroglyce- in brain
rine is due to the release of No·. In 1980, Robert Furch-
Mediates bactericidal
gott showed that "endothelium derived relaxing factor" actions of macrophages
(EDRF) is required for arterial dilatation. In 1987, Louis
F ig . 18.28: Nitric ,:,xide synthase (NOS) reaction. The enzyme
lgnarro showed that EDRF is chemically NO". They contains heme, FAD, FMN and tetrahydro biopterine. It utilizes
were awarded Nobel Prize in 1998. NADPH
Synthesis of Nitric Oxide There are 3 isof,orms of NOS, these are products of 3
different genes. J\11 forms are seen in almost all tissues.
Nitric oxide is formed from arginine by the enzyme nitric Neuronal NOS: NOS-1 or nNOS or neuronal NOS
oxide synthase (NOS) (Fig. 18.28). It contains heme, is seen in central and peripheral neurons. Nitrogenic
FAD, FMN and tetrahydrobiopterine. The enzyme utilizes neurons are seen especially in cerebellum and gastro-
NADPH and molecular oxygen. Calmodulin is required to intestinal tract.
modulate its activity. The guanidino nitrogen of arginine is MacrophagH NOS: NOS-2 or iNOS or inducible
incorporated into No·.
NOS or macrophage NOS is mainly seen in macropha-
Metabolic Fate ges and neutrophils. It is induced by cytokines (interleu-
kin-1 and tumor necrosis factor).
No· combines with oxygen to form NO2 . These nitrites Endothelial NOS: NOS-3 or eNOS or endothelial
are excreted through urine. On exposure to superoxide NOS is seen in endothelial cells, platelets, endocardium
Chapter 18: Aliphatic Amino Acids 281
~ [I] ~ [I]
Serine Ethanolamine Choline 18
Tyrosine Tyramine 19
DOPA Dopamine 19
Tryptophan Tryptamine 19 Protein Protein Protein Protein
5-0H-tryptophan Serotonin 19 from food from muscles from food from muscles
Histidine
Ornithine
Histamine
Putrescine
19
18
I
Branched-chain
I
Branched-chain
Lysine Cadaverine 18 Amino acids (BCAAs) Amino acids (BCAAs)
Cysteine Taurine 18
developmental milestones . There is history of sibling The disease is inherited as autosomal recessive.
death at Day 15 of life. The clinician reported abnor- Diagnostic tests are sodium nitroprusside test; cystine
mal urine odor. Laboratory analysis revealed ketonuria stones are detected by X-rays as they are radiopaque
and metabolic acidosis. HPLC analysis of amino acid or by CT of kidneys. Traditionally intravenous pyelogram
showed elevated leucine, isoleucine and valine. Child (IVP) was used. Microscopy reveals flat hexagonal crys-
died immediately afterward. tals of cystine.
Patient 2 presented at Day 12 with metabolic aci- Treatment is aimed at eliminating stones and pre-
dosis, abnormal urine odor, ketonuria and hepatosple- venting new stones. Dietary sodium and protein restric-
nomegaly. Blood and urine studies revealed elevated tion increases cystine excretion. Urine alkalinization
levels of valine, isoleucine and leucine. Aggressive improves cystine solubility. Severe cases need surgical
treatment was started including branched chain amino intervention.
acid restricted diet and supplementation. Patient has In the condition cystinosis, excess cystine crystals
survived until 3 years of age, without any episode of accumulate in eyes and kidneys. This is a lysosomal
exacerbation afterward. Levels of leucine, isoleucine storage disorder and is also inherited as autosomal
and valine came down to normal level. recessive condition. There is mutation in the gene
What is the diagnosis? What is the significant diffe- CTNS, which codes for the protein cystinosin, the lyso-
somal cystine transporter. Symptoms include excessive
rence between the two patients?
urination, followed by poor growth, photophobia and
renal failure by age 6. Cystinosis is the common cause of
¥ Clinical Case Study 18.1 Answer renal Fanconi syndrome. There is loss of large amounts
of salt and other minerals in urine. Definitive diagnosis
The likely cause is cystinuria. Cystinuria is characterized is by measurement of WBC cystine levels. The drug
by build-up of cystine stones or crystals in kidneys and cysteamine is used to clear cystine. Replenishment of
bladder. Patients with cystinuria cannot properly reab- lost fluid and electrolytes as well as high doses of vita-
sorb cystine into their bloodstream and the amino acid min D and phosphorus is needed.
accumulates in their urine. As urine becomes more con-
centrated, excess cystine forms crystals that can lodge
in bladder or kidneys. Cystine can also combine with • · Clinical Case Study 16.2 Answer
calcium to form larger stones. They can block passage Likely cause is homocystinuria. Classical homocysti-
of urine and this can also lead to infections. nuria is due to the deficiency of enzyme, CBS. It is
Symptoms are recurrent nephrolithiasis and obs- inherited as an autosomal recessive condition. Com-
truction of urine flow. This can produce severe, sudden mon clinical features are failure to thrive, developmen-
onset of flank pain, blood in urine, infection which can tal delay, ectopia lentis (subluxation of lens), severe
produce fever, WBC in urine and in advanced cases, myopia, cataracts, retinal detachment and optic atrophy
renal failure. during infancy and early childhood. Progressive mental
284 Section B: General Metabolism
retardation may be there, but in some individuals intel- 8. Glutathione helps in amino acid transport, acts as a
ligence may be normal. Skeletal abnormalities may re- coenzyme for reduction reactions, maintains RSC
semble Marfan syndrome. Generalized osteoporosis membrane integrity and helps in detoxification.
may be present. Thromboembolic episodes involving 9. Cystinuria is an inborn metabolic disorder result-
large and small vessels, especially those of the brain, ing from deficiency of amino acid transporter.
are common and may be seen at any age. Elevated Symptoms include crystalluria, uropathy and secon-
homocysteine level (hyperhomocysteinemia) is an inde- dary infections.
pendent risk factor for coronary artery disease, cerebra- 10. Cystathionuria is due to cystathionase deficiency.
vascular diseases, peripheral arterial disorders as well Mental retardation, anemia and endocrinopathies
as deep vein thrombosis. During pregnancy, it can lead accompany this condition.
to neural tube defects in embryo and pre-eclampsia in 11 . Arginine is tlhe precursor of Nitric oxide (NO ' ) by the
pregnant mother. action of NO synthase.
Diagnosis is by amino acid screen in urine and blood 12. NO" synthase has three isoforms: neuronal, indu-
(homocysteine and methionine are elevated, cystine cible and erndothelial.
will be low), liver biopsy and enzyme assay, skeletal 13. NO" is a potent vasodilator and participates in
X-ray, skin fibroblast enzyme assay, standard ophthal- macrophagB mediated antimicrobial activity. No· is
mic testing and genetic testing. Treatment with high
used in treatment of pulmonary hypertension and
doses of pyridoxine, folic acid, 8 12 and betaine reduces
erectile dysfunction.
homocysteine levels.
14. Glycine cleavage system, a multienzyme complex
$2. can break cjown glycine to CO2, NH 3 and one-
¥ Clinical Case Study 18.3 Answer carbon unit (N5- N10 methylene THFA).
15. Glycine condenses with succinyl-CoA in the first
The diagnosis is maple syrup urine disease. In the case
step of heme synthesis.
of the first patient (Patient 1), diagnosis was delayed
16. Whole mole·cule of glycine is incorporated into the
and hence treatment could not be instituted and the
purine ring (C4,C5 and N7)
baby died. But in the second case (Patient 2), diagnosis
17. The tripeptidle glutathione, an antioxidant, is gamma-
and treatment was started early in life and outcome was
glutamyl cysteinyl glycine.
better. These two case studies indicate the importance
18. Glycine is used for conjugating bile acids to form
of early diagnosis and treatment in MSUD.
glycocholic ;acid.
19. Glycine is a neurotransmitter which opens chloride
l!:_EARNING POINTS, CHAPTER 18 specific channels.
1. Glycine may be formed from serine, threonine and 20. As a constituent of protein, glycine plays a crucial
by the action of the enzyme glycine synthase. role in maintaining the triple helical structure of col-
2. Glycine is used in the biosynthesis of heme, crea- lagen.
tine, purine and glutathione. 21 . Deficiency of glycine cleavage system can cause
3. Stored creatine phosphate acts as an immediate nonketotic hyperglycinemia.
source of energy in the muscle. ATP is generated 22. Primary hyperoxaluria, characterized by repeated
by its hydrolysis by Lohmann's reaction. occurrence of urin ary calculi, is due to a protein tar-
4. Serine occurs in the active sites of many proteolytic geting defect. The absence of alanine glyoxylate
enzymes, such as Trypsin and Chymotrypsin. Such aminotransf,erase in peroxisomes increases the
enzymes are called 'serine proteases'. glyoxylate pool which can be oxidized to oxalates.
5. Major metabolic role of alanine is to provide sub- 23. Addition of a one carbon group to glycine produces
strate for gluconeogenesis. serine.
6. Methionine is activated to S-adenosyl methionine 24. Serine by cleamination can be converted to Pyru-
(SAM). This participates in transmethylation reac- vate, a glucogenic intermediate.
tions. 25. Serine on clecarboxylation gives rise to ethanol-
7. Cysteine is a component of the tripeptide gluta- amine. Both serine and ethanolamine are constitu-
thione. ents of phospholipids.
Chapter 18: Aliphatic Amino Acids 285
26. Methylation of ethanolamine can give choline, used 28. The selenoproteins contain the 21 st amino acid
for acetylcholine and phospholipid synthesis. selenocysteine formed by replacement of the "O"
27. The hydroxyl groups of serine and threonine can by"Se".
be linked in O-glycosidic linkage to carbohydrate 29. Glucose-alanine cycle is one of the metabolic
residues of glycoproteins. adaptations during starvation.
18-1 . What are the important substances derived from glycine? Describe the steps by which creatine is synthesized.
18-2. Write the reaction s by which glycine is synthesized and catabolized. Name six important compounds derived
from glycine and indicate their functions.
18-3. Describe the metabolism of methionine. Explain the term t ransmethylation with suitable examples.
18-4. Describe the fate and functions of methionine and cysteine.
18-5. Describe the pathway of methionine metabolism.
18-6. What is the biochemical basis of homocystinuria? What test you will do to diagnose homocystinuria?
18-13. Glutathioneisusedinthebodyforallthefollowing 18-24. Which amino acid will give rise to an inhibitory
functions , except: neurotransmitter?
A. For ammonia trapping A. Histidine B. Glutamic acid
B. For reduction of methemoglobin to normal hemo- C. Ornithine D. Tyrosine
globin 18-25. Gamma-annino butyric acid (GABA) is derived from
C. To carry amino acids across membranes which amiino acid?
D. For removal of peroxidases A. Tyrosine B. Tryptophan
18-14. One salient clinical manifestation in homocystinuria C. Glutamic acid D. Serine
is: 18-26. Transamination of glutamic acid leads to:
A. Agitation, hyperkinesia A. Glutamine
B. Renal stones B. Alpha k:etoglutaric acid
C. Ectopialentis (subluxation of lens) C. Beta h~rdroxy beta methyl glutaric acid
D. Nystagmus D. Gamma amino butyric acid
18-15. The incidence of homocystinuria in the population 18-27. Transamination of aspartic acid gives rise to:
is about: A. Asparagine B. Malate
A. 1 in 1,500 B. 1 in 50,000 C. Oxalos1uccinate D. Oxaloacetate
C. 1 in 1,00,000 D. 1 in 2,00,000 18-28. Lysine is:
18-16. Example for a transport defect is :
A. Deficient in cereals (rice, wheat )
A. Cystinuria
B. Deficient in pulses (dal, bengalgram)
B. Homocystinuria C. Mainly ,glucogenic
C. Crigler-Najjar syndrome
D. A non-essential amino acid
D. Sulfituria
18-29. Glutaminase which hydrolyses glutamine to glu-
18-17. The urine of a patient with homocystinuria will be
tamic acid is used for:
positive for:
A. Ammonia trapping in brain
A. Benedict's test .:
B. Excretion of ammonium ions in renal tubules
B. Ferric chloride test
C. Purine :synthesis
C. Rothera's test
D. Transamination
D. Cyanide nitroprusside test •
18-30. Which amino acid does not undergo transamination?
18-18. Urine of a 12 years old boy gave a positive cyanide
A. Leucine B. lsoleucine
nitroprusside test. He had renal stones. He is likely
C. Lysine D. Alanine
to have:
18-31 . Lysine is required for the synthesis of all the fol-
A. Homocystinuria B. Cystinosis
lowing, exc;ept:
C. Hartnup disease D. Renal glycosuria
18-19. Name defective enzyme in homocystinuria:
A. Collage,n B. Cadaverine
A. Propionyl-CoA carboxylase C. Carnitine D. Carnosine
B. Methyl malonyl racemase 18-32. Alpha amino group of aspartic acid is incorporated
C. Tyrosinase into all the following compounds, except:
D. Cystathionine synthase A. Adenint3 B. Cytosine
18-20. Following constituents of urine listed below contain C. Urea D. Creati ne
Nitrogen derived from glycine, except: 18-33. Aspartic areid is used for the synthesis of which
A. Creatinine B. Uric acid compound?
C. lndican D. Urobilinogen A. Porphyrin B. Sphingomyelin
18-21. Alanine on transamination gives rise to: C. Pyrimidine D. Folic acid
A. Pyruvic acid 18-34. All are monoamines, except:
B. Para hydroxy phenylpyruvate A . Histamine B. Serotonin
C. Phenyl pyruvate C. Dopamiine D. Glutamine
D. Hydroxy pyruvate 18-35. Ornithine when decarboxylated will produce what?
18-22. 5-adenosyl methionine transfers methyl groups to A. Ethanol amine
which compound? B. Beta m13carpto ethanolarnlne
A. Homocysteine B. Methionine C. Histamine
C. Homoserine D. Nor epinephrine D. Putrescine
18-23. All the following are excretory products of metabo- 18-36. Branched c;hain keto acids are excreted in urine in
lism of methionine and cysteine, except: large quantities in:
A. Ethereal sulphates B. Inorganic sulphates A. Phenylketonuria B. Maple syrup urine disease
C. Sulphites D. Phosphates C. Tyrosinosis D. Hartnup disease
Chapter 18: Aliphatic Amino Acids 287
18-37. Which test will be positive with the urine of patient 18.38. Name the defective enzyme in maple syrup urine
with maple syrup urine disease? disease (branched chain ketonuria):
A. Dinitro phenyl hydrazine test A. Cystathionase
B. Benedict's test B. Phenyl alanine hydroxylase
,_ C. Ferric chloride test C. Homogentisic acid oxidase
D. Cyanide nitroprusside test D. Oxidative decarboxylase
18-1. Glycine is used for synthesis of what compounds? 18-10. Give examples of transmethylation reactions?
(Name biologically important substances derived Guanido acetic acid to creatine, Serine to choline,
from glycine). Nor-epinephrine to epinephrine, N-acetyl serotonin to
Serine, Creatine, Purines. Heme. Glutathione, Bile salts. melatonine
18-2. For creatine synthesis, which amino acids are used? 18-1 1. What is the methyl donor in transmethylatlon reac-
Glycine, arginine, methionine. tion? (What is active methionine)?
18-3. What is the significance of creatinine? S-adenosyl methionine
Excretion is increased in muscle dystrophy. 18-12. What is glutathione?
18-4. How creatinine is produced in the body? Gamma-glutamylcysteinyl glycine.
By spontaneous degradation of creatine phosphate. 18-13. What are the functions of glutathione?
18-5. What are the sources of oxalic acid in urine? Keeping RBC membrane integrity; carrying amino acids
Ascorbic acid and glycine.
across membranes; detoxification of peroxidases.
18-6. What is the cause of hyper oxaluria?
18-14. What is the test for homocystinuria?
Protein targeting defect.
Cyanide nitroprusside test will be positive in urine.
18-7. Choline is derived from which amino acid?
18-15. What is defective enzyme in homocystinuria?
Serine
Cystathionine synthase
18-8. Alanine is transaminated to what?
18-16. What are the characteristic features of homocys-
Pyruvic acid.
18-9. What is significance of glucose-alanine cycle? tinuria?
During starvation, alanine is released from muscle and Mental retardation, subluxation of lens, thrombosis.
is taken up by liver. In liver alanine is transaminatec '.- 18-17. What is defective enzyme in Cystathionuria?
pyruvate, and pyruvate under goes gluconeogenes1s. Cystathionase.
_ __ _ _Chapter 19
Aromatic Amino Acids
(Phenylalanine, Tyrosine, Tryptophan,
Histidine, Praline and Aminoacidurias)
Chapter at a Glance
The learner will be able to answer questions on the following topics:
(!5) Phenylalanine (Phe) (F) 0 Hypertyrosinemias
tO Tyrosine (Tyr) (T) 0 Tryptophan (Trp) (W)
I{) Phenylketonuria (PKU) 0 Histidine (His) (H)
.(} Alkaptonuria Praline (Pro) (P) and Hydroxyproline
Albinism Aminoacidurias
. ..-
-=---J~ Chapter 19: Aromatic Amino Acids 289
\...
\-\ a..._€ \4 0 tf\< b,... ) 0,:"\ C1 \. \ b<:l.al
1 = Phenylalanrne Jiydroxylase .:..--- Step 3: Production of Homogentisic acid
2 = Tyrosine
3 = Parahydroxyphenylpyruvate hydroxylase The n~ ~t .~ ~~ 3, Fig.19.2) is catalyzed b para-
4 = Homogentisic acid oxidase
5 = Maley! acetoacetate isomerase
hydroxyp!w~'lf>§hWate . It is a c pper
6 = Fumaryl acetoacetate liyarolase • containing enzyme. Interestingly, the'"fea9tion involves
, 'l'\.._'t ~\ ot,0-\-.tt
shifting of the sjde chajp posi-
u - tion. Ascorbic acid is helpful in this reaction.
Phenylalanine
(+}02~ J
4
1,
+Maleyl acetoacetic acid
Tyrosine
1. Melanin
r,~tx
2. Catecholamines (epinephrine)
Fumarylacetoacetic acid
3. Thyroxine
(+)H20 A6
(' Synthesis of Melanin
-OOC-CH2-C0-CH3 -OOC-CH=CH-COO
Melanin pigment gives the black color to the skin and
Acetoacetic acid Fumaric acid hair (Greek word Melan means black). There is only one
(Ketogenic pathway) (Glucogenic pathway)
enzyme involved.w hich catalyzes the first two steps. The
Fig. 19.2: Catabolism of phenyl~ ct.tyrosjoe..,,--,....--_.;; remaining reactions are non-enzymatic and occur spon-
¼::_J ~ , 4 - 21,., O> 'l""'.) ,taneously.
Catabolism of Tyrosine i. Formation of DOPA: The first step is the !)¥drogta-
(and Phenylalanine) ~?J~ ti.on of tyrosine by tyrosinase. It is a mono-oxyge- 0 $
. t· 4- !}, ;.;;
na:;:.:s~e.;.c=on.::
ta;:in.;in
~g~ ~
o i!!!ieiiirlL(.::
st~e;;p.,;1;.
, Fig. 19. 3) to form
Step 2 : Transamma 10n <i---0 ~·- ,. .
~ydroxypheny a arnne or DOP _ (Box 19.1 ).
Degradative pathway of phenylalanine and tyrosine ii. Formation of DOPA quinone: Tyrosinase again
are the same. Tyrosine is transa i ate to give para- acts on DOPA to form dopaquinone (step 2,
hydroxyphenylpyruvic acid by rosme transaminase Fig.19.3).
(step 2, Fig.19.2). It is pyridoxal phosphate dependent. iii. Formation of indolequinone: It is converted to
It is induced by glucocorticoids. indolequinone through a series of reactions. The
l
BOX 19.1: Tyrosinr1se and tyrosine hydroxylase
B~tb v,6e
enzymes ~II a,dd hyd.roxybg_r~ to-tyros!Ae to pro- HO- OCIH i-yH~COO- Tyrosine \ fl~
duce dihydroxyphenylalanine (D'?,p"l- "':::, NH3 (•)O, 1
Tyrosinase is present in melanoblasts. The enzyme produces '<;
DOPA, which is used for melanin synthesis. HO \.o.g._
DOPA CL. '!'JI)
Tyrosine hydroxylase is present in adrenal medulla and the DOPA
thus generated is used for catecholamine synthesis. Thus even in HOOCH2-9H~COO- (Dihydroxy- 1
phenylalanine) r
~-
tyrosinase deficient person (albinism) synthesis of the catechola- .. NH 3 \.
mines is normal. '6 0~ •
0 .t'I
ti]
0 O -cH 2-CH-C00- DOPA-qulnone
- I+
Q NH 3
elan~ in the deeper layers of epidermis synthesize
mefa~in in granular form in nosomes ~ 9r of
;,.o 0 -a::: I
. ,ar,
1
the ~kin depends the di the f>PJ ( crle ._g,_ (00 0 .-,:: N
concentration of melanin an I s state oicidation. The extra- lndolequinone
Melanin
cellular granules are later dispersed under the influence of
melanocyte st imulating hormone (MSH). Fig. 19.3: Melanin synthesis pathway; 1 and 2 steps ave the
Copper deficiency: Since tyrosinase is a copper containing same enzyme, tyros,inase :rncr ~\
enzyme, there may b _s turban~ i!'.1 pigm~~on during \,Lor€
n I C i symhesized at
• thf tim
of deficiency tyrosinase involved in melanin synthesis which
c ~ may be depigmented. If copper deficiency · ntermittent, al-
catalyzes a similar reaction (Fig. 19.3 and Box 19.1).
(- ternate black and white regions may be s in the hair (flag-
type of hair). The tyrosirne hydroxylase reguires tetrahydro-
may multiply to give bj_opterine and NADPH (similar to phenylalanine
rise to malignant melano I&.' hydroxylase ). -
~ ~ o n d l i MD@uch urine if kept in a test ii. OPA-dec r x.ylase : DOPA is decarboxylated to
~ • ~~ GE Refl et rbe lllbe bernrne5 b!ar;~ due to oxi- form Dopamine y DOPA-decarboxylase, a pyridox,JI
dation loelanin. c:..c,_,....vJ_ .. .-.00,
phosphate cjependent enzyme (step 2, Fig.19.4).
iv. Leukoderma: W~~ osinase or meram-/l'for~ s or
bot h are ab~ from _;}derrlJls, leukoderT~hi~es) It is a catecholamine.
results. .
v. Graying of hair is also due to the disa@ earance 9f....m~ano-
from thf nair root.ft
vi. Albinism: Albinism and leukoderma are different. In albi- tract.
nism, tyrosinase is absent in melanocytes all over the body.
In ParkJ nsonism , the dopamine content in
See later in this chapter.
QJ:ain.ls_red~d- As dopam~ ot enter into
indolequinoneisg£1_y~ zegJofQrrn..rnrua,nin.Melanin the brain cells, the orecurso~-l.90.P
is used as a
is a group of polymers of random structure formed drug in Parkinsonism. ,..
- =,s
from indolequinone. Melanin when reduced chang- ,Alpha 111ethyldop will inhibit d_opa _decar:boxyl-
es from black to a tan color. Clinical applications of ase and prevent production of epinephrine; so it is
,,
melanin are shown in Box 19.2. an antihyge :t~ e drug.~
Tyrosine
021
Epinephrine and adrenaline are two names for the same hor-
(+) mone. John Jacob Abel discovered "epinephrine• in 1901 . In
NADPH and 1 the same year, Japanese-born chemist Jokichi Takamine, work-
tetrahydrobiopterin
ing independently, isolated the same hormone. which he called
~ . , ; , DOPA f "adrenalin. It was first marketted as Adrenaline for therapeutic
Odr (d1hydroxy- use. Hence the word adrenaline is more u~ed in clinical practice,
~v- ph::~la:lne) while the term epinephrine is more favored in academic circles
CO2 ~
~ . . . , , Dopamine
t
HO
"lv~ Cu++
Vil. C
1 {j 3
Transaminase Reduction
Phenyl- 7' , • Phenyl- 7" , • Phenyl- Guthrie test was developed in 1961 by Robert Guthrie (1916-
+ +
alanine
a-KG
r
Glutamate l
pyruvate f +
NADH+H NAO
+ lactate
1995). It is a rapid screening test. Cert ain strains of ~ II~
!'---. co
~ ~ ine as an essential growth factor. Bac-
Decarboxylation teria cannot row in a medium devoid= hen.xJal~jJle. Bacte-
2
rial growth is proporuQ~I tp..tl)e.p e anine content in the
Phenylacetate patient's blood.
Conjugation lr-- Glutamine Guthrie was the father of a mentally challenged child and had a
niece with PKU. He developed a rapid diagnostic screening test
~ H20 forPKU.
Phenylacetyl glutamine
Fig. 19.5: Alternate pathways in phenylketonuria is elevate~aernate mmor pathwa~ are ~ned
4,~ ~-t~o,q..
level of excretion of VMA is =6 mg/24"fi. It is ln'i:reased in
G (Fig.19.5).
lactate and fi
I ketone (phenylpyruvate), ~ n I-
!acetate are ex~r ted in urine.
R e chrom I toma (epinephrine excess) and in !]SY.-
stoma (norepinephrine excess). Clinical Manifestations '1
The classical PKU child is mental!
© Synthesis of Thyroid
Lo~
Hormones
Tyrosine residues are 1-monoiodo-
~c,,\~IQ of 50. About ~ ~ mate::s:::;;=p~s=yc
may PKU. AgiSfon,
; ::1~
a r~1c hospitals
h . a~ty, t_r~ors and
tyrosine (MIT) and 3,5-~dotyrosina (DIT). These are conv'M"s1ons are often manifested. This may be
coupled to gi~~ ~.3'-triiodothyronine (T3) and becaus~ phenylala~ine interferes with neurotransmitter
3,5,3',5'-t~dothyron ne or thyroxine (T4) (see Chap- synthesis. The child often has o 1gmen a 10
ter 45). explained by the i_ghigJ,tion of ~sJ~e. Phen ctlactic
Tyrami U'rru).._'l'tx, \ \<,.Gll\~\.-o.-0..P sweat ~ ay lead lo..JJlousy body odor.
~We,,; ~ i > ~&l\ , Laboratory Diagnosis
Tyrosine i ca cboxylated to tyramin'e by intestinal
bacteria. Tyramine is present in chocolate, cocoa, wine, A. Blood phenylalanine: Normal level is 1 mgldl In
dried fish, processed meat, buttermilk, cheese, yeast, PKU, the level is >20 mg/dl. This may be demon-
beans, peas, papaya and peanut. These may precipitate strated by chromatography. Tandem mass spec-
an attack of mi raine in susceptible individuals. troscopy is the most reliable test; but is costly.
B. Guthrie test is a rapid screening test. See Box
I PHENYLKETONURIA (PKU)- - --
19.4 and Figure 19.6.
C. Ferric chloride test: Urine of the patient contains
Deficiency of phenylalanine hydroxylase (Fig.19.1) is p~enylketones about 500-3,000 mg/day. This could
the cause for this disease. The gene~mutation be be detected by adding a drop of ferric chloride to
such that either the enzyme is not syht?iesized ~ - the urine. A ransient blue-gteen color is a positive
f4:1Actiooal eQZ.ynle is synthesized. It is a recessiv condi- test. But this is a less reliable test.
tion. Frequency of PKU was considered to be 1 in 10,000 D. DNA probes are now available to diagnose the
Treatment
'.KU in lnd'.a is lesser th~t_,!W-n c ~ ies; only 1 Early detection is ve!Y importa11t. About 5 units of IQ
1n 25,000 births. There ar types of Pkll described, are lost for each 10-week delay in starting the treatment.
depending on which enzyme is de 1cient. 1.Y e 1 is the The treatment is to provide a diet containing IQ.w ghe-
classical one, due to deficiengy of t}ydroxylase enzyme. (10-20 mg/kg bo~ weight per day). Food
ioca (cassav ~ill have low phenylala-
Biochemical Abnormalities
nine conten . 1s special diet is to be continued during
Phenylalanine cannot be converted to tyrosine. So, phe- the first decade of life; after which the child can have a
~ lanin~cumulates. Phenylalanine level in blood normal diet.
f:G . - r""<"
\ I 1"'1 I Qxe.~\v-£~0j -
,C <)
C \.r b,_-~ a)
1
The specimen
on the left, which
has been After two
standing for hours, the
fifteen minutes,
shows some
darkening at the
surface. due tq. Vertebral disc~.-r.::.;_,.
the oxidation showing blackish
. of nomogentisic d1
·scolorat1·on
F 19. 19.6: Guthrie test. Robert Guthrie is shown on right side
, •
=-~
.,c,u. (alkaptone
,,. :\J1lw1f? C.. :"":
I ALKAPTONURIA
bodies
--- - 9 v -~n;.Qe.. r
\(,\.£1' l=ig.19.7: Left side, urine of alkaptonuria; right side alkatone bod-
ies deposited in vertebral disc of patient
The term alkaptonuria arises from the Arabic word alka-
t f
p on. ?r
" lk 1·" d G k
1 an
lie
d " t:r,t.lC,. +- \?oll.lm-. ~Cl! .
ree wor to s_ucK up flxyg~n In Diagnosis of Alkaptonuria ..:,,, "ILJ,,,'.
,..,..,,.\ n
n . \00--'__..
v..n""~
_t1..,. -~
alkali. This Is based on the observation that the~ rioe ~\~\.J
becomes b lack on standing when it be_oomes_alka- 1. Urine becomes black on standing due to con-
line. Sir Archibald Garrod in 1902 reported that patie~ _ tact with oxygen or when it becomes alkaline.
comp.lai.D.Jhat..llleiumde.rweacs ace getting...bla~~ Blackening is accelerated on exposure to sunlig~t
Garrod concluded that the disease is i(lherited and ii and oxygen. The urine when kept in a test tube will
is due to the d~ ofi&ie enZ'@fl re91,urngJor fur- start to blacken from the top layer (Fig. 19.7).
ther meta oli ~-·---·- · · · lea tonurl and 2. Ferric chloride test will be positive for urine.
are two inborn errors included in Garrod's 3. Benedict's test is positive with a ~~!=!::::~
tetrad; the other two bein pentosurla and cysti ~ ipitq~ Therefore, alkaptonuria comes under
' Garrod introduced the term "inborn errors of meta• &differential diagnosis of reducing substances in
bolism" in 1908. The condition had been vividly des- urine.
cribed by Zactus Luxtanus in 1649. Egyptian mummies 4. Dectection of homogentisic acid by GC/M~ (gas
dating back 2000 BC had pigmented cartilages due to chromatography/mass spectrometry).
I ALBINISM
alkaptonuria.
I HYPERTYROSINEMIAS
Black pigments are deposited over the conne~ v~
tis~s_iocluding_ joio.t crutities to produce F~ Hepat~ren~I Tyrosmem1a - -.- - -.- - -- -- - -
1 6
Phenylalanine - - - + Tyrosine - - • Hydroxyphenylpyruvic acid - - - -- ---+ Homogentisic acid
2t
DOPA ~ Dt A - - - - - - Thyroxine Maleyl acetoacer e
l
Dopamine
l
Dopaquinone Fumaryl acetoacetate
!
Norepinephrine l F,ma,ale (Gl•«>geolc~
+ Melanin
~ 77
~ C H 2 - f H :coo- "rNH 2 llNJ-cooH
NH 3
Ring opens
OH
3-hydroxy-
anthranillc acid l
Quinollnic acid
(-CO,)
~ COOH
N
~ COOH
ll.N
I
Ribose-5-phosphate
r " r - cooH Fig. 19.10 : Synthesis of niacin from t to han; PRPP= phosphori-
bosy\"yrophospha1e. = qu1nolinate phosphoribosyl transferase
l,eu o"e -:;ll"~)
CHO .J - NH2
HOOC ,.
Amino IJLUConat_e aldehyde Functions Serotonin
(- ~ t\
/' "'3. ~,, .) Serotonin is an important neurotransmitter in brain. 5-HT
Keto adipic acid ...,is an anti-depressant.
..J (-) 2C0212..-..@ When or Ina ro eIns are a all amino acids
1 are available in blood. This cause raffic jam in the
Ketogenic +-- CH3 -CO-CH 2-COOH Acetoacetyl-CoA ,,... amino acid transport systems in brain cells. Try12._tophan,
the b;:!lk~l,amino acid.is therefore t~ e i v slc>wiy. '/J'~
~ -- - - -- - -- - - - - -- ---t7in:~ lbcM However, when carbohydrate-rich diet is taken, •: ~o;
Fig. 19.9: MetabolisA1 ?ftryptophan l,.Q,r)\c:ui insulin secretion is increased, which will lower the amin~
l.-~ acid concentration in blood. So tryptophan easily enters
Serotonin@ ~: 'i~Ya.~iV\.L = ,.. 1011 Jre brain cells. When tryptophan is available in brain in
'])e,C.O,.,\>JO~~ iq excess quantity, serotonin may be generated to induce .~,::"
Serotonin (5-hydroxytryptamine) is produced in the sleep.(!ii,i:liiifu·drates wm induce sleep, w bl(e p.r.o-
brain, mast cells, platelets and gastrointestinal tract tein-nch foods will cause alert~
•mucosa. Tryptophan is first hydroxylated by !!YEto han ... Seroton~~ IE!vel is found to be low in patients~ith
l!Yj roxyl~ (step 1, Fig.19.11 ). This reaction is very p~ ~ p sycho~:-{,erotonin is involved in rl'Rlod,
much similar to phenylalanine hydroxylase (Fig. 19.1). ep, appetite an temf5'erature regulation. It increases
The coenzyme is tetrahydrobiopterine. The tetrahydro- gastrointestinal motility.
biopterine is regenerated using NAOPH. In the next step,
5-hydroxytryptophan is decar-boxylated to 5-hydroxy- Catabolism of Serotonin
tryptamine (SHT) (serotonin). The enzyme, decarboxy_. Monoamine OJ1cidase (MAO) converts serotonin to
lase requires pyridoxal phosp'hc:lft( (step 2, Fig. 19.11).'~ 5-hydroxyindolleacetic acid (HIAA) (step 3, Fig. 19.11 ).
296 Section B: General Metabolism
CCT
dff'
CH2-yH~COO
Tryptophan
NH3 Melanin is the pigroem pf baic and 5kil)· it is synthesized from
N Tyrosi ne (Fig. 19.3).
NADP:xTetrahydrobiopterin Melatonin is a neurotransmitter synthesized from tryptophan
0
(Fig.19.11).
NADPH+H Dihydrobiopterin
SAH
5-0H-tryptopha n
!
Kynurenine
+
1-C Unit N~NH
.-,~~.::::.,...,- CH=CH-COOH
NH,@1 1
3-hydroxykynurenine
Urocanic acid C\
Serotonin
+ +
J~
N NH
!
Alanine + 3-0H anthran11ic acid
(+) H20l 2
Glulgenic
CD
P
CH2-CH2-COOH
Acetoacetate 1.,
5-HIAA l H tmadazolone
Ketogenic
ni: acid \.N
{: ~:l: l
I HISTIDINE (HIS) (H) The major cells producing histamine are platelets,7 S. tf€
I
tions are minimal'(C CoA entering into the TCA cycle, is completely oxidized.
I AMINOACIDURIAS
is reduced, causing decreased strength of fibers, lead-
Aminoacidurias are clinically very important. It is esti-
ing to scurvy. The excretion of hydroxyproline in urine
mated that about 20 to 25% of the inmates of psychiatric
is increased in tumors infiltrating bones and in diabetic
hospjtals. ~ g fr'om one of the ai;!inoecid• Irias.
patients due to enhanced rate of protein catabolism. Most of them man~ s mental retardation. It is
important to remember that thei ntal retardation could
Fate of Carbon Skeletons be prevented, if the condition is · gnosed immediately
of Amino Acids after the birth, and adequate tre nt is started. Delay
During catabolism of carbon skeletons, amino acids may in diagnosis for each week will a preciably reduce the
enter into the TCA cycle and they can be converted to intelligence quotient.
glucose. In other words, those amino acids, which give
rise to citric acid cycle intermediates can be converted
:ji.. Clinical Case Study 19.1
to glucose. Hence, those amino acids entering into TCA
cycle, or at pyruvic acid level are called glucogenic A 50-year-old man with 2 years history of refractory
amino acids. This is shown in Figure 19.15. hypertension and occasional panic attacks reported to
Chapter 19: Aromatic Amino Acids 299
- - - - -- - ---
302 Section 8: General Metabolism
19-15. Which amino acid will give rise to a vitamin? 19-22. Try~~tn is excreted in large quantities in which
Tryptophan gives rise to pyridoxal. condition?
19-16. Serotonin is derived from which amino acid? Hartnup disease.
Tryptophan. 19-23. What is physiological importance of histidine?
19-17. What is serotonin? Histidine has a pK value of 6.8; and so it has the maxi-
It is derived from tryptophan. It is a vasoconstrictor. mum buffering capacity at physiological pH.
19-18. What is MAO (Monoamino oxidase)? 19-24. What is the precursor of histamine?
It deaminates monoamines. MAO inhibitors are used Histidine.
to treat hypertension and depression. 19-25. What is decarboxylation product of histidine?
19-19. Tryptophan is deficient in which food stuff? Histamine.
Maize and corn. 19-26. What is the clinical significance of histamine?
19-20. Pellagra is manifested in which conditions? It is a powerful vasodilator and mediator of anaphylaxis.
Niacin deficiency; Pyridoxal deficiency; Tryptophan 19-27. What are partially ketogenic and partially gluco-
deficiency; Carcinoid syndrome. genic amino acids?
19-21. What is Hartnup disease? Phenylalanine, tyrosine, tryptophan, isoleucine.
~J...a[omatic amino acids from in ~ 19-28. What are important aminoacidurias which cause
w ~~tsciotiqp fcoro regal t11b11les are~~./ mental retarrf;,~1-=n?
So tryptophan deficiency, and pellagra like symptoms Phenylketonuria, homocystinuria, maple syrup urine
are seen. disease.
.,______ __ _ Chapter 20
• Citric Acid Cycle
Chapter at a Glance
The learner will be able to answer questions on t he following topics:
Citric acid cycle Regulation of citric acid cycle
Significance of citric acid cycle Integration of metabol ism
Historical Perspectives
l
Before 1937, Car Martias, Fray Knoop and Albert Szent-Gyorgyi had elucidated most of the reactions described in this chapter.
The complete cycle was proposed by Sir Hans Krebs in 1937 (Nobel prize, 1953). The cycle is therefore named after him.
.. Please note that the name is Krebs cycle (there is no apostrophe). Krebs proposed the original name as TCA (tricarboxylic
acid) cycle, because he was not sure whether citric acid is a member of the cycle. Later, Ogston (1948) showed that the tricar-
boxylic acid in question is indeed citric acid, and so the name Citric Acid cycle was given later. Scheele in 1780 had isolated
citric acid from citrus fruits.
)'
H3C- C - 5--CoA
Acetyl-Co A CoA-SH
CHC.COO
I -
Ho- y-eoo
o=y-coo- 1 Citrate synthase
CHc-C~ O- Oxaloacetate (Irreversible) c;~~• ~
NADH+H+
ate dehydrogenase 2
HO- CK-COO- NAO+ • ~- ~ ~---~ CK-C00-
l f~;~H
2 Aconlt.aae 11 _
CH -COO Malate
2 Cis•Aconitate y-eoo _
• H ~~
FADH 2
CITRIC
6 Succlnate dehydrogenaae
ACID
CYCLE
O=C-COO-
Oxalosuccinate tH-C00-
3 lsocltnte tH 2-COO-
dehydrogenaae
5 Succinate thloldnase
1
NAO+
NADH•~• TPP / __,.,..-
Succinyl-CoA
Acetyt-CoA (2 carbon), enters the cyde. These are released as CO2 in steps 3 and 4. SoAcetyl-CoA is completely oxidized by
the time cycle reaches alpha ketoglutarate.
All reacllOns are reversible· extllpt 1st end 4th steps
Fig. 20.2: Krebs cycle or citric acid cycle or lricarboxylic acid cycle
g
Generating Steps in TCA Cycle marked in Figun3 20.5 and in Table 20.1 . The summary
is shown in Tablt3 20.2.
There are 3 NADH molecules generated during one
Note: Recent wiork shows that in the electron transport
cycle, each of them will give rise to 2½ ATPs on oxida-
chain, NADH produces only 2.5 ATPs and FADH only
tion by electron transport chain (ETC}; so altogether
they will give 3 x 2½ = 7½ (7.5) high energy phos- 1.5 ATPs. The old values are also given for comparison
phates. The FADH2 will generate 1½ molecules of ATP. in Table 20.1.
In addition, one molecule of GTP (equivalent to one Alpha ketoglutarate dehydrogenase reaction is the
molecule of ATP} is formed by substrate level phos- only one irrever.sible step in the cycle. The free energy
phorylation . Hence, per turn of the cycle, 10 high changes of the reactions of the cycle are such that the
energy phosphates are produced. These steps are cycle will operate spontaneously in the clockwise direction.
306 Section 8: General Metabolism
Only about 33% of liberated energy is trapped as Figure 20.9, all the major ingredients of food stuffs are
ATP. The rest is used to keep the body temperature at a finally oxidlzed through the TCA cycle.
higher level than the environment. Almost all the biochemical processes use ATP
for meeting energy needs-muscle contraction, active
~ Fina Common Oxidative Pathway
Citric acid cycle may be considered as the final com-
mon oxidative pathway of all foodstuffs. As shown in
a KG-dehydrogenase
Alpha ketoglutarate ? "\ " Hydroxy succinyl-TPP
+ Acetyl-CoA
i
TPP CO2
Trans-succinylase
Hydroxy succinyl-TPP 7 "\ " Succinyl lipoamide Aspartate Malate Citrate
CoA Dihydrolipoamide
(i., Complete oxidation of acetyl-CoA
2. ATP generatioin
DHL-Dehydrogenase v.3. Final common oxidative pathway
Dihydrolipoamide 7 " Lipoamide A.
.-5.
Integration of major metabolic pathways
Fat Is burned on the wick of carbohydrates
FAD FADH2
6. Excess carbohydrates are converted as neutral fat
FADH2 ---?....,._,.'\---+" FAD (z_ No net synthe:sis of carbohydrates from fat
(!!, Carbon skeletQns of amino acids finally enter the citric acid
NAO+ NADH + H+ cycle
Fig. 20.3: Alpha ketoglutarate dehydrogenase reaction; compare 9. Amphibolic p21thway
It with Figure 10.19 JO. Anaplerotic role.
4
Malate \
H
acetate ........,~
1 .X#
Citrate
(physiological
regulation)
i
/ \SATP 2
Fluoroacetate (toxic)
Aconita
Fumarate
Malonate (toxic)
FADH2 1.SATP
,
')
lsocitrate
1 = Citrate synthase
2 = Aconitase
3 = lsocitrate dehydrogenase
Succi: ate
A //
ATP
2 5,tl~NADH NADH inhibits;
ADP activates
4 = Alpha ketoglutarate (physiological)
~ ::. 25ATP
Oxalosuccinate
;{
dehydrogenase
5 = Succinic thioklnase
6 = Succinate dehydrogenase
7 = Fumarase 1)(__ 4 ~ ADH Alpha
8 = Malate dehydrogenase keto- ... CO
Arsenite (toxic)~ o 2
glutarate
2
Fig. 20.5: Summary of Krebs citric acid cycle. Enzymes are numbered. Reactions number 3 and 4 are carbon dioxide elimination
steps. Physiological regulatory steps are: Step No.1(citrate synthase) is physiologically inhibited by ATP. Step No.3 (ICDH) is inhibited
by NADH and activated by A DP. Steps where energy is trapped are marked with the coe,nzyme and the number of ATP generated dur-
ing that reaction. A total of 10 ATPs are generated during one cycle. Recent work shows that in the electron transport chain, NADH may
produce only 2 ½ ATPs and FADH only 1 ½ ATPs
Chapter 20: Citric Acid Cycle 307
l "'
Acetyl-CoA - - + Fatty acid synthesis
Tryptophan-+Jj,lanine /
W l'I
Aspartate Oxaloacetate Citrate Acetyl-CoA
I
Aspartate Oxaloacelate
Gl,oo,,.._
genesis ~
Jp0,2,
alate
Phenyl-
I( c'"\
/ J~j .
% alanine; - Fumarate o.-ketoglutarate
Tyrosine
Heme +-- Succinyl-CoA
Alpha keto-
glutaric acid
Ketolysis
!
Glutamic acid
Succinyl-
CoA
f 't',~ His~id_ine
!
GABA
,Vl<-l.'tlr4'
I'
Propionyl-CoA
f
Arginine
Praline
Odd chain
Valine. lsoleu~!ne, Methionine fatty acids
Fig. 20.8: Efflux of TCA cycle intermediates Fig. 20.9: Influx o'f TCA cycle intermediates
amino acids (Fig. 20.9). Glucogenic amino acids get are essential. Tllis is called anaplerotic role of TCA cycle
converted to intermediates of TCA cycle. (Greek word, ana = up; plerotikos = to fill). Anaplerotic
reactions are "fiilling up" reactions or "influx" reactions
TT-
r' Amphibolic Pathway or "replenishin!Q" reactions which supply 4-carbon units
All other pathways, such as beta-oxidation of fat or gly- to the TCA cycle (Fig. 20.9). The important anaplerotic
reactions are:
cogen synthesis are either catabolic or anabolic. But
a. Pyruvate to oxaloacetate by pyruvate carboxylase
TCA cycle is truly amphibolic (both catabolic and ana-
enzyme (sHe Fig. 10.21 ). It needs ATP.
bolic) in nature. (Greek, amphi = both). There is a con- b. Glutamate is transaminated to alpha ketoglutarate:
tinuous influx (pouring into) (Fig. 20.9) and a continuous and aspartate to oxaloacetate. Other important
efflux (removal) of 4-carbon units from the TCA cycle amino acids entering the TCA cycle are shown in
(Fig. 20.8). In a traffic circle, many roads converge and Figure 20.9.
traffic flows towards one direction. Since various com- c. Pyruvate can be carboxylated to malate by NADP•
pounds enter into or leave from TCA cycle, it is some- dependent malic enzyme.
times called as "metabolic traffic circle". Important
anabolic reactions related with citric acid cycle are: IREGULATION OF fa
a. Oxaloacetate is the precursor of aspartate ~ IC ACID CYCLE ~ c.o.l_
b. Alpha ketogutarate can be made into glutamate
Citrate and Citrate Synthase~ =
c. Succinyl-CoA is used for synthesis of heme
The formation a,f citrate from oxalo acetate and acetyl-
d. Mitochondrial citrate is transported to cytoplasm,
CoA is an important part of control (Step 1, Fig. 20.5).
where it is cleaved into acetyl-CoA, which then is the ATP acts as an allosteric inhibitor of citrate syn-
starting point of fatty acid synthesis (see Fig. 13.11 ). thase. Citrate inhibits PFK (key enzyme of glycolysis):
20-16. The amino acids entering TCA cycle as succinyl- C. Operates in the mitochondria close to ETC
CoA are all except: 0 . It is a cyclical process
A. Valine 20-19. Which of the intermediates listed is a dicarboxylic
B. Methionine hydroxy acid?
C. Tyrosine A. Succinate B. Fumarate
0 . lsoleucine C. Malato 0 . Oxaloacetate
20-17. Which of the reactions listed replenishes a TCA 20-20. Which amino acid can enter the TCA cycle as fuma-
cycle intermediate? rate and oxaloacetate?
A. Heme synthesis A. Aspartate B. Glutamate
B. Transamination of oxaloacetate C. Arginiine 0. Serine.
C. Carboxylation of pyruvate 20-21. From the pairs of inhibitors and enzymes, pick out
0 . Reutilization of ketone bodies. the mismatched pair.
20-18. The TCA cycle is the final common oxidative path- A. Alpha ketoglutarate dehydrogenase and arsenite
way beca use B. Malonate and succinate dehydrogenase
A. It provides large a fraction of energy
C. Fluorc,acetate and aconitase
B. Acetyl-CoA derived from all sources can be oxidi-
0 . lodoacetate and malate dehydrogenase
zed
20-1. What are the steps in which carbon dioxide is 20-6. How maniy ATPs are generated per one rotation of
liberated, during oxidation of glucose? the citric acid cycle?
Pyruvate dehydrogenase; lsocitrate dehydrogenase; 10ATP .
Alpha ketoglutarate dehydrogenase 20-7. What is tihe net yield of ATP from one molecule of
20-2. Acetyl-CoA is produced from what substrates? glucose in anaerobic glycolysis?
2ATP.
Pyruvate; Fatty acids; ketogenic amino acids
20-8. What is the net yield of ATP from one molecule of
20-3. Acetyl-CoA is used for what purposes?
glucose in aerobic glycolysis?
Oxidation in TCA cycle; fatty acid synthesis; choles-
?ATP.
terol synthesis; ketone body formation. 20-9. During cc>mplete oxidation, what is the net yield of
20-4. Give examples of substrate level phosphorylation. ATP from one glucose molecule?
1,3-bisphospho glycerate kinase; Pyruvate kinase; 32ATP.
Succinate thiokinase. 20-10. What is a1naplerosis?
20-5. Which is the substrate level phosphorylation step The reactions which involve replenishment of TCA
in the TCA cycle? cycle intermediates. For example, pyruvate gives rise
Succinate thiokinase to oxaloacetate.
Ir
_ _ _ _ _Chapter 2J
Biological Oxidation and
Electron Transport Chain
Chapter at a Glance
The learner will be able to answ er questions on the follow ing topics:
A Redox potentials E Organization of electro n t ransport chain
S Biological oxidation F Chemiosmotic t heory
C Enzymes and coenzymes 'i ATP synthase
J) High energy compound H Inhibit ors of )~TP synthesis
. ..Chapter- 1.
metabolism (intermediary) metabolism are descri~
(digestion) metabolism
Metabolism of - - - - A-H
N>
useful work
2. llG can predict the direction of a chemical reaction
3. Chemical reactionscan be coupled, which allowsan energeti-
H,0 0
<ADH•H o, cally unfavorable reaction to conclusion
4. llG measured under physiological conditions may be diffe-
ATP ADP + Pi rent from that at a standard state.
?~__..; r"
~ - b. Pyruvate kinase (see ~ cf\
Energy is used for: Muscle contraction Active
transport Biosynthesis
c. Succinate thiokinase(see Fig. 20.2).
----===----:-..
f"i:C--?
~o,.,._..,-__::!Y.=--
~ - - -- - - - - - - - - - -- - - - - - - - ' TP__ge eratlon is couI)led w ith a more exergonic
Fig. 21.2: ATP generation. Food is catabolized; energy from f~ metabolic reaction. ~n,6-0,'f'O-~
is trapped as ATP; it is then used for anabolic reactio~ ~ f-t-R _ _ ,·b, V\
t;,c cg."·,J...~ IOLQGICAL OXIDATION ~ mo~ t-o
The transfer of electron fr~.JIDhe, ~c~ ~ e~es T
through the respiratory chai o ox n is known as bio- t>.:>--
logical oxidation. Energy released during this process
is trapped as ATP. This coupling of oxidatiol'I with phos-
-
transferred to nucleoside diphosphate to form a tri-phos-
phate ithout the nelp of electron transport.chain, e.g. oxidation belong to the major class of xido~d~s.
a.
- ---
Bisphosphoglycerate kinase (see Fig. 10.11 ). They can be classified into the following 5 heai:rrngs:
L~,?E-tPr)
314 Section B: General Metabolism
@ oxidases
~ CO-NH 2
These enzym~talyze the removal of hydrogen from
substrates, but Cob~
0 x)(geo cao act as accept~of ijj
hydrogen, so that water is fo~ ed. R
AH + zS 0 RQTI\~ . ,,. ,_A+
2 2 IH
This group includes Cytochrome oxidase (termi- NADH
nal component of ETC), tyrosioase, polyphenol ~xida~. Fig. 21 .3: NAD· accepts H2
catechol ~1 <!§se and monoamine oxidase. f' ,
rn\ (MPtO)- r)~t tui~\e- vi. Pyruvate dehydrogenase P')'rl
~eJB.RLc Dehydrogenases ·~ ~dlj ~ vii. Alpha ketoglutarate dehydrogenase.o( 1';C7
These enzymes ca.tajyze the removal of hydrogen from b. NADP• linked dehydrogenases: NADPH cannot
a substrate, but oxygen can act as the acceptor. These be oxidized with concomitant production of energy.
enz mes are flavoproteins and the product is usually NADPH is used in reductive biosynthetic reac-
~~Alz, lions like f~tty acid syntheaj§ a~ ,,cholesterol syn~
- - - - --A+ H ~ ) l ' P sis, e.g. HMG-CoA ~ :..l ~ot~er example of
These flavoproteins contain eithe .f ~ r [1p
as Clt~ADPH linked dehydrogenase i~ _the glucose-
prosthetic group. Examples are L-a~ ~d d ox~ e 6-phosphate_de_h~ t ogenase (see Fig. 10.38:.
which catalyzes the o~ e deamination ~f L-amirJ.o c. FAD-linked dehydrogenases: When FAD 1s the
acids (see Chapter 17) and Xanthine oxidase (see coenzyme (unlike NAO•), both the hydrogen atoms
Chapter 38).. P,.0-'flVlr-."- ?;iu ">r\.£> , ' ·,j_ are attached t8 tQ~ ~avin~g. Examples:
.Qp.g~- i. Succinatl aehyd~~nase (see st~~~El~-J..9.:2)
@)
~,?GU\'\-\
Anaerobic Dehydrogenases ii. Fatty acyl-CoA ctehya16g~ s ,(see ~ - 8 )
~ mes catalyze the removal of hydrogen from iii. Glycerolphosphate dehydrogenase.CISPl))
a substrate but O!l_gen cannot act as the hydrogen d. Cytochromes: All the cytochromes, except cyto-
acceptor. They therefore require coenzy'ines as accep- chrome oxidase, are anaerobic dehydrogenases.
(Cytochrome oxidase is an oxidase, see above).
tors of the hydrogen atoms. When the substrate is
All cytochromes are h ~ m · s having iron atom.
oxidized, the coenzyme is reduced. - l'f"'I
a. NAO• linked dehydrogenases: NAD• is derived Cyto_c hro~~ c~ochr~m c and cyto romr( c J
. . . ·ct b f th ·t . B are In m1tochondna while cytol rom P-45 ana
from n1cotm1c ac1 , a mem er o e v1 amm com-
\&OJ
in "
plex (see Chapter 33). When the NAD• accepts the cytochrom~ l l Q Pplas ic reJLcu
two hydrogen atoms, one of the hydrogen atom~ HIGH ENERGY COMPOUNDS 0-1:t~e-v
is removed from the substrate as such. The other
4-H~trP These compounds when hydrolyzed will release a large
hydrogen atom is split into one hydrogen ion and
one electron. The electron is also accepted by the quantity of energy. High energy compounds are listed
NAO• so as to neutralize the positive charge on the in Table 21 .2.
/'~lO'(l'}
coenzyme molecule. The remaining hydrogen ion i denosine Triphosphate (ATP) ~c;r 0
release int in medium Fi . 21.3). COf"fi f'fl')l~
i. ATP is the universal currency of energy within the
H - H +H• +e- •
f,of"
~n
\a-"''~ L ~
;H2 + NAO·-+ A+ NADH + H' ..
II.
living cells. Structure of ATP is shown in Fi~~ ~-3.
The hydrolysis of ATP to ADP releases - ,U Tccal/
g' tOl" The NAO• linked dehydrogenases are: mol. The energy in the ATP is used to drive all
~~.,.;~ y eeraldehyde-3-phosphate dehydrogeriase g.pi;r e.Q..derg~ ic {bi~ yntlJ,etic) reactions. The energy
_ . ii. lsocitrate dehydrogenase '1 c: ?"' ( tJ\,e) } Re c1.U. efficiency of the cell is comparable to any machine
'bier-
=
•;
iii . Malate dehydrogenase {l.fl,::>1-) l~':J0-1") ""'faf! so far invented. ATP captures the chemical energy
~ iv. Glutamate dehydrogenase ('""''t) \ 1t~&. '" released by the combustion of nutrients and trans-
ca\,l.O.\o... v. Beta hydroxyacyl-CoA dehydrogenase oo"jJ fers it to synthetic reactions that require energy.
r~ ·fl\--;~
Pf>~ = ftT\>
fl>,~ ·
9kld1<:\ , U.,'6~U'1\\y ~e,
?Et' O.I'\..¢- c.,µ.,"'§b d,.~N'-.( Chapter 21: Biological Oxidation and Electron Transport Chain 315
--
carrier of chemical energy in the cell and coined tl'le In the,,E!ectron ,tran ort chain1 or esQiratory chain,
m()w..o, "'-lLVJ \,t 0-. uaJ~ ll'I
expression "e.oergy rich phosphate bmlds". Alexander the electrons are transferr ci from ADH to a chain
Todd (Nobel Prize 1957) elucidated its structure. of electron carriers. The electrons flow from the more
316 Section 8 : General Metabolism
I'
NADH Succinate
TC Complex I
-,ane.~
_ ""' c.,,. w
NAO+
.,,
'~Hz.;
It is also called N'..;;A=D=H
=-=C3o~ ~ ===:::-
Fig. 21.7: Complex I or NADH-CoQ reductase (NADH
genase complex) (!} drogenase complex. It is
@ NADH Generation
The NADH is generated during intermediary metaboli
2. l
~ the m oc
omplex II
ndna.
,,,-'/
.
A-1,::: ~1,4...C~}o~~ ,
'l
---------Cc, --------------
Summary:
--------
0-+ Fe-S-+ cyt. b-+ cyt. c1 -+ cyt. c
l
electrons to Complex II are:
of the enzy~s a single protein, ~ referred to as
Succinate dehydrogenase (see step 6, Fig. 20.2)
cytochrom~~ ,-
Fatty acyl-CoAdehydrogenase (see step 1, Fig. 13.8)
The sequential arrangement of members of electron
Mitochondrial glycerol plibsphate dehydrogenase.
1::-A-P ~n\;u:l ~e.v
>,:J (_r:,s:>~") transport chain iis shown in Box 21 .2 and Figure 21.11 .
Coenz me Q PTP ¼ ~ d - f=TTPf'IS~ \ <3'i\d"
The~ uinone Q is reduced successively t o ~ Current Concept, Energetics of IT'\ f'U0'-\-~
Q! · ) an mally to quinol (QH2 ) . It accepts a ATP Syntheisis
pair of electrons from NADH or'f-ADH2 through Complex I
o~ lex II respectively (Figs. 21 .6 and 21.11 ). The energy of electron transfer is used to drive protons
~ atrix by the ~ ~ s , I, Ill and IV __that
C lex Ill or Cytochrom ctase are]o \~e) The proton gradient thus created is
This contai s e b and c ochro e c1 , both maintained across the inner mitochondrial membrane till
contain heme prosthetic group. The sequ effectrons are..tr.,aruuecced to._px~oJoJ:m-water. The
tion inside the Complex Ill is shown in Fi electrochemical potential of this gradient is used to syn-
free energy change is - 10 kcal/mol; an rotons are
~
p~.gut. JU_ C.
esis of o A.~T n - . ..... 1.
ecuJe_js driven by the
flow o 3 pr on•~ through the TP s_ynthase (see below). ,
Cyto~ When NADH is ,oxidized 10 h dro en i s (proto [ '++'-4- 4111
It contains(one heme rosthetic group. The term cyto- pumped out (Fig . 21.11 ). According to recent findings, +-lj I
chrome is derived from Greek, meaning cellular colors. one NADH may generate only 2.5 ATP; and one FADH2
Axel Theorell (Nobel Prize, 1955) isolated it. Cytochrome may generate only 1.5 ATP. So, one molecule of glucose
c_collects electro1:ns from c _,mplex Il l and delivers them will generate only 32 ATPs. The traditional values and
to~ lex IV. ..... \-tl = @ ~the new values are compared in Table 21 .3.
Site 2 Site 3
4 protons pumped out 2 protons pumped out
MRcf\ ~) _1 c_o>,. 1
Complex I Complex Ill J Complex
Co Q --+ FeS -+ Cyt b -+ Cyt c1 Cyt c --+ IV 2 H+
nner
.membrane ------f
FMN--+ FeS
t
Complex II
- - ~1- ~ Cyt0 3, 1' l
Fe-S ti\ ~\l"\ r • f •
r
2. lsocitrate f t 8. Succinate
1/2 0 2 H20
3. Malate
4. Glutamate Fp (FAD)
f
FAo) 9. Ac yl-CoAl fo..tly )
10. Glycerol-3-phosphate
~ -
· -=: haveh
e,otn\\z.,,t.
h>
5. Beta hydroxyacyl-CoA
Lipoate
Co-~ o0 - v;:.t~<JJ\);
t
6. Pyruvate
7. Alpha ketoglutarate
718\e
is known that ATP synthesis actually occurs when the
· 0 G i; • - !IIOMJOblffi
proton gradient is dissipated, and not when the protons
Further, outside is- pos'ltive
are pumped out (Fig. 21 .13).
~ lative to the insiide (+0.14 V) (Fig. 21 .13).
, Mecco..
/ e,u-qH~
t
•iijY
High pH
LowH Matrix
concentration
F ig . 21 .13: Summary of ATP synthesis. One mitochondrion is depicted, with inner and outer memberanes. ETC complexes will push
hydrogen ions from matrix into the intermembrane space. So, intermediate space has more H' (highly acidic) than matrix. So, hydrogen
ions tend to leak into matrix through Fo. Then ATPs are synthesized. I, 11, 111, IV = cqmponents of ETC \ · , .~ r..-mAlt•cll'tQ
we.. o..u..nt" \ l"4 TI\()~ v-.y I..)'-'"" i; \v, "'--""""'. J'
i-1\. ~ll' V\.O\d.l~ €;'.~,~ ~~~"'fl\i .
reg11irajfp'ftbe roofor-
F1 Unit: It projects~ m~ t ~ talyzes
synthesis (Fig. 21.12). ATP synthesis requires
TP
15nal
syn~nesis\6f ATP, but enerfi;'. is
changes.
----=~--~
c.o,"\~
Th egulates the process. When
synthesis (oxi- . ATP level is low and P level is high, oxidative phos-
i.50.
dative phosphorylation) is through the. intecactjop of f 1
C
Zr 1/2 «,.. c, ~,..
cawes ¥~
phorylation proceeds at a rapid rate. This is called respl-
ratory control or acceptor control. The major source
of NADH and FADH 2 is the citric acid cycle, th, 3!
mding Change Mechanism ~ ~,'(\~ ,af>,N
4' 7 Under resting conditions, ' §ii Sdiiklllffl,
The fwpgjga c;han9e mechanip proposed by ~ I
(Nobel Prize, 1997) explains the synthesis of ATP So, flow of protons back into the mitochondria through
by the proton gradient. The ATP synthase is a •~ u- ATP synthase is minimal. The energy charge, or more
la~a..fbine". CQO'.lpacable to a "water-driven hammer, precisely ADP concentration, normally determines the
minting coins". S IL, · of electron transport.
Succinate +I
Malonate
Complex II I BAL
AntJmycin
--+-- - - - Carboxin
t
DNP Amobarb1tal DNP DNP
CCCP Rotenone CCCP CCCP
~ Ol19omycm
ADP+Pi ATP
Fig. 21 .14: Inhibitors of electron transport chain and oxidative phosphorylation. Abbreviations are shown in Table 21.4.
? ..l~.-.-.-.-....=. ".--.-.--------------- -l
iii. Barbiturates (amobarbital), sedative {f'<r · gradient (Table 21 .4; Fig. 21.14).
iv. Cb!,o rpromazine, tranquilizer ~/
v. Piericidin, antibiotic
n animals and
=
.=
<:=:.
. .
i. Carboxin ____h_u_m_a~ infants, the liberation of heat energy
is required to maintain body temperature. In brown
i. BAL (British anti-lewisite), antidote of war gas adipose tissue, thermogenesis is achieved by this '
ii. Naphthoguinone
iii. AntimycilJ
a protein present in the mitochondria
, , 11 • I , • t
of is also
' ·' i.
ii.
~ onoxid~, inhibits cellular respiration
Cyanide (CNj known to act as
iii. Azide (N3-J
iv. H_ydrogen~sulfide (H2S}
·••·--• How Does Shivering Produce Heat?
i. Carbox(n, inhibits transfer of ions from FADH2 Shivering is due to muscle contraction, it increases
Ii. Malonate, competitive inhibitor of succinate DH ATP hydrolysis. During proton entry for ATP synthesis,
>1) electron transport chain is stimulated.
I. Atractylosid~, inhibits translocase
a : sa a ::c:: &&61t1diil;;elJI nas-
ii. Oligomycin, inhibits flow of protons t hrough Fo
lono hores, e.g. Valinomycin , ~-l5Q•t,.;,cj&;'('\ - --··-··- .. ..------··· __,,.
~,.,e,, er.akf.
t" W:!;> I'(\ ~kt,g,(i
Bro~ Adipose Tissue and
c'!,\~~i~ - - - --
The uncoupling of proton flow releases the en rgy f the
electrochemical proton gradient as heat. Thi
-------
es
-- ..
Thermogenin in brown adipose tissue is a normal physjo!ogjcal function of brown
tissue. Brown adipose tissue gets its color fro
iii. f · di o e lls.
The mitochondria in brown fat contain thermo~enin.
It acts as a channel in the inner mitochondrial membrane
~ hapter 21: Biological Oxidation and Electron Transport Chain ~21
:'f~
.,.. H
•~- free radicals or any other form of stress. As the mem- (:{)u
brane permeability increases, there is opening of a
release of the mitochondrial permeability transition pore (MPTP). It
enerf as . results in dissipation of mitochondrial proton gradient,
,, . ~,. t!!fll-
&
ATP depletion and release of cytochrome c. Thisrc a.Q-
ecific Inhibitors of.ETC chrome C acts_as._a tngger QI apfiptosis by forming an
an drug, but is cardiotoxic. It ; poptosomr c~ pl e~~h other pro-apoptotic factors.
inactivates oclfrorl~ Mase, affects ion pumps and The lm!@tor ~s then activated leading to activa-
inhibits ATP synthase. l(lficreases free radicals leadlng tion of effec_tou~spases, and finall)l--the cell death.
to mitochondrial membrane damage. End effect can be (i!> - ·c .---=== ' ------ - --
co-~ mein hfitochond1rial Dysfunction and
Insulin Resistance D~
- - • .L• ..------. • .l - ~- ;
~v. J.J.
© assav contains cyanogens, and improper ero-
in animal studies. There is also a decrease in insulin-
stimulated glucose uptake by skeletal muscle in elderl
cess1ng of cassava can result in toxicity. HCN is released
from cx.aoogellS..J>_y 11.
glucosida~ present in plants
people. Cumulative damage of mrtocilo dria b OS ?}
may be the reason. Therefore
, Cyanoglycosid~ mygdalin may also be found in
edible plants, such as al~ onds, ap~icots, pefches, plJ'ms,
chdrries, sorghum, soybeans, and bamboo shoots.
• i)! insulin resistance may o
chondrial biogenesis and
Diseases Associated with Mitochondria
Mitochondrial DNA is inherited cytoplasmically and is 0 'Dt ~c..y\. G.~c.W'\,
therefore transmitted maternally. OXPHOS (oxidative ·• Clinical Case Study 21 .1
A 68-year-old female in a hypertensive crisis is being
. treated in the intensive care unit (ICU) with intravenous
r o
blin ness i o males. It is caused by a single base nitroprusside for 48 hours. The patient's blood pressure
io tJA QH Coenzyme Q reductase. (C'omp~'i) was brought baick down to normal levels; however, she
in'tochondrial Permeability was complaining of a burning sensation in her throat
T ·t· p (@JP) and mouth followed by nausea and vomiting, excessive
rans, 10n ore ) sweating. (d.1apt1ores1s
. ) , ag1 -
-1a110n, and dyspnea. An arte-
C ochrome also the ru.e:§3tor of apapto$is (pro- rial blood gas revealed a significant metabolic acidosis. A
gramme cell death}. This can happen in response to serum test sugg1ests a metabolite of nitroprusside, thiocy-
anate, is at toxic levels.
1. What is tho likely cause of her symptoms?
2. What is the biochemical mechanism of this prob-
lem?
3. What is tho treatment for this condition?
0
Axel HT PeterD PaulD John E •• Clinical Case Study 21 .2
Theore/1 Mitchell Boyer Walker
NP 1955 NP 1978 NP 1997 NP 1997 A 55-year-old man was treated in the ICU with intrave-
1903-1982 1920-1992 b. 1918 b. 1941 nous nitropruss;ide for hypertensive crisis for 48 hours.
322 Section B: General Metabolism
BP was restored, but he had a burning sensation in his mining, electroplating, jewelry manufacture and X-ray
throat and mouth, followed by nausea and vomiting, film recovery. It can occur during fumigation of ships,
excessive sweating, agitation and dyspnea. There was warehouses, etc. and are also used commonly as sui-
a sweet almond smell in his breath and arterial blood cidal agents, espeicially by terrorists and healthcare and
gas analysis revealed severe metabolic acidosis. What laboratory workers. Cyanide affects all body tissues •
is the likely condition? How is it treated? What is the and attaches to many metalloenzymes, rendering them
pathogenesis? inactive.
Treatment includes administration of amyl nitrite,
sodium nitrite and sodium thiosulfate, increasing oxygen
8 · Clinical Case Study 21.1 Answer concentration in inspired air and sodium bicarbonate
Diagnosis: Cyanide poisoning from toxic dose of nitro- therapy. Amyl and sodium nitrites induce methemo-
prusside. globin formation , it combines with cyanide and reduces
its toxicity. Sodium thiosulfate converts cyanide to thio-
Biochemical mechanism: Cyanide inhibits mitochon-
cyanate and whicl1 is excreted in urine. Hydroxycobala-
drial cytochrome oxidase, blocking electron transport
min combines with cyanide to form cyanocobalamin
and preventing oxygen utilization. Lactic acidosis results
which is excreted! through urine. Sodium bicarbonate
secondary to anaerobic metabolism.
reduces lactic acidosis.
Treatment: Supportive therapy, oxygen, and antidotal
therapy with sodium nitrite, and sodium thiosulfate. (hEARNINGi POINTS, CHAPTER 21
Clinical correlation: Malignant hypertension is diag-
1. Oxidation of food stuff occurs in 3 stages-primary
nosed when there is elevated blood pressure (systolic
metabolism where macromolecules are converted
levels of 220 mm Hg and/or diastolic blood pressures
to smaller units, secondary metabolism where
exceeding 120 mm Hg). The symptoms may include
reducing equivalents are formed and tertiary meta-
severe headache, neurological deficits, chest pain, or
bolism where energy is released.
heart failure. Hypertensive emergencies require imme-
diate lowering of the blood pressure to lower levels.
2. Oxidation is loss of electrons and reduction is gain "
One hazard of abruptly lowering the blood pressure of electrons. A pair that exists in both oxidized and
is causing hypotension and subsequent ischemia to the reduced state is a redox couple.
brain or heart. Sodium nitroprusside induces a smooth 3. In substrate level phosphorylation, energy from
fall in blood pressure. One side effect of sodium nitro- high-energy compound is directly transferred to
prusside is that its metabolite is thiocyanate, and with NOP to form INTP without the help of electron trans-
prolonged use, cyanide poisoning may result, which port chain.
inhibits the electron transport chain. Thus, in clinical 4. Transfer of electrons from reduced coenzymes
practice, short-term nitroprusside is used. through respiratory chain to 0 2 is known as bio-
logical oxidation.
5. The energy released is trapped as ATP. This cou-
• Clinical Case Study 21.2 Answer pling of oxidation with phosphorylation is called
Patient is most probably suffering from cyanide poison- Oxidative phosphorylation. All enzymes of biologi-
ing. Cyanide inhibits mitochondrial cytochrome oxidase, cal oxidation are oxidoreductases. ..
blocks the electron transport chain and prevents oxy- 6. Electron flow occurs through successive dehydro-
gen utilization. Lactic acidosis is secondary to anaero- genase enzymes (located in the inner mitochondrial
bic metabolism. Cellular oxygen metabolism is impaired membrane), together known as Electron Transport
and can produce death within minutes. Nltroprusside Chain; the el,ectrons are transferred from higher to
therapy, which is the drug of choice for hypertensive lower potenti;al.
emergency, on prolonged usage can produce cyanide 7. NADH is imp,ermeable to mitochondrial membrane.
poisoning. Hence, in clinical practice, nitroprusside is Hence it is transferred via malate-aspartate shuttle
used only for short term. in liver, kidneiy and heart as NADH reducing equi-
Causes for cyanide poisoning include smoke inha- valents and in skeletal muscles as FADH 2 through
lation from residential or industrial fires, metal trades, glycerol 3-phosphate shuttle.
Chapter 21: Biological Oxidation and Electron Transport Chain 3~!3
8. The ETC has 4 distinct multiprotein complexes-- 9. Inhibitors of oxidative phosphorylation include atracty-
viz; Complex I, 11 , Ill and IV connected by two loside and oligomycin. Cyanide inhibits terminal cyto-
mobile carriers to Co Q and cytochrome c. chrome and brings cellular respiration to stand still.
21-1 . All contain high energy bond, except: 21 -8. Which is the inborn error due to a mutation in
A. ATP 8 . Glucose-6-phospahte NADH-0 reductase?
C. Acetyl-CoA D. Phosphoenolpyruvate A. Amyotrophic lateral sclerosis
21-2. All the following phosphate esters are high energy 8 . Leber's oplil; myopathy
compounds, except: C. Ragged red fiber disease
A. Cretine phosphate D. Duchenne muscular dystrophy
8 . Carbamoyl phosphate 21-9. Valinomycin inhibits oxidative phosphorylation,
C. 2,3-bisphosphoglycerate because:
D. 1,3-bisphosphoglycerate A. It inhibits ATP synthase
21-3. Coenzyme Q catalizes electron transport between 8 . It inhibits cytochrome oxidase
A. FADH and cytochrome b C. It forms a complex with NADH
D. It makes mitochondria permeable to potassium
8. It is the last member in the electron transport chain
21-10. Which is true with regard to oligomycin?
C. NADH and ubiquinone
A. It inhibits oxidative phosphorylation
D. Cytochrome Q and cytochrome C
8 . It is an uncoupler
21-4. Which contains copper?
C. It inhibits translocase
A. Cytochrome oxidase
D. It acts as an ionophore
8 . Cytochrome b5
21-11 . Which is true with cytochrome reductase?
C. Coenzyme Q
A. It catalyzes the electron transport between FADH
D. Cytochrome P-450 and cytochrome b
21-5. Death due to cyanide poisoning is a result of: 8 . It is the last member in the electron transport chain
I
A. Cyanide hemoglobin complex formation C. It catalyzes the electron transport between NADH
8 . Cyanide inhibiting complex I of respiratory chain and ubiquinone
C. Cyanide inhibiting cytochrome oxidase
..
D. It catalyzes the electron transport between Co Q
D. Cyanide blocking oxygen transport in blood and cytochrome C
21-6. All are true with ATP synthase, except: 21-12. Which is an NAO• linked dehydrogenase?
A. It has two subunits, F1 and Fo A. Succinate dehydrogenase
8 . F1 subunit has catalytic activity 8 . Fatty acyl-CoA dehydrogenase
C. F0 subunit serves as a proton channel C. Malate dehydrogenase
D. Proton flow is from F1 to Fo D. Glucose-6-phosphate dehydrogenase
21-7. Oxidative phosphorylation is inhibited by the 21-13. Which of the following compound does not con-
following, except: tain a high energy bond?
A. Oligomycin 8. Carbon monoxide A. Fructose-1 ,6-bisphosphate
C. Hydrogen cyanide D. Pyrophosphate 8 . 1,3-bisphosphoglycerate
324 Section B: General Metabolism
C. Succinyl-CoA C. Peroxiclases
D. Creatine phosphate D. Reducatses
21-14. Allare true with regard to mitochondria, except: 21-18. Which of tlhe following is NOT a high energy com-
A. Glycolytic enzymes are in mitochondrial matrix pound?
B. Inner mitochondrial membrane contains enzymes A. creatinB phosphate
B. 1,3-bisphosphoglycerate a
of electron transport chai n
C. Fluid matrix contains enzymes of TCA cycle C. Phosphoenolpuruvate
D. Acyl-CoA synthetase is localized in the outer D. Glucos,e-6-phosphate
21-19. The high energy phosphate with highest free r
mitochondrial membrane
energy of hydrolysis is:
21-15. ETC is located in the:
A . PEP B. ATP
A. Outer mitochondrial membrane
C. GTP D. Succinyl-CoA
B. Inner mitochondrial membrane
21-20. Wh ich of the electron carriers is soluble and
C. Mitochondrial matrix
mobile?
D. Nucleus
A . CoQ B. Cytochrome c
21-16. When electron transport occurs:
C . Cytochrome a D. Cytochrome b
A. Electrons are transferred from more electro- 21-21. Which of t he following is NOT true regarding ATP
negative to electropositive complexes synthesis'il
B. Electrons get tightly bound to the complexes A . ATP is formed by phosphorylation of ADP
C. Energy is required for the transport of electrons B. Proton gradient is dissipated
D. Electrons flow freely from a high redox potential to C . Oxidati,on is coupled to phosphorylation
low redox potential D. Inner membrane pores are opened to release ATP
21-17. The enzymes of ETC belong to the following 21-22. Which of lthe hormones listed can uncouple oxi-
classes, except: dative phosphorylation in high concentrations?
A. Oxidases A. Insulin B. Cortisol
B. Dehydrogenases C. Thyroxiine D. Glucagon
21 -1. Give examples of high energy compounds. 21-6. What are FAD linked dehydrogenases?
ATP, GTP, Creatine phosphate, 1,3-bisphosphoglyce- Succinate dehydrogenase; acyl-CoA dehydrogenase.
rate, Phosphoenolpyruvate, Acetyl-CoA, Succinyl-CoA. 21-7. Which cytC)chromes contain copper?
Cytochrome oxidase.
21-2. What are the activities taking place inside mito-
21 -8. Wh ich is complex V of respiratory chain?
chondria?
ATP synthase.
Citric acid cycle; Electron transport chain; Beta oxi- 21-9. What is Valinomycin ?
dation fatty acid. It acts as an ionophore; dissipates the proton gradient:
21 -3. Where are enzymes of citric acid cycle located? and so inhilbits ATP synthesis.
Fluid matrix. 21 -10. What is Atractylocide? •
21-4. Cytochrome oxidase is present in which complex? It inhibits tr:anslocase; and inhibits ATP synthesis.
21-11 . What is Oligomycin?
Complex IV.
It inhibits o:(idative phosphorylation.
21-5. What are NAO• linked dehydrogenases?
21 .12. Name inhilbitors of oxidative phosphorylation.
Glyceraldehyde-3-phosphate dehydrogenase; Pyru- Oligomycin; Carbon monoxide; Cyanide.
vale dehydrogenase; alpha ketoglutarate dehydroge- 21-13. What is the cause for death due to cyanide poi-
nase; lsocitrate dehydrogenase; Malate dehydroge- soning?
nase; Beta hydroxyacyl-CoA dehydrogenase. Cyanide inhibits cytochrome oxidase.
_ _ _ _ _Chapter 22
Heme Synthesis and
I "
Breakdow1n
Chapter at a Glance
The learner w ill be able to answer questions on t he following topics:
St ructure of heme Bilirubin metabolism
Biosynthesis of heme Plasma biliriubin
Porphyrias Jaundice
•
Red blood cells (RBCs) are biconcave discs, with a
diameter of about 7 microns. RBCs live for about
ISTRUCTURE OF HEME
Heme is usually pronounced as "heem".
120 days in peripheral circulation, during which time
Heme is a derivative of the porphyrin. Por-
they traverse about 160 km! In a 70 Kg person, there will
phyrins are cyclic compounds formed
be about 25 x 1012 RBCs and 750 g of hemoglobin (Hb).
by fusion of 4 pyrrole rings linked by
100 ml blood contains about 14.5 g of Hb. Mature RBC
methenyl (=CHI-) bridges (Fig. 22.1).
is non-nucleated; have no mitochondria and does not Since an atom o,f iron is present, heme is
contain TCA cycle enzymes. However, the glycolytic Hans Fischer
a ferroprotoporphyrin . The pyrrole rings NP 1930
pathway is active which provides energy Erythropoietin are named as I, 11 , 111, IV and the bridges 1881-1945
is the major stimulator of erythropoiesis. It is synthesized as alpha, beta, gamma and delta. The
in kidney. RBC formation in the bone marrow requires possible areas of substitution are denoted as 1 to 8 (Fig.
amino acids, iron, copper, folic acid, vitamin 8 12 , vita- 22.2 and Table 22.1 ). Type Ill is the most predominant
min C, pyridoxal phosphate, and pantothenic acid; they in biological systems. Hans Fischer synthesized heme
are used as hematinics in clinical practice. in laboratory in 1920 (Nobel Prize, 1930). The usual
Heme is present in: substitutions am:
a. Hemoglobin a. Propionyl (- CH2- CH2- COOH) group
b. Myoglobin b. Acetyl (--CH2- COOH) group
c. Cytochromes C. Methyl 1 :-c H3} group
d. Peroxidase d. Vinyl (- C H=CH2 ) group.
e. Catalase Complete structure of heme is shown in Figure 22.3.
f. Tryptophan pyrrolase
g. Nitric oxide synthase. IBIOSYNTHESIS OF HEME
Heme is produced by the combination of iron with Heme can be synthesized by almost all the tissues in the
a porphyrin ring. Chlorophyll, the photosynthetic green body. Heme is synthesized in the normoblasts, but not
pigment in plants is magnesium-porphyrin complex. by the matured erythrocytes.
326 Section B: General Metabolism
0 N
H
2
3
Fig. 22.1 : Pyrrole ring
•
4
7
Ill
6 5
The pyrrole rings are numbered I to IV; the bridges named as alpha
to delta and the possible sites of substitutions are denoted from
1 to 8. (For brevity, the bridges and double bonds are sometimes
omitted, as shown on the right).
V = Vinyl Succinyl-CoA
= Proplonyl coo- ALA coo- ALA coo-
Fig. 22.3: Structure of heme
I synthase
(+) PLP
I synthase I
CH2 CH2 (+) PLP CH2
- I
coo_
6
PPG-111 - -.. PP __:_. Heme 1 !
Glycine + Succinyl-CoA
COO CH
I I 2
Mito-
chondria t fi ALA
... CH2 CH2 CPG-111
' D
C HN
NH+
I 2
Cytoplasm~CPG-II I
CO2 ~I N\3
l
ALA
2
3
2 ~ 3
ALA+ALA Porphobilinogen UPG-111 •4-----c==---- PBG
(PBG)
Fig. 22.6: Summary of heme biosynthesis. The numbers denote
4 x Porphobilinogen the enzymes. Pant of synthesis is in mitochondria, and the rest in
c ytoplasm
A p
Step 6: Generation of PP
A J LA
The protoporphyrinogen-III is oxidized by the enzyme
p~p Uroporphyrinogen Ill
PA l
(UPG-111)
Acetyl
protoporphyrino!gen oxidase to protoporphyrin-I11 (PP-Ill).
The methylene bridges (-CH2 ) are oxidized to methenyl
1'--
4 bridges (-CH= ) and colored protoporphyrin-9 is thus
Mp 4CO, Jhyl formed.
,
M
JLM
p~ p
Coproporphyrinogen Ill
(CPG-111)
NADP
Step 7: Generation of Heme
The last step ,s the attachment of ferrous iron to the
NADPH+H+ protoporphyrin to form the heme. The enzyme is heme
PM synthase or fe,rrochelatase.
Propionyl
i
Vinyl
Iron atom is coordinately linked with 5 nitrogen
atoms (4 nitrog,en of pyrrole rings of protoporphyrin and
M V 1st nitrogen atom of a histidine residue of globin). The
MJLM
P ~V
Protoporphyrinogen Ill
(PPG-Ill)
remaining valency of iron atom is satisfied with water or
oxygen atom (Fig. 22.7). A summary of the pathway is
shown in Figure 22.6.
When the f,errous iron (Fe++) in heme gets oxidized
p M 6~ 4H
to ferric (Fe•++) form, hematin is formed , which loses the
Protoporphyrin 111 property of carrying the oxygen. Heme is red in color, but
hematin is dark brown.
easier. The rate-limiting enzyme is in the mitochon- c. Porphyrias with both erythropoietic and hepatic
dria. The steps 1, 5, 6, and 7 are taking place inside abnormalities.
mitochondria, while the steps 2, 3 and 4 are in This classification is based on the major site, where
cytoplasm. the enzyme deficiency is manifested. The clinical
4. Drugs like barbiturates induce heme synthesis. manifestations vary. Porphyrias in general, are not •
Barbiturates require the heme containing cyto- associated with anemia.
chrome P450 for their metabolism. Out of the
total heme synthesized , two thirds are used for
Acute Interm ittent Porphyria (AIP) '
cytochrome P450 production. It is inherited as an autosomal dominant trait. PBG-
5. The steps catalyzed by ferrochelatase and ALA deaminase (uroporphyrinogen-I-synthase) is deficient
dehydratase are inhibited by lead. So lead toxicity (Table 22.2 and Fig. 22.8). This leads to a secondary
causes anemia. increase in activiity of ALA synthase, since the end-
product inhibition is not effective. The levels of ALA
and PBG are elevated in blood and urine. As they are
DISORDERS OF HEME SYNTHESIS
colorless compoU1nds, urine is colorless when voided,
Porphyrias are a group of inborn errors of metabolism but the color is increased on standing due to photo-
associated with the biosynthesis of heme. (Greek oxidation of PBG to porphobilin. Hence, urine samples
'porphyria' means purple). These are characterized by for PBG estimatiion should be freshly collected and
increased production and excretion of porphyrins and or transported in dark bottles. Porphyrins are not excreted
their precursors (ALA + PBG). Most of the porphyrias or elevated in blood; so there is no photosensitivity.
are inherited as autosomal dominant traits (Table 22.2). As the name indicates, the symptoms appear inter-
Porphyrias may be broadly grouped into 3 types: mittently and they are quite vague. Hence, it is at times
a. Hepatic porphyrias called the "little imitator". Most commonly, patients pre-
b. Erythropoietic porphyrias sent with acute abdominal pain . The patients often land
up with the surgeon as a case of acute abdomen and on
several instances exploratory laparotomies are done.
Pyrrole-N N-Pyrrole An attack is precipitated by starvation and symptoms
are alleviated by a high carbohydrate diet. Drugs like
barbiturates, whic:h are known to induce ALA synthase,
can precipitate an1attack.
Pyrrole-N N-Pyrrole Another group may have neurological manifesta-
tions like sensory ;and motor disturbances, confusion and
N-Histidine (proximal)
agitation. Some patients may present with psychiatric
Fig. 22.7: In the he me molecule, iron atom is coordinately linked problems and may be treated accordingly. It is said that
with nitroge n atoms King George Ill ('1760- 1820) was suffering from mania
(PBG = Porphobilinogen; CP = Coproporphyrin; ALA = delta amino levulinic acid; UP = uroporph,yrins). (Enzyme numbers are given as shown in
Fig ure 22.8. See also Figure 22.9 for appearance of porphyria cutanea tarda)
Chapter 22: Heme Synthesis and Breakdown 329
l G)
Glycine + Succinyl-CoA
ALA-synthase
• l® ALA-dehydratase
Porphobilinogen (PBG)
t
Uroporphyrinogen (UPG)
Porphyria
© UPG-decarboxylase - - - -·I~ -cutanea
- --tarda
---
t
Coproporphyrinogen (CPG)
® CPG-oxidase
·I
Hereditary
coproporphyria
Protoporphyrinogen (PPG)
t@
j_
PPG-oxidase
·I Porphyria variegata
t
Protoporphyrin
due to porphyria. Many of his obstinate decisions, inclu- manifestations relate to the skin due to the photosensi-
ding the ones which led to war of American independence, tization by the presence of porphyrins in the capillaries.
were made when he had acute attacks of intermittent Reactive oxygen species (free radicals) are the cause
porphyria. for cell destruction (see Chapter 30). Repeated attacks
of dermatitis and scarring lead to a typical facial
ALA Synthase (ALAS) Deficiency deformity often r,eferred to as 'monkey face'. Repeated
It is the key enzyme of the pathway. ( See enzyme ulceration and scarring may cause mutilation of nose,
No.1 in Fig. 22.8) There is excessive production of ear and cartilagB. This may mimic leprosy. When UV
heme intermediaries causing neurological porphyrias. light is reflected ,on to teeth a red fluorescence is seen;
Porphyrin precursors and porphyrins in urine and feces this is called erythrodontia.
are normal. Definitive diagnosis is by demonstration of
mutation in erythroid ALA synthase. Porphyrias in General
' Congenital Erythropoietic Porphyria
Accumulation of porphyrin precursors, ALA and por-
phobilinogen leads to neurovisceral manifestations.
It is inherited as an autosomal recessive trait. (Table Accumulated uroporphyrin and coproporphyrin cause
22.2). (See enzyme No. 3b in Fig . 22.8) Normally the delayed bullous lesions. On the other hand, being more
type Ill isomer is produced in larger amounts, but in this lipophilic, protoporphyrin associates with cell membranes
condition, type I isomer is formed considerably. They and causes burning sensation and inflammatory reac-
are converted to porphyrins type I. This would lead to tion in skin exposed to sun. Hence biochemical diagnosis
ineffective feedback inhibition which further increases of porphyria will be considered based on the major
the rate of formation of type I porphyrins. clinical features, namely; a) neurovisceral and b)
Their level in blood increases leading to photo- cutaneous manifestations. The salient features of diffe-
sensitivity. Their excretion in urine makes the urine rent types of porphyrias are given in Table 22.2. See
dark red in color (portwine appearance). The major also Figure 22.9.
330 Section B: General Metabolism
ALA
Urine µg/24 hr
4000
Fecesµg/g
Nil AIP BOX 22.1: Bile . .. . .. .. .
Bile pigments arie Bilirubin and Biliverdin. They are the b reak-
PBG 1500 Nil AIP
down products of heme; they are useless excretory products.
CP 200 so CEP, HCP
Bile salts are the sodium salts of bile acids (glycocholate and
UP 25 so Acquired porphyria, CEP
taurocholate). ThEiy are produced from cholesterol; they help in
PP Nil 100 Acquired porphyrias t he absorption of fat.
(AIP = acute interm ittent porphyria; CEP = congenital erythropoietic Both bile pigments and bile salts are p resent in the bile.
porphyria; HCP= hereditary coproporphyria)
Hemoglobin Heme
(H me oxygenase system:
} - . Globin -+ amino acid pool
Cytochrome C and NADPH)
Heme Ring Carbon monoxide released
5 opens
•
!. . .__,.
l~:;~,i~tennase (NADPH; Cytochrome c) Iron liberated
Iron -+ Iron re-utilized
M V MP PM M V
• Biliverdin
i
Bilirubin
IV I I
OH
Fig. 22.10: Catabolic pathway of hemoglobin
Biliverdln
NADPH + H+
UDP-glucuronyl-transferase verdin reductase
Bilirubin / -........._ • Bilirubin NADP+
r monoglucuronide
Bilirubin
UDP-glucuronic UDP
M V M p p M M V
acid
Bilirubin UDP-glucuronyl-transferase
mono- • Bilirubin
glucuronide / dlglucuronide
UDP-glucuronic UDP
acid
Fig. 22.12: Production of bilirubin diglucuronide Fig. 22.11: Breakdown of heme
displace bilirubin from albumin. Hence, care should be colorless tetrapyrrole urobilinogen (UBG). It is further
taken while administering such drugs to newborn babies reduced to stercobilinogen (SBG). The SBG is mostly
• to avoid kernicterus. Liver takes up bilirubin by a carrier excreted through feces (250-300 mg/day) (Fig. 22.13).
mediated active process.
Enterohepatic Circulation
Conjugation in Liver
About 20% of the USG is reabsorbed from the intes-
Inside the liver cell, the bilirubin is conjugated with glucu- tine and returned to the liver by portal blood. The UBG
ronic acid, to rnake it water-soluble. (Fig. 22.12). About is again re-excreted (enterohepatic circulation) (Fig.
80% molecules are in the diglucuronide form, while 22.13). Since the USG is passed through blood, a small
20% are monoglucuronides. Drugs like primaquine, fraction is excreted in urine (less than 4 mg/day).
novobiocin , chloramphenicol, androgens and pregnane-
diol may interfere in this conjugation process and may Final Excretion
cause jaundice.
USG and SBG are both colorless compounds but are
Excretion of Bilirubin to Bile oxidized to colored products, urobilin or stercobilin res-
RBC lysis
... rt1es of conjugated and free b1llrub1n
Hemoglobin released Free bilirubin Conjugated bi/irubin
In water Insoluble Soluble
Heme
In alcohol Soluble Soluble
Reticuloendothelial
Normal plasma level
In b ile
0.2-0.7 mg/ dl
Absent
0.1 -0.4 mg/dl
Present
•
system
In urine
Absorption from GIT
Always absent
Absorbed
Normally absent
Not absorbed
•
Diffusion into tissue Diffuses Does not diffuse
Bilirubin diglucuro nide van den Berg's test Indirect positive Direct positive
I HYPERBIL.IRUBINEMIAS
Fouchet's; Gmelin's; Ehrlich's Schlesinger's
Bile pigments van den Bergh test test
Bilirubin +ve - ve - ve
Depending on the nature of the bilirubin elevated , the
Bilinogens (UBG) - ve +ve -ve
condition may be grouped into conjugated or uncon-
Bilins (UB + SB) - ve - ve +ve
jugated hyperbilirubinemia. Based on the cause it may
also be classified into congenital and acquired.
while conjugated bilirubin is only 0.1-0.4 mg/dl. If the
plasma bilirubin level exceeds 1 mg/dl , the condition Congenital Hyperbilirubinemias
is called hyperbilirubinemia. Levels between 1 and
They result from abnormal uptake, conjugation or exc-
2 mg/dl are indicative of latent jaundice. When the
retion of bilirubin due to inherited defects.
bilirubin level exceeds 2 mg/dl , it diffuses into ti ssues
producing yellowish discoloration of sclera, conjunctiva, Crigler-Najjar Syndrome
skin and mucous membrane resulting in jaundice.
Here the defect is in conjugation. In type 1 (congenital
lcterus is the Greek term for jaundice.
nonhemolytic jauindice), there is severe deficiency of
UDP-glucuronyl transferase. The disease is ofte n fatal
Van den Bergh Test for Bilirubin
and the children dlie before the age of 2. Jaundice usually
Properties of bilirubin are shown in Table 22.5. Bilirubin appears within the first 24 hours of life. Unconjugated
reacts with diazo reagent (diazotized sulfanilic acid) to bilirubin level increases to more than 20 mg/dl , and
produce colored azo pigment. At pH 5, the pigment is hence kernicterus results.
Chapter 22: Heme Synthesis and Breakdown 333
The type 2 disease is a milder form; only the second second pregnancy, the Rh antibodies will pass from
stage of conjugation is deficient. When barbiturates are mother to the fetus. They would start destroying the
given, some response is seen and jaundice improves. fetal red cells even before birth. Sometimes the child is
born with severe hemolytic disease, often referred to as
Gilbert's Disease erythroblastosis fetalis . When blood level is more than
It is inherited as an autosomal dominant trait. The defect 20 mg/dl, the capacity of albumin to bind bilirubin is
(; ,, is in the uptake of bilirubin by the liver. Bilirubin level exceeded. In young children before the age of 1 year, the
is usually around 3 mg/dl, and patient is asymptomatic, blood-brain barrier is not fu lly matured, and therefore free
except for the presence of mild jaundice. bilirubin enters the brain (Kernicterus ). It is deposited
in brain , leading to mental retardation , fits, toxic ence-
Dubin-Johnson Syndrome phalitis and spasticity. If the child develops hemolytic
It is an autosomal recessive trait leading to defective disease, child may be given exchange transfusion along
excretion of conjugated bilirubin; so conjugated bilirubin with phototherapy and barbiturates. Phototherapy with
in blood is increased. There is defect in transport of blue light (440 nm wavelength) isomerizes insoluble bili-
conjugated bilirubin into bile. The bilirubin gets deposited rubin to more soluble isomers. These can be excreted
in the liver and the liver appears black. The condition is through urine without conjugation.
referred to as Black liver jaundice.
Hemolytic Diseases of Adults
Rotor Syndrome This condition is seen in increased rate of hemolysis. It
Bilirubin excretion is defective, but there is no staining of usually occurs in adults. The characteristic features are
the liver. It is an autosomal recessive condition. increase in unconj ugated bilirubin in blood, absence
of bilirubinuria and excessive excretion of UBG in urine
Acquired Hyperbilirubinemias and SBG in feces (Table 22.6). Common causes are:
i. Congenital spherocytosis
Physiological Jaundice ii. GPO deficiency
It is also called as neonatal hyperbilirubinemia . In all iii. Autoimmune hemolytic anemias
new born infants after the 2nd day of life, mild jaundice iv. Toxins like carbon tetrachloride.
appears. This transient hyperbilirubinemia is due to
an accelerated rate of destruction of RBCs and also Hepatocellular Jaundice
because of the immature hepatic system of conjugation The most common cause is viral hepatitis, caused by
of bilirubin. In such cases, bilirubin does not increase Hepatitis Viruses A, B, C, D or E. In pure hepatocellular
above 5 mg/dl. It disappears by the second week of life. disease, conjugation in liver is decreased and hence
free bilirubin is increased in circulation. However,
Breast Milk Jaundice inflammatory edema of cell often compresses intra-
cellular canaliculi at the site of bile formation and this
In some breast-fed infants, prolongation of the jaundice
produces an element of obstruction. When conjugated
has been attributed to high level of an estrogen derivative
bilirubin level also increases, mixed type of jaundice
in maternal blood, which is excreted through the milk. results. Bilirubinuria also occurs. The UBG level in urine
This would inhibit the glucuronyl transferase system. may be normal or decreased in hepatocellular jaundice
Sulfa and such other drugs may release bilirubin from (Table 22.7).
albumin, and may cause jaundice in newborn.
Obstructive Jaundice
Hemolytic Jaundice Conjugated bilirubin is increased in blood, and it is
Hemolytic Disease of the Newborn excreted in urine. If there is complete obstruction, UBG
will be decreased in urine or even absent (see Fig. 22.13
This condition results from incompatibility between and Tables 22.7). In total obstruction of biliary tree, the
maternal and fetal blood groups. Rh positive fetus may bile does not enter the intestine. Since no pigments are
produce antibodies in Rh negative mother. In Rh incom- entering into the gut, the feces become clay colored.
patibility, the first child often escapes. But in the The common causes of obstructive jaundice are:
334 Section B: General Metabolism
22-1 . All the following oxygen handling proteins contain 22-8. Urine of a patient with acute intermittent porphyria
heme as a prosthetic group, except: is likely tc, contain:
A. Cytochromes B. Peroxidase A. Porphobilinogen
C. Superoxide dismutase B. Uroporphyrinogen
D. Tryptophan pyrrolase C. Protop10rphyrinogen
2.2-2. The methenyl bridge of protoporphyrin is derived D. Bilirubin
from : 22-9. Degradati,on of heme to bilirubin releases one mole-
A. Alpha carbon atom of glycine cule of:
B. Alpha carbon atom of succinyl-CoA A. Carbon dioxide B. Water
C. Carboxyl group of succinyl-CoA C. Ammonia D. Carbon monoxide
D. Carboxyl group of glycine 22-10. Heme Is converted to bilirubin in:
22-3. Rate limiting enzyme in heme synthesis is: A. Mitoch1ondria B. Microsomes
A. Heme synthase C. Golgi bodies D. Plasma membrane
B. ALA dehydrase 22-11 . Bilirubin in serum can be measured by:
C. Uroporphyrinogen synthase A. Van den Bergh reaction
D. ALA synthase B. Ehrlich's reaction
22-4. All the following are precursors of heme, except C. Schlesinger's reaction
A. Porphobilinogen B. Urobilinogen D. Fouchet's reaction
C. Uroporphyrinogen D. Protoporphyrinogen 22-12. Which disease is associated with conjugated
22-5. Which enzyme of heme blosynthesis is inhibited hyperbilirubinemia?
by lead? A. Gilbert's disease
A. ALA dehydratase B. Crigler-Najjar syndrome
B. UBG I synthase C. Dubin--Johnson's syndrome
C. UBG decarboxylase D. Conge,nital spherocytosis
D. PPG oxidase 22-13. All the following have a regulatory effect on heme
22-6. Acute intermittent porphyria (AIP) is: synthesis, except:
A. Characterized by photodermatitis A. Cellular heme pool
B. An autosomal dominant trait B. Low calorie diet
C. More common in men than women C. Compartmentalization of enzymes
D. Hemolytic anemia is associated D. Hemat:ocrit value
22-7. Congenital erythropoietic porphyria Is caused by 22-14. Which int,e rmediate of heme synthetic pathway is
defi ciency of: not a tetrc1Ipyrrole?
A. ALA synthase A. Uroporphyrinogen
B. Uroporphyrinogen Ill cosynthase B. Coproporphyrinogen
C. Coproporphyrinogen oxidase C. Porphobilinogen
D. Uroporphyrinogen decarboxylase D. Protoporphyrinogen
Chapter 22: Heme Synthesis and Breakdown 337
22-15. Which of the following statements is NOT true? 22•27. Bilirubin is formed in all the tissues, except
A. ALA synthase requires energy for its activity A. Liver B. Spleen
B. ALA dehydratase is inhibited by lead C. Kidney D. Bone marrow
C. ALA synthase requires PLP for its activity 22-28. Which is inot involved in catabolism of heme?
D. Two molecules of ALA condense to form PBG A. Cytochrome a B. NADPH
-
22-16. Porphyrins are:
A. Monopyrroles
B. Colorless components
C. Showing fluorescence under UV light
C. Molecular oxygen D. Cytochrome c
22-29. Which enIzyme is not involved in catabolism of
heme?
A. Hemeoxygenase
D. Destroyed by sunlight B. UDPiilucuronyltransferase
22-17. In obstructive jaundice, prothrombin time: C. Biliverdin reductase
A. Remains normal B. Decreases D. Ferroc:helatase
C. Becomes normal when vitamin K is administered 22-30. All the following are produced from heme during
D. Increases when vitamin K is administered catabolisirn, except.
22-18. Which does not contain heme? A. Urobilnnogen B. Porphobilinogen
A. Myoglobin B. Cytochrome C. Bilirubin D. Biliverdin
C. Ferroxidase D. Peroxidase 22-31 . Normal color of feces is due to:
22-19. All are correct with regard to heme, except A. Bilirubin B. Stercobilin
A. Ferro protoporphyrin C. Porphobilinogen D. Biliverdin
B. Prosthetic group of cytochromes 22-32. All the following compounds undergo entero•
C. Synthesized by using glycine hepatic ciirculation, except:
D. Linear tetrapyrrole A. Urobil1inogen B. Iron
22-20. Which is required for synthesis of porphyrins? C. Cholic: acid D. Cholesterol
A. Ammonia B. Methionine 22-33. Increased level of urobilinogen In urine is observed
(
C. Glycine D. Carbon dioxide in all the !following conditions, except
22-21 . All are substituent groups present in heme (proto• A. Congemital spherocytosis
porphyrin), except: B. Mismatched transfusion
A. Methyl B. Vinyl C. Glucose-6-phosphate dehydrogenase deficiency
C. Propionyl D. Ethyl D. Lead poisoning
22-22. All are correct regarding heme synthesis, except 22-34. Congenital hyperbilirubinemia with a bilirubin level
A. Compartmentalized of 2.5 mg,/dl is suggestive of:
B. Stimulated by lead A. Criggler-Najjar syndrome
C. Regulated by end product inhibition B. Gilbert's syndrome
D. Induced by barbiturates C. Dubin-Jhonson's syndrome
22-23. All affect heme synthesis, except D. Rotor syndrome
A. Barbiturates B. Estrogens 22-35. Heme contains:
C. Glucose D. Oncogenes A. Magn,::!sium B. Calcium
22-24. A patient with acute intermittent porphyria should C. Iron D. Sodium
be given: 22-36. Heme is not found in:
A. High carbohydrate diet A. Cytochrome c
B. Barbiturates B. Cytochrome a
C. Aspirin C. Iron s1Ulphur proteins
D. Saline infusion D. Nitric oxide synthase
22-25. Congenital erythropoietic porphyria has all the 22·37. Bilirubin is:
features, except: A. Conjugated with glucuronic acid
A. Sensitivity to sunlight B. Transported free in plasma
B. Reddish discoloration of urine C. Normally excreted through urine
C. Erythrocyte porphyrin level elevated D. Bound to haptoglobin
D. Red fluorescence of teeth 22-38. UPG ;s cc>nverted to CPG by:
22-26. Presence of porphyrins in urine can be detected by: A. Cond13nsation B. Decarboxylation
A. UV fluorescence C. Dehydrogenation D. Reduction
B. Absorption band at 565 nm 22-39. Which of the following is colored?
C. Fouchet's test A. PBG B. UPG
D. Schlesinger's test C. CPG D. pp
338 Section B: General Metabolism
Chapter at a Glance
The learner wi ll be able to answer questions on the following topics:
Structure of hemoglobin O Hemoglobino pathies
0 Oxygen dissociation curve O Sickle cell anemia
Transport of carbon dioxide Thalassemia s
0 Fetal hemoglobin O Myoglo bin
Carboxy hemoglobin Anemias
M et-hemoglobin
Historical Perspectives
Marcello Malpighi described the RBCs in 1665. Felix Hoppe-Seyler in 1862 isolated pure hemoglobin. Christian Bohr in 1904
discovered that hemoglobin is the transporter of oxygen. In 1912, Kuster established the structure of hemoglobin. Hans Fischer
synthesized heme in laboratory in 1920 (Nobel Prize, 1930). Perutz (Nobel Prize, 1962) studied the three-dimensional structure
of hemoglobin.
"t /
is called Met-Hb; then iron is in ferric state and the oxygen
0 ca rrying capacity is lost.
Pyrrole-N N-Pyrrole
l"'
Fe by the oxygen dissociation curve (ODC) (Fig. 23.3).
Hemoglobin is oxygenated and not oxidized (Box
Pynole-N / N-P- - - -Arginine - 23.1). At the oxygen tension in the pulmonary alveoli,
the Hb is 97% saturated with oxygen. Normal blood
1 N-Histidine (proximal) with 15 g/dl of Hb can carry 1.34 x 15 = 20 ml of O/ dL
Arginine of blood. In the tissue capillaries, where the pO2 is
I only 40 mm Hg, theoretically, Hb saturation is 75%.
Thus under NTP conditions, blood can release only
Fig. 23.1: Linkage of heme with globin. Pink circle represents 22% (Fig. 23.3A). But actually in tissue capillaries,
the g lobin chain. Blue rectangle represents the protoporphyrin ring where pO2 is 40 mm Hg, the Hb is about
I TRANSPORT OF OXYGEN BY
HEMOGLOBIN
Hemoglobin has all the requirements of an ideal respira-
Heme-Heme Interaction and Cooperativity
The sigmoid shape of the oxygen dissociation curve
(ODC) is due to the A teric effect, or cooperativity.
tory pigment (Barcroft): The equilibr~ m of Hb with oxygen is expressed by the
a. It can transport large quantities of oxygen Hill equation7 AV Hill, Nobel Prize, 1922). The binding
b. It has great solubility of oxygen to one heme residue increases the affinity
c. It can take up and release oxygen at appropriate of remaining heme residues for oxygen
partial pressures interaction) (Fig. 23.38 ). This is calle<11•••
coopera-
d. It is a powerful buffer. tivity.
Affinity Affinity Affinity Affinity
Oxygen Dissociation Curve (ODC) 1 time 2 times 4 times 8 times
The ability of hemoglobin to load and unload oxygen at Hb - HbO2 - HbO4 HbO6 HbO8
physiological pO2 (partial pressure of oxygen) is shown (+)0 2 (+)02 (+)02 (+)02
\
Chapter 23: Hemoglobin 341
(+)02 (+)02:
Deocy-Hb; T conformatioin
B IIHb-02
2
Hb-0 4
2
Fig. 23.2: Diagramatic representation of the subunit interaction in hemoglobin. Pinik rec tangles represent Hb monomers. Black
Hb-0 8; R form
connection lines represent salt bridges. As oxygen is added, salt bridges are successively broken and finally 2,3-BPG is expelled.
Simultaneously the T (taught) confonnation of deoxy-Hb is changed into R(relaxed) c:onformation of oxy-Hb. Blue circle represents
2,3-bisphosphoglycerate (BPG)
Deoxy-Hb
+ 402 ---+ Oxy-Hb
••
Lungs
•rt_e_ry_..J ~ ~- _ .
~--
Venous
circulation
Archibald
Vivian Hill
NP 1922 Deoxy-Hb + 402 +-- Oxy-Hb
1886-1977
Fig . 23.4: In tissues Oxy-Hb releases oxygen
20 40 60 80
p02 in mm of Hg
A-Theoretical curve as per mass action.
B- Sigmoid curve , due to heme-heme
interaction (Hill effect). C-Further shift to right
due to carbon dioxide (Bohr effect) and BPG.
This curve represents the pattern under normal
conditions. D- Further shift to right when
Christian Bohr
temperature is increased to 42°C.
1855-1911 Joseph Joseph Lavoisier John
Fig. 23.3: Oxygen dissociation curve (ODC)
Priestley Black 1743-1794 Haldane
1733-1804 '1 728-1799 1860-1936
Chloride Chloride
enters into RBC comes out of RBC
Fig. 23.5: Chloride shift; reactions in tissues Fig. 23.6: Chloride shift; reactions in lungs
Carboxy-Hemoglobin (Carbon
Monoxy Hb) (CO-Hb)
Hb-Fe+++ y,,,- Cyto.b5-Fe.. NAO+
than that of oxygen. It is then unsuitable for oxygen Fig. 23.7: Met-hemoglobin reductase system
transport. When one molecule of CO binds to one
monomer of the hemoglobin molecule, it increases the . In
affinity of others to 0 2 ; so that the 0 2 bound to these persons with this enzyme deficiency, the condition may
monomers are not released. This would further decre- be manifested even with small doses of drugs. In such
ase the availability of oxygen to the tissues. persons, NADPHI is not available in the RBC. Therefore
CO is a colorless, in such individuals, disease is manifested easily (see
odorless, tasteless gas generated by incomplete Chapter 6).
combustion. CO poisoning is a major occupational
hazard for workers in mines. Breathing the automobile Hemin Crystals
exhaust in closed space is the commonest cause for When iron is oxidized to Fe++•, it has a net positive
CO poisoning. The carboxy-Hb level in normal people is charge. It can combine with negatively charged chloride,
0 .16%. An average smoker has an additional 4% of CO- to form hemin or hematin chloride. Hemin crystals can
Hb. One cigarette liberates 10-20 ml carbon monoxide be prepared from even very old blood stains in medico-
into the lungs. legal cases. Blood or eluted blood stains are heated with
Clinical symptoms manifest Nippe's fluid (1% solution of KCI, KBr and Kl in glacial
when carboxy-Hb levels exceed 20%. Symptoms are acetic acid) over a glass slide, when dark brown rhom-
breathlessness, headache, nausea, vomiting, and pain bic crystals are seen under the microscope. The test is
in chest. At 40-60% saturation, death can result. Admini- sensitive, but is answered by the heme part of blood of
stration of 0 2 is the treatment. In severe cases, oxygen all species.
under high pressure (hyperbaric oxygen) is helpful.
Glycated Hemoglobin
Met-Hemoglobin (Met-Hb) Described in detail in Chapter 11 .
When the ferrous (Fe·•) iron is oxidized to ferric (Fe•••)
state, met-Hb is formed. Small quantities of met-Hb HEMOGLOBIN (GLOBIN CHAIN)
formed in the RBCs are readily reduced back to the VARIANTS
ferrous state by met-Hb reductase enzyme systems. - -- - -
About 75% of the reducing activity is due to enzyme Hemoglobin1opathies
system using - and cytochrome b5 (Fig. 23.7). These are a group of heterogeneous disorders affecting
Another 20% of the reducing activity is due to globin chain synthesis. Common symptoms like anemia,
dependent system. met-Hb- splenomegaly and vase-occlusive episodes in sickling
reductase accounts for the rest 5% activity. disorders overlap in these conditions.
Qualitative defects in globin chain synthesis, i.e.
Met-Hemoglobinemias abnormal globin chains with altered amino acid sequence
Normal blood has only less than 1% of met-hemoglobin. (abnormal hemoi~lobins or hemoglobin variants) result
It has markedly decreased capacity for oxygen binding in hemoglobinopathies. These are structural variants of
and transport. An increase in met-hemoglobin in blood, hemoglobins. Huindreds of such hemoglobin variants
(met-hemoglobinemia) is manifested as have been discovered (Box 23.3). The variants may be
Causes may be congenital or acquired . either alpha chain variants or beta chain variants (Table
23.2).
Acquired or Toxic Met-Hemoglobinemia Quantitative defects in globin chain synthesis, i.e.
Drugs which produce met-hemoglobinemia are-aceta- normal globin chains in abnormal amounts res~ t in
minophen, phenacetin, sulfanilamide, amylnitrite, and thalassemias; alp1ha or beta thalassemia depending on
sodium nitroprusside. the deficiency of the chain.
Chapter 23: Hemoglobin 345
Fig. 23.SA: Left side; normal RBCs. Right side, sickle cells
HbA
Val-His-Leu-Thr-Pro-Glu-Glu-Lys
12345 6 78
HbS
Val-His-Leu-Thr-Pro-Val-Glu-Lys
12345 6 78 QOIIHIM8M_of___
HbC
Sticky sickle cells
r~ ~~;ands
::..
Val-His-Leu-Thr-Pro-Lys-Glu-Lys
12345 6 78
I 5 1-"§.
Normal RBC Result
0
--+ 008 Malaria:
0 00 000
Oxy-Hb Deoxy-Hb Oxy-HbS Deoxy-HbS Two deoxy-HbS stick dJ 0
RBC tysis
Fig. 23.9: Sticky patches on HbS molecule Malarial parasite enters and multiplies
+ve pole
!D - ve pole
--+ No RBC lysis
Sickle
cell trait
' • Sand A
Parasite enters, but cannot multiply Normal health
•
Fig. 23.10: Sickle cell trait protects from malaria
Sickle
cell anemia D s
I
Linus
Pauling Parents
Normal D A NP 1954
hemoglobin 1901- 1994
Offspring
Hemoglobin D Normal
Sickle HbC HbS-C
It does not produce sickling. HbD Punjab results from trait trait disease
replacement of beta 121 glutamic acid by glutamine I= Normal gene Q=Sickle gene
(HbD Punjab) (see Table 23.2). HbD migrates similar
to HbS on electrophoresis. HbSD disease is a severe
I= HbC gene
condition . HbD Punjab is the commonest Hb variant Fig. 23.12: Inheritance of HbS-C disease
seen in Punjabi population.
and are less likely to produce impairment of red cell
M-Hemoglobins (Hb M) function (codominant inheritance). Thus beta chain
These are a group of variants, where the substitution disease is more common and more severe than alpha
occurs in the proximal or distal histidine residues of alpha chain disease.
or beta chains. As a result, the heme has a tendency to Globin chain genes are located on two different
get oxidized to hemin, forming • • • • • •· Oxygen chromosomes: alpha and similar chains on chromo-
binding is decreased. This would result in some 16; beta and similar chains on chromosome 11 .
Genetic defects in hemoglobinopathies are mainly point
Inheritance of Hemoglobin Variants mutations resulting in substitution of a single amino acid.
They are inherited as ... -~.i . l
The abnormal phenotype is decided by the type and
One beta chain gene is inherited from each parent. If location of the substituted amino acid. If the properties of
both parents are heterozygous for S, (the abnormal the substituted amino acid are different from the original
gene), there is 50% chance that the child will be AS, one, clinical manifestations can occur even in heterozy-
25% chance for AA and 25% chance for SS genotype gous state.
(inheritance is described in Chapter 42). If the genotype
is SS , all the Hb molecules produced are abnormal, and THALASSEMIAS
hence disease is manifested .
If one parent is heterozygous for HbS and another The name is derived from the Greek word, "thalassa" ,
for C or beta thalassemia , 25% chances are that the which means "sea". Greeks identified this disease pre-
child will be a double heterozygote (Fig . 23.12). An sent around Mediterranean sea. It is the most common
individual inherits only 2 beta chain genes; but genetic disease and has a carrier frequency of about
j a dre inherited. So, the alpha chain variants 7% across the globe. The disease is also prevalent
constitute only 25% of the total hemoglobin in circulation in lndia.Thalassemia may be defined as the normal
348 Section B: General Metabolism
IANEMIAS symptoms?
2. What is the biochemical rationale for 100% 0 2
In India, anemia is the most common medical problem. being the treatment of choice?
Perhaps about 75% of patients attending a primary
health center may have signs and symptoms directly or 0
indirectly related to anemia. Anemia results whe,, the •• · Clinical Case Study 23.2
Hb concentration in blood is reduced. Normal value for A 15-year-old Indian aboriginal girl presents to the
Hb in normal male is 14 to 16 g/dl and in female 13 to emergency room with complaints of bilateral thigh and
15 g/dl. If the Hb level is below 10 g/dl, it is a severe hip pain. The pain has been present for 1 day and is
condition. steadily increasing in severity. Acetaminophen and
The most common cause for anemia in India, is iron ibuprofen have not relieved her symptoms. She denies
deficiency which is described in Chapter 34. A list of any recent trauma or excessive exercise.
other causes is given in Box 23.4. Common symptoms She reported fatigue and burning sensation during
of anemia are shown in Figure 23.15. urination. She reports having similar pain episodes in
the past, sometimes requiring hospitalization. On exami-
0 nation, she is afebrile. Her conjunctiva and mucosa!
• • Clinical Case Study 23.1
membranes are slightly pale in color. She has non-
During a winter day morning, an elderly couple were specific bilateral anterior thigh pain with no abnormali-
taken to the emergency department, when their son ties appreciated. The remainder of her examination is
350 Section 8 : General Metabolism
completely normal. Her white blood cell count is ele- autosomal recessive fas hion. The sickled red blood cells
vated at 17,000/mm3 , and her hemoglobin (Hb) level is cause infarction of bone, lung, kidney, and other tissue
decreased to 7.1 g/dl. The urinalysis demonstrated an from vaso-occlus.ion.
abnormal number of numerous bacteria. The patient's description of pain is typical of a
1. What is the most likely diagnosis? sickle cell pain crisis. Often infection is a trigger, most
2. What is the molecular genetics behind this disorder? commonly pneumonia or a urinary tract infection. Here,
3. What is the pathophysiologic mechanism of her symptoms are consistent with a urinary tract infection,
symptoms? indicated by burning with urination (dysuria). Her white
blood cell count is elevated in response to the infection.
0 The low hemoglobin level is consistent with sickle cell
• • Clinical Case Study 23.3 anemia. Since she is homozygous (both genes coding
A 25-year-old female presents to her obstetrician at for sickle hemoglobin), both her parents have sickle cell
12 weeks gestation. This is her first pregnancy, and she trait (heterozygo1us) and thus do not have symptoms.
is concerned about her baby and the risk of inheriting a The diagnosis can be established with hemoglobin
"blood" disease like others in her family. Her brother had electrophoresis.
died at age 10 due to severe anemia. 0
Her ultrasound confirmed an intrauterine pregnancy •• Clinical Case Study 23.3 Answer
at 12 weeks gestation. The patient's hemoglobin level
Molecular genetics: Impaired production of 13-globin
shows a hypochromic, microcytic anemia. Hemoglobin
peptide chain.
electrophoresis demonstrated increased fetal hemoglobin
level, a pattern consistent with a-thalassemia minor. Likely test: Oligonucleotide probe. After chorionic villus
sampling is performed, a radioactive probe can be used
1. What is the molecular genetics behind this disorder?
and hybridized with specific genetic mutations in the
2. What was the likely test and what is the biochemical
fetus' deoxyribonucleic acid (DNA), allowing for prompt
basis?
detection and prematal diagnosis.
0 The most common cause of anemia is iron defi-
•• · Clinical Case Study 23.1 Answer ciency. Another common cause of microcytic anemia
is thalassemia. Certain ethnic groups have higher inci-
Most likely cause: Carbon monoxide poisoning
dence of thalassBmia .
(increase carboxyhemoglobin level).
The microcyltic (small red blood cell size) anemia
Rationale for treatment: Administration of 100% 0 2 with elevated hemoglobin Az and F is consistent with
displaces CO from hemoglobin. 13-thalassemia miinor. Patients with 13-thalassemia major
Carbon monoxide binds very avidly to hemoglobin. (Cooley anemia) typically have severe anemia requiring
It is a colorless and odorless gas and may arise from frequent transfusions and shortened life expectancy.
internal combustion engines, fossil-fueled home appli- Infants will appear healthy after birth, but as the hemo-
ances (heaters, furnaces, stoves). It does not give globin F levels fall, the infant becomes severely anemic.
warning signs. The patient generally has confusion and
symptoms of 0 2 deprivation, but not the symptoms of LEARNING POINTS, CHAPTER 23
dyspnea , since the hemoglobin is saturated. The lips
1. Hemoglobin (Hb) is a globular protein containing
are a distinct red color as a result of the hemoglobin
2 alpha 2 b13ta (HbA), 2 alpha 2 gamma (HbF), 2
being "oxygenated." However, because CO binds so
alpha 2 delta (HbA2). Alpha chain has 141 amino
avidly to the hemoglobin, no transfer of 0 2 occurs in the
acids while beta, gamma and delta have 146 amino
peripheral tissue. Treatment is thus 100% 0 2 to displace
acids.
the CO from the hemoglobin.
2. The ability cit Hb to load and unload 0 2 at physio-
0 logical pO2 iis shown by the 0 2 dissociation curve
• Clinical Case Study 23.2 Answer and its sigmoid shape is due to the allosteric effect
(co-ope rativiity).
Most likely diagnosis: Sickle cell disease (pain crisis 3. The influence of pH and pCO2 to facilitate oxygena-
Biochemical mechanism of disease: Single amino acid tion of Hb in lungs and deoxygenation at the tissue
substitution on hemoglobin beta chain , inherited in an is known as Bohr effect.
Chapter 23: Hemoglobin 351
4. Entry of Cl from plasma to cells to establish 11 . The positive co-operative effect is exhibited by
neutrality for the HCO3- out to the plasma is called different subunits where by binding of 0 2 by one
Chloride shift. subunit favors binding of 0 2 to other subunits. In a
5. HbF moves slower than HbA on electrophoresis, similar manner 0 2 release also shows a co-operative
is resistant to alkali denaturation, has decreased interaction referred to as heme-heme interaction.
interaction with 2,3-BPG. , 12. Transport of CO2 by is called isohydric transport,
6. Hb S is sickle cell Hb resulting from a Glu-Val which helps in buffering and the elimination of vola-
substitution at the 6th position on the beta chain. tile acid, carbonic acid. In the tissues where pCO2
HbS gives protection against malaria. is high, CO2 diffuses into RBCs and combines with
7. Thalassemias are caused due to functional abnor- Hp to form carbonic acid catalyzed by carbonic
mality of alpha or beta chains of Hb. Homozygous anhydrase. The H+ formed by dissociation of H2 CO3 ,
states exhibit Thalassemia major while heterozy- is buffered by HHb, which can take up H+when oxy-
gous state exhibit Thalassemia minor. gen dissociates.
8. Alpha chain gene is on chromosome 16 and beta, 13. A fraction of CO2 is transported as carbamino Hb.
(gamma, delta) chain genes on chromosome 11 . 14. Fetal hemoglobin has a higher affinity for 0 2 than
9. The factors which affect oxygen binding and Hb A, since it does not bind 2,3-BPG. This is bene-
release are pO2 , pCO2 , pH and 2,3-BPG concentra- ficial in oxygen transport and delivery to tissues in
tion in RBCs. foetal life.
10. Arterial blood is 97% saturated at pO2 of 100 mms 15. The fetal Hb level falls after birth and reaches adult
of Hg and can carry about 40 mms of Hg. In venous levels by 2 years of age (<2%).
blood, where pO 2 is about 40 mms of Hg, Hb is only 16. In anemic children and children with beta chain
60% saturated. Net effect is a release of 40% 0 2 to defects, Hb F level remains high as a compensatory
tissues. mechanism.
23-5. Which factor causes a shift in the ODC (oxygen 23-15. The common molecular defects in beta thalassemia
dissociation curve) to left? include the following , except
A. High pO2 B. Low pH A. Frame shift mutations
C. Low pO2 D. High temperature B. Nonsense mutations
23-6. All the following manifestations are seen in sickle C. Suppressor mutations
cell anemia (HbS disease), except D. Improper splicing of hnRNA
A. Pain and swellings in joints 23-16. Unstable hemoglobins will produce:
B. Hemolytic anemia A. Erythrocytosis B. Heinz body anemia
C. Sickled cells in peripheral circulation C. Polycythemia D. Sickling
D. Inclusion bodies in RBCs 23-17. Presence of an abnormal hemoglobin is suspected
23-7. A person with HbS trait is likely to have: if a patient has any of the signs, except
A. About 90% of hemoglobin as HbS A. Nonspherocytic hemolytic anemia
B. A single band on electrophoresis B. Hypochromic microcytic anemia
C. Sickling at high altitudes C . Polycythemia
D. Irreversibly sickled cells in peripheral smear D. Cyanosis without cardiopulmonary dysfunction
23-8. All are features of isohydric transport, except: 23-18. Hemoglobin will not copolymerize with HbS?
A. CO2 is transported in plasma as bicarbonate A . HbC B. HbA
B. Carbonic anhydrase is the major enzyme involved C. HbD D. HbE
C. Chloride shift is a result of CO2 transport 23-19. Which phenomenon is not related to functional
D. CO2 is bound to amino terminal group properties of hemoglobin?
23-9. Methemoglobinemia may be caused by all the A. Bohr effect
following, except B. Haldane effect
A. Methemoglobin reductase deficiency C. Pasteur effect
B. Presence of HbM D. Donnan effect
C. Ingestion of nitrites 23-20. In homozygous beta thalassemia:
D. Deficiency of ascorbic acid A. HbH is the major hemoglobin
23-10. Carbon dioxide is carried in blood with all the B. Symptoms appear al birth
forms, except C. Persistence of HbF is observed
A. Dissolved form D. Patients have a normal life span
B. lsohydric transport 23-21 . Beta thalassemia can result from, except
C. Carboxyhemoglobin A. Terminator codon mutations
D. Carbaminohemoglobin B. Abnormal splicing of mRNA
23-11. All are true with regard to HbF, except C. Defect in post-translational processing
A. Poorly binds 2,3-BPG D. Mutation in suppressor genes
B. Copolymerises with HbS 23-22. Myoglobinuria is observed in all the following con-
C. Affinity for oxygen is more ditions, except:
D. Is a tetramer of 2 alpha and 2 gamma chains A. Severe muscular exercise
23-12. Regarding myoglobin, all are true, except: B. Crush injuries of limbs
A. It has 4 polypeptide chains C. Myasthenia gravis
B. Binds oxygen D. Muscle wasting diseases
C. Oxygen binding affinity is higher than that of hemo- 23-23. Which hemoglobin derivative is normally found in
globin blood?
D. Contains one heme residue per molecule A. Deoxyhemoglobin
23-13. Regarding HbS and HbA, all are true, except: B. Sulfhemoglobin
A. The chain length of HbS is smaller than HbA C. Acid hemalin
B. Solubility of deoxy HbS is lower than deoxy HbA D. Cyanmethemoglobin
C. HbS is slower moving on electrophoresis than HbA 23-24. Methemoglobinemia may be caused by all the
D. Structural difference is with regard to beta chain following , except:
23-14. Anemia can result from deficiency of all the A. Methemoglobin reductase deficiency
following, except B. Presence of HbM
A. Iron B. Cobalamin C. Ingestion of nitrites
C. Cholecalciferol D. Pteroylglutamic acid D. Deficiency of ascorbic acid
Chapter 23: Hemoglobin 353
23-25. All are the characteristics of HbS, except it: 23-32. All are true with regard to hemoglobin M variants,
A. Is an abnormal hemoglobin except:
. phenomenon
D. Chronic hemolytic anemia is the usual manifestation
C. Blood gives a strong absorption band at 630 nm
D. Cyanosis improves on giving 1/V methylene blue
SECTION
Clinical and Applied
Biochemistry
i
_____Chapter 24
Liver and Gastric
Function Tests
Chapter at a Glance
The learner will be able to answer questions on the following topics:
D Serum and urine bilirubin Gastric function and HCI secretion
D Tests based on synthetic function D Gastric juice analysis
D Enzymes indicating hepatocellular damage Pancreatic function tests
Group II: Liver enzyme panel (see Chapter 6) (These are markers of
liver injury and/or cholestasis)
i. Alanine ami not ransferase (ALT) Fig. 24.1: Jaundice
ii. Aspartate aminot ransferase (AST)
iii. Alkaline phosphatase (ALP)
Detoxification Functions
iv. Gamma-glutamyltransferase (GGT)
Group Ill: Plasma proteins (see Chapter 26) (Tests for synthetic func- Exogenous substances : Toxic substances entering
tion of liver)
from gut and parenteral route are mainly detoxified in the
i. Total prot eins
liver by different reactions. The cytochrome P450 enzyme
ii. Serum albumin, globulins, NG ratio
system of hepatocyte is mainly concerned with drug me-
iii. Prothrombin time
Group IV: Special tests
tabolism (Chapter 36).
i. Ceruloplasmin (see Chapters 26 and 34) Endogenous s1...bstanc9s: Disposal of bilirubin is
ii. Ferritin (see Chapter 34) already discussed. Ammonia produced from amino acid
Iii. Alpha-1-antit rypsin (AAT) catabolism is detoxified by the liver to form less toxic
iv. Alpha-fetoprotein (AFP) (see Chapter 48) urea. The key urea cycle enzymes are located entirely
B. Classification based on clinical aspects
in liver.
Group I: Markers of liver d ysfunction
i. Serum bilirubin, total, conjugated
ii. Urine: BIie pigments, bile salts and UBG
Excretory Functions
iii. Total protein, serum albumin and N G ratio Substances detoxified by the liver are excreted through
iv. Prothrombln ti me
bile. About 3 liters of bile is produced daily and out of this
v. Blo od ammonia, w hen indicated
1 Lis excreted and the rest is reabsorbed and circulated
Group II: Markers of hepatocellular injury
i. Alanine amino transferase (ALT)
in the enterohepatic circulation. The bile contains bile
ii. Aspartate aminotransferase (AST) salts, conjugated bilirubin, phospholipids and cholesterol.
Group Ill: Markers of cholestasis Major functions of the liver are summarized in Box 24.1.
i. Alkaline phosphatase
I CLINICAL MANIFESTATIONS
ii. Gamma-glutamyltransferase
OF LIVER DYSFUNCTION
----
Lipid Metabolism Jaundice
Fatty acids will be catabolized to release acetyl-CoA, Jaundice is the yellowish discoloration of sclera , skin and
and further to produce energy. Acetyl-CoA is also used mucous membrane. It is characteristic of liver disease
for fatty acid synthesis or cholesterol production. Dietary but it will occur when rate of hemolysis is increased
lipids are repackaged and secreted into the systemic leading to elevation of serum bilirubin.
circulation as lipoproteins. The protein parts of the lipo-
proteins, apoproteins are synthesized by the liver only. Portal Hypertension
Lipoproteins are described in Chapter 14.
The entire venous drainage of gastrointestinal tract, the
spleen, the pancreas and the gallbladder constitutes
Bilirubin Metabolism
portal circulation with a pressure of 5 mm of Hg. Any
Heme is catabolized to bilirubin, which is transported as obstruction in the course of portal circulation will cause
bilirubin-albumin complex. In the liver, bilirubin is conju- portal hypertension.
gated with glucuronic acid, and fi nally excreted through The major cause of portal hypertension is cirrho-
bile (Chapter 22). sis. Portosystemic shunting leads to deterioration of the
Chapter 24: Liver and Gastric Function Tests 359
l
tors, many globulins)
b. Synthesis of cholesterol
c. Synthesis of triacyl glycerol Hypoalbuminemia, leading to decreased colloid intravascular
pressure, oozing of protein rich fluid into the intraperitoneal
d. Lipoprotein synthesis
space, increased lntraperitoneal colloid pressure
2. Metabolic function
a. Carbohydrates: Glycolysis; glycogen synthesis; glyco-
gen breakdown; gluconeogenesis
l
Decreased central blood pressure
b. Ketogenesis; fatty acid synthesis and breakdown
c. Protein catabolism
!
Decreased renal perfusion, leading to
d. Citric acid cycle, production of ATP activation of renin angiotensin system,
sodium retention, water retenbon
3. Detoxification and excretion
a. Ammonia to urea i
Further fluid escapes into the peritoneal cavity
b. Bilirubin (bile pigment)
c. Cholesterol Fig. 24.2: Pathogenesis of ascites
d. Drug metabolites
4. Homeostasis: Blood glucose regulation
BOX 24.2: lnd1cat1ons for liver function tests
5. Storage function: Vitamin A, D, K, B12
1. Jaundice
6. Production of Bile salts; help in digestion
2. Suspected liver metastasis
3. Alcoholic liver disease
metabolic functions of the liver. Failure of detoxification
4. Any undiagnosed chronic illness
of ammonia by urea synthesis leads to hyperammone-
5. Annual checkup of diabetic patients
mia and hepatic encephalopathy. Decrease in albumin
6. Coagulation disorders
synthesis leads to hypoalbuminemia.
7. Therapy with statins to check hepatotoxicity
Ascites
complex) (free bilirubin) (indirect bilirubin) varies from
It is due to effusion of serous fluid into the abdominal
0.2-0.7 mg/dl and conjugated bilirubin (direct bilirubin)
cavity. It is a common presenting feature of cirrhosis.
Ascites may be due to different causes. If the ratio of
0.1- 0.4 mg/dl. A rise in serum bilirubin above 1 mg/dL
serum albumin : ascitic fluid albumin is > 1.1 , it is diag- is abnormal (latent jaundice); but jaundice appears only
nostic of portal hypertension as the cause. Figure 24.2 if the level goes above 2 mg/dl.
explains the pathogenesis of ascites. Box 24.2 gives the The bilirubin is estimated by van den Bergh reac-
indications of liver function tests. A detailed classification tion, where diazotized sulfanilic acid (sulfanilic acid in
of the liver function tests (LFT) is shown in Table 24.1 . HCI and sodium nitrite) reacts with bilirubin to form a
Important liver function tests are described below: purple colored complex, azobilirubin. When bilirubin is
conj ugated , the purple color is produced immediately
Markers of Hepatic Dysfunction on mixing with the reagent, the response is said to be
van den Bergh direct positive. When the bilirubin is
Measurement of Bilirubin
unconjugated, the color is obtained only when alcohol
(Test of Excretory Function of Liver) is added, and this response is known as indirect
Bilirubin is the excretory product formed by the catabo- positive. If both conjugated and unconjugated bilirubin
lism of heme. It is conjugated by the liver to form bilirubin are present in increased amounts, a purple color is pro-
diglucuronide and excreted through bile (see Chapter duced immediately and the color is intensified on adding
22). Measurements of bilirubin is an important liver func- alcohol. Then the reaction is called biphasic.
tion test. In Hemolytic jaundice, unconjugated bilirubin is
Normal serum bilirubin level varies from 0.2 to 1.0 increased. Hence van den Bergh test is indirect positive.
mg/dl. The unconjugated bilirubin (bilirubin-albumin In obstructive jaundice, conjugated bilirubin is elevated,
360 Section C: Clinical and Applied Biochemistry
5. General diseases: Wilson's disease, Hemochromatosis, AAT Hepatitis D (delta) HBsAg and anti-HOV
deficiency, Porphyrias, Sarcoidosis, Amyloidosis Hepatitis E Anti-HEV
6. Neoplasm: Hepatocellular carcinoma, Metastatic liver d isease,
Lymphoma
7. Bacterial infections:TB, Leptospirosis, Brucella, Abscesses In a small fraction of such cases, development of
8. Parasites: Helminths, Amebiasis, Plasmodia, Leishmania hepatocellular caircinoma is also noticed. Thus HBV is an
9. Drugs: Salicylate, Tetracyclines, Methotrexat e, lsoniazid,
Rifampicin, Halothane, Methyld opa, Valproate
oncogenic virus. Medical personnel, including medical
students, doctors, nurses and technicians are advised to
take Hepatitis B vaccination. Hepatitis viruses type A, B,
C, D, E and Gare identified. Box 24.5 gives the serology
to define the type of viral hepatitis.
TABLE 24.6: Overview of liver function tests BOX 24.8: Liver regeneration
Parameter Remarks Prometheus was punished for stealing fire from the Gods. As
Serum albumin punishment he was chained to rocks and his liver was pecked by
Increased in chronic liver d isease
vultures everyday. However, he survived. How?
Serum globulins Increase in chronic hepatitis
About 90% of liver can be removed and still the remaining liver
PT Prolonged in liver disease
cells divide replacing the lost cells within weeks. The story tells
PT + vitamin K Prolonged in hepatocellular us that this ability of liver cells to regenerate was known even
If PT normal, cholestasis
from ancient days.
Al pha-fetoprotei n Increase in carcinoma
Ceruloplasmin Decrease in Wilson's
disease, Menke's disease Mechanism of HCI Secretion
Transthyreti n To assess nutritional stat us
The daily volume of gastric secretion is around 2000 ml.
Alpha-1-antitrypsin Decrease in neonatal
cholestasis, progressive juvenile The HCI secreted by the parietal cells is of high con-
cirrhosis, micronodular cirrhosis
centration (0.1 5 M) with a pH as low as 0.8. The parietal
Haptoglo bin, Transferrin Severe hepatocellular disease, cirrhosis.
cells transport protons against a concentration gradient. It
Amino acids Increased aromatic amino acids
+ branched chain aminoacids is an energy requiring process. The K• activated ATPase
in hepatic coma; both increased is necessary for the production of HCI (Fig. 24.3). The H+
in cirrhosis
ions are generated within the cell by ionization of carbonic
Serum bilirubin See Table 24.4
acid. The carbonic anhydrase is active in the parietal cells.
Urine bilirubin See Table 24.4
One molecule of ATP is hydrolyzed for every molecule
Urine urobilinogen See Table 24.4
of W secreted. The hydrolysis of ATP is coupled with
Plasma bile acids Post-prandial rise in hepatic
dysfunction; increased fasting an exchange of K• for W. The hydrogen ions are then
level in portosystemic shunting secreted into gastric lumen.
Urine bile salts Positive in posthepatic jaundice and
Side by side with the H+ to K• exchange, bicarbonate
hepatic jaundice
Ammonia Increase in cirrhosis, portocaval
to chloride exchange is also taking place (Fig. 24.3).
anastamosis When the bicarbonate level within the cell increases
Transamlnases (formed from H2C03) , it is reabsorbed into blood stream,
Viral hepatitis ALT and AST i ncreased in exchange for Cl-. The chloride is then secreted into the
Chronic active hepatitis N or slight increase
lumen to form HCI. This would account for the alkaline
Cholestasis Slight increase
tide of plasma and urine, following hydrochloric acid
Alcoholic hepatit is ALT/ AST ratio reversed
secretion, immediately after meals.
ALP Increase in cholestasis
GGT Increase in cholestasis
Regulation of Acid Secretion
Most commonly employed liver function tests in clini- i. Gastrin, the gastrointestinal peptide hormone
cal practice are serum bilirubin (total and direct), albu- secreted by G cells, stimulates secretion of HCI.
min, ALP, ALT, AST and GGT. Cholesterol level in blood The secretion of gastrin is cut off by acidic pH by a
is also increased in obstructive jaundice due to defective feedback regulatory control.
excretion through bile. In general, ALT and ALP distin- ii. The most potent stimulus for acid secretion is
guishes the pattern of liver disease. Albumin determines hi~tamine, which acts through specific H2 receptors
the chronicity and prothrombin time determines the on the gastric mucosa.
severity of liver dysfunction (Box 24.8).
bicarbonate and enzymes. This secretion is under the (vi) Crohn's disease or (vii) Whipple's disease. Pancreatic
control of the hormones, Secretin and Cholecystokinin. disease can leacj to defective digestion. The following
Secretin is produced under the stimulation of gastric HCI. tests are useful to assess the malabsorption states.
Secretin produces a secretion with high bicarbonate Fat balance studies: The estimation of fat in stool is
content. Gastrin stimulates production of cholecystokinin done. When feces contains split fatty acids, it points to a
(CCK), which in turn produces pancreatic secretion rich normal pancreatic function, but defective absorption. On
in enzymes. The major enzymes present in pancreatic the other hand, if the fat excreted is neutral fat, it is due
juice are amylase, lipase and proteolytic enzymes to defective digestion, and is more in favor of pancreatic
(trypsin, chymotrypsin, carboxypeptidase, elastase) as disease.
their zymogens (see Chapter 17).
0
•• Clinical Case Study 24.1
Assessment of Pancreatic Function
A 45-year-old maile with history of cirrhosis of the liver is
Measurement of pancreatic enzymes: Amylase or
brought to the emergency center by family members for
alpha-1,4-glucosidase is the major enzyme which
acute mental derangements, disorientation, alterations in
digests starch. Normal amylase level in serum is 50-120 personality and confusion over the last few days. Patient
units. The level rises within 5 hours of the onset of acute is vomiting blood. On examination, he is disoriented with
pancreatitis and the level reaches a peak within 12 evidence of icteric sclera. His abdomen is distended
hours. But the level need not parallel the severity of the and fluid shift si~1n is noted. His urine drug screen and
disease. Within 2-4 days of the attack, the level returns ethyl alcohol (EtOH) screen are both negative. A blood
to normal. ammonia level was elevated, and all other tests have
Amylase level in blood is mildly increased in cases been normal.
of cholecystitis. No significant change or only mild What is the most likely cause of the patient's symp-
elevation is noticed in chronic pancreatitis . toms? What was the likely precipitating factor of the
Serum lipase is the major lipolytic enzyme which patient's symptoms? What is the cause for fluid in abdo-
hydrolyzes glycerol esters of long chain fatty acids. The men?
level in blood is highly elevated in acute pancreatitis 0
and th is persists for 7-14 days. Thus lipase remains :ji. Clinical Case Study 24.2
elevated longer than amylase. Moreover, lipase is
A 45-year-old female presents to the clinic with mid-
not increased in salivary diseases. Therefore, lipase
epigastric pain and nausea/vomiting after eating "greasy
estimation has advantage over amylase.
meals". The symptoms gradually disappear, and return
Lundh test: The test meal is composed of milk powder, after some days. She denies any hematemesis. She
vegetable oil and glucose to make 6% fat, 5% protein had elevated cholesterol levels. On examination, she
and 15% carbohydrate. After aspirating the duodenal is afebrile with normal vital signs. Her physical exami-
contents, 500 ml of fl uid meal is given. Then duodenal nation is completely normal with no evidence of abdo-
secretions are collected at 30 minutes intervals for minal pain. An abdominal ultrasound is performed and
2 hours. The tryptic activity of duodenal aspirates are revealed a few gallstones in the gallbladder. What
measured. In chronic pancreatitis, the tryptic activity is factors would you need to consider to assess the need
decreased, but not in carcinoma of pancreas. for cholecystectomy? What are gallstones made of?
Can gallstones be seen on abdominal X-ray?
l.§_TUDIES ON MALABSORPTION 0
Malabsorption may result from defective digestion or • • Clinical Case Study 24.3
fau lty absorption or from both. Reduction of absorptive A 26-year-old female at 35 weeks gestation presents
surface may result from (i) Celiac disease; (ii) Gluten to the clinic with complaints of generalized itching (pru-
sensitive enteropathy; (iii) Tropical sprue; iv) ldeopathic ritis ). She denies any change in clothing detergent,
steatorrhea; (v) Extensive surgical removal of ileum; soaps, or perfumes. She denies nausea and vomiting.
Chapter 24: Liver and Gastric Function Tests 367
On physical examination, there are no rashes apparent fat. Gallstones form when the solutes in the gallbladder
on her skin. Blood test reveals slightly elevated serum precipitate. Cholesterol stones are usually yellow-green
transaminase and bilirubin levels. What is the patient's in appearance and account for approximately 80% of
likely diagnosis? What are treatment options? What is gallstones. Stones made of bilirubin appear dark in
the cause of the patient's generalized itching? color. Patients may have pain from the gallstones, usu-
ally after a fatty meal. The pain is typically epigastric
0
or right upper quadrant and perhaps radiating to the
• • Clinical Case Study 24.1 Answer
right shoulder. If the gallbladder becomes inflamed or
The likely cause is Cirrhosis. infected, cholecystitis can result. The stones can also
Patient presents with cirrhosis, most probably, travel through the bile duct and obstruct biliary flow
secondary to hepatitis virus infection, with acute mental leading to jaundice, or irritate the pancreas and cause
status change coinciding with recent onset of hemat- pancreatitis.
13mesis. Patient has an elevated serum ammonia level 0
and otherwise negative workup. Hepatic encephalopathy 6 Clinical Case Study 24.3 Answer
{disorientation, etc.) is secondary to elevated ammonia
Cholestasis of Pregnancy
levels. The precipitating factor is increased nitrogen load
from upper gastrointestinal bleeding. Etiology: Cholestasis of pregnancy is a condition in
Cirrhosis is a chronic condition of the liver with diff- which the normal flow of bile from the gallbladder is
use parenchymal injury and regeneration leading to dis- impeded , leading to accumulation of bile salts in the body.
tortion of the liver architecture and increased resistance Generalized itching and, possibly, jaundice may result. It
of blood flow through the liver. The patient usually is speculated that the hormones such as estrogen and
manifests malaise, lethargy, palmar erythema, ascites, progesterone, which are elevated in pregnancy, cause
jaundice, and hepatic encephalopathy in the late sta- a slowing of the gallbladder function, leading to this dis-
order. Uncomplicated cholestasis is usually diagnosed
11es. Toxins accumulating in the bloodstream affect the
clinically by generalized itching in a pregnant woman,
patient's mental status. The most common etiologies of
usually in the third trimester without a rash. Elevated
cirrhosis are toxins such as alcohol, viral infections such
serum bile salts, bilirubin and transaminase may also be
as hepatitis B or C infection, or metabolic diseases in
seen. The usual treatment includes antihistamine medi-
children (Wilson disease, hemochromatosis, or alpha-
cations for the itching. More severe cases may require
·1 -antitrypsin deficiency). As liver functions are reduced,
bile salt binders such as cholestyramine or corticosteroids.
albumin synthesis is lowered, which leads to ascites
(fluid in abdomen).
lh_EARNING POINTS, CHAPTER 24
0
,8 Clinical Case Study 24.2 Answer 1. Bilirubin is estimated by van den Bergh reaction.
Normal serum does not give a positive van den
Gallstones Bergh reaction.
Hurgical removal of gallbladder (cholecystectomy) 2. When bilirubin is conjugated, the purple color is
is done when there is frequent and severe attacks. produced immediately on mixing with the reagent,
Components of gallstones are cholesterol, calcium the response is said to be van den Bergh direct
bilirubinate, and bile salts. positive. When the bilirubin is unconjugated, the
Mixed stones are much easier to see on plain film color is obtained only when alcohol is added, and
secondary to calcifications, comprising approximately this response is known as indirect positive.
- 0% of gallstones. This individual fits the "classic" 3. The most common cause for hepatocellular jaundice
patient with gallbladder disease- female, middle-aged, is the infection with hepatitis viruses (viral hepatitis).
overweight. The gallbladder stores bile salts produced 4. Elevated levels of Gamma-glutamyltransferase
by the liver. The gallbladder is stimulated to contract (GGT) are observed in chronic alcoholism, pan-
when food enters the small intestine; the bile salts then creatic disease, myocardial infarction , renal failure,
travel through the bile duct to the duodenum. The bile chronic obstructive pulmonary disease and in dia-
salts act to emulsify fats, helping with the digestion of betes mellitus.
368 Section C: Clinical and Applied Biochemistry
ALT or AST> 3 times normal value and ALT or AST < 3 times normal value and
..
ALP < 2 times value
Hepatocellular disease
ALP > 2 times
..
Cholestatic disease
I I
+
Albumin normal
+
Albumin low
+
Albumin 1normal
+
Albumin low
... ...
Acute cholestasis Chronic cholestasis
Acute hepatitis Chronic hepatitis
24-1 . Enumerate liver function tests and describe in detail any two of them ,with clinical significance.
24-2. Classify jaundice. Give an account of the biochemical tests which will help in differentiat ing the types of
jaundice.
24-3. Name the bile pigments. Give the significance of their presence in blood and urine. How are they detected in
blood and urine?
24-4. Classify jaundice. How do you investigate a case of jaundice?
24-5. Discuss the biochemical alterations seen in blood and urine in differeint types of jaundice.
24-6. Enzymes used as liver function tests. 24-9. Clinical significance of serum b ilirubin level.
24-7. Albumin globulin ratio. 24-10. Fractional test meal.
24-8. van den Bergh test. 24-11 . Hyperchk>rhydria.
24-1 . What is the normal serum bilirubin level? 24-10. What are important liver function tests?
0.2 to 1.0 mg/dl. Serum bilirubin, albumin, alkaline phosphatase; Urine
24-2. What is normal level of unconjugated bilirubin? bile salts, bile pigments and urobilinogen.
0.2-0.7 mg/dl. 24-11 . What are the features of obstructive jaundice?
24-3. What is normal level of conjugated bilirubin? Elevated conjugated bilirubin in plasma; presence of
Less than 0.4 mg/dL. bile salts and bile pigments in urine; increased alkaline
24-4. What is latent jaundice? phosphatase level in blood; increased cholesterol in
Serum bilirubin between 1 mg/dL and 2 mg/dl. blood.
24-5. Jaundice appears at what level of bilirubin? 24-12. Which tests will be positive in a urine of a patient
Jaundice appears if the serum bilirubin goes above with obstructive jaundice?
2 mg/dl. Gmelin's test, Hay's test, Fouchet's test.
24-6. How jaundice is classified? 24-13. What are the enzymes useful in diagnosis of liver
(i) Prehepatic or Hemolytic. (ii) Hepatocellular. (iii)
diseases?
Posthepatic or Obstructive jaundice.
Alanine aminotransferase; Alkaline phosphatase; Gam-
24-7. What type of bilirubin is present in hemolytic jaun-
ma-glutamyltransferase.
dice?
24-14. What is the characteristic laboratory finding in
Unconjugated bilirubin is increased in serum . Hence
chronic alcoholism?
van den Bergh test is indirect positive.
Elevation of gamma-glutamyltransferase level in serum.
24-8. What happens in obstructive jaundice?
In obstructive jaundice, conjugated bilirubin in serum is 24-15. What is alkaline tide?
elevated, and van den Bergh test is direct positive. When HCI is produced in stomach, bicarbonate level
24-9. What is the picture in hepatocellular jaundice? within the cell increases (formed from H2C03 ) , it is
In hepatocellular jaundice, a biphasic reaction is reabsorbed into blood stream. This would account for
observed, because both conjugated and unconjugated the alkaline tide of plasma and urine, immediately after
bilirubins are increased in serum. meals.
_ _ _ _ _Chapter 25
Kidney Function Tests
Chapter at a Glance
The learner will be able to answer questions on the follow ing topics:
0 Glomerular functions Clearance tests: lnulin, creatinine and urea
Tubular functions Proteinuria
Abnormal constituents of urine Tests for tubular functions
The major functions of the kidneys are to excrete meta- Glomerular Function
bolic waste products as well as to maintain water, pH, elec-
trolyte balance, production of calcitriol and erythropoietin When the blood is perfused through the Bowman's cap-
sule, an ultrafiltrate of the blood is produced in glome-
(Box 25.1 ). A decrease in kidney function is due to a reduc-
rulu s, while the cells and proteins are retained in the
tion in the performance of nephrons. The functional unit
blood. The sieves of the glomeruli are such that hemo-
of the kidney is the nephron, which is composed of the
globin (mol wt 67,000 D) is passed through to be excre-
Bowman's capsule with the glomerular tuft of capillaries,
ted in urine, while albumin (mol wt 69,000 D) is retained
the proximal convoluted tubule (PCT), loop of Henle,
in the blood. Therefore, the earliest manifestation of the
distal convoluted tubule (OCT) and collecting tubules.
abnormal fu nction of the glomeruli is the appearance of
IRENAL FUNCTION TESTS albumin in urine.
TABLE 25. 1: Handling of solutes by the renal tubules TABLE 25.2: Threshold value of some common substances
excreted through urine
Relative
Compound Mode of handling by tubules concentration Substance Threshold value plasma level
Creatinine Not reabsorbed; secreted in GF = Urine 1. Glucose 180 mg/dL
small amounts 2. Lactate 60 mg/dL
Uric acid 90% is first absorbed in PCT; GF ;:: Urine 3. Bicarbo nate 28 mEq/ L
but later secreted in OCT
4. Calcium 10 mg/dL
Urea About 40% rea bsorbed in GF > Urine
PCT
Sodium Partially reabsorbed GF > Urine Renal Threshold and Tubular Maximum
Glucose Completely reabsorbed GF >> Urine
Compounds whose excretion in urine are dependent
Amino acid Completely reabsorbed GF >> Urine
on blood level are known as threshold substances.
(PCT: Proximal convoluted tubules; OCT: Distal convo luted tubules)
At normal or low plasma levels, they are completely
reabsorbed and are not excreted in urine. But when the
blood level is elevated, the tubular reabsorptive capa-
TABLE 25.3: Main functions of kidney tubules
city is saturated, so that the excess will be excreted in
Segment ofnephron Reabsorption of Secretion of
urine (Table 25.2). The renal threshold of a substance is
Proximal Sodium (85%), H+,
convoluted Chloride (85%), Acids
the plasma level above which the compound is excreted
tubule (PCT) Bicarbonate (85%), and bases, in urine. For glucose, the renal threshold is 180 mg/dl.
Glucose (100%), NH;, In other words, glucose starts to appear in urine when
Amino acids (100%), Diodrast,
Uric acid, water (obligatory). PAH blood level is more than 180 mg/dl. Table 25.2 gives a
Loop of Henle Na+, Cl·, Ca++, Mg++
list of threshold substances. In abnormal conditions, the
renal threshold may be lowered so that even at lower
Distal convoluted Na',CI·, H+, K+, NH;,
tubule (OCT) Water (facultative) Uric acid blood levels, compounds are excreted in urine, e.g.
renal glucosuria (glucose); and renal tubular acidosis
(bicarbonate).
Glomerular Filtration Rate (GFR)
GFR is decreased when BP is below 80 mm of mercury. Reabsorption of Water
The GFR is reduced when there is obstruction to the The osmolality of urine can vary between 60- 1200
renal flow (calculi, enlarged prostate, etc.). It also mosmol/kg (specific gravity = 1.003 to 1.032), depend-
decreases with age. The renal blood flow is about 700 ml ing on the water intake and state of hydration. The GFR
of plasma or 1200 ml of blood per minute. The glome- is about 125 ml/min. In the proximal convoluted tubules,
rular filtration rate (GFR) is 120-125 ml per minute in a most of this is reabsorbed. Since Na+, c1- and HCO3-
person with 70 kg body weight. Glomerular filtrate formed ions are absorbed, water has to move along with the
is about 170 to 180 liters per day, out of which only solutes to maintain the osmolality. Hence, this is called
1.5 liters are excreted as urine. This means that most of obligatory reabsorption of water. By the time it reaches
the water content of glomerular fi ltrate is reabsorbed. the loop of Henle, the filtrate is only 25 ml/min. Here
sodium is again reabsorbed, but water absorption is less
Functions of the Tubules so that, urine, is hypotonic at this level.
By the time urine reaches distal tubules, the flow
When the glomerular filtrate is formed, it contains almost rate is reduced to 16 mUmin. Here again water is reab-
all the crystalloids of plasma. In the proximal convoluted sorbed, but it is under the influence of ADH. Therefore,
tubules, about 70% water, Na• and Cl- as well as 100% this is called facultative reabsorption of water. ADH
glucose, amino acids and K• are reabsorbed . Urea, phos- secretion, in turn, is controlled by hypothalamic osmo-
phate and calcium are partially absorbed. (Table 25.1 ). receptors. The osmolality of plasma is the stimulus for
Table 25.3 shows the functions of different parts of the modulating ADH secretion. Thus, when urine reaches
renal tubules. the collecting ducts, the flow rate is only about 1 ml/min,
372 Section C: Clinical and Applied Biochemistry
TABLE 25.S: Abnormal1t1es detected 1n d1pst1ck may refer the practical textbook. The routine analysis of
Test and normal range Interpretations urine is the most popular test in hospital practice. The
1. Specific gravity Low SG in renal tubular dysfunction; following param1:iters are usually checked when report-
1.005-1.025 diabetes insipidus; polydypsia. High ing on a urine sample.
SG in inadequate water intake; volume
depletion
Physical Characteristics of Urine •
2. pH 5.5-6.5 Low pH in high protein diet and acidosis,
recent meal-alkaline tide, high pH in low
protein diet
i. Volume: Th1:i average output of urine is about 1.5 liters
3. Blood Menstruation, t raumatic catheterizat ion,
per day. Urine volume may be increased in excess
glomerulonephritis, stones, tumor and water intake, diuretic therapy, diabetes mellitus and
trauma of urinary t ract, hemoglobinuria,
hemolysis
in chronic irenal diseases. Urine volume may be
4. Protein Fever, exercise, orthostatic proteinuria; decreased in excess sweating, dehydration, edema
<150 mg/day glomerulonephritis, urinary tract infection, of any etiolo~JY, kidney damage. Urine volume 1.5 U24 h;
tubu lar diseases
typical in health, oliguria < 400 ml, anuria < 100 ml,
5. Glucose Diabetes mellitus, renal glucosuria;
Fanconi's syndrome polyuria > 3,000 ml
6. Ketone bodies Diabetes mel litus, starvation ii. Specific gravity: Described under tests for tubu-
7. Bil irubin Hepatitis, obstructive jaundice lar function . A summary of the findings is listed in
B. Urobilinogen <4 Concentrated urine; hepatitis; intravascular Tables 25.4 and 25.5.
mg/day hemolysis; low in obstructive jaundice
9. Bile salt Obstructive jaundice Chemical Clharacteristi cs of Urine
10. Nitrite Urinary tract infection Reaction to Litmus
11. Leukocyte esterase Urinary tract infection, fever
The pH of urine varies from 5.5 to 7.5. If diet is rich in
proteins, sulfuric and phosphoric acids are produced
and the urine is hypertonic. Clinical applications of diu-
from amino acids, and the urine becomes acidic. If the
retics are shown in Box 25.3.
diet is rich in vegetables, urine is alkaline because the
organic acids (citric and tartari c) present in vegetables
ABNORMAL CONSTITUENTS OF
are converted to bicarbonate in the body.
URINE
In clinical biochemistry, urine is tested and report is Proteins
given on a urine sample. The procedure is called uri ne Proteinuria is an important index of renal diseases. In
analysis or urinalysis. For details of the tests, the student normal urine, protein concentration is very low, which
Chapter 25: Kidney Function Tests 373
TABLE 25.8: Normal reference values TABLE 25.9: Factors affecting serum creatinine
Serum crearinine GFR Factors reducing serum crearinine Factors increasing serum crearinine
Adult male 0.7 - 1.4 mg/ dL 95 - 115 ml/min Low muscle mass Old age
Adult female 0.6 - 1.3 mg/dL 85 - 110 ml/min Females Males
Children 0.5 - 1.2 mg/dl Malnutrition Renal diseases
Medicines Glomerulonephrltis
Thiazide Pyelonephritis
The kidney reserve is such that about 50% kidney
Vancomycin Renal failure
function must be lost before creatinine level in blood is
Urinary obstruction
raised. Serum level usually parallels the severity of the
Congestive cardiac failure
disease.
Dehydration. shock
Creatinine level more than 1.5 mgldL indicates
Medicines
impairment of renal function. Creatinine is quantitated by
Amphotericin B
Jaffe's test (alkaline picrate). Test kit based on specific
Cephalosporins
enzymatic reaction is also available.
Kanamycin
Reference values for creatinine level in blood and
the creatinine clearance are shown in Table 25.8. When
corrected for surface area, the creatinine clearance in healthy individuals, nor in children and obese people.
value will become comparable between males, females The accuracy of plasma creatinine estimation is a major
and children, which is about 100 mUmin/1.73 sq meter. deciding factor in both equations.
TABLE 25.1 0: Grading of chronic kidney disease TABLE 25.11 : K1dnP-y failure early symptoms
State Grade GFR mUmt/1.73 m' Polyu ria (passing more urine)
Minimal damage with normal GFR 1 >90 Nocturia (passing more urine during night) I
Mild damage with slightly low GFR 2 60- 89 Pedal edema, puffiness of face
Moderately low GFR 3 30-59 High blood pressure I
Severely low GFR 4 15-29 Unexplained anemia I
Kidney failure s < 15 Fatigue, lassitude and tiredness I
Microalbuminuria
a better test for kidney function (GFR) than serum Mild elevation of serum creatinine I
BOX 25.5: Causes for increased blood urea involves administration of an extraneous compound, this
procedure is not used routinely.
1. Prerenal conditions:
Dehydration: Severe vomiting, intestinal obstruction, diarrhea lnulin clearance (GFR) = 125 mUmin and urea
Diabetic coma and severe burns clearance = 75 mUmin .
Fever and severe infections
inulin clearance - urea clearance 125-75 = 0 4
2. Renal diseases: 125 .
glomerular filtration rate
Acute glomerulonephritis
Nephrosis In other words, 40% of urea present in the glomeru-
Malignant hypertension lar filtrate is reabsorbed in the tubules.
Chronic pyelonephrltls
3. Postrenal causes:
Stones in the urinary tract
Diodrast Clearance
Enlarged prostate Diodrast is a contrast medium usually used in taking X-
Tumors of bladder
ray of urinary tract. Diodrast and PAH (para-amino hip-
4. Medications:
ACE inhibitors purate) are filtered and excreted, so that these substan-
Diuretics ces are removed by one passage of the blood through
NSAIDs kidney. PAH clearance is a measure of renal plasma
flow . It is about 700 ml of plasma or 1200 ml of blood
,vhile the same value in a person of 60 years can be per minute and is about 114th of the total cardiac output.
:onsidered as perfectly normal. Causes for increased Since, renal plasma flow is 700 mUmin and the
blood urea are enumerated in Box 25.5. GFR is 125 mUmin , it is obvious that about 115th of the
plasma brought to the glomeruli becomes the glomeru-
Renal Diseases lar filtrate. This is called the filtration fraction .
Proteinuria
Azotemia
It may be of the following types:
Increase in the blood levels of NPN is referred to as
azotemia and is the hallmark of kidney failure.
Glomerular Proteinuria
/nu/in Clearance Plasma proteins are absent in normal urine. When glo-
lnulin is a polysaccharide of fructose. It is not appre- meruli are damaged or diseased, they become more
ciably metabolized by the body. It is neither absorbed nor permeable and plasma proteins may appear in urine.
secreted by the tubules. Therefore, inulin clearance is a The smaller molecules of albumin pass through dam-
measure of GFR. The test needs continuous infusion of aged glomeruli more readily than the heavier globulins.
inulin so as to keep the plasma level adequate. Since it Albuminuria is always pathological.
378 Section C: Clinical and Applied Biochemistry
BOX 25.6: lnd1cat1ons for quant,tal1011 of proteinuna through normal glomeruli and, therefore, if it exists in
1. Diagnosis of nephrotic syndrome- Nephrotic syndrome is a free form (as in hemolytic conditions), hemoglobin can
triad of edema, hypoalbuminemia and proteinuria > 3g/day. appear in urine (hemoglobinuria). Similarly, myoglobi-
24 hours urine protein, creatinine clearance and sodium
should be measured for planning appropriate treatment. nuria is seen following crush injury of muscles.
2. Prognosis of progressive renal disease-It is a marker for asses- Yet another example is the Bence-Jones proteinu-
sing the progressive loss of renal function in renal disease;
diabet ic nephropathy, chronic glomerulonephritis, reflux ria. In about 20% cases of multiple myeloma (plasmacy-
nephropathy. Treatments that reduce proteinuria (like anti hy- toma), the light chains of immunoglobulins are produced
pertensive drugs) decrease rate of progression.
abnormally. Being of smaller molecular weight, they are
3. Diagnosis of early diabetic nephropathy-Early stages of dia-
betic nephropathy are characterized by increase in GFR, mi- excreted in urine. These are called Bence-Jones Pro-
croalbuminuria and hypertension. teins (monoclonal light chains produced by plasmacy-
tomas) (Chapter 46). When the urine is heated, at 45°C
Overnight first voided sample (early morning urine- they start precipitating, at 60°C there is maximum preci-
EMU) may be used for the measurement of protein. pitation , at 80°C these proteins start redissolving , and
Detection limit with Dipstick is 200- 300 mg/L. will form a clear solution at 100°C. The precipitate reforms
300 mg/day = Benign proteinuria on cooling. It is also detected by immunoprecipitation.
300 mg - 1000 mg = Pathological proteinuria
> 1000 mg/day = Glomerular proteinuria
Tubular Proteinuria
Indications for quantitation are shown in Box 25.6. The tubular reabsorption mechanism is impaired, so
low molecular weight proteins appear in urine. They are
Microalbuminuria retinol binding protein (RBP) and alpha-1-microglobulin.
The condition is also called minimal albuminuria or
Urogenic Proteinuria
paucialbuminuria. It is identified, when small quantity of
albumin (30-300 mg/day) is seen in urine. The test is This is due to inflammation of lower urinary tract, when
not indicated in patients with overt proteinuria (+ve dip- proteins are secreted into the tract.
stick). Early morning midstream sample is preferred.
Microalbuminuria is an early indication of nephropa- ITESTS FOR TUBULAR FUNCTION
thy in patients with diabetes mellitus and hypertension.
Specific Gravity of Urine
Hence, all patients who are known diabetics and hyper-
tensive should be screened for microalbuminuria. It is an Normal specific gravity of urine is 1.015-1 .025. Theo-
early indicator of onset of nephropathy. The test should retical extremes are 1.003 to 1.032. The specific gravity
be done atleast once in an year. will be decreased in excessive water intake, in chronic
nephritis and in diabetes insipidus. It is increased in
It is expressed as albumin-creatinine ratio; normal
diabetes mellitus, in nephrosis and in excessive per-
ratio being,
spiration . In chronic renal failure, the specific gravity of
Males < 23 mg/g of creatinine
urine is fixed at 1010. The earliest manifestation of renal
Females < 32 mg/g of creatinine
damage may be the inability to produce concentrated
Patients showing hig her values on more than one urine.
occasion are considered to have microalbuminuria. The simplest test of tubular function is the measure-
Confirmed by overnight urine collection and calculation ment of the specific gravity (SG) of urine. This is an indi-
of albumin excretion rate. A value more than 20 microg/ cation of osmolality.
min confirms microalbuminuria. Specific gravity depends on the concentration of
solutes, whereas osmolality depends on the number of
Overflow Proteinuria osmotically active particles. Hence, in cases of protein-
uria, the specific gravity is elevated considerably, but
When small molecular weight proteins are increased osmolality is only mildly elevated.
in blood, they overflow into urine. For example, hemo- The inability to excrete the waste products may
globin having a molecular weight of 67,000 can pass be counterbalanced by large urine output. Thus the
Chapter 25: Kidney Function Tests 379
earliest manifestation of renal disease may be diffi- TABLE 25.12: Summary of renal function tests
culty in concentrating the urine. Glomerular dysfunction Tubular dysfunction
Serum urea t Urine concentration ..i.
Measurement of Osmolality Serum creatinine t Dilution test Abnormal
The osmolality of urine samples vary widely from 60 lnulin clearance .!, Uric acid excretion ..i.
mosmol/kg to 1200 mosmol/kg. A random urine sample Creatinine clearance ..i. Blood uric acid t
may have an osmolality around 600 mosmols/kg and Urea clearance ..i.
it increases to 850 after 12 hours fluid restriction. The PAH clearance ..i. Acidification of ..i.
urine
normal value of plasma osmolality is 285- 300 mOsm/kg.
Proteinuria Present Aminoaciduria Present
revealed that he had similar symptoms 5 years back and disturbances are clinical features of ARF. Causes for
was diagnosed with hypertension and kidney failure. On ARF are usually classified as prerenal, instrinsic and pos-
examination, temperature was 36.8°C, respiratory rate trenal causes. Risk factors for ARF include hypertension,
was 22/min , pulse rate 64/min, BP was 170/100 mm Hg, congestive heart failure, diabetes, multiple myeloma,
marked pallor was present, chest and lungs showed bi- chronic infection and myeloproliferative diseases.
lateral basal rales, abdomen was soft, flat and tender. Laboratory investigations include blood urea (or
No other abnormality was detected. Patient was an BUN, blood urea nitrogen), serum creatinine, myo-
occasional alcoholic, and a chronic smoker. globin, free hemoglobin, uric acid, serum electrolytes
Laboratory investigations showed-Blood urea (hyponatremia, hyperkalemia, hyperphosphatemia ,
65 mg/dl, serum creatinine 2.4 mg/dl, serum calcium 6.4 hypocalcemia, hyper-magnesemia and metabolic acido-
mg/dl, serum potassium 4.9 mg/dl, and serum sodium
sis are seen in ARF), serological tests for antinuclear anti-
139 mmol/L. Urine examination results were - Color straw
bodies (ANA}, hepatitis, antistreptolysin-O (ASO), cross
colored, pH 5.0, specific gravity 1.020, appearance turbid,
reacting antibody produced by streptococci and comple-
volume 900 mU24 h, albumin 3+, sugar negative, pus
ments, urine analysis (casts, myoglobin, hemoglobin,
cells 1-3/HPF, RBC - 1-2/HPF, and epithelial cells rare.
proteins, RBC and WBC or their casts, eosinophils, uric
What is the probable diagnosis?
acid, calcium oxalate crystals) and urine electrolytes.
Renal biopsy is indicated in patients in whom prerenal
•ii• Clinical Case Study 25.1 Answer and postrenal causes have been ruled out and cause for
Proteinuria, hypoalbuminemia and hypercholesterolemia instrinsic ARF is unclear.
along with renal failure and edema are the classi-
cal presentation of nephrotic syndrome (nephrosis). •• Clinical Case Study 25.3 Answer
Patient has diabetes mellitus and hypertension, and these
The patient is suflfering from chronic renal failure (CRF),
may have caused nephrotic syndrome. Confirmation of
also known as chronic kidney disease (CKD). The history
diagnosis can be done by renal biopsy. Hypercoagula-
of hypertension and kidney failure goes against the
ble state, hypovitaminosis D and immunodeficiency are diagnosis of ARF.
associated features seen.
Decreased n:inal function interferes with kidneys'
Causes of nephrotic syndrome are; (1) Primary - ability to maintain fluid and electrolyte balance. The abi-
minimal change nephropathy, focal glomerulosclerosis, lity to concentrate urine declines early and is followed
membranous nephropathy, hereditary nephropathies,
by inability to excrete phosphate, acids and potassium.
and (2) Secondary - Diabetes mellitus, lupus erythema- In advanced stagies, urine cannot be diluted and urine
tosus, amyloidosis, paraproteinemia viral infections, like osmolality is "fixeid". Plasma levels of urea and creati-
hepatitis B, C, HIV, etc., pre-eclampsia, vasculitis, drugs. nine are elevated rapidly, and abnormalities of calcium,
Treatment is based on treating protein loss, edema, phosphate, PTH, vitamin D, and renal osteodystrophy
hyperlipidemia, hypercoagulable state, associated are seen. Moderate acidosis is seen. Anemia is
nutritional deficiencies and protection from infections. normochromic-normocytic in nature. CKD is rarely rever-
Prognosis depends on cause, person's age and type and sible and leads to progressive decline in renal function.
degree of renal damage caused at the time of initiating Diagnostic tests are electrolytes, blood urea nitrogen
treatment. (BUN) or blood urea, creatinine, phosphate, calcium,
CBC and urinalysis. The definitive diagnostic tool is
• · Clinical Case Study 25.2 Answer renal biopsy, but 1is not recommended when ultrasound
The patient is suffering from acute renal failure (ARF), indicates small, fibrotic kidneys.
also referred to as acute kidney injury (AKI). A brief Treatment includes: (1) Control of underlying dis-
history of bacterial infection, rising urea and creatinine eases, (2) Restriction of dietary protein, phosphate and
values, oliguria, edema and failed response to diuretics potassium, (3) Vitamin D supplements, (4) Treatment
all point towards with diagnosis. Retention of nitroge- of anemia and heart failure, and (5) dialysis for severe
nous waste products, oliguria, electrolyte and acid-base decrease in renal function.
Chapter 25: Kidney Function Tests 381
25-1. Normal specific gravity of urine is: 25-6. Renal plasma flow is measured by:
A. 1.003 to 1.010 B. 1.010 to 1.015 A. Creatinine clearance
C. 1.015to1 .025 D. 1.025 to 1.035 B. lnulin clearance
25-2. Polyuria is a characteristic feature of all the follow- C. Para amino hippurate clearance
ing, except D. Urine output
25-7. A patient with chronic renal failure will excrete:
A. Diabetes mellitus B. Glomerulo nephritis
A. Acid urine
C. Diabetes insipidus D. Chronic renal failure
B. Hypertonic urine
25-3. Specific gravity of urine increases in:
C. Urine with specific gravity more than 1018
A. Chronic glomerulonephritis
D. Urine with low creatinine concentation
B. Diabetes mellitus
25-8. Which indicates an abnormal renal function?
C. Liver diseases
A. Blood urea 30 mg/di
D. Intake of vegetables =
B. GFR 125 ml/min
25-4. Proteinuria is seen in all the following conditions, C. Serum creatinine 8 mg/di
except: =
D. Urine pH 6.8
A. Acute glomerulo nephritis 25-9. Urea clearance is lowered in:
B. Diabetes insipidus A. Acute nephritis
C. Pyelonephritis B. Chronic liver failure
D. Multiple myeloma C. Chronic cardiac failure
25-5. Which of the following compounds have a clea- D. Benign hypertension
ranee value higher than GFR? 25-10. Normal value of blood urea is:
A. Uric acid B. Diodrast A. 3-4 mg/100 ml B. 4-8 mg/100 ml
C. Urea D. lnulin C. 8-16 mg/100 ml D. 20-40 mg/100 ml
382 Section C: Clinical and Applied Biochemistry
25-11 . Failure of concentrating capacity of urine is as- A. Ehrlich's test
sessed by: B. Rothera's test
A. Urea clearance C. Jaffe's test
B. Water deprivation test D. Schlesinger's test
C. Specific gravity of urine after giving water 25-23. Which of the following constituents of urine does
D. Creatinine level in urine not contain nitrogen?
25-12. Urine of a patient with obstructive jaundice will A. Urobilinogen B. Uric acid
give a positive test for all the following, except C. Aceto acetic acid D. Urea
A. Gmelin's test B. Hay's test 25-24. To calculate the creatinine clearance, all the fol-
C. Fouchet's test D. Benzidine test lowing data are required, except
25-13. Urine will have abnormal color in all the following A. Plasma creatinine level
conditions, except B. Patient's height and weight
A. Melanuria B. Porphyria C. Daily urine output
C. Creatinuria D. Alkaptonuria D. Specific gravity of urine
25-14. All the following statements regarding glomerular 25-25. Urea level in blood may be elevated in all the fol-
filtration is correct, except: lowing conditions, except:
A. GFR is approximately equal to endogenous creati- A. Dehydration B. Renal failure
nine clearance C. Hepatic failure D. Hypertension
B. Measurement of GFR is a test of renal tubular 25-26. Renal stones may contain all the following organ-
function ic constituents , except:
C. Glomerular filtrate has a specific gravity of 101 O A. Cystine B. Uric acid
D. Glomerular filtrate is formed by ultrafiltration of C. Cholesterol D. Xanthine
plasma 25-27. All the following laboratory data are suggestive of
25-15. Clearance values of all the following compounds acute renal failure, except
are used to test renal function , except: A. Plasma sodium - 150 mmol/L
A. Creatinine B. lnulin B. Plasma potassium - 5.6 mmol/L
C. Urea - 220 mg/di
C. Glucose D. Urea
D. Creatinine - 3.2 mg/di
25-16. When 15N (radio-active nitrogen) labelled glycine
25-28. Which ion is decreased in renal failure :
is given, the label will appear in all the following
A. Phosphate
compounds in urine, excep t
B. Potassium
A. Urobilinogen B. Urea
C. Bicarbonate
C. Urocanic acid D. Uric acid
D. Hydrogen ions
25-17. Normal urine contains all following, except
25-29. Renal tubular function can be assessed by:
A. Glucose B. Ethereal sulfate
A. Urea clearance
C. Bicarbonate D. Creatinine
B. Plasma electrolytes
25-18. Opalescent urine is seen in?
C. Specific gravity of early morning sample of urine
A. Porphyria B. Alkaptonuria
D. Plasma urea
C. Chyluria D. Creatinuria
25-30. Plasma urea is increased but creatinine is normal
25-19. Which anions are not normally present in urine?
in all the following, except:
A. Phosphate B. Sulfate
A. High protein intake
C. Chloride D. Proteinate B. Gastrointestinal bleeding
25-20. All are functions of the kidney, except C. Starvation
A. Excretion of hydrogen ions D. Muscle wasting
B . Detoxification of alcohol
25-31 . Which of the following cannot pass through the
C. Stimulation of erythropoiesis normal glomerular membrane?
D. Formation of 1,25 DHCC A. Creatinine B. Albumin
25-21 . A patient with chronic renal failure may have all the C. Myoglobin D. Uric acid
following features , except: 25-32. Which substance is not normally present in urine?
A. Urine with fixed specific gravity of 101 0 A. Creatinine B. Glucose
B. Protein creatinine ratio more than 3 C. Uric acid D. Urobilinogen
C. Positive heat and acetic acid test 25-33. Excretion of which substance is not under control
D. Nocturnal polyuria of hormones?
25-22. All the following tests are answered by normal A. Calcium
urine, except: B. Potassium
Chapter 25: Kidney Function Tests 383
25-1. What is glomerular filtration rate? expressed as milliliter per minute. It is the ml of plasma
120-125 ml per minute. which contains the amount of that substance excreted
25-2. What is the function of glomerulus? by the kidney within a minute.
An ultrafiltrate of the blod is produced in glomerulus, 25-13. What is the best method for assessing glomerular
while the cells and proteins are retained in the blood. filtration rate?
25-3. What is the normal specific gravity of urine? Creatinine clearance test.
1.015 to 1.025. 25-14. What is the advantage of creatinine test?
25-4. What is the minimum and maximum specific gra- Creatinine is formed spontaneously (non-enzymatic),
vity of urine? so the blood level and excretion rate of creatinine is a
1.003 to 1.032. constant.
25-5. What is the specific gravity of urine in chronic 25-15. What is the significance of creatinine clearance?
renal failure? A decreased creatinine clearance is a very sensitive
Fixed to 1.010. indicator of a red uced glomerular filtration rate.
25-6. Specific gravity of urine increased when? 25-1 6. What is the normal creatinine level in blood?
Diabetes mellitus and acute glomerulonephritis. For adult males, 0.7-1.4 mg/di; for adult females,
25-7. Polyuria is seen in which conditions?
0.6-1.3 mg/di.
Diabetes mellitus, diabetes insipidus, chronic renal failure.
25-17. What is creatinine coefficient?
25-8. Proteinuria is seen in which conditions?
It is the utrinary creatinine expressed in mg/kg body
Acute glomerulonephritis, nephrotic syndrome, pyelo-
weight.
nephritis, multiple myeloma.
25-18. Urea clearance is lowered in which condition?
25-9. What is the test to detect urinary protein?
Chronic liver failure.
Heat and acetic acid test.
25-19. What is tlhe normal blood urea level?
25-10. What is microa lbuminuria?
When small quantities of albumin (30-300 mg/day) is 20-40 mg/di
seen in urine.
25-20. What is tlhe clinical significance of urea level?
25-1 1. What are Non-protein nitrogen (NPN)? Urea level is increased in renal failure.
Urea, uric acid, creatinine. In kidney diseases, the lev- 25-21 . How is tubular concentrating capacity measured?
els of these substances in blood are increased. By measutring urine specific gravity.
25-12. What is meant by renal clearance? 25-22. What are the tests to assess tubular function?
Clearance is defined as the quantity of blood or plasma Measurement of specific gravity, concentration test,
completely cleared of a substance per unit time and is dilution test. acidification test.
_ _ _ _ _Chapter 26
Plasma Proteins
Chapter at a Glance
The learner will be able to answer questions on the following topics:
0 Plasma proteins Transport proteins in blood
O Electrophoresis Acute phase proteins in blood
0 Albumin, functions, clinical significance Ceruloplasmin
0 Hypoalbuminemia O Alpha-1 antitrypsin
0 Globulins, alpha, beta, gamma Clotting factors
Point of application
! 13
[JO 00 0 · ,
y a2 a1 Albumin -ve 2 3 4 5 6 7 8 9 10
Start point
Gamma
laJ~D oo o I· , Beta
Alpha-2
-I DO OO I I· ,
Alpha-1
Albumin
+ve
-IOo ID D I· ·
terminal
Lanes 2,4,8, 10 = Normal pattern. Lane 1 = Nephrotic syndrome;
hypoalbuminemia, prominent alpha-2 band. Lane 3 = Cirrhosi~;
o o oo o I· ,
hypoalbuminemia with beta-gamma bridging. Lane 5 = Chrome
infection, broad based increase in gamma region; general Increase In
alpha-1 and alpha-2 bands, comparative reduction of albumin. Lanes
6,7 = Multiple myeloma; monoclonal ba~d (M-band) betwee~ beta
I om 00 I I·
Fig. 26.1B: Serum electrophoretic patterns
Multiple myeloma
Normal pattern Inflammatory
Monoclonal band
I I I
unconjugated bilirubin can cross the blood brain barrier Albumin-Gl,obulin Ratio
and get deposited in brain. The brain of young children
are susceptible; free bilirubin deposited in brain leads to In hypoalbuminemia, there will be a compensatory
kernicterus and mental retardation (see Chapter 22). increase in globulins which are synthesized by the reti-
culoendothelial system. Albumin-globulin ratio (NG
Drug Interactions ratio) is thus altered or even reversed. This again leads
to edema.
When two drugs having high affinity to albumin are
administered together, there may be competition for Hypoproteiinemia
the available sites, with consequent displacement of
Since albumin is the major protein present in the blood.
one drug. Such an effect may lead to clinically significant
any condition causing lowering of albumin will lead to
drug interactions, e.g. phenytoin-dicoumarol interaction.
reduced total proteins in blood (hypoproteinemia).
Protein-Bound Calcium
Hypergammaglobulinemias are Seen in
Calcium level in blood is lowered in hypoalbumine-
mia. Thus, even though total calcium level in blood is Low Albumin Level
lowered, ionized calcium level may be normal, and When albumin level is decreased, body tries to com-
so tetany may not occur (see Chapter 34). Calcium is pensate by incneasing the production of globulins from
lowered by 0.8 mg/dl for a fall of 1 g/dl of Albumin . reticuloendothelial system.
1. Albumin: It is an important transport protein, which conditions. Such proteins are acute phase proteins.
carries bilirubin, free fatty acids, calcium and drugs Important acute phase proteins are described below:
(see above).
2. Pre-albumin or Transthyretin: It is so named C-Reactive Protein (CRP)
because of its faster mobility in electrophoresis than
So named because it reacts with C-polysaccharide of
albumin. It is more appropriately named as Trans-
capsule of pneumococci. It is synthesized in liver. It can
thyretin or Thyroxin binding pre-albumin (TBPA),
stimulate complement activity and macrophage phago-
because it carries thyroid hormones, thyroxin (T4)
cytosis. When the inflammation has subsided, CRP
and tri-iodo thyronine (T3). Its half-life in plasma is
quickly falls, followed by ESR (erythrocyte sedimenta-
only 1 day.
tion rate). CRP level, especially high sensitivity C-reac-
3. Retinal binding protein (RBP): It carries vitamin
tive protein level in blood has a positive correlation in
A (see Chapter 32). It is a negative acute phase
predicting the risk of coronary artery diseases (see
protein.
Chapter 15).
4. Thyroxine binding globulin (TBG): It is the spe-
cific carrier molecule for thyroxine and tri-iodo thy- Ceruloplasmin
ronine. TBG level is increased in pregnancy; but
Ceruloplasmin (cp) is blue in color (Latin, caeruleus =
decreased in nephrotic syndrome.
blue). It contains 6 to 8 copper atoms per molecule.
5. Transcortin: It is also known as Cortisol binding
Ceruloplasmin is mainly synthesized by the hepatic
globulin (CBG). It is the transport protein for cortisol
parenchymal cells. Ceruloplasmin is also called Ferroxi-
and corticosterone.
6. Haptoglobin: Haptoglobin (for hemoglobin), Hemo- dase, an enzyme which helps in the incorporation of
iron into transferrin (see Chapter 34 ). It is an important
pexin (for heme) and Transferrin (for iron) are
antioxidant in plasma. About 90% of copper content of
important to prevent loss of iron from body.
plasma isboundwithceruloplasmin, and 10o/owithalbumin.
Copper is bound with albumin loosely, and so easily
LACUTE PHASE PROTEINS exchanged with tissues. Hence, transport protein for
The level of certain proteins in blood may increase 50 to copper is Albumin. Lowered level of ceruloplasmin is
1000 folds in various inflammatory and neoplastic seen in Wilson's disease, malnutrition, nephrosis, and
Chapter 26: Plasma Proteins 389
cirrhosis. Ceruloplasmin is an acute phase protein. and is an important in vivo anti-coagulant. Its concen-
Increased plasma Cp levels are seen in hemochroma- tration is markedly increased in Nephrotic syndrome,
tosis, and obstructive biliary disease, pregnancy, estro- where other proteins are lost through urine.
gen therapy, inflammatory conditions, collagen disorders
and in malignancies. Drugs increasing the ceruloplas- Negative Acute Phase Proteins
min level are, estrogen and contraceptives. Reference During an inflammatory response, some proteins
blood level of normal adult male is 22-40 mg/dl.
are seen to be decreased in blood; those are called
Wilson's Disease negative acute phase proteins. Examples are albumin,
transthyretin (pre-albumin), retinal binding protein
Ceruloplasmin level is reduced to less than 20 mg/dl
in Wilson's hepatolenticular degeneration. It is an and transferrin. Transferrin is described in Chapter 34,
inherited autosomal recessive condition. Incidence of under iron metabolism.
the disease is 1 in 50,000. The basic defect is a mutation A comprehensive list of normal values for the sub-
in a gene encoding a copper binding ATPase in cells, stances present in blood is given in Appendix II.
which is required for excretion of copper from cells. So,
copper is not excreted through bile, and hence cop- CLOTTING FACTORS
per toxicity. Please also see Chapter 34, under copper
The word coagulation is derived from the Greek term,
metabolism. Increased copper content in hepatocyte
"coagulare" = to curdle. The biochemical mechanism of
inhibits the incorporation of copper to apoceruloplasmin.
clotting is a typical example of cascade activation.
So ceruloplasmin level in blood is decreased.
The coagulation factors are present in circulation as
inactive zymogen forms. They are converted to their
Clinical Features
active forms only when the clotting process is initiated.
Accumulation in liver leads to hepatocellular degenera- This would prevent unnecessary intravascular coagula-
tion and cirrhosis. Deposits in brain basal ganglia leads tion. Activation process leads to a cascade amplifica-
to lenticular degeneration and neurological symptoms. tion effect, in which one molecule of preceding factor
Copper deposits as green or golden pigmented ring activates 1000 molecules of the next factor. Thus within
around cornea; this is called Kayser-Fleischer ring. seconds, a large number of molecules of final factors
are activated. The clotting process is schematically rep-
Alpha-1 Antitrypsin (AAT) resented in Figure 26.4 and the characteristics of coagu-
It is otherwise called alpha-anti-proteinase or protease lation factors are shown in Table 26.2. Table 26.3 gives
inhibitor. It inhibits all serine proteases (proteolytic the assays of clotting factors.
enzymes having a serine at their active center), such as Several of these factors require calcium for their
plasmin, thrombin, trypsin, chymotrypsin, elastase, and activation. The calcium ions are chelated by the gamma
cathepsin. Serine protease inhibitors are abbreviated carboxyl group of glutamic acid residues of the factors,
as Serpins. prothrombin, VII, IX, X, XI and XII. The gamma car-
The AAT is synthesized in liver. It is a glycoprotein boxylation of glutamic acid residues is dependent on
with a molecular weight of 50 KO. It forms the bulk of vitamin K (see Chapter 32).
molecules in serum having alpha-1 mobility. The inci-
dence of AAT deficiency is 1 in 1000 in Europe, but
Prothrombin
uncommon in Asia. Bacterial infections in lung attract It has a molecular weight of 69,000 D. The plasma con-
macrophages which release elastase. In the AAT defi- centration is 10-15 mg/dl. The prothrombin is converted
ciency, unopposed action of elastase will cause dam- to thrombin by Factor Xa, by the removal of N-terminal
age to lung tissue, leading to emphysema. About 5% of fragment.
emphysema cases are due to AAT deficiency.
Fibrinogen
Alpha-2 Macroglobulin (AMG)
The conversion of fibrinogen to fibrin occurs by cleav-
It has a molecular weight of 725 KO. It is the major com- ing of Arg-Gly peptide bonds of fibrinogen. Fibrinogen
ponent of alpha-2 globulins. It is synthesized by hepato- has a molecular weight of 340,000 D and is synthe-
cytes and macrophages. AMG inactivates all proteases, sized by the liver. Normal fibrinogen level in blood is
390 Section C: Clinical and Applied Biochemistry
a -L
Xlla Tissue activation (PT)
~ 1__
• -*.__-~
Xia
l VIiia calcium
platelet factor
Xa
Va calcium
platelet factor
liMii:i·H:M - -~•--
FibrinP
Fibrin clot
200-400 mg/dl. The fibrin monomers formed are produce the active plasmin. Plasmin in turn, is inacti-
insoluble. They align themselves lengthwise, aggregate vated by alpha-2 antiplasmin.
and precipitate to form the clot. Fibrinogen is an acute Tissue plasminogen activator (TPA) is a serine
phase protein. protease present in vascular endothelium. TPA is relea-
sed during injury and then binds to fibrin clots. Then TPA
Fibrinolysis cleaves plasminogen to generate plasmin, which dis-
Unwanted fibrin clots are continuously dissolved in vivo solves the clots.
by Plasmin, a serine protease. Its inactive precursor Urokinase is another activator of plasminogen. Uro-
is plasminogen (90 kD). It is cleaved into two parts to kinase is so named because it was first isolated from
Chapter 26: Plasma Proteins 391
26-1 . Enumerate the major transport proteins of p lasma. Explain the t ransport of free fatty acids, bilirubin, iron and
calcium.
26-2. What are the important functions of albumin? Give the major causes and manifestations of hypoalbuminemia.
26-3. Hemopexin carries: 26-13. Albumin globulin ratio is reversed in all the follow-
A. Free hemoglobin B. Free heme ing conditions, except
C. Free bilirubin D. Free iron A. Cirrhosis liver
26-4. All the following are acute phase reactant proteins, B. Primary immune deficiency
except: C. Nephrotic syndrome
A. C-reactive protein (CRP) D. Multiple myeloma
B. HDL 26-14. It is not safe to give salicylate to infants having
C. Ceruloplasmin hemolytic disease, because:
D. Haptoglobin A. Bilirubin and salicylate compete each other for
26-5. Polymorphism is exhibited by all the following binding on albumin
B. Salicylate is toxic to children
proteins, except:
C. Hemolysis is produced by salicylate
A. Haptoglobin B. Transferrin
D. Both bilirubin and salicylate are metabolized by
C. Albumin D. Ceruloplasmin
the same enzyme systems in liver
26-6. Wilson's hepatolenticular degeneration is charac-
26-15. All are transport proteins of blood, except:
terized by:
A. Albumin B. Transcortin
A. Ceruloplasmin level in blood is increased C. Transferrin D. Ceruloplasmin
B. Copper is accumulated in liver to produce cirrhosis 26-16. One of the plasma proteins listed below Is not a
C Copper is deposited in skin to produce bronze transport protein (carrier protein):
color A. Transferrin B. Haptoglobin
D. Autosomal dominant inheritance C. Albumin D. Alpha-1-antitrypsin
26-7. All are true with regard to alpha-1 antitrypsin (AAT), 26-17. All are true with ceruloplasmin, except
except: A. It is a copper containing protein found in serum
A. It is a protease inhibitor B. It is an acute phase protein
B. It shows polymorphism C. Blood level Is increased in Wilson's disease
C. Deficiency leads to emphysema in lungs D. It has ferroxidase activity
D. Deficiency is associated with edema 26-18. All are correct with hemophilia, except:
26-8. Albumin level in blood is estimated by: A. Inherited as an x-linked recessive trait
A. Jaffe's picric acid reaction B. Inherited by son from father
B. Bromo cresol green reaction C. Daughters are likely to be carriers
C. Diacetyl monoxime method D. Prenatal diagnosis is important
D. Chromatography 26-19. Hemorrhagic tendency is observed in all the follow-
26-9. Which reaction takes place exclusively in liver: ing conditions, except:
A. Gluconeogenesis A. Hemophilia
B. Glycolysis B. Vitamin K deficiency
C. Glycogen synthesis C. Thrombocytopenia
D. Albumin synthesis D. Phenylketonuria
26-10. In blood, allarebound with albumin, except 26-20. Which does not show X-linked inheritance?
A. Glucose-6-P dehydrogenase deficiency
A. Non-esterified fatty acids
B. Hemophilia
B. Bilirubin
C. Von Willebrandt's disease
C. Iron
D. Christmas disease
D. Salicylate
26-21 . The anticoagulant found in the body is:
26-11 . Electrophoretic separation of proteins is of dia-
A. Potassium oxalate B. Sodium citrate
gnostic value in all conditions, except:
C. Heparin D. EDTA
A. Nephrotic syndrome 26-22. Which protein is not present in plasma?
B. Multiple myeloma A. Albumin B. Fibrinogen
C. Alpha-1 antitrypsin deficiency C. Hemoglobin D. Globulins
D. Acquired immunodeficiency syndrome 26-23. The protein present in highest concentration in
26-12. Edema due to hypoproteinemia may be seen in all plasma is:
the following clinical conditions, except A. Fibrinogen B. Gamma globulins
A. Rheumatoid arthritis C. Albumin D. Alpha globulins
B. Cirrhosis liver 26-24. Electrophoretic separation is based on:
C. Malnutrition A. Charge on the protein molecule
D. Nephrotic syndrome B. Molecular weight of the protein
394 Section C: Clinical and Applied Biochemistry
26-1 . What is normal serum albumin level? Retinol binding protein, Thyroxine binding globulin,
3.5 to 5 g/dl. Transcortin (cortisol); Haptoglobin (hemoglobin); Trans-
26-2. What is normal value of total proteins in serum? ferrin (iron); Hemopexin (free heme).
26-10. Name some acute phase proteins.
6 to 8 g/100 ml.
C-reactive protein; Ceruloplasmin; Haptoglobin.
26-3. Edema due to hypoproteinemia is seen in which
26-11 . Polymorphism is exhibited by which proteins?
conditions?
Haptoglobin; Transferrin; Ceruloplasmin.
Cirrhosis liver; Malnutrition; Nephrotic syndrome. 26-12. What is the clinical manifestation of Alpha-1-anti-
26-4. Albumin carries what substances? trypsin deficiency?
Free fatty acids; Bilirubin; Salicylate; Calcium. Emphysema and chronic lung infections.
26-5. What are the functions of albumin? 26-13. What is ceruloplasmin?
It maintains colloidal osmotic pressure of plasma; it It is a copper containing enzyme (ferroxidase) seen in
transports non-esterified fatty acid and bilirubin. blood. It is an acute phase protein.
26-6. Albumin is synthesized in which organ? 26-14. What is the significance of ceruloplasmin?
Ceruloplasmin level in blood is decreased in Wilson's
Liver.
hepatolenticular degeneration.
26-7. Where are gamma globulins synthesized?
26-15. What is the carrier protein of copper?
By reticuloendothelial system (Spleen. lymph nodes).
Albumin.
26-8. Albumin globulin ratio is reversed in which condi- 26-16. Hemopexin carries what?
tions? Free heme.
Cirrhosis; Chronic infections; Nephrotic syndrome; 26-17. How Hemophilia is transmitted?
Multiple myeloma. It is inherited as an x-linked recessive trait; males are
26-9. Name some transport proteins. affected; females are carriers.
1----------Chapter 27
Acid-Base
Balance and pH
Chapter at a Glance
The learner will be able to answer questions on the following topics:
Hydrogen ions (H+) are present in all body compart- BOX 27.1: Terms explained
ments. Maintenance of appropriate concentration of Term Definition and explanations
hydrogen ion (H+) is critical to normal cellular function. pH Negative logarithm of hydrogen ion concentra-
The acid-base balance or pH of the body fluids is main- tion. Normal value 7.4 (range 7.38- 7.42)
Acids Proton donors; pH < 7
tained by a closely regulated mechanism. This involves
Bases Proton acceptors; pH > 7
the body buffers, the respiratory system and the kidney. Strong acids Acids which ionize completely; e.g. HCI
Some common definitions are given in Box 27 .1. Weak acids Acids which ionize incompletely, e.g. H,CO3
pKvalue pH at which the acid is half ionized; Salt:Acid
I ACIDS AND BAS~ = 1:1
Alkali reserve Bicarbonate available to neutralise acids;
Definition Normal 24 mmol/ L (range 22-26 mmol/L)
Buffers Solutions minimize changes in pH
The electrolyte theory of dissociation was proposed by
Arrhenius (Nobel Prize, 1903). According to the definition
proposed by Bronsted, acids are substances that
are capable of donating protons and bases are
those that accept protons. Acids are proton donors
and bases are proton acceptors. A few examples are
shown below:
Acids Bases
HA:;::::::::==W+A- NH 3 +H+:;::::::::==NH/
SPL Svante Arrhenius Johannes N
HCI :;::::::::== w + c1- Hco; + H+ :;::::::::== H2C03
Sorensen NP 1903 Bronsted
H2C03 :;::::::::== H+ + HC03 1868-1939 1859-1927 1879-1947
396 Section C: Clinical and Applied Biochemistry
Weak and Strong Acids TABLE 27. 1: Reli:lt1on between hydrogen ions hydroxyl ions
and pH of aqueous solutions Ionic: product of water~ [H·]
The extent of dissociation decides whether they are [OH]= 10 '
strong acids or weak acids . Strong acids dissociate
[OH"] [H'J log -log(H'J
completely in solution, while weak acids ionize incom- molsAiter molsAiter [H'] =pH pOH Inference
pletely, for example,
1 X 10 13 1 X 101 -1 1 13 Strong acid
HCI - - w+c1- (Complete)
1 X 1010 1 X 10_... -4 4 10 Acid
H2CO3 W + HCO3- (Partial)
1 X 107 1 X 10-7 -7 7 7 Neutral
In a solution of HCI , almost all the molecules disso- 1 X 10 4
1 X 10 10
-10 10 4 Alkali
ciate and exist as H+ and Cl- ions. Hence the concentra- 1 X 10 1 1 X 10 13 - 13 13 1 Strong alkali
tion of W is very high and it is a strong acid. But in the
case of a weak acid (e.g. acetic acid), it will ionize only
partially. So, the number of acid molecules existing in Thus the pH value is inversely proportional to the
the ionized state is much less, may be only 50%. acidity. Lower the pH, higher the acidity or hydrogen
ion concentration while higher the pH, the
Dissociation Constant
acidity is lower (Table 27.1 ). At a pH of 1,
Since the dissociation of an acid is a freely reversible the hydrogen ion concentration is 1o times
reaction, at equilibrium the ratio between dissociated that of a solution with a pH 2 and 100 times
and undissociated particle is a constant. The dissocia-
that of a solution with a pH of 3 and so on.
tion constant (Ka) of an acid is given by the formula,
The pH 7 indicates the neutral pH. KA Hasse/batch
[H•] [A] 1874-1962
K a = - -- - The Effect of Salt Upon the
[HA]
Dissociation
Where [W] is the concentration of hydrogen ions, [A-J =
the concentration of anions or conjugate base, and [HA] The relationship between pH, pKa, concentration of acid
and conjugate base (or salt) is expressed by the Hen-
is the concentration of undissociated molecules.
derson-Hasselbalch equation,
The pH at w hich the acid is half ionized is called
pKa of an acid which is constant at a particular tempera- [base] [salt]
ture and pressure. Strong acids will have a low pKa and pH = pKa + log [acid) or pH = pKa + log [acid]
weak acids have a higher pKa.
When (base]= [acid]; then pH = pKa
Acidity of a Solution and pH Therefore, when the concentration of base and
The acidity of a solution is measured by noting the acid are the same, then pH is equal to pKa. Thus,
hydrogen ion concentration in the solution and obtained when the acid is half ionized, pH and pKa have the same
by the equation. values.
_ K [acid] [HA]
[H+l - a - - or - -
[base] A- IBUFFER_S_ _ _ __ _
where Ka is the dissociation constant. Definition
To make it easier, Sorensen expres-
Buffers are solutions which can resist changes in pH
sed the W concentration as the negative
when acid or alkali is added.
of the logarithm (logarithm to the base 10)
of hydrogen ion concentration, and is
Composition of a Buffer
designated as the pH. Therefore, Lawrence J
Henderson Buffers are of two types:
pH = - log [W] = log _ 1_ 1878-1942 a. Mixtures of weak acids with their salt with a strong
[W] base or
Chapter 27: Acid-Base Balance and pH 397
b. Mixtures of weak bases with their salt with a strong The Henderson-Hasselbalch's equation, therefore
acid. A few examples are given below: has great practical application in clinical practice in
i. H2 CO/ NaHCO3 (Bicarbonate buffer) assessing the acid-base status.
(carbonic acid and sodium bicarbonate)
ii. CH3 COOH/CH 3COONa (Acetate buffer) Effective Range of a Buffer
(acetic acid and sodium acetate) A buffer is most effective when the concentrations of salt
iii. Na2 HPOj NaH2 PO4 (Phosphate buffer) and acid are equal or when pH = pKa. The effective
range of a buffer is 1 pH unit higher or lower than pKa.
Factors Affecting pH of a Buffer
Since the pKa values of most of the acids produced in
The pH of a buffer solution is determined by two factors: the body are well below the physiological pH, they
a. The value of pK: The lower the value of pK, the
immediately ionize and add H• to the medium. This would
lower is the pH of the solution. necessitate effective buffering. Phosphate buffer is
b. The ratio of salt to acid concentrations: Actual
effective at a wide range, because it has 3 pKa values.
concentrations of salt and acid in a buffer solution
may be varying widely, with no change in pH, so
~ CID-BASE BALANCE
_ __ _ _
long as the ratio of the concentrations remains the
same. Normal pH
Factors Affecting Buffer Capacity The pH of plasma is 7.4 (range 7.35-7.45). In normal
On the other hand, the buffer capacity is determined by life, the variation of plasma pH is very small. The pH of
the actual concentrations of salt and acid present, as plasma is maintained within a narrow range. The pH
well as by their ratio. of the interstitial fluid is generally 0.5 units below that of
Buffering capacity is the number of grams of strong the plasma.
acid or alkali which is necessary for a change in pH of
one unit of one litre of buffer solution. Acidosis
The buffering capacity of a buffer is defined as the If the pH is below 7.35, it is called acidosis. Life is threa-
ability of the buffer to resist changes in pH when an tened when the pH is lowered below 7.25. Acidosis
acid or base is added.
leads to CNS depression and coma. Death occurs when
pH is below 7.0.
How Do Buffers Act?
Buffer solutions consist of mixtures of a weak acid or Alkalosis
base and its salt.
To take an example, when hydrochloric acid is When the pH is more than 7.45, it is alkalosis. It is
added to the acetate buffer. the salt reacts with the acid very dangerous if pH is increased above 7.55. Alkalo-
forming the weak acid, acetic acid and its salt. Similarly sis induces neuromuscular hyperexcitability and tetany.
when a base is added, the acid reacts with it forming salt Death occurs when the pH is above 7.6.
and water. Thus changes in the pH are minimized.
CH3- COOH + NaOH -+ CH3-COONa + Hp Volatile and Fixed Acids
CH3-COONa + HCI -+ CH3-COOH + NaCl During the normal metabolism , the acids produced may
The buffer capacity is determined by the absolute be volatile acids like carbonic acid or nonvolatile (fixed)
concentration of the salt and acid. But the pH of the acids like lactate, keto acids. sulfuric acid and phosphor-
buffer is dependent on the relative proportion of the salt
ic acid. The metabolism produces nearly 20,000 milli
and acid (see the Henderson-Hasselbalch's equation).
equivalents (mEq) of carbonic acid and 60-80 mEq of
Application of the Equation fixed acids per day. The sulfoproteins yield sulfuric acid
and phosphoproteins and nucleoproteins produce phos-
The pH of a buffer on addition of a known quantity
of acid and alkali can therefore be predicted by the phoric acid. On an average about 3 g of phosphoric acid
equation. and about 3 g sulfuric acid are produced per day. The
398 Section C: Clinical and Applied Biochemistry
Buffers Act Quickly, but Not Permanently The reverse occurs in the lungs during oxygenation
and elimination of CO2 • When the blood reaches the lungs,
Buffers can respond immediately to addition of acid or the bicarbonate re-enters the erythrocytes by reversal
base, but they do not serve to eliminate the acid from of chloride shift. It combines with W liberated on oxyge-
the body. They are also unable to replenish the alkali nation of hemoglobin to form carbonic acid which disso-
reserve of the body. For the final elimination of acids, ciates into CO 2 and Hp. CO2 is thus eliminated by the
the respiratory and renal regulations are very essential. lungs.
The Second Line of Defense The activity of the carbonic anhydrase increases in
acidosis and decreases with decrease in H' concentration.
This is achieved by changing the pCO2 (or carbonic acid,
the denominator in the equation). The CO2 diffuses from
the cells into the extracellular fluid and reaches the lungs
I RENAL REGULATION OF pH
An important function of the kidney is to regulate the pH
through the blood. When there is a fall in pH of plasma
of the extracellular fluid. Normal urine has a pH around
(acidosis), the respiratory rate is stimulated resulting
6; this pH is lower than that of extracellular fluid (pH =
in hyperventilation. This would eliminate more CO2 ,
7.4). This is called acidification of urine. The pH of the
thus lowering the H2 CO3 level (Box 27.3). However, this
urine may vary from as low as 4.5 to as high as 9.8,
cannot continue for long . The respiratory system res- depending on the amount of acid excreted. The major
ponds to any change in pH immediately, but it cannot renal mechanisms for regulation of pH are:
proceed to completion. A . Excretion of H' (Fig. 27.2)
B. Reabsorption of bicarbonate (recovery of bicarbo-
Action of Hemoglobin nate) (Fig. 27.3)
The hemoglobin serves to transport the CO2 formed C. Excretion of titratable acid (net acid excretion)
in the tissues, with minimum change in pH (see iso- (Fig. 27.4)
hydric transport, Chapter 23). Side by side, it serves to D. Excretion of NH/ (ammonium ions) (Fig. 27.5).
400 Section C: Clinical and Applied Biochemistry
- - ---=--- Na•
HCOr3 + H• - -----'- (-Hydrogen
_. H+ Ions
excratad)
Fig. 27.2: Excretion of hydrogen ions in the proximal tubules; CA Fig. 27.3: Reabsorption of bicarbonate from the tubular fluid ;
= Carbonic anhydrase CA= Carbonic anhydrase
Pasma Tubular cell Tubular lumen Plasma Tubular cell Tubular lumen
Na2HPO4 (pH 7.4)
. A
(+)H 20
Glutamir,~ Glutamic Acid
~i
(Glutaminase)
Na ' -- ----"- Na + NaHPO4
-J
NH3 . NH
3
+
Na Na•
)
H2C03 NaH:zPC)4 NH"
f CA !(pH 5.4)
H2 C03
jcA (H.llb$Ped
'
andexc:Nted)
H O+CO Excreted CO2 + H20
Excretion of H•; Generation of the urine is normally bicarbonate free. The bicarbonate
Bicarbonate combines with H' in tubular fluid to form carbonic acid.
It dissociates into water and CO2• The CO2 diffuses into
This process occurs in the proximal convoluted tubu- the cell , which again combines with water to form car-
les (Fig. 27.2). The CO2 combines with water to form bonic acid .
carbonic acid, with the help of carbonic anhydrase. The In the cell, it again ionizes to H+ that is secreted into
H2CO3 then ionizes to H' and bicarbonate. The hydrogen lumen in exchange for Na•. The HCO3- is reabsorbed
ions are secreted into the tubular lumen; in exchange for into plasma along with Na•. Here, there is no net excre-
Na• reabsorbed. These Na• ions along with HCO3- will tion of W or generation of new bicarbonate. The net
be reabsorbed into the blood. There is net excretion of effect of these processes is the reabsorption of filtered
hydrogen ions, and net generation of bicarbonate. bicarbonate. But this mechanism prevents the loss of
So this mechanism serves to increase the alkali reserve. bicarbonate through urine.
The term titratable acidity of urine refers to the num- ~ ELLULAR BUFF_E_
RS_ _ __
ber of milliliters of N/10 NaOH required to titrate 1 liter
of urine to pH 7.4. This is a measure of net acid excre- Cytoplasmic pH varies from 6.8 to 7.3. Intracellular pH
1ion by the kidney. The major titratable acid present in modulates a variety of cell functions:
1. The activity of several enzymes is sensitive to
the urine is sodium acid phosphate. As the tubular fluid
changes in pH.
passes down the renal tubules more and more W are
secreted into the luminal fluid so that its pH steadily falls. 2. Reduction in pH reduces the contractility of actin
The process starts in the proximal tubules, but continues and myosin in muscles.
up to the distal tubules. Due to the Na• to W exchange 3. The electrical properties of excitable cells are also
affected by changes in pH.
occurring at the renal tubular cell boarder, the Na2 HPO4
(basic phosphate) is converted to NaH2 PO4 (acid phos- Intracellular buffers are depicted in Figure 27.1.
The major tissues involved in cellular buffering are
phate) (Fig. 27.4). As a result, the pH of tubular fluid
bone and skeletal muscle. The buffering of acid is
falls. The acid and basic phosphate pair is considered
achieved by the exchange of W that enters into the
as the urinary buffer. The maximum limit of acidifica-
tion is pH 4.5. cells for Na• or K· ions.
(+) H (+) H (+) (--) rmal anion gap metabolic ac1dos1s (NAGMA)
+ + +
I I,.. I I 1s)
K ,4- 12 AG K AG K AG
1- Cause Remarks
Hco,-
25 HC0 - Diarrhea, intestinal fistula Loss of bicarbonate and cations.
3 Hco,- Sodium or Potassiu m or both
Na+ 138 105 Cl- Na• Cl
- Na• Cl
- Renal Tubular Acidosis Defective acidification of urine
Normal Acidosis Hyperchlo- Due to inability t o reabsorb
remlc bicarbonate or due to inability to
acidosis excrete hydrogen ions
Compensatory increase in chloride
The figures are the concentration in mEq/L or mmol/L. The red (hyperchloremic acidosis)
shaded area denotes AG (anion gap). In acidosis, the bicarbonate
is reduced causing an increase in the anion gap. In hyperchloremic Carbonic anhydras,e Loss of bicarbonate, Na and K
acidosis, there is no change in the anion gap, but as a compensation, inhibitors Similar to proximal RTA
chloride ions are increased.
Ureterosig mioidosltomy Loss of bicarbonate and reabsorption
Fig. 27.7: Gamblegram showing cations on the left and anions of chloride. Hyperchloremic acidosis
on the right side. Such bar diagrams were first depicted by Gam-
Drugs Antacids containing magnesium,
ble, hence these are called Gamble grams
lithium, polymixin B
TABLE 27.5: High anion gap metabolic ac1dos1s (HAGMA) <10 mmol/L. In such conditions, there is depressed
(organic acidosis) myocardial contiractility.
Cause Remarks
Renal failure Sulfuric, phosphoric, organic anions. Decreased Treatment cif Metabolic Acidosis
ammonium ion formation. Na•/H+ exchange results Treatment is to stop the production of acid by giving IV
in decreased acid excretion
fluids and insulin. Oxygen is given to patients with lactic
Ketosis Acetoacetate; beta hydroxy butyrate anions. Seen
acidosis. In all cases, potassium status to be monitored
in diabetes mellitus or starvation
Lactic
closely and promptly corrected.
Lactate anion. It accumulates w hen the rate of
acidosis production exceeds the rate of consumption Bicarbonate re1quirement: The amount of bicarbonate
Salicylate Aspirin poisoning required to treat acidosis is calculated from the base
Amino Acidic metabolic intermediates deficit. In cases ,of acidosis, mEq of base needed= body
acidurias Accumulation due to block in the normal metabolic wt in Kg x 0.2- base excess in mEq/L.
pathway
Organic Organic acids (methyl malonic acid, propionic
Metabolic Alkalosis
acidurias acid, etc.) excreted
Drugs Corticosteroids, Furosemide, Methanol, Nitrates, Primary excess of bicarbonate is the characteris-
Salicylates, Thiazides tic feature. Alkalosis occurs when (a) excess base is
added, (b) base excretion is defective or (c) acid is lost.
Associated hyperkalemia is commonly seen due to All these will lead to an excess of bicarbonate, so that
a redistribution of K• and H+. The intracellular K• comes the ratio becomes more than 20. Important causes and
out in exchange for H+ moving into the cells. Hence, care findings are givein in Table 27.7. Loss of acid may result
should be taken while correcting acidosis which may from severe vomiting or gastric aspiration leading to loss
lead to sudden hypokalemia. This is more likely to hap- of chloride and acid. Therefore, hypochloremic alka-
pen in treating diabetic ketoacidosis by giving glucose losis results. H)rperaldosteron!sm causes retention of
and insulin together. sodium and loss of potassium.
Hypokalemia is closely related to metabolic alkalo-
Clinical Features of Metabolic Acidosis sis. In a!kalosis, there is an attempt to conserve hydro-
The respiratory response to metabolic acidosis is to gen ions by kidney in exchange for K•. This potassium
hyperventilate. So there is marked increase in respira- loss can lead to hypokalemia. Potassium from ECF will
tory rate and in depth of respiration; this is called as enter the cells in exchange for H+. So, in alkalosis, pH
Kussmaul respiration. The acidosis is said to be of urine remains, acidic; hence this is called paradoxic
dangerous when pH is < 7.2 and serum bicarbonate is acidosis.
Chapter 27: Acid-Base Balance and pH 405
No abnormality or
0 mixed acid-base
• · Clinical Case Study 27.1 disorder
High Low
Interpret the data and give the type of acid base distur- pC02 pC02
bance. Blood pH - 7.12, pCO2 - 80 mm Hg, Plasma
Bicarbonate- 26 mEq/L, H2CO3 - 20.7 mEq/L. What are Respiratory Respiratory Metabolic
acidosis acidosis acidosis
the causes for the condition?
Chapter 27: Acid-Base Balance and pH 407
27-1 . What is the normal pH of blood? Explain the role of plasma buffers and renal mechanisms in the maintenance
of acid base balance of the body.
27-2. Name the important buffer systems in the body. Describe the role of kidlney and lungs in the maintenance of acid
base balance.
27-3. What is titratable acidity of urine? What is the role of kidney in maintaii ning acid base balance?
27-4. What is metabolic acidosis? Enumerate its causes. What are the compensatory mechanisms?
27-1. When pH falls by 1 unit, what is the change in the In acidosis, there is hyperventillation, so CO2 is low-
hydrogen ion concentration? ered, and H 2C03 is lowered.
Increases 10 times. 27-12. Glutaminase enzyme is used for what purpose?
27-2. Relationship between pH and pK is given by which For production of ammonia in kidney tubules Figure 27 .5.
equation? 27-13. What is metabolic acidosis?
Henderson-Hasselbalch's equation. Primary d eficit of bicarbonate.
27-3. What determines the pH of buffer? 27-14. What are the causes of metabolic acidosis?
By the ratio of salt to acid. Diabetic ketosis, chronic renal fail ure, diarrhea.
27-15. What are the features of diabetic ketoacidosis?
27-4. What determines the buffering capacity?
Lowered bicarbonate; elevated plasma chloride;
The absolute concentrations of the salt.
increased anion gap.
27-5. What is meant by buffering capacity?
27-16. How to calculate anion gap?
The buffering capacity of a buffer is defined as the abil-
(sodium+ potassium) minus (chloride + bicarbonate).
ity of the buffer to resist changes in pH when an acid or
27-17. What is tine cause for high anion gap acidosis?
base is added.
Diabetic ketoacidosis, chronic renal failure, renal tubu-
27-6. Buffer is most effective at which pH?
lar acidosis, lactic acidosis.
When the pH is nearer to the pK of the acid.
27-18. What is metabolic alkalosis?
27-7. In the blood, which buffer is most effective? Primary excess of bicarbonate.
Bicarbonate buffer. 27-19. What are the causes of metabolic alkalosis?
27-8. What are the mechanisms for maintaining the nor- Prolonged vomiting; gastric aspiration; and ingestion
mal pH of plasma? of antacids.
Buffers of plasma; Lung mechanism; Kidney mechanism. 27-20. What is r,espiratory acidosis?
27-9. What is the alkali reserve of the body? Primary excess o f carbonic acid.
Bicarbonate is the alkali reserve. 27-21 . What are the causes of respiratory acidosis?
27-10. What is the normal ratio of bicarbonate to carbonic Bronchial asthma; bronchopneumonia; narcotic poisoning.
acid in blood? 27-22. What is r,espiratory alkalosis?
Bicarbonate to carbonic acid ratio is 20. Primary deficit of carbonic acid.
27-11 . What is the respiratory mechanism of pH regu- 27-23. What is tlhe cause for respiratory alkalosis?
lation? Hyperventillation, as in hysteria, salicylate poisoning.
_ _ _ _ _Chapter 28
Electrolyte and
Water Balanice
Chapter at a Glance
The learner will be able to answer questions on the following topics:
0 Intake and output of water O lsotonic/hypotonic/hypertonic contraction, ECF
0 0smolality of extracellular fluid O lsotonic/hypotonic/hyperton ic expansion, ECF
0 Electrolyte composition of body fluids O Sodium metabolism
0 Regulation of sodium and water balance O Potassium metabolism
Renin-angiotensin system O Chloride meltabolism
The maintenance of extracellular fluid volume and pH The thirst center is stimulated by an increase in the
are closely interrelated. The body water compartments osmolality of blood, leading to increased intake.
are shown in Box 28.1. Body is composed of about 60- The renal function is the major factor controlling the
70% water. Distribution of water in different body water
rate of output. Thie rate of loss through skin is influenced
compartments depends on the solute content of each
by the weather, the loss being more in hot climate (per-
compartment. Osmolality of the intra- and extracellular
spiration) and less in cold climate. Loss of water through
fluid is the same, but there is marked difference in the
solute content. skin is increased to 13% for each degree centigrade rise
in body temperature during fever.
IINTAKE AND OUTPUT OF WATER OSMOLALITY OF ECF
One gram carbohydrate, during oxidation produces
0.6 ml of water, 1 g protein releases 0.4 ml water and Osmolarity means osmotic pressure exerted by the
1 g fat generates 1.1 ml of water. Intake of 1000 kcal number of moles, per liter of solution. Osmolality is the
produces 125 ml water (Table 28.1 ). The major factors osmotic pressum exerted by the number of moles per
controlling the intake are thirst and the rate of meta- kg of solvent. Crystalloids and water can easily diffuse
bolism. across membranes, but an osmotic gradient is provided
Total body water (42 l ) (60% of body weight) Intake pei· day Output per day
Mag nesium with anions 1. At equilibrium, the osmolality of extracellular fluid (ECF) and
intracellular fluid (ICF) are identical
Urea 5 8%
2. Solute content of ICF is constant
Glucose 5 3. Sodium is retained only in the ECF
Protei ns 4 . Total body solute d ivided by t otal body water gives the body
292 fluid osmolality
by the non-diffusible colloidal (protein) particles. The col- reduced (dilutional hyponatremia). Hence, the plasma
loid osmotic pressure exerted by proteins is the major sodium is a reliable index of total and effective osmola-
factor which maintains the intracellular and intravascular lity in the normal and clinical situations. See summary in
fluid compartments. Albumin is mainly responsible in Box 28.2.
maintaining this osmotic balance (see Chapter 26). The
composition of each body fluid compartment is shown in Regulation of Sodium and Water Balance
Figure 28.1 and Table 28.2.
Since osmolality is dependent on the number of The major regulatory factors are the hormones (aldo-
solute particles, the major determinant factor is the sterone, ADH) and the renin-angiotensin system.
sodium. Therefore, sodium and water balance are Aldosterone secreted by the zona glomerulosa of
dependent on each other and cannot be considered the adrenal cortex regulates the Na• --> K• exchange and
separately. The osmolality of plasma varies from 285 Na• --> H· exchange at the renal tubules. The net effect
to 295 mosm/kg (Table 28.3). It is maintained by the kid- is sodium retention .
ney, which excretes either water or solute as the case
may be. Antidiuretic Hormone (ADH)
When osmolality of the plasma rises, the osmoreceptors
Effective Osmolality of hypothalamus are stimulated, resulting in ADH secre-
Permeable solutes, such as urea and alcohol enter into tion. ADH will increase the water reabsorption by the renal
the cell and achieve osmotic equilibrium. On the other tubules. Therefore , proportionate amounts of sodium
hand , if impermeable solutes like glucose, mannitol, and water are retained to maintain the osmolality.
etc. are present in ECF, water shifts from ICF to ECF When osmolality decreases, ADH secretion is inhi-
and extracellular osmolality is increased. So, for every bited. When ECF volume expands, the aldosterone
100 mg/dl increase in glucose, 1.5 mmol/L of sodium is secretion is cut off.
412 Section C: Clinical and Applied Biochemistry
!~
Angiotensin II ACE
Factor
trol of sodium and water
Acting through Effect
Hypo-
thalamus
l i cortex
Adrenal
Extracellular
osmolality
Thirst and ADH Water intake;
Rea bsorpt ion of
water from kidney
Hypovolemia Stimulation of Retention of w ater
L
thirst and ADH
-do- Stimulates aldosterone Retention of sodium
Thirst and ADH Vasoconstriction Aldosterone
Expansion of ECF Inhibits Reabsorption of
Increase in
ECFvolume
1 Kidney Hypo-osmolality
aldosterone
Inhibits ADH
secretion
sodium
Reabsorption of water
BOX 28.5: Assessment of sodium and water balance • urbances of fluid volume
BOX 28.6: Clinical features of electrolyte imbalance b. Vomiting an1d excessive sweating can also cause a
1. Hypo-osmolatiy and hyponatremia go hand in hand similar situation.
2. Hypo-osmolality causes swelling of cells and hyper-
c. The increase in osmolality will stimulate thirst and
osmolality causes dehydration of cells
3. Hyponatremia of ECF causes symptoms only when asso-
increase in the water intake. ADH secretion occurs
ciated with hyperkalemia and urine volume decreases.
4. Fatigue and muscle cramps are the common symptoms of
electrolyte depletion Isotonic Expansion
5. Hypo-osmolality of gastrointestinal cells causes nausea,
vomitin and paralytic ileus. Water and sodium retention is often manifested as
edema and occurs secondary to hypertension or car-
diac failure. Heimodilution is the characteristic finding .
Isotonic Contraction
Secondary hyp1eraldosteronism often results from
This results from the loss of fluid that is isotonic with
hypoalbuminemia (edema in nephrotic syndrome, protein
plasma. The most common cause is loss of gastroin-
malnutrition , etc.). In these cases, the water retention
testinal fluid. Since equivalent amounts of sodium and
causes ADH secretion. The intravascular volume can-
water are lost, the plasma sodium is often normal.
Hemoconcentration is seen. In severe cases, hypoten- not be restored since the low colloid osmotic pressure
sion may occur. Hypovolemia may be seen. Com- tends to drive the fluid out into the extravascular space,
pensatory mechanisms will try to restore the volume. aggravating the edema.
Renin-aldosterone system is activated, and selective
sodium reabsorption occurs. ADH secretion leads to Hypotonic Expansion
reabsorption of equivalent amounts of water. There is water retention either due to glomerular dys-
function or ADH excess. The water excess will lower the
Hypotonic Contraction
osmolality. Hyponatremia persists due to the inhibition
There is predominant sodium depletion. Most important of aldosterone secretion by the expanded ECF volume.
cause is infusion of fluids with low sodium content like
dextrose. The hypo-osmolality will inhibit ADH secretion Hypertonic Expansion
resulting in water loss. Since only the excess fluid is
lost, the plasma sodium tends to return to normal. Thus, It can occur in cases of Conn's syndrome and Cush-
osmolality is restored, but at the expense of the volume. ing's syndrome. The excess mineralocorticoid would
Therefore in postoperative cases, care should be pro-duce sodium retention. There is associated hypoka-
taken to adequately replace sodium by giving sufficient lemia which often leads to metabolic alkalosis. Extracel-
quantity of normal saline. lular hypertonicity may lead to brain cell dehydration,
leading to coma and death.
Hypertonic Contraction
It is predominantly water depletion.
a. The commonest cause is diarrhea, where the fluid Sodium level is intimately associated with water balance
lost has only half of the sodium concentration of the in the body. Sodium regulates the extracellular fluid
plasma. volume. Total body sodium is about 4000 mEq. About
414 Section C: Clinical and Applied Biochemistry
50% of it is in bones, 40% in extracellular fluid and 10% retention of wat,er. This vicious cycle is broken when
in soft tissues. Sodium is the major cation of extra- aldosterone anta1gonists are administered as drugs.
cellular fluid.
Sodium pump is operating in all the cells, so as Hypernatrern ia
to keep sodium extracellular. This mechanism is ATP Increased sodium in blood is known as hypernatremia.
dependent (see Chapter 2). Sodium (as sodium bicar- Symptoms of hypernatremia include dry mucous mem-
bonate) is also important in the regulation of acid-base brane, fever, thirst and restlessness. Causes of hyper-
balance (see Chapter 27). natremia are Cushing's disease, prolonged cortisone
Normal level of Na+ in plasma 136-145 mEq/L and therapy and prennancy, where steroid hormones cause
in cells 12 mEq/L. sodium retention in the body. Other causes are enume-
Normal diet contains about 5-10 g of sodium, mainly rated in Box 28.7.
as sodium chloride. The same amount of sodium is daily
excreted through urine. However, body can conserve Hyponatremia
sodium to such an extent that on a sodium-free diet Decreased sodiuim level in blood is called hypona tremia.
urine does not contain sodium. Ideally dietary sodium Clinical signs and symptoms of hyponatremia include
intake should be lower than potassium, but processed dehydration, drop in blood pressure, lethargy, confusion,
foods have increased sodium intake. tremors and corna. Causes of hyponatremia are shown
Normally kidneys are primed to conserve sodium in Box 28.8, most important causes being vomiting, diar-
and excrete potassium. Sodium excretion is regulated at rh ea, and adrenal insufficiency.
the distal tubules. Aldosterone increases sodium reab- Hyponatremia due to water retention is the com-
sorption in distal tubules. Antidiuretic hormone (ADH) monest biochemical abnormality observed in clinical
increases reabsorption of water from tubules. practice. This has to be treated with care, using diuretics
Different mechanisms are: (a) Sodium hydrogen and restricted flu id administration. Isotonic fluids have
exchanger located in the proximal convoluted tubules the same concentration of solutes as cells. So, cells will
and ascending limb; (b) Sodium chloride cotransporter remain viable in isotonic solutions. Hypertonic fluids
in the distal tubules (ascending limb); and (c) Sodium have a higher concentration of solutes (hyperosmolality)
potassium exchanger in the distal tubule. These are than is found inside the cells. This causes cells to shrink.
explained in Chapter 27, under renal regulation of pH. Hypotonic fluids have a lower concentration of solutes
(hypo-osmolality) than is found inside the cells, which
Edema causes fluid to flow into cells and out of the extracellular
In edema, along with water, sodium content of the body spaces. This causes cells to swell and possibly burst.
is also increased. When diuretic drugs are administered, Cellular dehydration and swelling are harmful, as both
they increase sodium excretion. Along with sodium, affect the brain cells.
water is also eliminated.
In the early phases of congestive cardiac failure, Treatment of Hyponatremia
hydrostatic pressure on venous side is increased; so Effects of administered sodium should be closely moni-
water is primarily retained in the body. This causes dilu- tored, but only after allowing sufficient time for distribu-
tion of sodium concentration, which triggers aldosterone tion of sodium, a minimum of 4 to 6 hours. Water res-
secretion . This is known as secondary hyperaldostero- triction, increased salt intake, fu rosemide and anti-ADH
nism. Thus sodium is retained, along with further drugs are the basis of treatment for hyponatremia.
Chapteir 28: Electrolyte and Water Balance 415
Hypokalemia Hyperkalernia
This term denotes that plasma potassium level is below Plasma potassium level above 5.5 mmol/L is known
3 mmol/L. A value less than 3.5 mmol/L is to be viewed as hyperkalemia. Since the normal level of K• is kept
with caution. Mortality and morbidity are high. Box 28.9 at a very narrow margin , even minor increase is life-
shows the causes of hypokalemia. threatening.
416 Section C: Clinical and Applied Biochemistry
1. Decreased renal excretion of potassium Increases K• entry Impairs K• entry into cells
into cells leading or exit of K• from cells;
- Renal failure
to hypokalemia hyperkalemia
- Severe volume depletion (heart failure)
Insulin Glucagon
2. Entry of potassium to extracellular space Alkalosis Acidosis
- Increased hemolysis Increased osmolality
.. ..
- Tumor lysis after chemotherapy
- Excess potassium supplementation
BOX 28.11 : Lab ' e I I • .
3. Redistribution of potassium to extracellular space mahties
- Metabolic acidosis
1. Serum potassium estimation
- Tissue hypoxia 2. Urine potassium: Low value (< 20 mmol/L) is seen in poor
4. Transmembrane shift intake, GIT loss or transmembrane shift. High (> 40 mmol/L)
5. Pseudohyperkalemia is seen in renal diseases
- Factitious (K• leaches out when blood is kept for a long 3. Sodium and Osmolality of spot urine: Low sodium (< 20
time before separation) mmol/ L) and high potassium indicate secondary hyper-
aldosteronism.
- Improper blood collection (hemolysis)
4. ECG in all cases
6. Hyperkalemic periodic paralysis
7. Drugs
-
-
Spironolactone
ACE inhibitors
LcHLoR,oE (._c _,_
n _ __
- Beta blockers Intake, output and metabolism of sodium and chloride
run in parallel. The homeostasis of Na•, K• and c 1- are
interrelated. Chloride is important in the formation of
In hyperkalemia, there is increased membrane hydrochloric acid in gastric juice (see Chapter 24).
excitability, which leads to ventricular arrythmia and Chloride ions are also involved in chloride shift (see
ventricular fibrillation, bradycardia and cardiac arrest. Chapter 23).
ECG shows elevated T wave, widening of QRS complex Chloride concentration in plasma is 96-106 mEq/L
and lengthening of PR interval. and in CSF, it is about 125 mEq/L. Chloride concentra-
Causes of hyperkalemia are shown in Box 28.10. tion in CSF is higher than any other body fluids. Since
True potassium excess results from decreased urinary CSF protein content is low, Cl- is increased to maintain
output, increased hemolysis and tissue necrosis. Donnan membrane equilibrium.
Redistribution occurs in metabolic acidosis, insulin Excretion of Cl- is through urine, and is parallel
deficiency and tissue hypoxia (Table 28.6). to Na•.
Pseudohyperkalemia is seen in hemolysis, throm-
bocytosis, leukocytosis or polycythemia; in these condi- Hyperchloremia is seen in
tions, potassium from within the cells will leak out into 1. Dehydration
plasma when the sample is collected. 2. Cushing's syndrome. Mineralocorticoids cause
Box 28.11 shows the conditions in which potassium increased reabsorption from kidney tubules
estimations are required. 3. Severe diarrhea leads to loss of bicarbonate and
compensatory retention of chloride
Treatment of Hyperkalemia
4. Renal tubular acidosis.
If serum potassium is > 6.5 mmol/L, emergency treat-
ment as intravenous glucose and insulin , should be Causes for Hlypochloremia
given. This stimulates glycogen synthesis. When 1 g 1. Excessive vomiting. HCI is lost, so plasma Cl- is
of glycogen is stored , 0.3 mM of K• is simultaneously lowered. ThE!re will be compensatory increase in
trapped intracellularly. So the serum K• is rapidly plasma bicarbonate. This is called hypochloremic
decreased. Continuous ECG monitoring should be done, alkalosis.
as sudden hypokalemia can occur. If patient is acidotic, 2. Excessive sweating.
give sodium bicarbonate. This will correct acidosis and 3. In Addison's disease, aldosterone is diminished,
help in shifting K• into the cells; but volume overload is renal tubular reabsorption of Cl- is decreased, and
to be monitored. more Cl- is e:(creted.
Chapter 28: Electrolyte and Water Balance 417
e
productive cough not responding to antibiotics. There
was no history of fever, but there was abdominal dis-
tension, difficulty to pass stool and vomiting . History
,a. Clinical Case Study 28.5
revealed that the child frequently passed bulky, foul- A 65-year-old female complaining of vomiting and diarrhea
smelling stools. No diarrhea was present. He had many over the past few days presented to the clinic. Her physi-
relatives with chronic lung and "stomach" problems and cal examination revealed dry mucous membranes, pos-
some had died at a young age. tural hypotension was present. Pulse 140/min, serum
On examination, child was ill looking , slender and in sodium 132 mmol/L, potassium 2.7 mmol/L, chloride
moderate distress. Lung examination and chest X-ray 90 mmol/L, pCO2 - 31 mm Hg, Blood urea and serum
revealed poor air movement in base of lungs, bilateral creatinine were normal. Interpret the findings.
and coarse rhonchi throughout lungs and bronchopneu- e
monia. A quantitative pilocarpine iontophoresis sweat ,a. Clinical Case Study 28.1 Answer
test was done and serum chloride was 70 mEq/L. Repeat The most probable diagnosis is cystic fibrosis, a disease
testing after a few days yielded same results. with defective cl1loride ion channels of exocrine glands
What is the diagnosis? in acinar cells of pancreas, sweat glands and mucous
What is the mechanism involved? glands of respiratory, digestive and reproductive tracts.
There is mutation in the CFTR gene; more than 1400
e mutations have been identified and 230 mutations are
• Clinical Case Study 28.2
associated with clinical features. CFTR ~508 mutation
A 70-year-old woman was admitted with anorexia, accounts for 70% of cases. Cystic fibrosis is comparati-
weight loss and anemia and diagnosed to have carci- vely rare in India, but more common in western countries.
noma of the colon. Biochemical results were Serum Clinical findings are (1) Lungs- Thickening of
sodium 123 mmol/L, Potassium 3.8 mmol/L, Chloride mucus and depletion of periciliary liquid leading to adhe-
88 mmol/L, Bicarbonate 21 mmol/L. Serum osmolality sion of mucus to airway surface; infections of airways,
was 247 mOsm/kg and urine osmolality was 176 mOsm/ (2) GI tract- Damage to exocrine pancreas and des-
kg. Urea and creatinine were normal. truction of pancreas, desiccated intestinal intraluminal
What is the probable diagnosis? contents, obstruction of small and large intestines,
418 Section C: Clinical and Applied Biochemistry
thickened biliary secretions, focal biliary cirrhosis, bile Clinical features of hypokalemia are muscle weakness,
duct proliferation, chronic cholecystitis, cholelithaisis, polyuria and cardiac hyperexcitability. Hypokalemia is
(3) Sweat gland - normal volumes of sweat with defec- also common among hospitalized patients. Causes are
tive chloride content is hallmark of CF. shown in Box 28 .9.
Laboratory findings are; (1 ) Hypoxemia and in
advanced cases, chronic compensated respiratory aci- ILEARNING POINTS, CHAPTER 28
dosis. Pulmonary function shows mixed obstructive and 1. Osmolarity means osmotic pressure exerted by the
restrictive pattern. (2) Elevated chloride in sweat on number of moles per liter of solution.
two tests on different days is diagnostic. Normal sweat 2. Osmolality is the osmotic pressure exerted by the
chloride level does not rule out diagnosis of CF. (3) DNA number of moles per kg of solvent.
analysis (PC R test). 3. Major determinant factor of osmolality is sodium.
4. Major regulatory factors of sodium and water
•• · Clinical Case Study 28.2 Answer balance am aldosterone, ADH and renin-angio-
tensin system.
The patient has dilutional hyponatremia. Normal urea
5. The body water contributes about 60-70% of body
and creatinine exclude significant sodium depletion
weight (42 L). Intracellular compartment has 40%
and absence of edema exclude increase in total body
of body We'ight (28 L) and extracellular compart-
sodium. The results are classical of "syndrome of inap-
ment has 20% (14 L). Extracellular water is distrib-
propriate ADH secretion" (SIADH), due to secretion of
uted between intravascular (4%) and extracellular
AVP in response to nonosmotic stimuli. Hyponatremia
compartment (20%).
is the most common electrolyte disturbance, and there
6. Osmotic balance is maintained between the extra
is marked presence of hyponatremia in hospitalized pa-
and intracellular fluid compartments even though
tients (30% of patients in ICUs may have hyponatremia).
there is a dilfference in solute content and composi-
Causes are described in Box 28.8.
tion.
7. Kidney is tlhe major organ regulating water and
• Clinical Case Study 28.3 Answer electrolyte balance.
8. Osmolality of plasma varies from 285 to 295 milli-
The patient possibly has prerenal uremia and severe
moles/kg.
hypernatremia. Patient might be suffering from water
9. Colloid osmotic pressure of plasma proteins keeps
deprivation. Serum potassium is normal. It is important
the water wi1thin the vascular compartment.
to exclude nonketotic diabetic coma and blood glu-
10. Regulation of sodium and water balance is by ren-
cose and ketones should be estimated for this purpose.
nin angiotensin system.
Causes are described in Box 28.7.
11 . Any fall in ECF volume stimulates the juxtaglomerular
cells to secrete renin. Renin in turn cleaves angio-
• Clinical Case Study 28.4 Answer tensinogen to angiotensin I. Angiotensin I convert-
ing enzyme cleaves angiotensin I to angiotensin II .
Patient has severe hyperkalemia due to release of
Angiotensin II will stimulate the adrenal cortex to
potassium from the damaged tissues. Clinical features
secret aldosterone. Retention of sodium and water
are neuromuscular; muscle weakness, cardiac toxicity,
leads to restoration of ECF volume.
and may produce ventricular fibrillation and asystole.
12. ADH secretion and re nin production are under
Causes are shown in Box 28.10.
feedback control by changes in osmolality.
13. Gain or loss of water and sodium and relative
&'· Clinical Case Study 28.5 Answer amounts of each decides the tonicity of ECF.
There is hypokalemia due to severe diarrhea. Diarrhea ECF volume Herriatocrit Nature ofECF Plasma sodium
has also produced loss of fluid and sodium chloride. Contraction High Isotonic Normal
Main cause of hypokalemia in this patient is extracellular Contraction High Hypertonic High
volume depletion (ECVD). Contraction High Hypotonic Low
Hypokalemia is caused by deficit of potassium Expansion Low Isotonic Normal
stores or abnormal movement into cells. Common Expansion Low Hypertonic High
causes are excess losses from kidneys and GI tract. Expansion Low Hypotonic Low
Chapter 28: Electrolyte and Water Balance 419
28-16. Which of the following causes hypokalemia? 28-17. Hyponatrennia is seen in:
A. Hemolysis A. Addison's disease
B. Polycythemia B. Conn 's syndrome
C. Leukemia C. Diabetes insipidus
D. Alkalosis D. Compulsive water drinking
Chapter at a Glance
The learner will be able to answer questions on the following topics:
Milk D Cerebrospinal flu id
D Colostrum D Amniotic fluid
Proteins in Milk
TABLE 29.1: Composition of milk
A comparison of protein content in milk of different
Constituent Human Cow Buffalo Goat
species is shown in Table 29.1. The protein content is
. .
Water (%) 87.5 87.2 83.6 87.5
Total solids(%) 12.5 12.8 16.4 12.5
: .. tase deficiency leads to lactose intolerance
Proteins (g/ dl) 1.1 3.3 4.3 3.7
Many infants develop diarrhea and skin manifestations due to
Lipids (g/d l ) 3.8 3.8 6.0 3.5 lactose intoleranoe. (It may also be due to allergy to milk proteins).
Carbohydrate (g/dl) 7.5 4.4 5.3 4.7 These children arei to be fed with lactose free formulae or soybean
Calcium (mg/dl) 34 150 160 170 proteins.
422 Section C: Clinical and Applied Biochemistry
TABLE 29.4: Normal composition of amniotic fluid 7. The best source of calcium and phosphorus for the
Early growing child is milk.
gestation Pre-term 8. CSF is produced as an ultrafiltrate of plasma by the
Volume 450- 1200 ml 500- 1400 ml choroid plexus.
Creati nine 0.8-1.1 mg/ 1.8-4.0 mg/ dl 9. Major constituents of CSF are proteins, glucose
dl
and chloride.
Lecithin-sphingomyelin (US) ratio <1:1 >2:1
10. The protein content is 10-30 mg/di of which albumin
Prot ein 0.6-0.24 g/ dl 0.26- 0.19 g/ dl
contributes more than 80%.
11. A small quantity of globulin is also present which
surfactant (surface tension lowering complex) concen- becomes significant in diseases like multiple
tration in amniotic fluid. In late pregnancy, the cells lining sclerosis.
the fetal alveoli start synthesizing d ipalmitoyl-lecithin so 12. The glucose level in CSF is about 75% of the level
that the concentration of lecithin increases, whereas that in blood .
of sphingomyelin remains constant. As a result, as the 13. Chloride content of CSF is higher than plasma to
fetal lung matures, the lecithin-sphingomyelin (US) ratio maintain Gibb's-Donnan equilibrium due to low
protein co ntent.
rises. An US ratio of 2 is taken usually as a critical value.
14. In bacterial meningitis, the protein content increases,
Chapter at a Glance
The learner will be able to answer questions on the following topics:
Reactive oxygen species Clinical significance
Generation of free radicals Lipid peroxidation
Damage produced by free radicals Antioxidants
Free radical scavenger enzyme systems
The outermost orbital in an atom or molecule contains Reactive oxygen species or ROS . The following are
two electrons, each spinning in opposite directions. The members of this group: (See Fig. 30.2)
chemical covalent bond consists of a pair of electrons, i. Superoxide anion radical (02- )
each component of the bond donating one electron each. ii. Hydroperoxyl radical (HOO·)
iii. Hydrogen peroxide (HP)
Definition iv. Hydroxyl radical (OH')
A free radical is a molecule or molecular fragment that v. Lipid peroxide radical (ROO-)
contains one or more unpaired electrons in its outer vi. Singlet oxygen (102 )
orbital (Fig. 30.1 ). Free radical is generally represented vii. Nitric oxide (NO')
by a superscript dot, (Rl viii. Peroxynitrite (ONOO-·).
Oxidation reactions ensure that molecular oxygen Out of this, hydrogen peroxide and singlet oxygen
is completely reduced to water. The products of partial are not free radicals (they do not have superscript dot).
•• .. -
reduction of oxygen are highly reactive and create
havoc in the living systems. Hence, they are also called
•o: o:
•• ••
•o: o:
•• • •
•• • •
H
••
: O : H
•
0 0 Su peroxide Hydroperoxyl Hydroxyl
•
anion (02•) radical (Hoo·) radical (OH")
e e
e
::,
• e
: ••
o ••
: 0 •
•• •• • H
Hydrogen
peroxide (H20 2)
••
o ::
•• ••
••
o
Oxygen
(02)
••
: 0 : R
••
Hydroxyl
ion (OH- )
Fig. 30.1: Left side = normal oxygen atom with all paired elec- Fig. 30.2: Some free radicals. Please compare hydroxyl radical
trons; one electron Is in the process of jumping out. Right side = (free radical) with hydroxyl ion, which is not a free radical. Also
free radical, with an unpaired electron compare oxygen with superoxide anion
426 Section C: Clinical and Applied Biochemistry
H
;:r pathwai
•••
UV Radiation Cigarette
Macrophage and PMN
(Respiratory burst)
i OH• • o-•
2
NADPH~ 02 O H.
IWMR:C-tftiEMJ NO.
I
.___ _ _ _ _ __.
. Bactena
killed
Mitochondria 1
OH•
03
Fig. 30.3: Generation of ROS in m acrophages. (GPD: Glucose-
White Air
6-phosphate dehyrogenase; SOD: Superoxide dismutase; MPO:
blood 10 pollution
Myeloperoxidasee) cell 2
Iron 0
Glutathione Reductase
The oxidized glutathione, in turn, is reduced by the
glutathione reductase (GR), in presence of NADPH (Fig.
30.6). This NADPH is generated with the help of glu-
NADP•
R NADPH ..
GPD
~ - -- Glucose
Free radicals are extremely reactive. Their mean effec- Respiratory Diseases
tive radius of action is only 30 A. Their half-life is only a
Breathing of 100% oxygen for more than 24 hours
few milliseconds.
produces destruction of endothelium and lung edema.
When a free radical reacts with a normal compound,
This is due to the release of free radicals by activated
other free radicals are generated. This chain reaction
neutrophils.
leads to thousands of events (see propagation phase
In premature newborn infants, prolonged exposure
below). to high oxygen concentration is responsible for bron-
Peroxidation of PUFA (poly unsaturated fatty cho-pulmonary dysplasia.
acids) in plasma membrane leads to loss of membrane Adult respiratory distress syndrome (ARDS) is
functions. Lipid peroxidation and consequent degrada- characterized by pulmonary edema. It is produced when
tion products such as malondialdehyde (-CHO-CH2- neutrophils are recruited to lungs which subsequently
CHO-) are seen in biological fluids. release free radicals.
428 Section C: Clinical and Applied Biochemistry
Xanthlne oxidase
ROS Protein damage;
loss of function
ATP - - - • Hypo,aa~;a, ( ) ,~ • x,.~;o,
! 0 0 000
~
Lipid peroxidation
membrane damage 11'0D !
ffJ_____ Mitochondrial II Block HzOz____. OH"
permeability transition
Fig. 30.8: Explanation for reperfusion injury
- - - - - ' - - - DNA damage; cell death;
mutation; cancer
Ageing Proce,ss
Reactive oxyge1n metabolites (ROM) play a pivotal
Fig. 30.7: Damages by reactive oxygen species role in degenerative brain disorders such as Parkin-
sonism, AlzheimBr's dementia and multiple sclerosis.
Cigarette smoke contains free radicals. Soot Cumulative effects of free radical injury cause gradual
attracts neutrophils to the site which releases more free deterioration in ,ageing process. A summary of free
rad icals, leading to lung damage. radica l toxicity is shown in Figure 30.9.
Eye - - - - - - - -- ------.
Diabetic retinopathy of prematurity, - - - - - - Brain
cataracts, age-related macular degeneration Parkinson's disease, Alzheimer's disease, amyotrophic lateral
sclerosis
Air tube - - - - -- - - - - - - ,
Bronchial asthma, inhalation injury
- - - - - Chest
Arrhythmia, cardiac infarction, high blood pressure
Abdomen- - - - - - - - - - - H ~
Gastric ulcer, ischemic colitis, fatty liver Age spots
wrinkles, sagging
- - -- Lower abdomen
Kidney failure, uremia
Body
Aging, diabetes, allergy, rheumatic
disease, cancer, arterial sclerosis
3. Ceruloplasmin can act as an antioxidant in extra- peroxyl, lipid peroxide, nitric oxide and peroxy
cellular fluid (see Chapter 34). nitrite radicals.
4. Cysteine, glutathione, carotenoids, flavonoids and 5. Enzymatic antioxidants are SOD, peroxidase, GSH
vitamin A are minor antioxidants. reductase, catalase and peroxidase most of which
5. Food items containing good quantity of antioxidants are preventive antioxidants as well.
are: (a) Spices used in ordinary Indian cooking con- 6. Peroxidation of PUFA leads to damage to mem-
tain highest quantity of antioxidants. (b) Curcumin. branes compromising membrane integrity and
(c) Fruits and vegetables such as berries, broccoli, function.
spinach, asparagus and green tea, which contain 7. DNA can also be damaged by free radicals and
flavonoids, flavones, isoflavones and anthocya- unless repaired promptly, the change may be
nins. (d) Resveratrol present in grapes. perpetuated.
8. Cataract formation and reperfusion injury (after
1
30-1. Which of the following is NOT a property of free 30-4. Which of the following processes make use of free
radicals: radical effects?
A. Produced in a well controlled manner A. Cell adhesion B. Phagocytosis
B. Have a very short half life C. Contact inhibition D. Transcytosis
30-5. Which of the following diseases are NOT attributed
C. Highly reactive molecules
to free radical injury?
D. Can trigger the production of other free radicals
A. Hypothyroidism
30-2. All cells produce free radicals except
B. Cataract
A. Macrophages B. Neutrophils C. Reperfusion injury
C. Erythrocytes D. Basophils D. Retrolental fibroplasia
30-3. The organelle In the body mainly concerned with 30-6. Which of the following enzymes produce a free
free radical scavenging is: radical in macrophages?
A. Lysosomes B. Golgi bodies A. SOD B. Catalase
C. Nucleolus D. Peroxisomes C. GSH peroxidase D. NOS
Chapter 30: Free Radicals and Antioxidants 431
Chapter at a Glance
The learner will be able to answer questions on the following topics:
D Electrophoresis D Anticoagulants and preservatives
D Adsorption chromatography D Quality Control in the laboratory
D Partition chromatography D Accuracy, precision, specificity and sensitivity
D Ion exchange chromatography D Quality control charts
D Gel filtration chromatography D External quality assurance
D Enzyme Linked lmmunosorbent Assay (ELISA) D Prenatal diagnosis
D Colorimeter 0 AFP, hCG, uE3, DIA, PAPP-A
D Spectrophotometer D Newborn screening
D Mass spectrometry D Investigations for metabolic disorders
D Preanalytical variables
Electric connection
Agarose slide
.»
- . . ~,. ~">. . ;
~ : .· Fig. 31.2: Polyacrylamide gel electrophoresis
' I~ . Albumin
Ame Wilhelm Mikhail Archer Richard
Tiselius Semenovich John Martin L Synge Albumin a 1 a2 P Y
NP 1948 Tswett
I· I
NP 1952 NP 1952
1902- 1971 1872-1919 1910-2002
Point of application i
·I
microscopic slides and allowed to cool. A small sample
(few microliters) of serum or biological fluid is applied by
cutting into the gel with a sharp edge. The electrophoretic
run takes about 90 minutes. This technique is modified
for immunoelectrophoresis which is described later. Fig. 31.3: Electrophoresis of normal serum sample
Agarose gel is used as support to separate different
types of protein mixtures as well as nucleic acids. lmmunoelectrophoresis
Polyacrylamide Gel Electrophoresis (PA GE) Here electrophoretic separation is followed by an anti-
gen-antibody reaction. The electrophoresis is carried out
It has a molecular sieving effect that makes separation first by applying the patient's serum into the wells cut out
very efficient. In agar gel electrophoresis, serum com-
in the agar or agarose gel. The proteins are now sepa-
::>onents are separated into 5 fractions; while in PAGE
rated. To visualize them, a specific antibody is placed in
;;erum will show more than 20 different bands. The
a trough cut into the gel and incubated. The precipitation
amount of cross linking and thereby the pore size can
arcs are formed where the antigen and antibody mol-
oe controlled (Fig. 31 .2).
ecules are at 1:1 ratio (Fig. 31.4). So it is much more
sensitive and specific than ordinary electrophoresis.
Visualization of Protein Bands
After the electrophoretic run is completed, the proteins Ultracentrifugation
are fixed to the solid support using a fixative such as This technique was developed by Svedberg (Nobel
acetone or methanol. Then it is stained by using dyes Prize, 1926). Large molecules can be sedimented at
(Amido Schwartz, naphthalene black, Ponceau S or high centrifugal forces whereas small molecules cannot.
Coomassie Blue) and then destained by using dilute The rate of sedimentation is a function of the size and
acetic acid. The electrophoretogram can be scanned shape of the molecule and is a constant for a particular
using a densitometer and each band quantitated. In the molecule. It also depends on the difference in the
densitometer, light is passed through the agar gel plate; density of solute particles and the solvent. When solute
the absorption of light will be proportional to the quantity particles are lighter than the solvent, they would float
of protein present on a band. The electrophoretic pattern and vice versa. Sedimentation constant is expressed in
of serum proteins on agar gel are shown in Figure 31.3. Svedberg (5) units. Substances with different S units
Abnormal patterns are shown in Figures 26.1A and B will separate from one another as separate bands. See
and 26.2. Chapter 14 for ultracentrifugation of lipoproteins.
434 Section C: Clinical and Applied Biochemistry
*
the trough and
incubated; antigen-
antibody reaction
I takes place
+ Fig. 31 .5: Adsorption chromatography
Trough containing antiserum
Precipitate
depending on tt,e partition coefficient (solubility) of the
pattern is now particular substances. This results in separation of the
clearly visible
components of the mixture.
Albumin lgM lgA lgG
Plate
Sample apply
B •• V
l
Solvent
•
••
Capillary
t
Two hours later turn plate column
90 degree: different
solvent
El • ,- Helium Incubator
Fig. 31. 7: Gas liquid chromatography
• • •H
allow small molecules to enter into the gel. But larger
molecules could not enter into pores of the gel and so
Two hours
• -
•---~-
later are excluded. The small molecules can enter the gel
particles, then come out, re-enter into another particle.
Bl Thus small molecule has to travel a long distance inside
the gels. Small molecules are held back. But the large
Figs. 31.6A to C: Thin layer c hromatography (two-dimensional)
molecules cannot enter the pores and sidetrack the gel
particles; so they move in the column rapidly (Figs. 31.8A
Importance of Rf Value to C). In short, larger molecules will come out first,
The spots may be identified by the Rf value of the while smaller molecules are retained in the column.
unknown substance and comparing with those of pure
standards. (Rf= ratio of fronts). The Rf value is the ratio High Pressure Liquid
of the distance travelled by the substance (solute) Chromatography (HPLC)
to the distance travelled by the solvent. The Rf value
Incompressible silica or alumina microbeads are used
is a constant for a particular solvent system at a given
as the stationary phase. This allows high flow rates.
temperature.
The liquid phase passes through this column under
Gas-Liquid Chromatography (GLC) high pressure (1000 times atmospheric pressure).
The method is therefore based on the same principle
Here the stationary phase is a liquid and the mobile
as for those types already described, but separation is
phase is gas. The stationary liquid phase is supported
by a column of inert material such as silica in a long achieved with better resolution and high speed (within
narrow column. The mixture is made volatile by an inert minutes) (Fig. 31 .9). HPLC can resolve mixtures of lipids
carrier gas like nitrogen. The fractions emerging from whose solubility differ only slightly. In reversed phase
the column are detected and quantitated by a detecting HPLC, hydrophobic polymers are used as stationary
device. This is more suitable for compounds (e.g. lipids) phase; this is generally used to separate peptides.
which resist degradation at high temperature (Fig. 31 .7).
Ion Exchange Chromatography
Gel Filtration (Size Exclusion) In this method, the separation is based on electrostatic
Chromatography attraction between charged biological molecules to
It is also called molecular sieving. Hydrophilic cross oppositely charged groups on the ion exchange resins.
linked gels are used for separation of molecules based These resins are cross linked polymers containing ionic
on their size. The gel is packed in a column. The groups as part of their structure.
436 Section C: Clinical and Applied Biochemistry
•••••
1. Gly 6. Glu 11 . Val
00••• 00
2. Ser/Asn
3. Asp
4 . Gin
7.
8.
9.
Cys/Lys
His
Pro
12.
13.
14.
Met
Tyr
lie
5
00 00 4
5. Ala/Thr 10. Arg 15.
16.
Leu
Phe
00 00 3
11
17. Trp
00 C,G 7 13
0-C~
12 14
00 15 16 17
00 00
0 0
00 gfj
m • 0 5 10 15
~ dmixture
.__B
Finally ant.igens are eluted
+-
Enzyme TABLE 31 .1: Color of filter and color of solution are comple-
•
4
mentary
0 Color
5 Color offilter
3 Enzyme Wavelength Color ofsolution
* reaction
linked Violet 420 Brown
antibody
Blue 470 Yellowish brown
Green 520 Pink
Yellow 580 Purple
Hormone 2
Red 680 Green/ blue
Well in the
microplate
-- -----------------
Ion beam
Digest
' Ionization of
Source molecules
Protein
vaporized
biomolecules
.....
..... Identification - ----
-.N
v-.. l
Energy
. .. -IV
)
Smaller - ii l
molecule rh i l spectrum
urh \-
U[:J
Bigger
molecule Fig. 31 .16: Tandem mass spectrometry
Ion detectors
BOX 31.2: Types of preanalyt1cal variables
Fig. 31.1 5: Mass spectrometry
Test conducted: The appropriate test should be requested and
performed.
mass spectrum in the second instrument (Fig. 31.16). Patient identification: The labeling of specimen may be imp-
MS-MS is used for identification and quantification of roper. Corrected by bar coding.
proteins, drug screening, pesticides and pollutants and Turnaround time (TAT): The time required from the specimen
screening of inborn errors of metabolism, especially reaching the laboratory and the result being dispatched should
be kept minimum. Time of arrival, complet ion of test and
organic acidurias. dispatch should be noted.
Laboratory logs: Entry of patient and test details in laboratory
I REFERENCE VALUE_S_ registers and computers.
Transcription errors: Substantial number at different levels
A patient's results have to be compared with the ref- especially if sample number is high. Electronic identification and
tracking of specimens. Check digits, units and test correlation.
erence values to either confirm a clinical diagnosis or
Patient preparation: Improper standardization of the collection
exclude a particular disease. Reference values and time and manner of collection.
<
normal values are not the same. The term "normal" Specimen collection: Container, anticoagulant time taken to
was used arbitrarily in the past, but was found to be send specimen to laboratory, corrected by using vacutainer
ambiguous when used in a statistical sense. As per tubes and collection of samples by laboratory personnel.
then only a correct interpretation is possible. Preanalyti- The possibility of enzyme inhibition especially creatine
cal variables may be controllable so that their effect can kinase, ALP, ACP, amylase and LOH are observed with
be minimized.The terminologies, preanalytical as well as several of these anticoagulants. Oxalates are unsuitable
analytical variables are recently re-designated as pre- for estimation of sodium and potassium also.
examination or examination variables. Pre-analytical Heparin is the most widely used anticoagulant for
variables include time of collection , posture, prolonged clinical chemical analysis. It interferes the least with test
bed rest, exercise, circadian variations and diet. procedures.
Ethylenediarnine tetraacetic acid (EDTA) is a chelating
Noncontrollable Variables agent, and is particularly useful for hematological exami-
Factors like age, sex, race, pregnancy, lactation, men- nation because it preserves cellular components of the
strual cycle, altitude, temperature, stress, obesity, fever, blood. It may affeict some of the clinical chemistry tests.
trauma and transfusion can all cause significant varia- Sodium fluc,ride is usually used as a preservative for
tion in several specific parameters. Separate reference blood glucose b}I inhibiting the enzyme systems involved
standards are maintained for children. adult and aged. in glycolysis. Without an anliglycolytic agent, the blood
glucose concentration decreases about 10 mg/dl per
~ PECIMEN COLLECTION hour at 25°C andl false results may be obtained. However,
such serum should not be used for enzymatic assays.
Plasma values are higher than serum values in the
Citrate is widely used for coagulation studies. Oxa-
case of calcium and chloride. Plasma values are less late inhibits blood coagulation by forming insoluble
than serum values for albumin, ALP, AST, bicarbonate, complexes with calcium ions. Potassium oxalate may
creatine kinase, urea, uric acid, sodium, glucose (5%), be used at a concentration of 1-2 mg/ml; at higher con-
phosphate (7%) and potassium (8.4%). centrations, oxalate may cause hemolysis.
Arterial blood is used for ABG (arterial blood gas)
analysis. Most of the other investigations use venous Storage and Preservation of Specimens
blood. However capillary blood gives higher values Plasma or serum should be separated from the cells
than venous for glucose and potassium, lower values for as soon as possible, and certainly within 2 hours. If the
bilirubin, calcium, chloride and sodium, but no difference specimen cannot be analyzed at once, the separated
for urea and phosphate. serum should be stored in capped tubes at 4°C. When a
sample is to be :stored beyond a week, it should be kept
Hemolysjs at -20°C in a freiezer.
Vigorous suction by the syringe during blood collection,
or forceful transfer from the syringe to the container may Care of lnfe,cted Specimens
cause hemolysis of blood. Even minimal hemolysis will Samples from patients, who are positive for HBsAg
alter the values of potassium. The presence of visually (hepatitis B su1face antigen) or HIV, should be kept
appreciable hemolysis is seen when the hemoglobin separately, in protected bags. The specimen is disposed
level is more than 200 mg/L. Enzymes like aldolase, LOH, off properly. An), serum specimen may be from patients
ACP, ICD and electrolytes potassium, magnesium and of these disea:ses. Therefore, all serum or blood
phosphate are elevated by the presence of hemolysis. samples should be handled as if they are infected
Hemolyzed samples give falsely low values for bilirubin and adequate precautions should be taken.
and special care is to be taken when blood is collected
for neonatal bilirubin estimation . Urine Colleiction
An early morn1ing fasting specimen is generally the
Anticoagulants most concentrated specimen. Therefore, it is preferred
for microscopic examination and for the detection of
Serum from coagulated blood is the specimen of choice
proteins and beta chorionic gonadotropin.
for many assay systems. Commonly used anticoagu-
lants are heparin , EDTA, oxalates, citrate and fluoride.
Timed Urin1e Specimen
Of these, lithium heparin is best suited for most of the
biochemical estimations. All other anticoagulants chelate Usually, urine sample is collected for a 24-hour period.
calcium and hence unsuitable for calcium estimation. This will minimize the influence of short-term biological
442 Section C: Clinical and Applied Biochemistry
is used for solutions having a few gram of protein/dl. BOX 31.3: Common medical indications for a referral to a
Spectrophotometric method is useful to detect a few genetic counselor
milligram of protein/dl , while ELISA method is employed 1. Advanced maternal age (greater than 35 years)
f the solution has only microgram of protein/dl. Thus 2. Positive maternal serum screening
3. Patient or family member with a known Mendelian disorder
::LISA method is most sensitive.
4. Prior pregnancy with a chromosomal disorder
The sensitivity of an assay is the fraction of those
5. Family history of mental retardation or birth defect
with a disease that the assay correctly predicts. Speci-
6. Fetal anomalies or markers by sonogram
4city is the fraction of those without the disease that the 7. Recurrent pregnancy loss/stillbirth
assay correctly predicts. A test should be both sensitive 8. lnfertilit
and specific. Generally speaking, as the sensitivity is
increased, specificity is decreased.
Genetic Counseling
Quality Control Charts This process involves an attempt by trained persons to
These are used to compare the observed control values help the individual or family to:
with the control limits and provide a visual display which 1. Comprehend the medical facts including the dia-
can be quickly reviewed. A daily QC chart should be gnosis, probable course of the disorder, and the
available in the laboratory. The control chart belps to available management,
detect accuracy problems shift in mean and recision 2. Appreciate the way heredity contributes to the
problem shift in SD). The values will indicate if the disorder and the risk of recurrence in specified
relatives,
analytical run is in control (acceptable) or out of control
(unacceptable). If any of these changes are noticed, 3. Choose a course of action which seems to them
prompt action is warranted. Commonly employed charts appropriate in view of their risk, their family goals,
and their ethical and religious standards and act in
in the laboratory practice are Levey-Jennings chart,
accordance with that decision, and
Westgard multirule chart and Cumulative sum chart.
4. To make the best possible adjustment to the disorder
in an affected fam ily member and/or to the risk of
External Quality Assurances (EQAs)
recurrence of that disorder. Indications for referring
Typically, a national organization will send the same a patient to a genetic counselor are shown in
sample to different laboratories; the laboratories will Box 31 .3.
send the results to the organization, which will measure Ultrasound is the main diagnostic tool for prenatal
l1ow similar the results are. Internal QC maintains diagnosis of congenital disorders. Ultrasound screening
the accuracy and precision of the analytical method, is offered routinely to all pregnant women. It is usually
whereas EQAs is necessary for maintaining long-term performed at 18- 23 weeks of pregnancy.
accuracy of analytical methods.
Amniocentesis
lf._RENATAL DIAGN_
O_S_IS_ _ __ Prenatal diagnosis of inborn errors of metabolism can
About 2% of live births are associated with a genetic be made by enzymatic assays of cultured amniocytes.
defect. In addition, genetic disorders are also a major
cause of pregnancy loss as well as perinatal mortality Chorionic Villi Sampling (CVS)
and morbidity. Taking a detailed family history is very
The most common indications for CVS are advanced
important in prenatal genetic evaluation, permitting the
maternal age, or a biochemical or genetic disorder indi-
counselor or physician to identify problems for which a
cated by molecular markers. The genetic makeup of the
couple may be at risk. One of the most important of these
placenta is identical to that of the fetus.
1s a three-generation family history analysis (pedigree
analysis). Details to be obtained include miscarriage,
Cordocentesis
neonatal or early life death, consanguinity as well as
-specific information of mental retardation, anemia and Fetal blood sampling (cordocentesis) can be performed
congenital anomalies. at 20 weeks gestation.
444 Section C: Clinical and Applied Biochemistry
Biochemical Genetics individuals with S gene was found to be 15.1 %. The inci-
dence of GPO deficiency was reported as 28% in males
Biochemical tests for diagnosis of inherited metabolic
and 1% in females.
disorders consist of identification of abnormal metabo-
Screening newborn infants for phenylketonuria
ites or abnormal levels of metabolites or the defective
(PKU) was the first, large-scale genetic screening initia-
or deficient gene product. Fetal tissues (chorionic villi
tive to be widely adopted. High-risk individuals should
and fetal liver biopsy) or cells (trophoblasts, amniotic
be detected by a simple, inexpensive test with high sen-
fluid cells, fetal erythrocytes, and leukocytes) are used
sitivity (the proportion of affected infants with a positive
·'or analysis of the enzyme or other protein primarily
screening test), specificity (the proportion of unaffected
nvolved. Testing of parents, the index case and unaffec-
infants with a negative test), and predictive efficiency
'!ed siblings can provide valuable information.
(ratio of true-positive to false-positive tests). For screen-
ing tests for PKU , see Chapter 19, under phenylketon-
Enzyme Assays and Molecular uria.
Techniques
Direct demonstration of abnormality or deficiency of the Screening Technology
gene (molecular techniques) or gene product (biochemical The commonly employed screening tests are: 1 . Test
techniques) is the preferred diagnostic approach . These for PKU (phenylketonuria), 2. for TSH (congenital hypo-
are carried out in trophoblast or amniotic fluid cell cul- thyroidism); 3. 17-alpha-hydroxy progesterone (for
tures. congenital adrenal hyperplasia), and 4. Tandem mass
Molecular genetics techniques like qRT-PCR, spectrometry (useful for most other disorders).
Southern blotting, linkage analysis as well as mutation The sensitivity of tandem MS-MS testing in screen-
analysis and a variety of PCR-based techniques have ing for PKU is greater than the sensitivity of any other
been used. tests. Newborn screening programs tend to focus on
Molecular biology techniques are described in three groups of metabolites: amino acids, fatty acid
Chapter 44. oxidation intermediates, and short chain organic acids.
BOX 31 .5: Class1f1cat1on of disorders of amino acid BOX 31 .6: Disorders of organic acid metabolism
metabolism 1. Alkaptonuria
1. Hyperphenylalaninemias 2. Branched chain organic acidurias (e.g. MSUD)
2. Hypertyrosinemias 3. Propionic acidurla, methylmalonic acidu ria
3. Disorders of histidine metabolism
4. Defect in lysine oxidation: 2-keto adipic acidemia and glu-
4. Disorders of proline and hydroxyproline
taric acidemia
S. Hyperornithinemias
S. Gamma-glutamyl cycle disorders
6. Urea cycle disorders
7. Errors of lysine metabolism 6. Lactic acidemias
8. Disorders of branched chain amino acids and keto acids 7. Mitochondrial fatty acid oxidation disorders
9. Disorders of trans-sulfuration 8. Oxidative phosphorylation disorders
10. Nonketotic hyperglycinemia 9. Glutanc acidemia type II (respiratory chain).
11 . Ot her disorders
-
TABLE 31.2: Urinary mucopolysaccharides in different muco- BOX 31.7: General pnnc1ples of genetic disease
polysacchandoses (MPS ) management
Heparan Keratan 1. Enhancing anabolism, depressing catabolism
Disease sulfate I sulfate 2. Correcting primary imbalance In metabolic relationships
Hurler's 3. Enhancing excretion of accumulated products
++++ + +
4. Providing alternate metabolic pathways
Hunter's +++ + +
5. Using metabolic inhibitors
Sanfilippo's - +++ + 6. Supplying products of blocked secondary pathways
Morquio's ++ + 7. Stabilizing altered enzyme proteins
Maroteaux- +++ ± + 8. Replacing deficient coenzymes
Lamy's 9. Artificially inducing enzyme production
Sly's ++ ± ++ 10. Replacing enzymes
Normal ± ± ± + 11. Transplanting organs.
12. Correcting underlying DNA defect.
and results in pale skin, blonde hair and pink iris as 3. Chromatography is the technique used to separate
seen in this patient. Visual impairment is also typical of all classes of biomolecules.
this condition. Other symptoms include photophobia, 4. HPLC and GiC form two powerful chromatographic
nystagmus and functional blindness. The disease does techniques that can resolve mixtures with high sen-
not affect lifespan. sitivity and specificity.
Albinism can be tyrosinase positive or negative. 5. Affinity chromatography relies upon the biological
In tyrosinase positive type, enzyme is present but the property of the molecule to be separated. E.g. Affinity
melanocytes are unable to produce melanin due to a of an enzymH to its substrate, ligand to its receptor.
variety of reasons. In tyrosinase negative type, enzyme 6. Enzyme linked immunosorbent assay (ELISA) is
is absent or non-functional. In oculocutaneous type, skin, used in clinical diagnostics. It has the advantage
eyes and hair lack melanin, whereas in ocular albinism, of not being radio hazardous and having a longer
only eyes lack melanin. Ocular albinism may have shelf life.
X-linked inheritance and hence may be more common 7. Colorimeters work in accordance with the Beer-
in male offspring. The other type has equal chances in Lambert law.. As per the Beer's law, the intensity of
both male and female. the color is directly proportional to the concentration
Genetic testing confirms diagnosis. Treatment invol- of the colored particles in the solution .
ves protecting skin and eyes from direct sunlight. 8. Preanalyticall variability may be due to (a) pre-
collection caiuses, or (b) blood collection causes.
lh_EARNING POINTS, CHAPTER 31 9. Accuracy is tile closeness of a result to the true value.
1. The term 'Electrophoresis' refers to the movement 10. Precision refers to the reproducibility of the result.
of charged particles, through an electrolyte when 11 . Specificity of a reaction denotes that only one
subjected to an electric field. substance will answer that particular test.
2. PAGE is useful in determining relative molecular 12. Sensitivity indicates that whether the method could
weights of proteins. be utilized to test a very dilute solution.
31-1 . Give the salient features of electrophoresis. What are the abnormalities that you could detect in serum
electrophoresis?
31-2. Describe the principle and applications of ELISA.
31-1 . Which is the anticoagulant of choice for biochemi- 31 -3. Which of thte following is not an emergency investi-
cal estimations in general? gation?
A. EDTA A. PotassilJm B. Fasting glucose
B. Oxalate-fluoride mixture C. Creatinine D. Calcium
C. Lithium heparin D. Citrate 31-4. Which of the following parameters is not affected
31-2. Which of the following is not a point of care test even if the, assay is carried out after 6 hours of
(POCT)? collection?
A. ABG analysis B. Plasma electrolytes A. Glucose B. Potassium
C. Plasma glucose D. Plasma proteins C. Carbondioxide D. Albumin
Chapter 31: Laboratory Techniques, Quality Control and Metabolic Diseases 449
31-5. Which of the following parameters is not affected 31 -9. The quick.est method for separation of protein:
by using a tourniquet while collecting blood? A. Electrophoresis
A. Calcium B. Ammonia B. High performance liquid chromatography
C. Glucose D. Lactate C. Ion ex,change chromatography
31-6. Mobility of particles during electrophoresis is D. Thin layer chromatography
based on all the following factors, except: 31-10. Ultracentrifugation is useful for all the following ,
A. Current voltage and ampere except:
B. Quality of supporting medium A. Isolation of mitochondria
C. pH of buffer medium B. Preparation of blood group antigens
D. Solubility of particular protein C. ldentifi,cation of lipoproteins
31-7. A polysaccharide made up of D-galactose and D. Separation of proteins
anhydrogalactose units, which are used as matrix 31-11 . Which of the following has maximum absorption at
for electrophoresis is? 340 nm?
A. Cellobiose B. Dextran
A. Heme B. Proteins
C. Polystyrene D. Agarose
C. Nuclei,c acid D. NADH
31-8. In a protein molecule, normal glutamic acid is sub-
31-12. All the techniques listed below measures proteins,
stituted to valine. Both normal and mutated proteins
except
are electrophoresed at pH 8.6. The mobility will be:
A. Nephe lometry B. Flame photometry
A. Mutated protein moves faster than normal protein
C. ELI SA D. Radial immunodiffusion
towards positive pole 31-13. lmmunosC)rbent assays will use antibody conju-
B. Mutated protein moves slower than normal protein gated to a II the following reagents, except:
towards positive pole A. Alkaline phosphatase (ALP)
C. Mutated protein remains at the point of application B. Horse radish peroxidase (HRP)
D. Mutated protein moves to negative pole and nor- C. Riboflavin
mal protein to positive pole D. Biotin
Chapter at a Glance
I
D Vitam in A D Deficiency of vitamin D
D Wald's visual cycle D Vitamin E
D Deficiency of vit amin A D Vitamin K
fD Vitamin D
Vitamins may be defined as organic compounds occur- TABLE 32.1: Comparison of two types of vitamins
ring in small quantities in different natural foods and Fat soluble vitamins Water soluble vitamins
necessary for growth and maintenance of good health in Solubility in fat ~ uble Not soluble
human beings. Vitamins are essential food factors, which Water solubility Not soluble ~ luble
are required for the proper utilization of the proximate Absorption Along with lipids ~ sorption simple
Requires bile salts
principles of food like carbohydrates, lipids and proteins.
Carrier proteins l,fesent *No carrier proteins
"A vitamin is a substance that makes you ill if you don't Storage l~ ored in liver *No storage
eat it" (Albert Szent-Gy6rgyi , Nobel Prize winner, 1937). Excretion Not excreted .i<creted
Discovery of vitamins started from observation of Deficiency <CA(nifests only when *Manifests rapidly as
stores are depleted there is no storage
deficiency manifestations, e.g. scurvy, rickets, beriberi,
Toxicity ~ ervitaminosis may ¥nlikely, since excess is
etc. The vitamin theory was suggested by Hopkins in result excreted
1912 (Nobel Prize, 1929). The term "vitamine" was Treatment of ~ gle large doses may Regular dietary supply
coined from the words vital + amine, since the earlier deficiency prevent deficiency is required
identified ones had amino groups. Later work showed Major vitamins A, D, Eand K Band C
*Vitamin 8 12 is an exception.
that most of them did not contain amino groups, so the
last letter 'e' was dropped in the modern term of vita~
The vitamins are ~ai~ly classified into two:
1. The fat soluble vItamIns are A, D, E and K
& .
VITA_M_I_N
_ A_____________
Mccollum, Simmonds and Kennedy isolated vitamin A
2. Water soluble vitamin s are named as B complex in 1913. Richard Kuhn (Nobel Prize, 1938) identified
and C. The major differences between these two carotenes. Paul Karrer in 1931 elucidated the structure
groups of vitamins are given in Table 32.1. of vitamin A, (Nobel Prize, 1937).
454 Section D: Nutrition
!
Retinol (alcohol) • Retinal (Aldehyde) (-CHO)
CHO
(-CH20H) I
NAO+ NADH+H•
Retinoic acid (COOH)
All trans-retinal
Fig. 32.2: lnterconv1ersion of vitamin A molecules
H3C CH3
(
12 lntestino cell
Intestinal Liver
lumen
Ir Beta-
l. ionone
ring
RE = Retlnol ester
Beta-carotene R = Retinol
RA = Retinoic acid Target cell
Fig. 32.1: Structure of vitamin A Chy = Chylomicrons
RBP = Retinol bincling protein
A) Chemistry Fig. 32.3: Vitamin A metabolism
Vitamin A is fat soluble. The active form is present only
in animal tissues. The provitamin, beta-carotene is pre•c)Transport froim Liver to Tissues
sent in plant tissues. Beta-carotene has two beta ionone
rings (Fig. 32.1). All the compounds with vitamin A activity The vitamin A from liver is transported to peripheral
. . . tissues as trans-r,etinol by the retinol binding protein
lT
are referred to as retmo1ds. They have beta-1onone
or
RBP h f . . Ad fi •
. In t e cas1e o vitamin
h RBP
e 1c1ency, t e 1eve 1
(cyclohexenyl) ring sys '.~ hree different compounds in blood falls.
with vitamin A ac~ y ar etinol (vitamin A alcohol),
)J
retinal (vitamin A aldehyde) and retinoic acid (vitamin At> ptake by Tissues
acid) (Fig. 32 .1 ). Th~ AV-t'IP.-f~~@_R]I The retinol-RBP complex binds to specific receptors on
by r · 1 readily reversible.
r
the retina, skin, gonads and other tissues. Vitamin binds
®_tinaj_is ox1a1zed to retinoic acid, which cannot ·be to cellul ar retingjc acjd binding._pwtejn.-4CR2P) and
converted back to the other forms (Fig. 32.2). The side finally to hormo~~responsive elewews-(1::iB.E.l-oLQ!::!A.
chain contains alternate double bonds, and hence many Thus genes are activated (Fig. 32.3).
isomers are possible. The all-trans variety of retinal ,E s·10chem1ca
. I RoIe of v ·t . A
I amm
also called vitamin A 1 is most common (Fig. 32.1 ). Vita-
min A2 is found in fish oils and has an e~ double bond l Wald's Visual Cycle
in the ring. Biologically important compound is 11-cis- '(§_emmrtlon of Ne,ve JmpuTs'e: Wald was awarded Nobel
retinal. Prize in 1967, fo r identifying the role of vitamin A in
S) Absorption of Vitamin A
Beta carotene is cleaved by a di-oxyge0gse, to form reti-
nal. The retinal is reduced to retinol by retinal reductase
present in the intestinal mucosa. Intestine is the major
site of absorption (Fig. 32 .3). The absorption is along
with other fats and requires bile salts. Vitamin is incor- Frederick Richard Paul Otto P Kurt
G Hopkins Kuhn Karrer Diels Alder
porated into chylomicrons and transported to liver. In the NP 1929 NP W38 NP 1937 NP 1950 NP 1950
liver, vitamin is stored a~ ~tinol palmitati.,) 1861- 1947 1900--1 967 1889-1971 1876-1954 1902- 1958
Chapter 32: r=at Soluble Vitamins (A, D, E and K) 455
Rods Cones
Rho, tpsin (Visuaxl pigment) ? Light
Pigment epitheli1um
Photo-
receptor
,;;lr;'
,§' Photoisomerization;
,z, ·~ Visual excitation els-retinal
Opsin
I\
l
11-cis-retinal All-trans-retinal OPSIN
Retinal
epithelium
l Retinal isomerase
in retina
els-retinal + - - - - - trans-retinal trans-retinal
I Blood J
7
OPSIN
Incident light
Fig. 32.5: Structure of retina showing rods and cones. The inset on
cis-retinal trans-retinal right side shows the structural alteration during photoisomerization
Liver J
---
retinal in the retina itself iny.aar~ by the enzyme, retinal NP 11981 NP 1981
isomer.a.~. The 11-cis-retinal can recombine withopsin b. 1924 1926-2013
456 Section D: Nutrition
.s.
Treatment of
ception in dim light. Rhodopsin present in rods is made i:1romy_elQc~
up of 11-cis-retinal + opsin. • • • • • • • • • will 1'1!!'ukem1a
lead to
-
brief display of saffron color pepduceS1Deasytabl~ elaxa-
lium . Infections niaµ.up.er:5.f:de.
tiw] jn mus,c~. ~ s:wa _on.e,.pe~ ul
-----
Fig. 32.8: A parody of the old proverb is "One carrot a day will
keep the Ophthalmologist away". Papaya, carrot and mango are
good sources of Vitamin A precursors
Fig. 32.7: Keratomalacia
Da•~Y Requirement of Vitamin A
vitamin A is given, as a prophylactic measwe, to children
The recommended daily allowance (RDA) for
below 1 year age.
i.Children =400-600mcg/day (C' , L\-.,., · 6~/c\.01j)
Skin and Mucous Membrane Lesions ii.Men = 750-1000 mcg/day
iii.Women =750mcg/day c_n.tt'"~/cl~)
t.=
Pregnancy= 1000 mcg/day ( 1m.it i:la.i.l)
i. - _, - 1 ; ~. • • • ffiSU~
iv.
from hyperkeratinization of the epithelium lining One international unit = 0.3 mcg of ret fnol. One
the follicles. The skin becomes rough. Keratinizing retinal equivalent = 1 microgram of retinal or 6 microgram
metaplasia of the epithelium of the respiratory, of beta carotene.
gastrointestinal and genitourinary tracts have been
observed. Dietary Sources of Vitamin A
ii. The alterations in skin may cause increased
Animal sources include roitt<, ~er, cre~m. cheese, egg
occurrence of generalized Therefore
in old literature, vitamin A is referred to as anti- y;,1fkand li\le'r.
. Vegetable sources
@fl9JII01atorµtam[!y
iii. ~ e,,. a synthetic variant of vitamin A is known contain the yellow pigment beta carotene. Carrot
to reduce the sebaceous secretions, hence it is used contains significant quantity of beta carotene (Fig. 32.8).
to preven during adolescence. Papaya, mango, pumpkins, green leafy vegetables
(spinach, amaranth
A activity
Causes for Vitamin A Deficiency
i. Decreased intake.
...
Therapeutic Use of Vitamin A
ii. Obstructive jaundice causing defective absorption.
Therapeutic dose is generally 20~r:mLs...higber..t.baQ_
-..;;. --=---;;
iii. Cirrhosis of liver leading to reduced synthesis of RBP
iv. Severe malnutrition, where amino acids are not
available for RBP synthesis. Hypervitaminosis A or Toxicity
v. Chronic nephrosis, where RBP is excreted through
urine. Excessive intake can lead to(foxicify since the vitamin is
stored . It has been reported in children where parents
have been overzealous in supplementing the vitamins.
Assessment of Deficiency
Eskimos refrain from eating the liver of polar bear due
a. Dark adaptation test- It is the time required to to its high vitamin A content. Symptoms of toxicity
adapt the eye to see objects in dim light. It is inc- include anorexia, irritability, headache, peeling of skin,
reased in vitamin A deficiency. drowsiness and vomiting.
b. RBP (retinal binding protein) level in serum is Hypercarotenemia can result from persistent exces-
decreased. (see Table 26.1 ). sive consumption of foods rich in carotenoEs. The
c. Vitamin A in serum is decreased. ~ . but n...9 staining of s ~ as in ja!J.Ddice
d. Normal blood level of vitamin A is 25 to 50 mg/dl. is observed.
458 Section D: Nutrition
22
26
27 27 - - +
UV rays in skin
Photolysis
HO
Cholecalciferol (D3}(trans)
Kidney
25-hydroxylase 1-alpha-hydroxylase
---
and Kurt Alder. Both were awarded Nobel Prize in 1950. l:i-'g3l. The ergosterol when treated with ultraviolet light,
ergocalciferol or vitamin 0 2 is produced. Ergocalciferol
@ Formation of Vitamin D differs in having an unsaturation in the side chain and an
extra methyl group (C28).
Vitamin Dis derived either from 7-dehydrocholesterol or
ergosterol by the action of ultraviolet radiations. 7-dehy Q ctivation of Vitamin D
drocholesterol, a derivative of cholesterol, is present in Vitamin o is a prohormone. The cholecalciferol is first
epidermis. In the skin, ultraviolet light breaks the bond transported to liver, where hydroxylation at 25th position
between position a.and 10 .of tbe steroid ring. So, the occurs, to form 25-hydroxy cholecalciferol (25-HCC)
ring B is opened, to form the provitamm, secosterol (Fig. 32.10). 25-HCC is the major storage form. In
(Fig. 32.9). The cis double bond between 5th and 6th plasma, 25-HCC is bound to "vitamin O binding protein"
carbon atoms, is then isomerized to a trans double bond (VOBP). In the kidney, it is further hydroxylated at th_e 1st
(rotation on the 6th carbon atom) to give rise to vitamin position. Thus 1, 25-dihydroxy cholecalciferol (DHCC) is
' (Fig . 32.9). So, vitamin O is called generated. Since it contains three hydroxyl groups at 1, 3
0 3 or cholecalciferol
the "sun-shine vitamin". and 25 positions, it is also called Calcitriol (Fig. 32.10).
The production of vitamin O in the skin is directly The calcitriol thus formed is the active form of vitamin; it
proportional to the e~ osyr.e to ~ 1!9,bt and inversejy is a hormone (Box 32.1 ).
proportional to the _Qigmenta..!!9n of skin. Vitamin PTH is released in response to low serum calcium
deficiency is seen in winter. and induces the production of calcitriol. The ..,alcitriol
Chapter 32: Fat Soluble Vitamins (A, D, E and K) 459
~ egulation of Calcitriol
1\1\I\I\I\I\I\
(C~ Formation by Feedback Control
The hormonal level of calcitriol is maintained by the
c a++ feedback control. The rate of production is modulated
by serum levels of calcium, phosphorus, PTH and cal-
Fig. 32.11: Calcitriol increases calcium absorption
= =
C Calcitriol; R R~¼i~ ~if S,alc{~ receptor mplex; CB citriol itself.
UL 'reb"\
"' Calbindin. (.
m,nv-- /
\._.V ' •
~ eficiency of Vitamin D
binds to its highly specific nuclear rece tor V The deficiency diseases are rickets in children a.nd
binds to VORE (Vitamin D response elements osteomalacia in adult§; Hence vitamin D is known as
and modulates the expression of more than 50 antirachitic vitamin.
T~ ctivation takes place within minutes. The optimal concentration of 25-hydroxy 0 3 is > 30
ng/ml, where as 20:29 ng/ml is considered insufficient ~h
Biochemical Effects of Vitamin D and 10-19 ng/ml is deficient. A level below 10 ng/ml '-''~
~
a. Intestinal villous cells
b. Bone osteoblasts
idney distal tubular cells.
J and 20-50% of children have hypovitaminosis D. Even in
affluent countries, vitamin D deficiency is very common.
Vitamin D deficiency was associated with poor bone
health, low calcium concentration, higher systolic blood
~ min D and Absorption of Calcium pressure and lower HDL cholesterol values; all of which
Calcitriol promotes the absorption of calcium and phos- are risk factors for heart disease.
phorus from the intestine. On the brush-border surface~
calcium is absorbed passively. From the intestinal ce~ auses for Vitamin D Deficiency
to blood, absorption of calcium needs energy. Catcitriot i. Deficiency of vitamin D can occur in people who are
acts like a steroid hormone. Transcriptional activation not exposed to sunlight properly, e.g . inhabitants of
of specific genes that code for calbindin occurs (Fig. northern latitudes, in winter months, in people who
32.11 ). Due to the increased availability of calcium bind- are bedridden for long periods, or those who cover
in~ tein, the absorption of calcium is increased. the whole body (purdah).
ii. Nutritional deficiency of calcium or phosphate may
E~ t of Vitamin D on Bone also produce similar clinical picture.
iii. Malabsorption of vitamin (obstructive jaundice and
Mineralization of the bone is increased by increasing steatorrhea). High phytate content in diet may also
'the activity of osteoblasts (see Chapter 34). Calcitriol reduce the absorption of vitamin.
coordinates the remodeling of bone and increases bone iv. Abnormality of vitamin o activation. Liver and renal
mineral density. diseases may retard the hydroxylation reactions.
Intracellular production of calcitriol by osteoblasts v. Deficient renal absorption of phosphates.
promotes their differentiation and mineralizati~
Osteoclastic bone re~rption and alkaline phosphatasWClinical Features of Rickets
activity of osteoblasts provide adequate calcium and Rickets is seen in children. There is insufficient minera-
phosphorus to promote mineralization . lization of bone. Bones become soft and pliable. The
460 Section D: Nutrition
bone growth is markedly affected. The classical features
of rickets are bone deformities. Weight bearing bones H3C
are bent. Continued action of muscles also cause bone
malformations. The clinical manifestations include bow
legs, knock-knee, rickety rosary, bossing offrontal bones,
and pigeon chest. An enlargement of the epiphysis
Fig . 32.1 2: Alpha tocopherol
at the lower end of ribs and costochondral junction leads
to beading of ribs or rickety rosary. Harrison's sulcus iii. Pregnancy, lactation = 1o mcg/day
is a transverse depression passing outwards from the iv. Above the age of 60 = 600 IU per day.
costal cartilage to axilla. This is due to the indentatio
of lo~ r ribs at the sj - c ; 1i aphragcn. <) ources of Vitamin D
ft:) . . . Exposure to sunligbLproduces cl"lo~ cal~ I. More-
\.Y Ch mca I Features of Osteomalac1a over, fi~ ver olffisn· and egg· yolk are good sources
The term is derived from Greek "9steon" = ~ ; and of the vitamin. Milk contains moderate quantity of the
"malakia" = softness. The bones are softened due to vitamin. The current recommendation is to fortify dairy
insufficient mineralization and increased osteoporosis. products with vitamin D and adequate exposure to~ -
Patients are more prone to ractures. It may be noted light without sunscreen before 10 am aod after 3 pm at
that vita · nc eve roduces se,yere.,byp_g- le ast 15..minutes a day (safe sun).
c;alc_e.mia. Tetany will not be anifested. Serum alkaline
phosphatase, bone isoenzyme, is markedly increased. ypervitaminosis D
(9 Different Types of Rickets Doses above 10,000 units per day for long periods
may cause toxicity. Symptoms include weakness,
1. The classical vitamin D deficiency rickets can be polyuria, intense thirst, hypertension and weight loss.
cured by giving vitamin D in the diet. Hypercalcemia leads to calcification of soft tissues,
2. Vitamin D resistant rickets is found to be associ- (metastatic calcification , otherwise called calcino-
ated withl Fanconi s yndrom;J where the renal sis ), especially in vascular and renal tissues. Although
tubular reabsorption of bicarbonate, phosphate,
vitamin D is toxic in higher doses, excessive expo-
glucose: and amino_acids ~re also deficie~t. . . sure to sunlight does not r esult in vitamin D toxicity,
3. Renal rickets: In kidney diseases, ~ en1f wtarrun because excess O is destroyed by sunlight itself.
3
q_ js ayaila~le, ~iPIJ.Q § ~- These
cases wi'.I respond to.admi~istration of calcitriol. i)._y.lTAMIN E /~
@ Other Actions of V1tamm D The active vitamin was isolated from wheat germ,..Q,!_I
1,25-DHCC has been found to have a modulatory effect and namec{fgcopherol \ta{Ws =,_c!Jild,JJidh: Qlleros.,= to
on immune-hematopoietic system. Therapeutic doses ~ : $J}. = alcohol). Initial studies of induced vitamin E
given to children with rickets have been found to correct deficiency in laboratory animals resulted in infertility and
the anemia. It has also been found to reduce the risk therefore the vitamin came to be known as a..Q.t i-infert[fily
of cancer and coronary vascular disease. A negative vitamin. Now vitamin E is known as the most potent bi~-
correlation between lgE and vitamin D concentration;
and a positive relation between vitamin D and lung
function were shown in children with asthma. Vitamin
D insufficiency is associated with metabolic syndrome,
----
loQical antioxid~ t.
Chemical Nature
A chromane ring (tocol) system, with an isoprenoid
,'":'\ insuljn resistwce and glucose iotGleraoce. side chain is present in all the eight naturally occurring
tocopherols. Of these, alpha tocopherol (5, 7,
\t:j/ Requirement of Vitamin D 8-trimethyl tocol) has greatest biological activity (Fig.
i. Children= 10 mcg (400 IU)/day 32.12). The structure of vitamin E was elucidated by Paul
ii. Adults= 5 to 10 mcg (200 IU)/day Karrer, who was awarded Nobel Prize in 1937.
Chapter 32: Fat Soluble Vitamins (A, D, E and K) 461
M-CH,
to poor nerve conducf n. These include neurR!fluscular
p~lems such as s ocerebellar ataxia, relrnopathy,
Renpheral neuropa\hy and m pathies. Deficiency can
~R
also cause anemia, due to oxidative damage to red
0
blood cells.
-------.~
R = 20C in (Phylloquinone) in K 1
R = 30C in (Menaquinone) in K2 Recommended Daily Allowance
R = H in Menadione
In rats, inability to produce healthy ovul.Jl and loss of structurally similar synthetic compound
motility of spermatozoa, hemolysis of red ceUs, acute having vitamin K activity is Menadione. It
hepatic necrosis and muscular dystrophy are observed. is water soluble synthetic vitamin, widely
In a normal adult, the body vitamin E stores can meet used in clinical practice. Henrik Dam
the requirement for several months. isolated vitamin K1 in 1929, while Edward Ed rd .
wa O01sy
No major disease states have been found to be Doisy isolated vitamin K2 in 1939. Both of NP 1943
associated with vitamin E deficiency due to adequate them were awarded Nobel Prize in 1943. 1893-1986
levels in the average diet. Vitamin E deficiency is seen
Biochemical Role of Vitamin K
in persons (a) who cannot absorb dietary fat, and (b) in
premature infants (birthweight less than 1500 grams). Vitamin K is necessary for coagulation. Factors
Vitamin E deficiency causes neurological problems due dependent on vitamin K are Factor II (prothrombin);
462 Section D: Nutrition
legs and swelling of costochondral junc~Laboratory 2. Retinals are polyisoprenoid compounds with vitamin
results were: ~ m calcium-8~2
g/d~rum phos- A activity, having the b ionone ring system.
phorus-2.8 ~ L and serum 720 U/L. What is 3. Active forms of the vitamin A include; Retinal,
the likely diagnosis? Retinal, Retinoic acid. The two important isomers
are all trans-retinal and 11-cis-retinal.
4. Vitamin A is transported with the help of Retinal
• • Clinical Case Study 32.1 Answer Binding Protein and this retinal-RBP complex has
specific receptors in various tissues.
Interpretation: Rickets. 5. Rhodopsin is a membrane protein made up of
Tests: Vitamin D, Calcium. opsin plus 11-cis-retinal and it is important in the
Vitamin D functions as both a vitamin and a prohormone. visual cycle.
Low levels of vitamin D are associated with increased 6. Rods are for dim light vision and cones for color vision.
mortality; excess as well as deficiency of vitamin D cau- 7. Decrease in number of cones/cone proteins lead
ses premature aging. Low vitamin D levels are associated C
to color blindness - -
8. Vitamin D is derived from 7 dehydrocholesterol by
with osteomalacia, rickets, falls and low bone mineral the action of UV rays.
density. Lower vitamin D levels also seem to be correlated 9. Vitamin D deficiency results in rickets and osteo-
with some cancers, bronchial asthma, heart palpitations, malacia. Different types of rickets are; vitamin D
multiple sclerosis, infections and neurodegenerative dis- resistant, and renal rickets.
eases. Serum ~hydroxy vitamin D l§V~s also have to 1a.Vitamin E is tocopheror. ,::.t:: ,s=a::;b::s==o==.rb:==e=-d-:---
a:= wm;-
lo-=-
n-=-
g
be maintained for bone and overall health. fats with the help of bile salts. It is transported as
chylomicrons and stored in adipose tissue.
32-1 . Describe sources, biochemical functions, requirement and deficiency manifestations of Vitamin A.
32-2. Describe the sources, biochemical functions, normal requirement and deficiency manifestations of Vitamin D.
32-1 . Daily requirement of vitamin A for a normal adult is: 32-3. Nyctalopia is due to the deficiency of:
A. 1 microgram B. 5 microgram A. Vitamin K B. Vitamin E
C. 100 microgram D. 750 microgram C . Vitamin 812 D. VitaminA
32-2. Deficiency of vitamin A leads to: 32-4. When kidney diseases are present, oral doses of
A. Night blindness B. Rickets vitamin D may not be effective in curing rickets,
C. Macrocytic anemia D. Microcytic anemia because:
464 Section D: Nutrition
A. Hydroxylation reaction is taking place in kidney 32-15. The structure of vitamin A contains:
which activates vitamin A. Chromane ring B. Beta ionone ring
B. Dehydrogenation of vitamin D is taking place in C. Thiazole ring D. Naphthoquinone ring
kidney 32-16. The structure of vitamin E contains
C. Hydroxylation of vitamin D is taking place in kidney A. Chromane ring B. Beta ionone ring
which destroys vitamin C. Thiazole ring D. Naphthoquinone ri ng
D. Vitamin D is stored in liver 32-17. All are good sources of vitamin A, except:
32-5.· Daily requirement of vitamin D is: A. Pumpkin B. Carrot
A. 1 microgram B. 10 microgram C. Mangoes D. Oranges
C . 100 microgram D. 750 microgram 32-18. Rickets may occur in all conditions, except:
32-6. Bleeding tendency is common in deficiency of all A. Chronic renal failure
the following, except: B. Liver diseases
A. Vitamin K B. Vitamin B 12 C. Under exposure to sun light
C. Vitamin C D. Platelets D. Prolonged antibiotic therapy
32-7. Cholecalciferol is synthesized in: 32-19. Richest source of vitamin Dis:
A. Liver B. Skin A. Fresh leafy vegetables
C. Kidney D. Intestinal mucosa B. Fish liver 011
32-8. The most important biological role for vitamin Eis: C. Egg yolk
A. To produce clotting factors D. Vegetable oils
8 . Antidote of selenium poisoning 32-20. Which is the enzyme specially involved in ossifica-
C. Anticoagulant tion of bone?
D. Antioxidant A. Alkaline phosphatase
32-9. Large doses of vitamin K in neonates may cause: B. Acid phosphatase
A. Rebound bleeding C. Hexokinase
B. Porphyria D. ATPase
C. Jaundice 32-21 . When kidney diseases are present, oral doses of
D. Cyanosis vitamin D may not be effective in curing rickets,
32-10. Deficiency of vitamin K can occur in the following because vitamin D is:
conditions, except: A. Stored in kidneys
A. Following gastrectomy B. Dehydrogenated in kidneys
8. Prolonged antibiotic therapy C. Destroyed in kidneys
C. Obstructive jaundice D. Activated in kidney
D. Admin istration of dicoumarol 32-22. If vitamin K is given to a patient with hemophilia:
32-11 . Biochemical function of vitamin K is for: A. Bleeding time prolonged, clotting time remains
A. Converting proline to hydroxyproline normal
8 . Conversion of prothrombin to thrombin B. Blotting time prolonged, bleeding time remains normal
C. Gamma carboxylation of clotting factors C. Both bleeding and clotting times prolonged
D. Inhibition of lipid peroxidation in biomembranes D. Both bleeding and clotting times decreased
32-12. Vitamin K is inhibited by: 32-23. All water soluble vitamins are excreted through
A. lsoniazid (INH) B. Methotrexate urine and are not stored in the body, except:
C. Dicoumarol D. Avidin A. Vitamin 81 2
32-13. Skin and mucous membrane are affected in defi- B. Vitamin 8 6 (pyridoxal)
ciency of all the vitamins listed below, except: C. Vitamin C
A. Retinol 8 . Niacin D. Niacin
C. Riboflavin D. Calcitriol 32-24. All these vitamins are required for normal bone
32-14. The active form of vitamin D is known as: formation, except:
A. Cholecalciferol A. Vitamin C
B. Cyclopentano phenanthrene 8 . Vitamin D
C. Calcitriol C. VitaminA
D. Lanosterol D. Vitamin E
Chapter 32: Fat Soluble Vitamins (A, D, E and K) 465
32-1. What is the major function of vitamin A 7 32-12. What is the function of vitamin 07
Important in vision, used in the Wald's visual cycle. It increases absorption of calcium from intestine; it also
32-2. What causes the nerve impulse in retina? increases mineralization of bone.
Photoisomerization of 11-cis-retinal to all trans retinal. 32-13. What are deficiency manifestations of vitamin D?
32-3. How is all-trans retinal regenerated? Rickets in children and osteomalacia in adults.
Trans retina l is taken to liver, where it Is made to trans- 32-14. In renal disease, oral doses of vitamin D may not
retinol. then isomerised to cis retinol and then to cis be effective, why?
retinal. Hydroxylation and activation of vitamin is taking place
32-4. What enzymes are required for regeneration? in kidney.
Alcohol dehydrogenase and retinol isomerase 32-15. What is the daily requirement of vitamin 07
(Fig. 32.4 ). 10-15 microgram.
32-5. What are the deficiency manifestations of vitamin 32-16. What is the function of vitamin E?
It is the most powerful antioxidant.
A?
32-17. How selenium and v itamin E are related?
Night blindness, xerophthalmia, keratomalacia, kerati-
They act synergistically as antioxidants.
nisation of epithelium.
32-18. What is the source of vitamin E?
32-6. What are the sources of vitamin A?
Vegetable oils e.g. wheat germ oil, sunflower oil, saf-
Carrot, mangoes, papaya, green leafy vegetables, fish
flower oil, cotton seed oil, palm oil.
oil.
32-19. What is the daily requirement of vitamin E?
32-7. What is the dally requirement of vitamin A?
15 mg or 33 international units.
750 to 1000 microgram.
32-20. What is the function of vitamin K?
32--8. How is cholecalciferolsynthesized?
Gamma carboxylation of clotting factors such as pro-
From 7-dehydro cholesterol in the Malpighian layer of
thrombin
epidermis, by the action of ultra violet rays. 32-21 . Deficiency of vitamin K can occur in which condi-
32-9. How is vitamin D activated?
tions?
Cholecalciferol from skin reaches liver. There it is Obstructive jaundice; antibiotic therapy; administration
hydroxylated to form 25-hydroxy cholecalciferol (25- of dicoumarol.
HCC ). It then reaches kidney, where further hydroxyla- 32-22. What is the mechanism of action of dicoumarol?
tion takes place to form 1,25-dihydroxy cholecalciferol It competitively inhibits vitamin K epoxide reductase.
(DHCC). 32-23. Dicoumarol is used for what purpose?
32-10. What is calcitriol? To prevent intravascular thrombosis.
1,25-dihydroxy cholecalciferol, or active vitamin D, 32-24. Excess dose of vitamin K in neonates may lead to
contains three hydroxyl groups at 1, 3 and 25 positions which condition?
(Fig. 32.9). Hemolysis and jaundice.
32-11 . Which vitamin acts as a pro-hormone? 32-25. Bleeding tendency is common in deficiency of?
Vitamin D is converted to calcitriol. Vitamin K; Vitamin C; Platelets; prothrombln.
_ _ _ _ _Chapter 33
Water
9
Soluble Vitan)!ns
(Thia 11line, Ribgflavin, Ni:t:in, Pyridoxine,
Pantot hen1c . ac1.d, e·1ot1n,
s~ . Fo 1·1cB.9ac1.d,
. 812
V 1tam1n• B an d Ascor C b.1c ac1.d)
12
Chapter at a Glance
The learner will be able to answer questions on the following to pics:
1D Thiam ine (Vitamin 81 ) GD Biotin
2D Riboflavin (Vitamin B2) an~ D TD Folic acid
3 D Niacin.@AD· anc(t}Ao p• ¥D Vitamin B12
D Pyridoxine (Vitamin B6) {.9 D Ascorbic acid (Vitamin C)
D Pant othenic acid and coenzyme A
ATP AMP
•
neuritis) or antiberberi factor. In 1900, Christian Eijkman I I
produced beriberi in chicken by feeding polished rice Thiamine Pyrophosphate
(Nobel Prize, 1929). Adolf Windaus (Nobel Prize, 1928)
elucidated the structure of the vitamin. Thiamine pyrophosphate (TPP)
and weai<ness.
4 7
early sy9Wtoms are anorexia, dy~ psia, heat rness "yellow enzyme'' of cellular respiration. Later Axel
¾°~"~ A\>~ ~ T h e o r e l l (Nobel Prize, 1955) isolated riboflavin. In 1935,
B. Wet beriberi: Here rdiovascula~station;, Paul Karrer (Nobel Prize, 1937) determined the structure.
are prominent. Edema of l~s. fas:e trun~ anp serous
cavit~s_are the main featu res. Palpitation,breathless- Structure of Riboflavin
ness and distended neck veins are observed. Death
._.__,;,;..iiiii.iii:::i=\Fig. 33.2). Riboflavin
occurs due to heart failure.
is converted to its ~'11£.§~ ~~w,g.JQlms (FMN and
C. Dry beriberi : In this condition, CNS manifestations
FA..Ql with the help of ATP (Fig. 33.4). Riboflavin is
are the major features. Walking becomes difficult.
Peripheral neuritis With sensory disturbance leads
to complete paralysis.
D. Infantile beriberi: It occurs in infants born to mothers
Coenzyme Activity of Riboflavin
suffering from thiamine deficiency. &_§_tlessoess Riboflavin exists in tissues tightly bound (but not
and sJeeple.ssness are observed. covalently) with enzymes. Enzymes containing riboflavin
E. koff syndrome: It is also called
as cerebral beribe i. Clinical features are those
of ejcephalopathy (opbthalmoplegia, rJ_Ystagmus,
cerebellar alaxia) plus psychosis. It is seen only
when the nutritional status is severely affected.
F. Polyneuritis: It is common in chronic alcoholics.
Alcohol utilization needs large doses of thiamine.
Christiaan Adolf Otto Axe/Hugo Otto
Alcohol inhibits intestinal absorption of thiamine,
Eijkman Windaus Theore/1 Warburg
leading to thiamine deficiency. Polyneuritis may NP 1929 NP 1928 NP 1955 NP 1931
also be associated with pregnancy and old age. 1858-1930 1876-1959 1903-1982 1883-1970
468 Section D: Nutrition
b
~':n:{~o
Dimethyl lsoalloxazlne
3 I Paul Karrer
CHr{CHOHlJ-CHrOH NP 1937
1889-1971 Ring of FAD
D-Ribitol I
Fig . 33.2: Riboflavin structure Fig . 33.3: Acceptance of hydrogen by FAD
\ . \.
Flavokinase FMN FAD synthase
Riboflavin
( ( FAD
Riboflavin Deficiency
ATP ADP ATP PPi Causes: Natural deficiency of riboflavin in man is
uncommon, because riboflavin is sy_nthesized b.y: the
Fig. 33.4 : Coenzymes FMN and FAD
inies*iRBl::flsra.
Manifestations: Symptoms are confined to skin and
are called flavoproteins. The two coenzymes are FMN
mucous membranes.
(flavin mono nucleotide) and FAD (flavin adenine
dinucleotide) (Fig. 33.4 ).
i. ~!s>tU ' s.,LC?jeek, glossa = tongue)
ii. ' Mal
enta coio'red tongue
FAD Accepts Hydrogen iii. Cheilosis (Greek, cheilos = lip)
iv. Angular stomatitis (inflammation at the corners of
During the oxidation process
mouth)
atoms from substrate. In turn,
v. Circumcorneal vascularization
The
vi. Proliferation of the bulbar conjunctiva! capillaries is
(Fig. 33.3).
the earliest sign of riboflavin deficiency.
FMN-Dependent Enzymes
Dietary Sources of Riboflavin
,,.
i. During the amino acid oxidation, FMN is reduced
(see Fig.17.8).
Good sources are Usb,
ii. In the resp.Jri ory chain, the NADH dehydrogenase
green leajy vegetables.
contains FMN. The electrons are transported in the
following manner (see Chapter 21 ):
NAO• _ _ ..,.. FMN - -- CoQ
Daily Requirement
Riboflavin is concerned maioJ_y with the metabolism of
FAD-Dependent Enzymes
carbohydrates and r;_~ uirerr1:1nt is related to calorie
These are _enumerated in Box 33.2. FADH2 when intake. Adults on sederlt~ • Ork require about 1.5
oxidized in the electron transport chain will generate 1.5 mg per day. During pregnancy, lactation and old aJie,
ATP molecules (see Chapter 21 ). additional 0.2 to 0.4 mg/day are required.
---
Chapter 33: Water Soluble Vitamins 469
N I<.O ' a.m{,l,...
'O'
2
UCOOH UCONH,
4
6
f
I I
&N I! r~-
,,,. NH2
I P-
~.'1 ~.t
-I lcv
Pyridine Niacinamlde
Niacin
r , o o
Fig. 33.5 : Structure of niacin
H H
- CH (YA )-\
6-CQ.NH, 6-CO-NH
, Alexander
N I N RTodd
NP 1957
I I 1907-1997
Ribose-5-P Ribose-5-P
Fig. 33.7: Acceptance of hydrogen BOX 33.3: NAO· dependent enzymes
1. Lactate dehydrogenase (lactate pyruvate) (see Fig. 10.14)
2. Glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-
~ IACIN @ N!COT">NIC. AC.1D_ 3-phosphate 1,3-bisphosphoglycerate) (see Fig. 10.10)
3. Pyruvate dehydrogenase (pyruvate acetyl-CoA) (see Fig.
Niacin and Nicotinic acid are synonyms . It is also called
10.19)
as Pertagra preventing factor of Goldberger. The term 4. Alpha-ketoglutarate dehydrogenase (alpha-ketoglutarate
nicotinic acid should not be confused with nicotine. succinyl-CoA) (see Fig. 20.2)
Nicotinic a~ ~ n icotine is the otent S. Beta hydroxyacyl-CoA dehydrogenase (beta hydroxyacyl-
poison tforn
tobacco~cinamict is the active form of CoA beta ketoacyl-CoA (see Step 3, Fig.13.8)
6. Glutamate dehydrogenase (Glutamate alpha-ketogluta-
the vitamin, present in tissu s. Warburg (Nobel Prize,
rate (see Fig.17.7)
1931) elucidated the structure of NAO• and Alexander
Todd (Nobel Prize 1957) demonstrated its function . C
ne Hydrogen Atom and One Electron
1stry of Niacin
i. In the ~ nitmge,ri of the nicotinamide
Niacin is pyridine-3-carboxylic..-acid. Niacinamide is the residue--ha~ ~ ~ 4 \ Hence the oxidized
acid amide (Fig. 33.5). In NA[Yo,NAOP+, the reactive form of coenzyme is usually wifflen as ~ .
site is the carbon alo.!Jl and the nitrogen atom of the ii. In the process of reduction, NAO• accepts one
nicotinamide ring (see numbering in Fig . 33.5). The
hydrogen atom fully. The other hydrogen is ionized.
CQenz@ is bound to the appepzyme. Only the electron is accepted (Fig. 33. 7). See the
positive sign in the molecule is removed.
Coenzyme Forms of Niacin
2H - - - • + H• + -
Niacin is converted to its coenz me forms, viz, Nicoti-
Thus NAO• accepts one H atom and one e· (elec-
namide adenine dinucleotide Ao• and Nicotinamide
tron), to form NAOH. The hydrogen ion (H•) is released
adenine dinucleotide phosphate AD ). The niacin is
into the surrounding medium. During the oxidation of
attached to a ribose phosphate to form a mononucleotide.
NAOH, the reaction is reversed .
It is then attached to AMP, to form the dinucleotide
. (Fig . 33.6). ®
NAO• Dependent Enzymes@) N PtD~
In the case of NAOP·, one more phosphoric acid is
attached to the ribose of the AMP (see the asterisk in They are so many, that an exhaustive ·1isting is no~
Fig. 33.6). attempted. A few examples are given in Box 33.3.
BOX 33.5: NADPH utilizing reactions
1. ~ a t e dehydrogenase in the hexose mono- 1. {Beta-ketoacyl-ACP beta
phosphate shunt pathway (Glucose-6-phosphate hydroxyacyl-ACP) {see Step 4, Fig. 13. 13)
6-phospho-glucono lactone) (see Fig. 10.38)
2. Alpha, b e 1 a ~-ACP >acyl-ACP (see Step 6, Fig.
2. ~ t e dehydrogenase in the shunt pathway 13.13) ( ~ ~ \ ~ \ CMc..)
(6-phosphogluconate 3-keto-6-phosphogluconate) (see
3. AMG-CoA reductase {HMG-CoA mevalonate (see Fig. 14.2)
Fig. 10.38).
hemoglobin
3. Cytoplasmic lsocftnttedeh)1frogenase
Folate reductase (Folate dihydrofolat e tetrahydrofolate)
4. t,iatrc (malate to pyruvate).
(see Fig. 33.15)
6. Phenylalanine ~ytme (Phenylalanine tyrosine) {see
Fig. 19.1 ).
® Niacin Deficiency
© Pellagra
Pellagra is an Italian word, meaning "rough skin". c.,
Pellagra is caused by the deficiency of Tryptophan as ietary Sources of Niacin
well as Niacin. The symptoms of pellagra are:
The richest natural sources of niacin are an eas ,
i. Dirmitltl._: In early stages, bright red erythema
rice J)OliShing, liver, peanut, who e cereals, legumes,
occurs, especially in the feet, ankles and face (Figs.
meat and fish. About half of the requirement is met by
33.8A and B). Increased pigmentation around the
the conversion of tryptophan to niacin. About 60 mg of
neck is known as CalaL'a:: . The dermatitis
to han w~ d ~of..wapin.
is precipitated by exposure to sunlight (Figs. 33.8A
and B). ecommended Daily Allowance {RDA)
-----
ii. Diarrhea: This may lead to weig~ oss. Nausea
Normal requirement is 20 mg/day. During lactation,
and vomiting may also be present.
additional 5 mg is required.
iii. Dementia: It is frequently seen in ron cases.
1
De~ um is common in acute pellagra. I · b'li ,
i~ li~ co centf.e_te am:l~ J ; Dory are more
common in mild cases. At~ia,_§ln__Q_ Sfil!Sticity are
~.,u..:L.W.~
also seen. ·
Chapter 33: Water Soluble Vitamins 471
CHO
HO-rl-~: -0
0
II
P- OH
HC-ll..)
I
OH
3
N
~ver·
Pyridine Pyridoxine Pyridoxamine Pyridoxal Pyridoxal phosphate (PLP)
Decar/joxylation G/ycogenolysis
All decarboxylation reactions of amino acids require Phosphorylase enzyme (glycogen to glucose-1-
PLP as coenzyme. A few examples are given below: phosphate) requires PLP. In fact, more than 70% of total
472 Section D: Nutrition
9
HO - CH - C - C- CO- NH - CH - CH-COOH
2 I I 2 2 amine + P
I CH3 H
Pantoic acid
11
+ Beta alanine
I
3· ,s·-Adenoslne- +
r
Phospho-
I I phosphate pantotheine
Fritz A
r
Pantolhenic acid
I
CoenzymeA
I Lipmann
NP 1953
1899-1986
Fig. 33.10: Structure of pantothenic acid Fig. 33.11 : Structure of Coenzyme A (CoA)
PLP content of the body is in muscles, where it is a part reduces the formation of PLP and causes vitamin
of the pfiosphorylase enzyme. B6 deficiency.
/ ii. Cycloserine: Acts as ~ ntag~ ist.
Deficiency Manifestations iii. Ethanol: B6 deficiency oe• 1ritis is quite common in
of Pyridoxine alcoholics.
@ Neurological Manifestations
Dietary Sources of Vitamin 8 6
enzymes
ffine, Rich sources are yeast, rice polismng, wheat germs,
cereals, legumes (pulses), oil seeds, - - illtKl:-meat
are not produced in adequate amounts. Neurological fish and
symptoms are therefore quite common in B6 deficiency.
In children, B 6 deficiency leads to convulsions due to Requirement of 8 6
decreased formation of lW~I\. PLP is involved in the
synthesis of ; so B6 deficiency leads to
Vitamin B6 requirements are rslated to protein int,!ke
and noJ ,tg c.,aJ,grie lD,!gls_e (Box 33.6). It is recommended
--
demyelination of nerves and conse uent peripheral
that adults need 1 .!£ 2 rngW§';Y· During pregnancy and
neuritis. This is reversible with high doses of B 6 •
lactation, the requirement is increased to 2.5 mg/day.
@ Dermatological Manifestations
Deficiency of B6 will also affect tryptophan metabolism.
I PANTOTHENIC-ACID
-------.----,---
Since niacin is produced from tryptophan, B6 deficienc~ tructure
in turn leads to nigcin deficiency, which is manifested as
Pantothenic acid (Fig. 33.10) and beta mercaptoethanol-
pellagra.
amine are parts of coenzyme A (CoA) (Fig. 33.11 ). The
HSCoA is a nucleotide derivative.
@;ematological Manifestations
In adults, hypochromic microcytic anemia may occu oenzyme Activity of Pantothenic Acid
due to the inhibition of heme biosynthesis. Impaired anti- The beta mercaptoethanol amine contains one thiol or
body formation is also reported. sulfhydryl (-SH) group. It is the active site where acyl
The metabolic disorders which respond to vitamin
groups are carried. Therefore, coenzyme A is sometimes
B6 therapy are xanthurenic aciduria and llomocysti-
abbreviated as CoA-SH to denote this active site. The
nuria. ')) XOM-~v..€.
thio ester bond in acyl-CoA is a high energy bond.
t)~ d.t..wo~\ These acyl groups are transferred to other acceptors,
Effect of Drugs on Vitamin 8 6 for example:
i. INH: lsonicotinic acid hydrazide (isoniazid) is an Acetyl-CoA + Choline _, Acetylcholine + CoA
antituberculosis drug. It inhibits pt oxal kinase; (enzyme is acetylcholine synthase)
\I , t .Bb QJ/1,ted/ ~l
Chapter 33: Water Soluble Vitamins 473
Pool
Steroids
lmidazole ring -+ ~
Thlophene ring-+ 1
ysine
Vincent du
Vigneaud
Ketone bodies S COOH NP 1955
1901-1 978
Acetylcholine
F ig. 33.13: Structure of biotin
Detoxification
l.-J
@ A
Prolonged use of antibacterial drugs may lead to biotin
N ~ C ~:rN~ ~ CO- NH
deficiency. = ---
H2N~N -.cl~ Biotin deficiency symptoms include dermatitis, atro-
1 @ CH phic glossitis, hyperesthesia, muscle pain, anorexia and
I 2
hallucinations. Injection of biotin 100-300 mg will bring
IH2
I
__P_te_ri_di_ne,--_ _ _ _ _P_A_BA
____.I about rapid cure of these symptoms.
I c~o- 1
IPteroic acid Glutamic acid I Requirement of Biotin
+
Pteroylglutamic acid or folic acid About 200-300 mg will meet the daily requirements.
Fig. 33.14: Structure of folic acid
Sources of Biotin
Propionyl-CoA Carboxy/ase Normal bacterial flora of the u will provide adequate
Propionyl-CoA +CO2 + ATP-+ quantities of biotin. Moreover, it is distributed ubiquitously
Methylmalonyl-C,,q + ADP + Pi in plant and animal tissues. L~ yeast, peanut,
(see Step 1, Fig. 13.10) tnvcc CJ>'?' soybean, ~g yol~ are rich sources.
Pyruvate Carboxylase
1£.0LIC A_C_ID_ _ _ _ _ __
Pyruvate + CO2 + ATP -> Oxaloacetate + ADP + Pi
(see Fig. 10.21 ). This is important in two aspects. One, Chemistry of Folic Acid
it provides the oxaloacetate, which is the sparking
The Latin word folium means leaf of vegetable. Folic acid
molecule for TCA cycle. Second , it is an important
is abundant in vegetables. It is composed of three consti-
enzyme in the gluconeogenic pathway.
tuents. The pteridine group link~d with para amino-
Biotin-Independent benzoic acid (PABA) is called pt~roic acid. It is then
Carboxylation Reactions attached to glutamic acid to form· pteroylglutamic acid
or folic acid (Fig . 33.1 4). Folic acid is soluble in water.
i. Carbamoyl phosphate synthetase, which is the
When expo~ rapidly destroyed.
stepping stone for 4rea_and:::Qy:cimidio~ synthesis
(see Step 1, Fig. 17.11).
Absorption of Folic Acid
ii. Addition of CO2 to form C6 in purine.Jing
iii. Malic enzyme, converting pyruvate to malate. Folic acid is readily absorbed by the upper part of
. =- "'=--~
jejunum.
Biotin Antagonists
Avidin, a protein present in egg white has great affinity Coenzyme Functions of Folic Acid
-
to biotin. Hence intake of raw (unboiled) egg may cause
A. The folic acid is first reduced to 7J.8-dihydrofolic
biotin deficiency. Biotin was originally named as anti-
acid and further reduced to 5,6,7,8-tetrahydro
egg-white-injury-factor.
Avidin is heat labile, and boiling of egg will neutralize folic acid (THFA) (Fig. 33.15). Both reactions are
the inhibitory activity. One molecule 9f avid in can combine catalyzed by ~ e n t folate reductase.
with four molecules of biotin. Egg white contains avidin B. The THFA is the carrier of one-carbon groups.
and egg yolk contains biotin. One carbon compound is an organic molecule that
The affinity of avidin to biotin is greater than most contains only a single carbon atom. The following
of the usual antigen-antibody reactions. Therefore, groups are one-carbon compounds:
avidin-biotin system is commonly utilized for detection i. Formyl (-CHO)
of pathogens by ELISA. ii. Formimino (-CH=NH)
Chapter 33: Water Soluble Vitamins 475
Folic acid
NADPH + H.
7,8-dihydrofolic acid
Fig. 33.16: NS, N 10-methenyl THFA. One carbon unit (red ring) is
NADPH + H• attached to N5 and N 10 groups (blue rings) of tetrahydrofolic acid
""K
5,6,7,8-tetrahydrofolic acid (THFA) NP 1988
1918-1999
Vi~mio Melhyl~balamioo
Fig. 33.15: Folate reductase
-y
PPi + Pi
to the 5th or to the 10th or to both 5th and 10th
nitrogen atoms of THFA (Fig. 33.16). The one-carbon 3
5-adenosyl S-adenosyl
metabolism and their interconversions are given methionine homocysteine
-----...
in detail in Figure 17 .1 4.
D. Methyl group in N5-methyl THFA s used for synthesis
C 3
cells in bon/riiarrow and intes!n"a1 mucosa are therefore Recommended Daily Allowance (RDA)
, most seriously affected. The requirement o f ~ mcg/day. In Q _
(r Macrocytic Anemia 0 nancy the re uirement is increased to 1QO mcq/dhd
durin actati to 00 mcg/day.
QNH, N '-::::-----r
0 N
Pyrrole ring lmidazole ring 1--1
N~NH
H
II /4 N-pyrrole-substituted
H2NAN Cl
Substituted pyrrole-N - + C o ~ N-pyrrole-substituted
NH2
Substituted pyrrole-N __... f - . . . . . __ N-benz-1midazole
Sulfanilamide Pyrimethamine
I substituted
Fig. 33.1 9: Folate antagonists R
Duodenum
< The production of methyl THFA is an irreversible step
.__.. _. ........ ---+ IF-Cbl
Ileum
i (see Fig. 17.14). Therefore, the only way for regeneration
of free THFA is step No. 1 in Figure 33.17. When 8 12 is
deficient, this reactio canoot ake place. This is called
f'.'.">T'1""a. 1"1\~r i : ~... r t
the meJ.byl folate trap. This le ds to he asso"tiated folic
\.;
acid scarcity in B12 deficiency. \ "' \ "l'e.t'
,f
~I
't> fr\ - r' o~eNi"
t>
'D
Causes of B12 Deficiency
~\,,,..o.,....'
Fig. 33.21 : Absorption and storage of vitamin B 12 • R = cobalo-
philin; Cbl = cobalamin; IF = intrinsic factor; TC = trans cobalamin Nutritional vitamin~ di ienc~ ·s v~ COJTimon in India,
. VK~~ 'ft\ ot . .
especially a.wang y o ~no"1!c
group. The only source for 8 12 in vegetarian diet is
is absorbed from ileum, curd/milk, and lower income group may not be able to
afford it.
Transport and Storage .
In the blood, methyl B12 form .1s .
predominant. Trans-® Decrease in Absorption--
cobalamin, a glycoprotein, is the specific carrier (Fig. Absorptive surface is reduced by gastrectomy, re§.ection
33.21 ). It is stored in the liver cells, as ado-B12 form, ~ m anc;l malabsorption syn<;l.IQmes.
in combination with transcorrin. Generally, 8 complex . r ~H · -\P-.L~'l O.• O\: rr w"11 \i If'\
vitamins are not stored in the body, is an excep io ddtSe[1lan Permctous AQ!;,;;:_f!J" > J. -~
Whole liver contains about 2 mg of 8 12 , which is sufficient It is~jv rare inIndra. but common in European countries.
for the requirement for 2-3 years. So, B12 deficiency is When it was described in 1849 by Thomas Addison, it
seen only years after gastrectomy. was pernicious (fatal), without any known remedy. It is
- :~ ~ \ e. manifested usually in persons over 40 years. It is an
Function I Role of B1 eo.}-h,.,, m autoimmune disease with a strong fa milial background.
\...> Q.14 u.& ~ ~ ~ ~ ~ntibodies are generated against IF. So, IF becomes
® Me\hylmalonyl-CoA
.,./CA.Cf\ 'lf' -l ~\..
lsomerase deficient, leading to defective absorption of 8 (Figs.
12
IC___,____ Vrtamin 8
12
ii.( Megaloblas ic anemia: In the peripheral blood,
megaloblasts and immature RBCs are observed
Intrinsic factor from stomach (Fig. 33.18).
iii. 'Abnorm~ homocY.steine le~~ In vitamin B,2
Ileum epithelium deficiency, step No. 2 (Fig. 33.17) is blocked, so
that homocysteine is accumulated , leading to horn-
=
E-• 4
1: 1- Antibody against IF
myelirts-heaths of nerves, dem~ rlnation and rtfuiro-
logical lesions.
v. Subacute combined degeneration : Damage to
nervous system is seen in 8 12 deficiency (but not in
folate def)Riency). There · em elinatlo affect-
ing~y reb~ ? cort a;,>Vell as dorsl:ll ~ orumn and
Antibody combines
with IF; IF and 8 12 p~ midal tract of s~inal cord. ~ _ senSQIY.,and
could not combine;
8 12 not absorbed
m...Q!~ct~are_2ffected, it is named as combi~ d
degenerati,on. s i mmetric_gj_paraesthesia of extre-
mities, alterations oU endon..and daep..seoses and
Figs. 33.22A to D: (A) Intrinsic factor secreted from stomach reflexes, l9ss ~ ~se, uosteadiness. in
reaches intestine; (B) Vitamin B,2 absorbed with the help of intrinsic gait, positive Romb.e(g.'s sign {falling when eyes
factor; (C) In pernicious anemia , antibody against IF is produced ;
-;; closed) and ROSiti~e Babinski's sign (extensorf
---
(D) In presence of antibody, absorption is not taking place
plantar reflex) are seen.
mucosal atrophy. In about 40% cases of iron deficiency vi. chlorh dria: Absence of acid in gastric juice is
anemia, superadded gastric atrophy is seen. associated with vitamin B 12 deficiency.
~ ~ \ - on -..1\t -B,~
NOC -V L c...€ a.. i n 1',,\~"ev-,.Y'E - ~~~'I"'>~ 'l.'C'i~ ¾
480 Section D: Nutrition
OH
Figs. 33.23A and B: (A) Choline, left side and (B) Myoinositol,
right side Henry Dale Otto Loewi Bernard Katz
NP 1936 NP 1936 NP 1970
1875-1968 1873-1961 1911-2003
Requirement of Vitamin 8 12
Normal daily re uirement is(1-2 mcgtciay.'Ouring preg- Acetylcholine esterase
Dietary Sources
IINOSITOL_ - -- --
Myo-inositol is seen in tissues (Fig. 33.23B). Inositol
Vitamin 8 12 is not ~nt in ve etables@¥er js tha deficiency is unknown to man.
riEnesf sou.@;curd is a because ~
b[ c]llus can synthesize B 12 • Biochemical Functions of Inositol
ICHOLINE_ --- 1. Inositol is a constituent of certain phospholipids.
2. Phosphatidylinositol is an important constituent
Choline is synthesized in the body (see Fig. 18.11) and
of cell membranes. In response to extracellular
therefore it is not a vitamin. But in view of its importance
signals, inositol triphosphate (IP3 ) is released from
in nutrition, conventionally, it is included as a member
phosphatidylinositol. IP3 is a second messenger (see
of vitamin B complex. Rice polishings, vegetables,
milk, egg and liver are good sources. It is synthesized Chapter 45) leads to cellular metabolic activation.
from serine. Structure of Choline is shown in Figure 3. It is a lipotropic factor and prevents fatty liver.
33.23A.
Biochemical Functions
Historical Perspectives
i. Myelin sheath: It is made by phospholipids. Demye-
A description of scurvy was found in the Ebers papyrus
lination causes severe diseases of nervous written in 1500 BC in Egypt. During the voyage of Vasco
system. da Gama, around the cape of Good Hope to India in 1498,
ii. Fatty liver: In choline deficiency, neutral fat and he lost two-thirds of the crew due to scurvy. The French
explorer, Jacques Cartier, in 1536, during the voyages to
cholesterol esters accumulate in liver, leading to fatty discover eastern parts of Canada, was laid up with scurvy.
liver. Choline is able to prevent fatty liver and cirrhosis A friendly native gave an extract from the leaves of spruce
(see Chapter 13). tree, which produced remarkable cure to scurvy (Fig. 33.24).
James Lind published "Treatise on Scurvy", in 1753. These
iii. Transmethylation reactions : Choline can donate observations led to compulsory rationing of lime or lemon
three methyl groups to the one-carbon pool (see juice to all the crew of the British Royal Navy from 1795
one-carbon metabolism in Chapter 17). Finally, onwards. So the British sailors were nicknamed as "Limeys".
However, it helped to eliminate scurvy from the British Navy,
these methyl groups are transferred to homo- I
while opponents continued to suffer. No wonder, in course
cysteine to produce methionine, which is used for of time, Britain had the colonies in which the sun never set.
transmethylation reactions (see Chapter 18). In 1907, Holst and Trochlich produced scurvy in guineapigs.
The factor was isolated in 1930 and named as "Hexuronic
iv. Acetylcholine (ACh) synthesis: acid" by Albert Szent-Gyorgi (Nobel Prize, 1937). In 1933,
Choline acetylase Haworth established the molecular structure. He renamed it
as ascorbic acid (Nobel Prize, 1937).
Choline + Acetyl-CoA-- - Acetylcholine + CoA
Chapter 33: Water Soluble Vitamins 481
Chemistry of Vitamin C
Biochemical Functions of Vitamin C
It is V{at~ b l e and is e2§ily destroyed by heat. alkali
and~e. In the process of ~ki~g, 70% of vitamin Reversible Oxidation-Reduction
C is lost. It can change between ascorbic acid and dehydro-
The structural formula of ascorbic acid closely ascorbic acid. Most of the physiological properties of the
resembles that of carbok9drates (Fig. 33.26). The strong vitamin could be explained by this redox system.
red~Ci!).IJ 12,[0!WrtY qW£i.1an1i~J1epends on the double-
bonded (enediol) carbons. Hydroxylation of Praline and Lysine
Ascorbic acid is necessary for the post-translational
antiscorbutic activity. -ascorb acid has no activity. hydroxylation of praline and lysine residues. Hydroxy-
- - ~~\e, proline and hydroxylysine are essential for the formation
Biosynthesis of Ascorbic of cross links in the collagen, which gives the tensile
Acid in Animals strength to the fibers. This process is absolutely neces-
sary for the normal production of supporting tissues such
Most animals and plants can s~tbesize ascorbic acjd
as osteoid, collagen and intercellular cement substance
from~ The pathway is described in Figure 10.44. of capillaries.
Man, higher primates, guinea pigs and bats are the
only species which caQJl.Ot syolbesize ascorbic acid Tryptophan Metabolism
(block in g ~ ~ i E , , - . ~- ~y lack the
Ascorbic acid is necessary for the hydroxylation of
genes responsible fol~~ rs nzyme. The tryptophan to 5-hydroxytryptophan . This is required for
vitamin, therefore, should be su plied in the diet of these the formation of serotonin (see Fig.19.11 ).
species.
Tyrosine Metabolism
Metabolism of Ascorbic Acid Vitamin C helps in the oxidation of parahydroxyphenyl
Ascorbic acid is readily absorbed from gastrointestinal pyruvate to homogentisic acid (see Fig. 19.2).
tract. The vitamin is excreted in urine. Since vitamin C is a
strong r~ t . the Benedict's test will be posi-
Iron Metabolism
tive in the urine sample after the vitamin administration. Ascorbic acid enhances the iron absorption from the
Oxidation of ascorbic acid yields dehydro ascorbic intestine (see Chapter 34). Ascorbic acid reduces ferric
acid, which is oxidized further to oxalic acid through iron to ferrous state, which is preferentially absorbed.
diketo-L-gulonic acid (Fig . 33.26). Ascorbic acid is partly
excreted unchanged and partly as oxalic acid. Most of Hemoglobin Metabolism
the oxalates in urine are derived from ascorbic acid, and It is useful for re-conversion of met-hemoglobin to
the rest from glycine metabolism. hemoglobin.
482 Section D: Nutrition
Hemorrhagic Tendency
In ascorbic acid deficiency, collagen is abnormal
and the intercellular cement substance is brittle. So
capillaries are fragile, leading to the tendency to bleed
even under minor pressure. Subcutaneous hemorrhage
may be manifested as petechiae in mild deficiency
and as ecchymoses or even hematoma in severe
conditions.
Internal Hemorrhage
In severe cases, hemorrhage may occur in the
conjunctiva and retina. Internal bleeding may be seen
Figs. 33.25A to C: (A) Gingivitis and bleeding gum in vitamin as epistaxis, hematuria or melena.
C deficiency; (B) Lime and (C) Gooseberry are good sources
of vitamin C Oral Cavity
In severe cases of scurvy, the gum becomes painful ,
Folic Acid Metabolism swollen , and spongy (Fig. 33.25A) and finally teeth are
lost. Wound healing may be delayed .
Ascorbic acid is helping the enzyme folate reductase
to reduce folic acid to tetrahydrofolic acid (Fig. 33.15). Bones
Thus it helps in the maturation of RBC .
In the bones, the deficiency results in the failure of the
osteoblasts to form the intercellular substance, osteoid.
Steroid Synthesis
Without the normal ground substance, the deposition of
Large quantities of vitamin C are present in adrenal bone is arrested. The resulting scorbutic bone is weak
cortex. The ascorbic acid is depleted by ACTH and fractures easily. There may be hemorrhage into joint
stimulation. So the vitamin has some role in adrenal cavities. Painful swelling of joints may prevent locomo-
steroidogenesis. Vitamin C helps in the synthesis of bile
tion of the patient.
acids from cholesterol.
Anemia
Antioxidant Property
In vitamin C deficiency, microcytic, hypochromic anemia
As an antioxidant (see Chapter 30). Daily intake of
is seen. The reasons for anemia may be:
vitamin C reduces this risk for cancer.
a. Loss of blood by hemorrhage
Cataract b. Decreased iron absorption
Vitamin C is concentrated in the lens of eye. Regular
Dietary Sources of Vitamin C
intake of ascorbic acid reduces the risk of cataract
formation. Rich sources are amla (Indian gooseberry) (700 mg/
100 g), guava (300 mg/100 g), lime, lemon and green
Deficiency Manifestations of Vitamin C leafy vegetables (Figs. 33.25B and C).
O=C O=C-H
0=1 7
I
OH-! 7 O=C O=C
11 o I o I
-2H
H-C
1
OH- _ J O=, _ J
H-C
O=C
I
H-C-OH
Walter N Albert
I I I Haworth Szent-Gyorgyi
OH-C-H OH-C-H HO-C-H - - + COOH NP 1937 NP 1937
I
CH2OH
I I I 1883- 1950 1893-1 986
CH2OH CH - OH
2 COOH
L-ascorbic Dehydro- Diketo-l- Oxalic acid
acid ascorbic acid gulonic acid
ii. Because of its power to heal wounds, vitamin C gaze is noted bilaterally. His gait is very unsteady. The
has been recommended for treatment of ulcer, urine drug screen was negative and he had a positive
trauma, and burns. Except in scurvy, the therapeutic blood alcohol level. The emergency room physician
use of vitamin is not specific. administers thiamine. What is the most likely diagnosis?
Summary of all water soluble vitamins are shown in
Table 33.1.
!t
•• · Clinical Case Study 33.2
Sl
u:, Clinical Case Study 33.1 A 65-year-old chronic smoker and alcoholic suffered
from nonspecific symptoms like painful swallowing, inso-
A 59-year-old male is brought to the emergency depart- mnia, epigastric discomfort and recurrent diarrhea. On
ment after a family member found him extremely confused examination , he had disorientation, stomatitis, glossitis,
and disoriented, with an unsteady gait. The patient has esophagitis and exfoliative dermatitis. Laboratory
been known in the past to be a heavy drinker. He has no measurements revealed leukocytosis, elevated ALT,
known medical problems. On examination, he is afebrile AST and GGT and there was ultrasound evidence of
with a normal blood pressure. He is extremely disoriented fatty liver. Treatment with 500 mg daily nicotinamide
and agitated. Horizontal rapid eye movement on lateral was started when rashes improved and other symptoms
484 Section D: Nutrition
were reduced. He restricted alcohol and smoking and demonstrated megaloblastic anemia. What is the most
increased consumption of other B complex vitamins and likely diagnosis? What is the most likely underlying
food sources of B complex vitamins. What is the likely problem for this patient? What are the two most common
condition? What is the pathophysiology of the findings? causes of megaloblastic anemia and how would this
patient's history and examination differentiate the two?
0 0
• • Clinical Case Study 33.3 • • Clinical Case Study 33.6
A 32-year-old female is being treated with methotrexate A 45-year-old man presented with loss of appetite,
for a recently diagnosed choriocarcinoma of the ovary, fatigue, muscle weakness and emotional disturbances.
and presents with complaints of oral mucosal ulcers. Physical examination showed enlarged liver that was
About 5 weeks ago the affected ovary was surgically firm and nodular, mild jaundice and smell of alcohol in
removed. The patient has been taking methotrexate breath . Hematological examination showed macrocytic
for 2 weeks. On examination, patient was afebrile but anemia, and bone marrow showed presence of
appeared ill. Several mucosal ulcers were seen in her megaloblasts. Serum folate was reduced and vitamin
mouth. The patient also had some upper abdominal B 12 and iron were normal. What is the cause of
tenderness. Her platelet count was decreased at megaloblastic anemia in this patient?
60,000/mm3 (normal 150,000 to 450,000/mm3 ) . What is 0
the most likely etiology of her symptoms? What is the •'ii• Clinical Case Study 33.1 Answer
biochemical explanation of her symptoms? What part of
Wernicke-Korsakoff syndrome (thiamine deficiency)
the cell cycle does me th0trexate act on? often associated with chronic alcoholics.
33-1. Describe the source biochemical functions normal requirement and deficiency manifestations of thiam ine.
33-2. Describe the sources, biochemical functions, normal requirement and deficiency manifestat ions of pyridoxal
phosphate.
33-3. Describe sources, biochemical functions, requirement and deficiency manifestations of folic acid.
33-4. Describe sources, biochemical functions, requirement and deficiency manifestations of vitam in B 12.
33-5. Describe sources, biochemical functions, requirement and deficiency manifestations of vitamin C.
33-1 . In thiamine deficiency, serum levels of the following 33-5. All the following vitamins are produced from
compound is increased precursors in the human body, except:
A. Glycine B. Pyruvic acid A. Niacin B. Ascorbic acid
C. Glutamic acid D. Transketolase C. Vitamin D D. Vitamin A
33-2. Thiamine deficiency is first manifested as 33-6. Neuritis is a manifestation of the deficiency of all
A. Reduced transketolase activity in RBC the following vitamins, except:
B. Increased glucose-6-phosphate dehydrogenase A. Thiamine B. Vitamin B 12
activity C. Pantolhenic acid D. Vitamin C
C. Absence of aldolase in RBC 33-7. Which vitamin is required for oxi dative decarboxy-
D. Altered lactate dehydrogenase isoenzymes in RBC lation?
33-3. Beriberi is due to the deficiency of A. Pyridoxal phosphate
A. Niacin B. Thiamine B. Thiamine
C. Ribonavin D. Vitamin B,2 C. Biotin
33-4. Daily requirement of vitamin B1 (thiamine) for a D. RiboOavin
normal healthy adult is 33-8. Glossitis is due to the deficiency of
A. 1 microgram B. 5 microgram A. Niacin
C. 100 microgram D. 1 milligram B. Thiamine
Chapter 33: Water Soluble Vitamins 487
33-55. All manifestations are seen in Vitam in 8 (pyrido- 33-56. Cystathionuria may be seen in the deficiency of all
6
xal phosphate) deficiency, except: the following, except:
A. Skin rashes as in pellagra A. Vitamin B 12
·- B. Macrocytic anemia B. Folic acid
C. Peripheral neuritis C. Pyridoxal phosphate
D. Convulsions in children D. Vitamin K
33-1 . What is the source of thiamine? 33-11 . What are the dietary sources of riboflavin?
Aleurone layer of cereals (food grains) is a rich source Rich sources are liver, d ried yeast, egg, and milk.
of thiamine. Whole wheat flour and unpolished rice 33-12. What is the daily requirement of riboflavin?
and yeast are other good sources. 1.5 mg per day.
33-2. Thiam ine pyrophosphate is required for? 33-13. Which vitamin is synthesized in the body?
Transketolase; Pyruvate dehydrogenase; Alpha ketog- Niacin.
lutarate dehydrogenase. 33-14. What are the coenzyme function of niacin?
33-3. Which vitamin is required for oxidative decarbo- NAO• and NADP•
xylation? 33-15. Name some NAO• dependent enzymes.
Thiamine pyrophosphate. Lactate dehydrogenase; Glyceraldehyde-3-phosphate
dehydrogenase; Pyruvate dehydrogenase; Beta
33-4. In thiamine deficiency, what alterations are seen in
hydroxyacyl-CoA dehydrogenase; Mitochondrial
the blood?
isocitrate dehydrogenase.
Increased pyruvic acid level and increased trans-
33-16. Name the NADPH generating reactions.
ketolase.
Glucose-6-phosphate dehydrogenase; 6-phospho-
33-5. What are the c linical manifestations of thiamine
gluconate dehydrogenase.
deficiency?
33-17. What are important NADPH utilizing reactions?
Beriberi, Wernick syndrome. polyneuritis.
Beta keto acyl ACP dehydrogenase; Alpha, beta unsa-
33-6. Beriberi is due to deficiency of which vitam in?
turated ACP dehydrogenase; HMG-CoA reductase;
Thiamine.
Met-hemoglobin reductase; Dihydrofolate reductase
33-7. What is the daily requirement of thiamine? (Box 33.5).
1 to 1.5 milligram. 33 -1 8. Pellagra is seen in deficiency of which vitam in?
33-8. What is the coenzyme function of riboflavin? Niacin.
FMN and FAD. 33-19. What are the salient features of pellagra ?
33-9. Name some FAD dependent enzymes. Dermatitis, diarrhea, dementia.
Succinate dehydrogenase; Acyl-CoA dehydrogenase; 33-20. What is the precursor of niacin?
Xanthine oxidase; Glutathione reductase; Pyruvate Tryptophan.
dehydrogenase; Alpha-ketoglutarate dehydrogenase. 33-21. Tryptophan will give rise to how much niacin?
33-10. What are the manifestations of riboflavin deficiency? About 60 mg of tryptophan will yield 1 mg of niacin.
Glossitis, cheilosis, angular stomatits, circumcorneal 33-22. Tryptophan is deficient in which food stuff?
vascularization. Maize and corn.
490 Section D: Nutrition
33-23. What are the conditions that will lead to symptoms 33-37. Deficiency of pantothenic acid leads to what?
of pellagra? Burning foot syndrome.
lsoniazid therapy; Low tryptophan content in diet; Niacin 33-38. What is the function of biotin?
deficiency; Hartnup disease; Carcinoid syndrome. Carboxylation reactions.
33-24. What is the dietary sources of niacin? 33-39. Name biotin dependent carboxylation reactions
Rice polishing, cereals, legumes, meat and fish Acetyl-CoA carboxylase; Propionyl-CoA carboxylase;
contain niacin. About half of the requirement is met by Pyruvate carboxylase.
the conversion of tryptophan to niacin. 33-40. What is the antagonist for Biotin?
33-25. What is the dally requirement of niacin? Avidin.
The R.D.A is 20 mg/day. 33-41 . What is the coenzyme form of folic acid?
33-26. Transamination reaction requires which vitamin? Tetrahydrofolic acid.
Pyridoxal phosphate. 33-42. What is the main function of folic acid?
33-27. Pyridoxal phosphate is required for what reactions? Tetrahydrofolic acid is the carrier of one-carbon units.
Transamination, decarboxylation of amino acids, ALA 33-43. What are the causes of folate deficiency?
synthase, glycogen phosphorylase. Pregnancy; defective absorption; anticonvulsant drugs
33-28. What is clinical significance of transaminase?
(dilantin, phenobarbitone); hemolytic anemias, dietary
Blood level of ALT is increased in liver diseases.
deficiency.
33-29. Give examples of decarboxylation reactions.
33-44. What is the major manifestation of fol ic acid
Glutamate to GABA (gamma aminobutyric acid); Histi-
deficiency?
dine to histamine; 5-hydroxytryptophan to serotonin;
Macrocytic anemia.
Serine to ethanol amine.
33-45. What is the complication of folic acid deficiency in
33-30. What are the manifestations of pyridoxal deficiency?
pregnancy?
Infantile convulsions; peripheral neuritis; pellagra;
Felic acid deficiency during pregnancy may lead to
anemia.
neural tube defects in the fetus.
33-31 . What is the reason for peripheral neuritis in
33-46. What is the daily requirement of folic acid?
..
pyridoxal deficiency?
The RDA of folate is 200 mg/day. In pregnancy the
PLP is involved in the synthesis of sphingolipids; so
B6 deficiency leads to demyelination of nerves and requirement is increased to 400 mg/day.
consequent peripheral neuritis. 33-47. What is the mechanism of action of sulphonamides?
33-32. What is the reason for pellagra like disease in They have structural similarity with PABA. Therefore
pyridoxal deficiency? they competitively inhibit the enzyme responsible for
Niacin is produced from tryptophan. One enzyme of the incorporation of PABA into folic acid. So, they kill
this pathway is PLP dependent. So B6 deficiency in turn bacteria.
leads to niacin deficiency. It is manifested as pellagra. 33-48. What is mechanism of action of methotrexate?
33-33. Give an example of one vitamin deficiency leading It inhibits folate reductase, and is a powerful anticancer
to another vitamin deficiency? drug.
PLP deficiency in turn leads to niacin deficiency which 33-49. What are inhibitors of folic acid?
is manifested as pellagra. Methotrexate; Pyrimethamine; Sulphonamide.
33-34. What is the daily requirement of pyridoxal 33-50. Name a water soluble vitamin , which Is stored in
phosphate? the body.
1 to 2 mg/day. Vitamin B12.
33-35. What is coenzyme form of pantothenic acid? 33-51. What is the metal present in vitamin B12 ?
CoenzymeA. Cobalt.
33-36. What is the function of CoA? 33-52. How vitamin B 12 Is absorbed?
Oxidation of pyruvic acid and activation of acyl groups. With the help of Intrinsic factor of Castle.
i - - - - - - - -_ _ Chapter 34
'!..
Mineral Metabolism
and Abnormalities
Chapter at a Glance
Minerals are essential for the normal growth and main- BOX 34.1: Important minerals
tenance of the body. If the daily requirement is more Major elements Trace elements
than 100 mg, they are called major elements or macro- 1. Calcium 1. Iron
minerals. They are listed in Box 34.1. 2. Magnesium 2. Iodine
If the requirement of certain minerals is less than 3. Phosphorus 3. Copper
100 mg/day, they are known as minor elements or micro- 4. Sodium 4. Manganese
minerals or t race elements. They are shown in see 5. Potassium 5. Zinc
Box 34.1, in order of their essentiality: 6. Chloride 6. Molybdenum
The following minerals are necessary for the body; 7. Sulfur 7. Selenium
8. Fluoride
but their exact functions are not known . They are chro-
mium, nickel, bromine, lithium and barium. The following
minerals are seen in tissues, but are nonessential and vegetables are medium sources for calcium. Cereals
are contaminants in foodstuffs. These are rubidium, silver, (wheat, rice) contain only small amount of calcium. But
gold, and bismuth. The following minerals are toxic cereals are the staple diet in India. Therefore, cereals
and should be avoided: aluminium, lead, cadmium and form the major source of calcium in Indian diet.
mercury. Heavy metal poisons are discussed in Chapter 37.
CR +
Ca-bound calmodulin
Kinase
+ .
--------. Active kinase
+
Enzyme ---------------• Phosphorylated enzyme
l +
Biological effect
""""""" CB protejn
Fig. 34.2: Mechanism of action of calmodulin
/
lactate into surrounding medium which solubilizes cal-
t cium. Parathyroid hormone (PTH) also causes secre-
Calbindin Decrease urinary Calcitriol
calcium tion of collagenase from osteoclasts. This causes loss
Increase urinary phosphate
of matrix and bone resorption.
Fig. 34.4: Calcium homeostasis When serum calcium is low, PTH PTH and kidney : In kidney, PTH causes decreased
is stimulated, resulting in increased calcium release from bone renal excretion of calcium and increased excretion
and decreased renal calcium excretion. PTH also stimulates of phosphates. The action is mainly through increase in
increased production of calcitriol, which acts to increase absorption
reabsorption of calcium from kidney tubules.
of calcium from intestine
PTH and intestines: Parathyroid hormone stimu-
hormone-receptor complex interacts with DNA and lates 1-hydroxylation of 25-hydroxycalciferol in kidney
causes transcription of specific genes that code to produce calcitriol. This indirectly increases calcium
absorption from intestine.
for Calbindin (see Fig. 34.1 ). Due to the increased
availability of calcium binding protein, the absorp- Calcitonin
tion of calcium is increased. Hence, blood calcium
level tends to be elevated. It is secreted by the thyroid parafollicular or clear cells.
Calcitonin and Calcitriol are different (Box 34.3). Calci-
iii. Vitamin D and bone: Vitamin D is acting indepen-
tonin is a polypeptide with 32-34 amino acids, depend-
dently on bone. Vitamin D increases the number and
ing on the species difference. Calcitonin secretion is
activity of osteoblasts, the bone forming cells. It also
stimulated by serum calcium. Calcitonin level is increa-
has a role in osteoclastogenesis. Calcitriol stimulates sed in medullary carcinoma of thyroid and therefore is
osteoblasts to secrete alkaline phosphatase. Due a tumor marker. Calcitonin decreases serum calcium
to this enzyme, the local concentration of phosphate level. It inhibits resorption of bone. It decreases the
is increased. The ionic product of calcium and phos- activity of osteoclasts and increases that of osteoblasts.
phorus increases, leading to mineralization and Calcitonin and PTH are directly antagonistic. The PTH
remodeling of bone (Fig. 34.4). and calcitonin together promote the bone growth and
iv. Vitamin D and renal tubules: Calcitriol increases remodeling (see Table 34. 1).
the reabsorption of calcium and phosphorus by renal
Calcitonin, Calcitriol and PTH Act Together
tubules, therefore both minerals are conserved.
(PTH conserves only calcium). When blood calcium tends to lower, PTH secretion is
stimulated and calcitonin is inhibited; bone deminerali-
Parathyroid Hormone (PTH) zation leads to entry of more calcium into blood. When
blood calcium is increased, PTH is inhibited and cal-
This hormone is secreted by the four parathyroid glands
citonin is secreted, causing more entry of calcium into
embedded in the thyroid tissue. The chief cells of the bone. These effects are summarized in Figure 34.4 and
gland secrete the PTH. Table 34.1 . Bone acts as the major reservoir of calcium.
PTH has 84 amino acids. The first 35 amino acids
of PTH are biologically active. Control of release of the Procalcitonin (PCT)
hormone is by negative feedback by the ionized calcium It is a relatively new marker that has been associated
in serum. The release of hormone is mediated by cyclic with inflammation and sepsis. It is the precursor to calci-
AMP. tonin. The levels have been shown to rise with severity
Chapter 34: Mineral Metabolism and Abnormalities 495
BOX 34.8: Requirements for growth of bone BOX 34.9: Functions of phosphate ions
1. Calcium 1. Formation of bone and teeth
2. Phosphorus 2. Production of high energy phosphate. compounds, such as
3. Vitamin D and Calcitriol ATP, CTP, GTP, creatine phosphate, etc.
4. Parathyroid hormone 3. Synthesis of nucleoside coenzymes, such as NAD and NADP
5. Calcitonin 4. DNA and RNA synthesis, where phosphodiester linkages
6. Vitamin A (for ground substance) form the backbone of the structure
7. Vitamin C 5. Formation of phosphate esters, such as glucose-6-phos-
phate, phospholipids
8. Sex steroids
9. Amino acids. 6. Formation of phosphoproteins, e.g. casein
7. Activation of enzymes by phosphorylation
8. Phosphate buffer system in blood. The ratio of Na2 HPO4:
BOX 34.10: Causes of hyperphosphatem1a NaH2 PO4 in blood is4:1. This maintains the pH of blood at 7.4.
1. Increased absorption of phosphate
Excess vitamin D
BOX 34.11 : Causes of hypophosphatem1a
2. Increased cell lysis
1. Decreased absorption of phosphate
Chemotherapy for cancer
Malnutrition
Bone secondaries
Malabsorption
3. Decreased excretion of phosphorus
Chronic diarrhea
Renal impairment
Vitamin D deficiency
Hypoparathyroidism 2. Intracellular shift
4. Hypocalcemia Insulin therapy, glucose phosphorylation
5. Massive blood transfusions Respiratory alkalosis
6. Drugs 3. Increased urinary excretion of phosphate
Chlorothiazide, Nifedipine, Furosemide. Hyperparathyroidism
4. Hereditary hypophosphatemia
5. Hypercalcemia
Serum Level of Phosphorus 6. Chronic alcoholism
7. Drugs
Serum level of phosphate is 3--4 mg/dl in normal adults Antacids, diuretics, salicylate intoxication.
and is 5-6 mg/dl in children. Fasting levels are higher.
The postprandial decrease of phosphorus is due to the
utilization of phosphate for metabolism. Monovalent and I MAGNESIUM (Mg)
divalent phosphate ions are present in plasma at a ratio Magnesium is the fourth most abundant cation in the
of 1:4 at pH 7.4. body and second most prevalent intracellular cation.
The whole blood phosphate is 40 mg/dl. This is Total body magnesium is about 25 g, 60% of which is
because RBCs and WBCs contain a lot of phosphates. complexed with calcium in bone. Magnesium orally
Hemolysis should be prevented when blood is taken for produces diarrhea; but intravenously it produces CNS
phosphate estimation . depression.
Serum phosphate level is decreased in hyper-
parathyroidism and rickets. Causes of hyperphosphatemia Requirement
are given in Box 34.10, and those of hypophosphatemia
The requirement is about 400 mg/day for men and
inBox34.11 .
300 mg/day for women. Major sources are cereals,
Phosphorus holds an inverse relationship with cal-
beans, leafy vegetables and fish.
cium. An excess of serum calcium or phosphate results
in the excretion of the other by the kidney.
Normal Serum Level of Magnesium
Like calcium, phosphate level in blood is controlled
by the parathyroid hormone. Parathyroid hormone inc- Normal serum level Mg++ is 1.8-2.2 mg/dl. Homeostasis
reases calcium and phosphate release from the bone is maintained by intestinal absorption as well as by
and decreases loss of calcium and increases loss of excretion by kidney. Magnesium is reabsorbed from
phosphate in the urine. loop of Henle and not from proximal tubules.
498 Section D: Nutrition
Hypomagnesemia Excretion
It is commonly seen in hospitalized patients. When serum All the sulfur groups are ultimately oxidized in liver to
magnesium level falls below 1.7 mg/dl, it is called sulfate (SO4 ) group and excreted in urine. The total
hypomagnesemia. Causes are shown in Box 34.12.
quantity of sulfur in urine is about 1g/day. This contains
Deficiency of magnesium leads to neuromuscular 3 categories.
hyper-irritability and cardiac arrhythemias. The mag-
i. Inorganic sulfates: It is about 80% of the total
nesium deficiency symptoms are similar to those of
excretion. This is proportional to the protein intake.
calcium deficiency; but symptoms will be relieved only
ii. Organic sulfate or ethereal sulfate: It is also
when magnesium is given. Acute symptomatic defi-
called as conjugated sulfate. It constitutes 10% of
ciency is treated by giving parenteral magnesium. Oral
urinary sulfates. Tryptophan is converted to phenol
therapy may lead to diarrhea, hence intravenous mag-
nesium sulfate is given. and indoxyl by intestinal bacteria. These are absor-
bed and conjugated with sulfates and excreted
Hypermagnesemia through urine. Therefore, this represents the putre-
factive activity in intestine.
Causes of hypermagnesemia are listed in Box 34.13.
Magnesium intoxication causes depression of neuro- iii. Neutral sulfur or unoxidized sulfur: This fraction
muscular system, causing lethargy, hypotension, respira- constitutes 10% of total sulfates. Sulfur containing
tory depression, bradycardia and weak tendon reflexes. organic compounds, such as amino acids. It is also
Hypermagnesemia induces decrease in serum calcium increased in amino acidurias.
by inhibiting PTH secretion .
IIRON (Fe)
I SULFUR (S) Distribution of Iron
Source of sulfates is mainly amino acids cysteine and Total body iron content is 3-5 g; 75% of which is in
methionine. Proteins contain about 1% sulfur by weight. blood, the rest is in liver, bone marrow and muscles. Iron
Inorganic sulfates of Na+, K• and Mg•+, though available is present in almost all cells. Heme containing proteins
in food, are not utilized. are shown in Table 34.2.
Blood contains 14.5 g of Hb per 100 ml. About 75%
Functions of Sulfur of total iron is in hemoglobin, and 5% is in myoglobin
Sulfur containing amino acids are important constituents and 15% in ferritin. Normal iron kinetics is shown in
of body proteins. The disulfide bridges keep polypeptide Figure 34.6.
Chapter 34: Mineral Metabolism and Abnormalities 499
Requirement of Iron (ICMR) ii. Children between 13-15 years need 20-30 mg/
day.
i. Daily allowance of iron for an adult Indian is 20 mg
iii. Pregnant women need 40 mg/day. Transfer of iron
of iron, out of which about 1- 2 mg is absorbed. In
and calcium from mother to fetus occurs mainly in
Western countries, requirement is less (15 mg/day)
the last trimester of pregnancy. Therefore, during
because the diet does not contain inhibitory sub-
this period, mother's food should contain surplus
stances.
quantities of iron and calcium.
iv. In the first 3 months of life, iron intake is negligible
because milk is a poor source of iron. During this
time, child is dependent on the iron reserve recei-
Heme containing proteins ved from mother during pregnancy. In premature
Hemoglobin 65,000 4 RBC babies, the transplacental transfer of iron might not
Myoglobin 17,000 Muscle have taken place. Hence such babies are at a risk
Cytochrome oxidase 180,000 2 Mito of iron deficiency. After 3 months of life, diet sup-
Cytochrome b 30,000 do plementation with cereals is essential for supplying
Cytochrome cl 37,000 do the iron requirement.
Cytochrome c 12,000 do
Cytochrome bS 15,000 ER Sources of Iron
Cytochrome P450 55,000 ER, Mito
i. Leafy vegetables (20 mg/100 g) are good sources.
Catalase 240,000 4 RBC
Pulses (10 mg/ 100 g) and cereals (5 mg/100 g)
Lactoperoxidase 93,000 Milk
contain lesser quantity of iron. In a typical Indian
Tryptophan pyrrolase 4 Cytosol
diet, the major quantity of iron is received from
Nitric oxide synthase Endothelium
cereals because of the bulk quantity taken, although
.. Iron-sulfur complexes
they contain iron only in moderate amounts.
Com plex Ill Fe-S 30,000 2 Mito
ii. Liver (5 mg/100 g) and meat (2 mg/100 g)
Succinate DH 27,000 4 M ito
Liver
iii. Jaggery is a good source for iron.
Xanthine oxidase 275,000 8
iv. Cooking in iron utensils will improve the iron con-
Nonheme iron containing proteins
Aconitase 66,000 2 TCAcycle tent of the diet.
110,000 2 Liver
v. Milk is a very poor source of iron , containing less
Phehydroxylase
Transferrin 77,000 2 Plasma than 0.1 mg/100 ml.
Ferritin 450,000 4,000 Tissues
Factors Influencing Absorption of Iron
Hemosiderin Many Liver
(Mito = Mitochondria; ER = Endo plasmic reticulum; DH = Dehydroge- Iron is absorbed by upper part of duodenum. The follow-
nase; Phe= Phenyl alanine) ing factors affect absorption of iron:
!
3
Food (Fe ·) (20mg) - -- -- -- -- - Upper GI tract {Fe •)
(1 mg)
Storage ferritin
+
Loss through skin Losa through
(600 mg) (10 µg) bleeding
Intestinal lumen
Fe<++ (ferric) form is not absorbed.
Divalent metal transporter
Ascorbic Acid Brush borde \V with Fe...
nnnnn nnnnnnnnnnnnnnn
M@
.@
Ferric ions are reduced with the help of gastric HCI,
Fe++ (ferrous) Intestine
ascorbic acid , cysteine and -SH groups of proteins. mucosal cell
-
Therefore, these will favor iron absorption. About
50-75 mg of ascorbic acid per day will be sufficient for Fe..'(ferric)
normal iron absorption.
Interfering Substances
Fe....Ferritin F0
Iron absorption is decreased by phytic acid (in cereals) Fe" crosses membrane Ill
and oxalic acid (in leafy vegetables) by forming insolu-
ble iron salts. An average Indian diet contains more than
20 mg of iron. But the phytates and oxalates in the diet
Fe...Transferrin
in bloodstream T
11111
0
reduce the absorption, and only about 1 mg of iron is 1= Fe- (ferric iron) in food. 2= Iron is reduced to Fe" (ferrous)
absorbed. In Western diet, even though iron content is state, and attaches to divalent metal transporter on the mucosal
surface. 3= Ferrous iron is internalized . 4= Iron is oxidized to ferric
about 10 mg, about 2 mg is absorbed. state. 5= Ferric iron binds with ferritin for temporary storage.
6=Ferric iron released, reduced to ferrous state crosses the cell
membrane, re-oxidized to ferric state by ceruloplasmin. In the
Other Minerals bloodstream, ferric iron is bound with transferrin,
Calcium, copper, lead and phosphates will inhibit iron Fig. 34.7: Absorption of iron from intestine
absorption. One atom of lead will inhibit absorption of
1000 atoms of iron .
When iron is in excess, absorption is reduced: this is the
Mucosa/ Block Theory basis of "mucosal block" (Fig. 34.7).
Duodenum and jejunum are the sites of absorption. Iron This mechanism of iron absorption from intestinal
metabolism is unique because homeostasis is main- lumen to the mucosal cell is different from the iron
tained by regulation at the level of absorption and not by release from intestinal cell to the bloodstream (see Fig .
excretion. No other nutrient is regulated in this manner. 34.7). Iron in the ferritin is released, then crosses the
In other words, iron is a one-way element. When iron mucosal cell with the help of a transport protein called,
stores in the body are depleted, absorption is enhanced. ferroportin. But this can happen only when there is free
When adequate quantity of iron is stored, absorption is transferrin in plasma to bind the iron. Iron crosses cell
decreased. This is referred to as "mucosa! block" of membrane in ferrous form. In blood it is re-oxidized to
regulation of absorption of iron. ferric state, and transported by transferrin.
Only ferrous (and not ferric) form of iron is absorbed. Absorption of iron needs divalent metal ion trans-
Ferrous iron in the intestinal lumen binds to mucosal porter and ferroportin. Synthesis of both these proteins
cell protein, called divalent metal transporter-1 (DMT-1). is downregulated by hepcidin, a peptide secreted by the
This bound iron is then transported into the mucosal cell. liver when body iron reserves are adequate. If there is
The rest of the unabsorbed iron is excreted. Inside the hypoxia or anemia, the synthesis of hepcidin is reduced;
mucosal cell, iron is oxidized to ferric state, and is com- so ferroportin synthesis will increase.
plexed with apoferritin to form ferritin . It is kept tempo-
rarily in the mucosal cell. If there is anemia, the iron is Iron Transport in Blood and
further absorbed into the bloodstream. If transferrin is Uptake by Cells
saturated with iron , any iron accumulated in the mucosal
cell is lost when the cell is desquamated. The fraction of Transport form of iron is transferrin. It is synthesized in
iron absorbed and retained is decided by the iron status. liver. Normal plasma level of transferrin is 250 mg/100 ml.
Chapter 34: Mineral Metabolism and Abnormalities 501
Laboratory Findings
Laboratory investigations generally used to diagnose
anemias are listed in Table 34.3.
i. Serum iron level: It is depressed in iron deficiency, Figs 34.9 A and B: (A) Peripheral blood smear. Iron deficiency
acute and chronic infections, hypothyroidism and manifests as microcytic hypochromic anemia; (B) Tired girl.
Apathy and poor scholastic performance are very characteristic of
Kwashiorkor. iron deficiency anemia
ii. Total iron binding capacity (TIBC): It is elevated
in hypochromic anemias, acute hepatitis and preg- granules, seen in spleen and liver. Prussian blue
nancy. reaction is positive for these pigments. Hemo-
siderosis occurs in persons receiving repeated
Treatment of Iron Deficiency
blood transfusions. Here the regulation at the
Oral iron supplementation is the treatment of choice. level of intestine is circumvented leading to iron
100 mg of iron + 500 mg of folic acid are given to overload . Hemophilic children require blood trans-
pregnant women, and 20 mg of iron + 100 mg folic fusion every 3 months. If whole blood is given every
acid to children. Iron tablets are usually given along time, by about 20 years of age, the patient will have
with vitamin C, to convert it into ferrous form, for easy hemosiderosis. This is the commonest cause for
absorption. Unabsorbed iron may generate free radicals hemosiderosis in India. Nowadays, component
and so, it is advisable to give vitamin E (to prevent separated plasma is available for transfusion,
free radical generation) along with iron. which eliminates the risk of hemosiderosis. Modern
treatment of hemophilia is to give cell free plasma;
Iron Toxicity this will avoid future hemosiderosis.
i. Hemosiderosis: Iron excess is called hemo- ii. Primary hemosiderosis: It is also called hereditary
siderosis.Hemosiderin pigments are golden brown hemochromatosis. It is an autosomal recessive
Chapter 34: Mineral Metabolism and Abnormalities 503
Ferroxidaze
Functions of Copper
T
Fe" (Transferrin) \ • Fe' '"' (Transferrin) 1. It is necessary for iron absorption and incorporation
cu- cu• of iron into hemoglobin.
(Oxidized ceruloplasmin) (Reduced ceruloplasmin) 2. It is necessary for tyrosinase activity.
Fig. 34.1 0: Function of ceruloplasmin 3. It is a co-factor for vitamin C requiring hydroxylations.
IMANGANESE (Mn) _ __
electrolytes, having much clinical applications. These
are described in chapter 28. A summary of the mineral
Requirement of manganese is 5 mg/day. metabolism is shown in Table 34.4. Lead, Cadmium,
Sources : Nuts are good sources and tea leaves are ex- Phosphorus and mercury are toxic minerals; these are
ceptionally rich in manganese. described in Chapter 37. Iodine metabolism is described
.. Functions: The following enzymes either contain or are in Chapter 45.
activated by manganese: Hexokinase, phosphogluco- 0
mutase, pyruvate carboxylase, isocitrate dehydrogenase, 8· Clinical Case Study 34.1
succinate dehydrogenase, arginase, glutamine synthe-
A 54-year-old grossly overweight woman presented
tase and Mn..-dependent superoxide dismutase. Man-
with complaints of cramps and spasms of both hands.
ganese is an integral part of glycosyltransferases,
She was depressed and had positive Trosseau's and
responsible for synthesis of glycoproteins and chon- Chvostek signs. Past medical history revealed history
droitin sulfate. Manganese is also required for RNA
of thyroidectomy for Grave's disease. The laboratory
polymerase activity. results obtained were as follows:
Deficiency leads to impaired growth and skeletal Serum Creatinine - 1.0 mg%, serum Calcium - 4.1
deformities. mg%, serum phosphate - 5.9 mg%, ALP - 60 IU/L,
ILITHIUM (Li)
serum albumin - 4.0 g%.
Comment on the laboratory results and give your
Lithium is used in treating manic depressive psycho- likely diagnosis. What additional tests are to be done to
sis (bipolar disorders), the dose being 25-500 mg/day. come to a conclusive diagnosis?
Lithium will counteract both mania and depression. 0
Therapeutically optimum concentration of u• in plasma • • Clinical Case Study 34.2
is 7- 10 mg/ml, while 12 micrograms is toxic. Since A 10-year-old girl presented with excessive tiredness,
margin of safety is narrow, the treatment requires constant poor appetite, inability to concentrate and tingling sen-
monitoring of blood level. Lithium elevates serotonin sations. On examination , there was pallor. Laboratory
levels and reduces catecholamines in brain tissue. examination revealed decrease in hemoglobin, ferritin
Lithium toxicity leads to hypothyroidism, hyperpara- and MCV. Total iron binding capacity (TIBC), transferrin
thyroidism and kidney damage. and ROW were increased. What is the likely diagnosis?
506 Section D: Nutrition
ae
zed as: stones (kidney), moans (abdominal pain),
Clinical Case Study 34.1 Answer groans (myalgias), bones (bone pain), and psychiatric
Hypocalcemia due to accidental removal of parathyroid overtones. Diagnosis can be established by finding
glands. Please see details under Clinical Case Study hypercalcemia and hypophosphatemia, with elevated
34.4 also. PTH levels. Symptomatic patients can be treated with
e parathyroidectomy.
.:ti-- Clinical Case Study 34.2 Answer (')
The symptoms are suggestive of iron deficiency ane-
.:ti• Clinical Case Study 34.4 Answer
mia, the commonest deficiency disease prevalent world- Muscle pain and cramps, stiffness, tingling of hands and
wide. Iron requirements are high during infancy, child- feet, carpopedal spasm and positive Trousseau's sign
hood, adolescence and last two trimesters of pregnancy. are indicative of hypocalcemia. Two common causes for
Iron is a critical element in the functioning of cells, but hypocalcemic tetany are hypoparathyroidism or chronic
Chapter 34: Mineral Metabolism and Abnormalities 507
renal failure. Abnormal renal function tests, arterial blood ( LEARNING POINTS, CHAPTER 3L
gases, hemogram and urine calcium/creatinine ratio
1. Factors favorably influencing calcium absorption
would point toward a renal cause for hypocalcemia.
are-calcitriol, PTH , acid pH, and arginine.
High serum alkaline phosphatase and low parathyroid
2. Factors decreasing calcium absorption are-phytic
hormones would indicate hypoparathyroidism. Antimicro-
acid, high phosphate content and malabsorption syn-
somal antibody may be elevated in the latter state.
dromes.
Hypoparathyroidism may be due to parathyroid aplasia
3. Calcium activates enzymes, such as protein kina-
or hypoplasia, parathyroid hormone gene mutations, ses, glycogen synthase, pyruvate carboxylase,
autoimmune parathyroiditis, hemosiderosis, Wilson's pyruvate dehydrogenase.
disease, or accidental removal during thyroid surgery. 4. Calcium mediates contraction of muscle fibers.
Additional tests include electrolytes (magnesium, 5. Calcium mediates secretion of insulin. PTH, calci-
phosphate and other electrolytes), renal function tests tonin, ADH and can act as a second messenger for
(urea and creatinine), arterial blood gases, liver function some hormones, e.g. Glucagon.
tests (albumin, coagulation studies and other liver func- 6. Calcium is known as Factor IV in blood coagulation.
tion tests), PTH and Vitamin D levels. Cardiac assess- 7. Hypercalcemia may occur in hyperparathyroidism,
ment may be necessary to rule out cardiac effects of multiple myeloma, bone cancer, and Paget's disease.
hypocalcemia. 8. Hypocalcemia leads to tetany as seen in renal tubu-
(") lar acidosis.
•ii• Clinical Case Study 34.5 Answer 9. Hypernatremia is seen in Cushing's syndrome and
after prolonged cortisone therapy. Hyponatremia is
The patient is suffering from hypoparathyroidism, due to seen in Addison's disease.
decreased activity of PTH . Treatment with 131 1decreased 10. Renal loss of potassium is seen in renal tubular
thyroid cells, but also killed neighboring parathyroid cells acidosis, tubular necrosis , metabolic alkalosis.
leading to persistent hypoparathyroidism in this patient. 11 . Hyperchloremia is seen in dehydration, Cush-
Carpopedal spasm is due to hypocalcemia and can be ing's syndrome, respiratory acidosis, renal tubular
treated by calcium and vitamin D supplementation. acidosis.
Causes of hypoparathyoidism are: (1) Primary- 12. Hypochloremia is seen in excessive vomiting,
which is permanent and irreversible, could be congenital Addison's disease, and respiratory alkalosis.
or acquired, (2) Iatrogenic-Excision of all 4 parathyroid 13. Iron homeostasis is maintained by regulation of
glands, as during surgery of thyroid or other neck surge- absorption and not by excretion.
ries, extensive irradiation of face, neck or mediastinum, 14. Transport form of iron transferrin. Storage form is
parathyroidectomy, (3) Autoimmune hypoparathyrodism. ferritin. Iron deficiency may be caused by hookworm
(4) Congenital, (5) Metal overload conditions like hemo- infection, nephrosis, lead toxicity, and leads to micro-
chromatosis and thalassemia, (6) Wilson's disease, cytic hypochromic anemia. Iron toxicity leads to hem-
(7) Hypomagnesemia, (8) Neonatal hypoparathyroidism, osiderosis.
and (9) Pseudohypoparathyrodism-due to resistance 15. Copper in plasma is bound to ceruloplasmin. It
to PTH . functions as co-factor for Vitamin C mediated
Clinical features are paresthesias, irritability, fatigue, hydroxylations.
anxiety, mood swings, personality disturbances, seizu- 16. Copper is essential for formation of Hb, deficiency
res, hoarseness of voice, wheezing and dyspnea, muscle leading to microcytic normochromic anemia.
cramps, biliary colic, electrolyte disturbances (hypomag- 17. Copper level in blood is lowered in Wilson's hepato-
' nesemia, hypokalemia and alkalosis) and tetany with posi- cellular degeneration.
tive Chvostek's sign and Trousseau sign. Hypocalcemia 18. Menkes Kinky hair syndrome, an X-linked disease
can produce a variety of associated features like extrapy- is caused due to decreased copper transport in the
ramidal choreoathetotic syndromes, spastic paraplegia, blood.
ataxia, papilledema, emotional disturbances, cataracts, 19. Zinc is stored in combination with metallothionein.
abnormal dentition, dry, puffy skin and effects on heart More than 300 enzymes are known to be zinc
including congestive heart failure. dependent.
508 Section D: Nutrition
34-18. The micromineral present in teeth is: 34-25. Hyperparathyroidism is characterized by all the
A. Calcium B. Iodine following, except
C. Fluorine D. Manganese A. Osteoporosis 8. Hypercalcuria
34-19. Zinc is present in all the enzymes, except: C. Alkalosis D. Recurrent urinary calculi
A. Al kaline phosphatase 34-26. Sodium level in serum is affected by all the follow-
8 . Amylase ing factors , except:
• C. Carbonic anhydrase A. Aldosterone
C. ADH
B. Cortisone
D. Cholecalciferol
D. RNA polymerase
34-20. Which of the following trace elements has an anti- 34-27. Which contains iron?
oxidant role? A. Ceruloplasmin
A. Chromium B. Zinc B. Xanthine oxidase
C. Selenium D. Nickel C. Albumin
34-21 . Accumulation of iron (hemosiderosis) leads to all D. Superoxide dismutase
the following clinical features, except: 34-28. All are true with selenium, except
A. Diabetes mellitus A. Action of selenium is complementary to vitamin E
B. Cirrhosis of liver B. Glutathione peroxidase contains selenium
C. Defective coagulation C. Selenium may cause hepatic necrosis
D. Hyper pigmentation D. Requirement is 50-100 microgram/day
34-22. Which of the following is not a feature of Wilson's 34-29. In a patient the serum calcium level was 12 mg/dL,
hepato lenticular degeneration? and serum inorganic phosphate was 2 mg/dL. The
A. Decreased excretion of copper in urine above findings are characteristic of:
B. Low ceruloplasmin level A. Excess calcitonin secretion
C. Keyser-Fleischer ring in cornea 8 . Vitamin D deficiency
t
D. Cirrhosis of liver C. Hyper parathyroidism
34-23. Which of the following is not true regarding the D. Hypervitaminosis D
absorption of calcium? 34-30. Fasting samples give lower values than post pran-
A. Absorption is increased by vitamin D dial samples for all the parameters, except:
B. Acidity favors calcium absorption A. Glucose
C. Deficiency of bile favours absorption B. Phosphate
D. Basic amino acids increase absorption C. Triglycerides
34-24. Parathyroid hormone regulates calcium homeo- D. Cholesterol
stasis by all the mechanisms, except: 34-31 . Hypoklaemia occurs in all conditions, except:
A. Bone resorption A. Rapid correction of acidosis
B. Secretion of calcitonin B. Infusion of insulin and glucose
C. Increased absorption of calcium from intestine C. Use of loop diuretics
D. Increased reabsorption from renal tubules D. Polycythemia
34-1 . What influences absorption of calcium from intes- 34-3. What are the sources of calcium?
tine?
Milk, egg, fish, vegetables.
Vitamin D; Calcitriol; Parathyroid hormone; phytic acid;
34-4. What is the function of calcium?
oxalate.
34-2. What is the daily requirement of calcium? Coagulation; Neuromuscular activity; Intracellular mes-
500 mg per day. senger; Activation of enzymes; Bone formation.
510 Section D: Nutrition
34-5. What is the normal level of calcium in blood? Hepcidin is secreted by liver, when body iron reserves
9-11 mg /dl . are adequate. In anemia, the synthesis of hepcidin is
34-6. Which will influence serum calcium level? reduced.
Calcitriol; Calcitonin; Parathyroid hormone. 34-23. What is the carrier protein in iron in blood?
34-7. How parathyroid hormone regulates calcium level Transferrin.
in blood? 34-24. What is the storage form of iron?
Bone resorption; increased absorption of calcium from Ferritin.
intestines; increased absorption of calcium from renal 34-25. What is hemosiderin?
tubules. Excess iron is loaded as hemosiderin.
34-8. Hypocalcemia results in what condition? 34-26. What is haptoglobin?
Tetany. It is the carrier of free hemoglobin.
34-9. What is the cause for tetany? 34-27. What is hemopexin?
Hypoparalhyroidism. It is the carrier of free heme.
34-10. What are the features of hyperparathyroidism? 34-28. Anemia is resulted in the deficiency of?
Osteoporosis; hypercalciuria; urinary calculi. Iron, copper, vitamin C, folic acid, vitamin 8 12, pyridoxal
34-11. What is the normal level of phosphorus in blood phosphate.
3-4 mg/dl. 34-29. What are the causes of iron deficiency?
34-12. What is the normal level of sodium in blood? Nutritional. hookworm infection, repeated pregnancy,
136- 145 m Eq/L. chronic blood loss, nephrosis.
34-13. What is the daily requirement of iron for a normal 34-30. How iron deficiency manifests?
adult male? Microcytic hypochromic anemia.
10-20 milligram per day. 34-31 . What are the features of hemosiderosis?
34-14. What are the dietary sources of iron? Cirrhosis of liver: diabetes mellitus; yellow color of skin
Green leafy vegetables; jaggery. 34-32. What is the major cause for hemosiderosis?
34-15. Which trace element is deficient in milk? Repeated transfusion of whole blood.
Iron. 34-33. Name some copper containing enzymes.
34-16. What are important iron containing proteins? Ceruloplasmin, cytochrome oxidase, cytochrome C,
Hemoglobin, myoglobin, cytochromes, catalase, trypto- tyrosinase, lysyl oxidase, super oxide dismutase.
phan pyrrolase, xanthine oxidase, transferrin, ferritin. 34-34. What are the dietary sources of copper?
34-17. Which will increase iron absorption from intestines? Cereals, meat, liver.
Gastric HCI, ascorbic acid, cysteine. 34-35. What is ceruloplasmin?
34-18. What are the factors which will retard iron absorp- It is ferroxidase; it promotes oxidation of ferrous ion to
tion? ferric form.
Phytic acid in cereals; oxalic acid in leafy vegetables; 34-36. What are the characteristic features of Wilson's
calcium, zinc, lead and phosphates. hepatolenticular degeneration?
34-19. How is iron absorbed? Ceruloplasmin level in blood is decreased; copper
Iron in the ferrous state is binding to divalent metal excretion is reduced; copper is accumulated in liver to
transporter (DMT-1) present in mucosa I surface of produce cirrhosis.
intestinal cell; thus iron is transported into the mucosa! 34-37. What is the daily requirement of iodine?
cell. 150-200 microgram.
34-20. What is mucosal block? 34-38. What is the dally requirement of zinc?
Iron homeostasis is maintained by regulation at the 15-20 milligram.
level of absorption and not by excretion. When iron 34-39. Which enzymes contain zinc?
stores in the body are depleted, absorption is enhanced. Alkaline phosphatase; Amylase; Carbonic anhy-
When adequate quantity of iron is stored, absorption is drase; RNA polymerase. Zinc is required for insulin
decreased. This is referred to as "mucosa! block". secretion.
34-21. How is this mucosa! regulation takes place? 34-40. What is the importance of selenium?
Absorption of iron needs DMT and ferroportin. Synthe- It is an antioxidant.
sis of both these proteins is downregulated by hepcidin. 34-41 . Name the selenium containing enzyme.
34-22. What is hepcidin? Glutathione peroxidase.
_ _ _ _ _Chapter 35
.. Energy Metabolism
and Nutrition
Chapter at a Glance
The learner will be able to answer questions on the following topics:
Calorific value, respiratory quotient Nutritional values of proteins
Basal metabolic rate (BMR) Limiting amino acids, supplementation
Specific dynamic action (SDA) Kwashiorkor and marasmus
0 Proximate principles Prescription of the diet
Nitrogen balance Glycemic index
.. A sound knowledge of the principles of nutrition is of para- TABLE 35.1: Calorific value of nutrients
mount importance. Dietetics is the science of food and Energy yield 0 1 consumed co,
nutrients, their action, interaction and balance in health Nutrient kcaVg Ug producedUg RQ
and disease. The main purpose of the food is to provide Carbohydrates 4 0.829 0.829 1
energy for muscular activity and also to supply basic Fats 9 2.016 1.427 0.71
body building materials such as essential amino acids. Proteins 4.2 0.966 0.782 0.81
Alcohol 7 1.429 0.966 0.66
ICALORIFIC VALUE
The energy content of food materials is measured in ii. RQ of carbohydrates is 1; RQ of fats is 0.7; that of
calories. One calorie is the heat required to raise ~lie proteins is 0.8 (Table 35.1 ).
temperature of 1 g of water through 1°C. Since it is a iii. For a mixed diet it is between 0.7 and 1, often
very small unit, in medical practice, the energy content around 0.82-0.85. When the rate of utilization of
is usually expressed in kilocalorie (kcal) which is equal fat increases in relation to carbohydrates, RQ falls.
to 1000 calories. [One kilo calorie is equal to 4.2 kilo- This happens in diabetes mellitus, when utilization
joules (kJ)]. The calorific value of nutrients otherwise of carbohydrate is reduced. The RQ is lowest when
known as "energy density" (energy yield per unit weight ketolysis is very active.
of food) is given in Table 35.1.
Energy Requirements
Respiratory Quotient of a Normal Person
i. Respiratory quotient (RQ) is defined as the ratio of i. Maintenance of basal metabolic rate (BMR)
volume of CO2 produced in Ug to the oxygen con- ii. Specific dynamic action or thermogenic effect of food
sumed in Ug. iii. Extra energy expenditure for physical activities.
512 Section D: Nutrition
iii. Temperature: BMR increases in cold climate as a If the person wants to get 1000 kcal, he should take food
compensatory mechanism to maintain body tempe- worth 11 00 kcal. Thus additional calories, equivalent
rature. Eskimos have a higher BMR. to SDA has to be added in diet.
iv. Exercise: The increase in BMR during exercise is The values of SDA are: for proteins 30%, for lipids
due to increased cardiac output. 15%, and for carbohydrates 5%. This means that out
v. Fever: About 12% increase in BMR is noticed per of every 100 grams of proteins consumed, the energy
degree centigrade rise in temperature. available for doing useful work is 30% less than the
Chapter 35: Energy Metabolism and Nutrition 513
TABLE 35.2: Energy requirement and occupation TABLE 35.3: Calculation for energy requirement ror a 55 kg
person. doing moderate work
Type ofactivity Occupation
Light Office workers, Shopworkers
For BMR = 24x ss kg = 1320 kcal
+ For activity = 40% ofBMR = 528 kcal
Moderate Industry workers, Farm workers
Very active Agricultural workers, Unskilled laborers, Athletes
Subtotal = 1320 + 528 = 1848 kcal
= 1848 X 10% = 184 kcal
• Heavy work Construction workers
+ Need for SDA
Total =1848+ 184 =2032 kcal
Rounded to nearest multiple of SO = 2050 kcal
TABLE 35.4: Carbohydrates in common foods
1. Cane sugar 100% 2. Cassava (Tapioca) 85%
3. Rice 80% 4. Honey 80% vitamins are also to be provided. Further, additional
5. Wheat 70- 80% 6. Cakes 55-65%
requirements for growth, pregnancy, lactation and conva-
7. Bread
lescence are to be provided in the food.
50-60% 8. Potatoes 25%
TABLE 3S.S: Cholesterol content of food items TABLE 3S.6: Fatty acids 1n oils
Food item Cholesterol content mg/ 100 g Mono- Poly-
Hen's egg, whole 300 Fotor oil Saturated (%) unsaturated(%) unsaturated(%)
Egg yolk 1330 Butter/ghee(*) 75 20 5
Liver 300-600 Safflower oil 9 12 79
2000 26 19
Brain
280
Cotton seed oil 65
•
Butter Coconut oil(') 86 12 2
Ghee 310 Ground nut oil 18 46 36
Meat and fish 40-200 (") Butter/ ghee contains short chain fatty acids and coconut oil
Milk 10 contains medium chain fatty acids, which are absorbed and meta-
bolized quickly
i
TABLE 3S.7: Recommended protein allowances Body proteins
TABLE 3S.8: L1m1llng amino acids in proteins TABLE 3S.9 : Comparison between the salient features of
Kwash1orkor and marasmus
Protein supplemented to
Protein Limiting amino acid rectify deficiency Marasmus Kwashiorkor
Rice Lys, Thr Pulse proteins Age of onset Below 1 year One to 5 years
Obesity can occur only as a result of ingestion of high levels of leptin in plasma. There is an "adipo-
food in excess of the body's needs. insular axis", with insulin promoting leptin secretion
and leptin inhibiting insulin release.
Diseases Related to Obesity 2. Neuropeptide-Y, a hypothalamic polypeptide, stimu-
Sensitivity of peripheral tissues to insulin is decreased. lates desire for carbohydrates. The action is to inhi-
The number of insulin receptors are decreased in adi- bit insulin secretion. ..
pose tissue cells. Plasma insulin levels may be elevated. 3. Ghrelin is secreted mainly by adipocytes. It stimulates
Obesity is associated with substantially increased cardio- hunger and appetite by acting on the hypothalamus.
vascular risk. Increased waist to hip ratio (abdominal Plasma level of Ghrelin is increased in fasting state,
obesity) is a greater risk. In metabolic syndrome, insulin which produces hunger signals.
resistance, hyperglycemia, hyperlipidemia (increased 4. Non-esterified fatty acids (NEFAs) are primarily
LDL and decreased HDL) and obesity are seen. released from adipose tissue during fasting . Circu-
The major ill effects of obesity are increased risk of lating NEFAs reduce glucose uptake, to promote
coronary artery disease, diabetes mellitus, hyper- lipid burning as a fuel source in most tissues, while
tension, metabolic syndrome and a reduced lifespan. sparing carbohydrate for neurons.
Calorie-fat-restricted diet may retard aging process and
extend the lifespan. l fRESCRIPTION OF DIET _
General Principles
Treatment of Obesity
Recommended dietary allowances (RDA) of nutrients
Lifestyle modification is the best suitable remedy. The are given in detail in Appendix V.
goal is to reduce the intake of calories and fat. Frequent i. The ideal body weight: The underweight person
small meals with lots of vegetables will make the food should be given more nutrients and the overweight
palatable and give a feeling of satiety. Controlled exer-
cise is very useful.
person should reduce calorie intake. The ideal body
weight is calculated from height.
•
ii. Protein requirement: 1 g per kg body weight for
Regulators of Appetite adults and 2 g per kg for children. During preg-
Hypothalamus has the central control of appetite. Psy- nancy and lactation, protein requirement is about
chologic, genetic, neural and humoral factors are invol- 2.5 g per kg.
ved in the control. Polypeptides that increase appetite iii. Calorie requirement: This depends on age, sex,
height, weight, health status and above all on the
are: Neuropeptide Y (NPY), Ghrelin, Polypeptide YY
physical activity and occupation. As a rule of thumb,
(PYY), Insulin and Cortisol. Appetite decreasing fac-
the calorie requirement for a person is taken as
tors are: leptin, melanocyte stimulating hormone (MSH),
30-35 kcal per kg of ideal body weight. For seden-
and Serotonin.
tary habits, lower values (30 kcal) and for moderate
muscular activities, higher values (35 kcal) are pre-
Adipose Tissue Talks to
scribed.
Brain through Factors
Considering the average weight of adult Indians
1. Leptin (Greek leptos = thin) is a hormone secre- between 55 and 65 kg, average calorie requirement
ted by adipocytes. It is mainly produced by white will be between 1700 and 2300 kcal per day.
adipose tissue. It functions as a satiety signal. It is An extra allowance of 300 kcal is necessary dur-
an index of the energy reserve in the body. When ing pregnancy and lactation periods.
the energy reserve is adequate, leptin levels are iv. Specific dynamic action : Extra energy is to be
increased and this would suppress further food supplied to account for SDA.
intake. Leptin inhibits neuropeptide-Y secretion, and v. Ratio: Balanced diet should contain calories from
so when fat depots are full, appetite is decreased. carbohydrate, proteins and fat in the ratio of
Obesity is associated with leptin resistance and 60:20:20.
Chapter 35: Energy Metabolism and Nutrition 519
TABLE 35.10: First step in the prescription of diet TABLE 35.11: Prescription of diet. 2nd step
Energy required + SDA 2000kcal Proteins : 60g
Protein : 60g Fats : 35 g
Calcium : 400mg Carbohydrates : 350g
Iron : 25 mg Calories : 2000 kcal
:
• Calcium
Iron :
400 mg
25 mg
TABLE 35.12: A diet for a 60 kg sedentary man
Item Quantity vegetarian Quantity non-vegetarian
Cereals 350g 350 g Fourth Step: Food Items
Pulses 75 g 60g
A judicious combination of cereals and pulses provide
Vegetable oil 40 ml 25 ml
all the essential amino acids (pulses are deficient in
Milk 250ml 150 ml
methionine, while cereals lack in lysine). (See mutual
Leafy vegetable 200g 200g
supplementation, described previously). An accepted
Sugar 25 g 25 g
formula is that the food should contain pulses and cereals
Fish/ meat - 60g
in the ratio of 1 :5 to provide good quality proteins.
First Step: Calorie Requirement Supplementing the cereals with animal proteins is
certainly good to improve the quality, but at a higher cost.
For a 60 kg sedentary man , the energy requirement is Fruits and vegetables will provide vitamins and minerals.
60 x 30 = 1800 kcal plus additional allowance for specific Vitamin A is available in ripe mango, papaya, carrot, beet
dynamic action (1800 x 10% = 180). Therefore the total root, and other leafy vegetables. B complex vitamins
requirement is roughly 2000 kcal. The recommended may be supplied by cereals (parboiled rice, whole wheat
dietary intake for a 60 kg sedentary man, based on the powder) and green leafy vegetables. Major sources of
above principles is given in Table 35.10. calcium are milk, cereals, pulses and green leafy vegeta-
t bles. Iron is mainly supplied by pulses, cereals, vegetab-
Second Step: Proximate Principles les, meat and liver. Jaggery is a good source for iron.
He requires 60 g proteins. This will give 60 x 4 = 240 kcal Jaggery is therefore superior to refined sugar, especially
of energy. His total requirement is 2000 kcal. Therefore for children. Milk is a very poor source for iron.
carbohydrates plus fats should provide (2000 - 240) =
1760 kcal. As a general rule, about 20% of total calo- Fifth Step: Prescribing a Normal Diet
ries are supplied by fat. Therefore, fats should supply The requirements shown in Table 35.11 may be trans-
1760 x 20% = 350 kcal which is provided by (350/9) = lated into Table 35.12. This will satisfy the requirements
about 35 g of fats. (About 30% of the total fat may be regarding protein (60 g); fats (45 g), calories (2000 kcal),
supplied as poly unsaturated fatty acids). calcium (400 mg) and iron (25 mg). See that cereals-
The rest 1400 kcal are supplied by 350 g of carbohy- pulses ratio is maintained at 5:1. When calories alone
drates. These calculations are based on the fact that 1 g are to be increased, as in the case of a person having
carbohydrate provides 4 kcal, 1 g fat supplies 9 kcal and severe muscular exercise, tubers and roots will serve
1 g protein gives rise to 4 kcal. Thus the requirements this purpose.
calculated in Table 35.10 may be rewritten as in Table The basic principles are summarized in Box 35.1 .
35.11 .
Diet for Patients with Diabetes
Third Step: General
Management of diabetes gives great emphasis for die-
Composition of Food tary control and exercise. The main aim is to keep the
The third step is to calculate how these proximate blood HbA 1c level to nearly normal values. Glucose level
principles are supplied as common foodstuffs. For this as near the normal values, for as many hours, as possi-
exercise we should familiarise with the nutritive value ble in a day. The calorie requirement is distributed for
of foodstuffs. Detailed values are shown in Appendix a diabetic patient: Carbohydrate 60-70%, fat 15-25%
VI of this book. and protein 15-20%.
520 Section D: Nutrition
0
a) Sugar, sweet and refined carbohydrates are avoided. • · Clinical Case Study 35.1
b) Leafy vegetables are increased, and tubers are res-
A 2-year-old boy was brought to the hospital. He was
tricted.
c) Frequent small meals are prescribed, with distribu- eating poorly for the last one month, had intermittent
tion of calories such that, breakfast 15%, mid-morn- diarrhea, and had become irritable and apathetic. On
ing snack 5%, lunch 30%, evening tea 10%, dinner examination, he was underweight for height and small
35%, bedtime snack 5%. for age. He was pale, weak, skin was flaky, hair was
brittle, abdomen was distended, liver was moderately
Glycemic Index enlarged and generalized edema was present. Labo-
It is assessed by the glycemic response after the particu- ratory tests were as follow: Hemoglobin-6.5 g/dL, Total
lar diet and comparing it with a reference meal. The refer- protein-4.0 g/dL, Albumin- 1.8 g/dL. What is the prob-
ence meal is always taken as 50 g of glucose (Fig. 35.4). able diagnosis?
Glycemic Index=
Area under glucose tolerance
a0 Clinical Case Study 35.2 I
now giving only formula milk (which was diluted). What that increase metabolic demands like infections, hyper-
is the probable diagnosis? thyroidism, burns, trauma, etc. Edematous PEM is
0 Kwashiorkor and non-edematous PEM is marasmus.
• • · Clinical Case Study 35.3 Another type is known as marasmic-Kwashiorkor PEM .
All organs are affected in malnutrition. Dietary pro-
A 35-year-old female presented with complaints of alter-
teins, energy and micronutrients become deficient.
nating diarrhea and constipation. She reported abdominal
Weight loss, immunodeficiency, neurological changes,
discomfort and bloating that were relieved by bowel move-
fatty degeneration and other effects are seen. Labora-
ment. The episodes were worse in times of stress. She
tory tests include CBC, measures of protein nutritional
denies any blood in the stools, weight loss and anorexia.
status assessment like serum albumin, retinal-binding
No history of fever or drug intake. Physical examination
protein, prealbumin, transferrin, creatinine and BUN
was within normal limits. She was prescribed a cellulose
levels, serum electrolytes, blood glucose, urinalysis and
containing dietary supplementation to increase the bulk of
culture and arterial blood gases (often metabolic acidosis
stools. What is the likely diagnosis? What is the effect of is present). If child has history of abnormal stools, stool
the treatment modality prescribed? specimens should be checked for ova and parasites.
0 Best treatment is oral balanced diet, if child can take
•• Clinical Case Study 35.1 Answer oral feeds. Severe PEM or prolonged starvation requires
The child is suffering from protein energy malnutrition treatment in a hospital with controlled diet.
(Kwashiorkor). This is a severe form of childhood mal- 0
nutrition characterized by edema, irritability, anorexia, •i i• Clinical Case Study 35.3 Answer
ulcerating dermatitis, enlarged liver with fatty infiltrates The patient is likely suffering from irritable bowel syn-
and when well advanced, there may be inadequate drome, a gastrointestin al disorder of unknown etiology.
growth, lack of stamina, loss of muscle tissue, increased Common symptoms include abdominal cramps or pain,
susceptibility of infections, vomiting, diarrhea, anorexia bloating and flatulence. There might be alternating peri-
and edema. Eventually there is stupor, coma and death. ods of diarrhea and constipation and passing mucus in
The condition may develop when mother weans the the stools. Vomiting, blood in stools, pain that interrupts
child from breast milk and replaces the diet with food sleep, fever and weight loss are rare.
rich in carbohydrates, and deficient in proteins. Along Diagnosis is made by excluding other causes. Stool
with protein deficiency, micronutrient and antioxidant bulking agents, antispasmodics, antidiarrheal agents,
deficiencies, like iron, folate, iodine, selenium, vitamin antidepressant drugs, serotonin receptor agonists and
C, glutathione, albumin, vitamin E and PUFA, may play antagonists and chloride channel activators are used for
an important role. Treatment includes treatment of dehy- therapy. Dietary fibers are the indigestible part of plant
dration, antibiotics, a diet providing adequate proteins, foods that make stool soft and enables smooth bowel
vitamins, minerals and electrolytes. movements, prevent constipation hemorrhoids and diver-
ticulosis. Soluble fibers lower total and LDL cholesterol
a0 Clinical Case Study 35.2 Answer and help to prevent ischemic heart disease and stroke.
Soluble fibers in excess can cause abdominal bloat-
• The child is suffering from nutritional marasmus, a type ing and flatulence, dehydration and pectins can reduce
of protein energy malnutrition (PEM). There is inade- absorption of cholesterol-lowering drugs like lovastatin.
t quate intake of proteins and calories leading to emacia- Insoluble fibe rs present in food include cellulose,
tion. Low body weight, diminished skin fold thickness, hemicelluloses, and lignins. Excess of insoluble fibers
and reduced arm muscle circumference reflect loss of can result in excessive gas , constipation and intesti-
fat and catabolism of proteins from the body. All avail- nal obstruction, diarrhea in sensitive patients, and can
able fat stores have been exhausted due to starvation. reduce absorption of calcium, iron, copper and zinc,
PEM might be (1) primary (due to lack of food) or (2) especially in children. Insoluble fibers consumed on
secondary, due to disorders affecting GI function, wasting empty stomach can aggravate symptoms of irritable
diseases like AIDS, cancer, renal failure, or conditions bowel syndrome.
522 Section D: Nutrition
35-1 . Define BMR. What are the factors that affect BMR?
35-2. What is a balance diet? How do you prepare a diet for a normal young adult male of sedentary habits?
35-3. What is the nutritional importance of dietary proteins? Explain how the dietary deficiency of proteins will affect
growing children.
35-4. An adult male is consuming 350 g carbohydrate, 70 g protein and 60 g lipid per day. Calculate the total caloric
content of his diet. Calculate the percentage of calories obtained from carbohydrates lipids and proteins. Are
the percentages of calories that have obtained from these macronutrients conforming to the requirements of a
balanced diet?
35-1.
35-8.
B
C
35-2.
35-9.
B
B
35-3.
35-10.
A
C
35-4.
35-11 .
C
C
35.5.
35-12.
B
D
35.6.
35-13.
B
C
35.7.
35-14.
D
C
..
35-15. D 35-16. A 35-17. D 35-18. C 35-19. A 35-20. D 35-21. C
35-22. D 35-23. C 35-24. A 35-25. A 35-26. A 35-27. C 35-28. B
35-1. What is the calorific value of carbohydrates? 35-15. What is the recommended daily allowance of pro-
4 kcal per gram tein for a normal adult?
35-2. How much calories are generated from fat ? 1 g/kg body weght.
9 kcal per gram of fat 35-16. Negative nitrogen balance is observed in which
35-3. What is the respiratory quotient? conditions?
It is the ratio of carbon dioxide produced to the oxygen Chronic infection; old age, malnutrition.
consumed. 35-17. Positive nitrogen balance is observed in which
35-4. What is the respiratory quotient of carbohydrates? conditions?
One. Pregnancy; convalescence, growth period.
35-5. The respiratory quotient for a mixed diet? 35-18. Lysine is deficient in which foodstuff?
About 0.82. Pulses.
35-6. What is specific dynamic action (SDA)? 35-19. Phenylalanine is deficient in which food stuff?
Increased heat production after intake of food. It is the Tapioca.
extra heat produced by the body, over and above the 35-20. Methionine is deficient in which food stuff?
calorie value of the given food . It is also called thermo- Cereals. •
genie action of food. 35-21 . How all amino acids are made available in a mixed
35-7. Wh ich foodstuff has maximum specific dynamic diet?
action? Mutual supplementation by combining cereals and
Proteins. pulses.
35-8. BMR (basal metabolic rate) is increased in which 35-22. What is protein calorie malnutrition (PCM)?
conditions? Deficiency of protein causes Kwashiorkor. Deficiency
Fever; Starvation; Cold climate; Increased thyroid hor- of calorie produces marasmus. Both are two spectra
mones. of PCM.
35-9. Increased basal metabolic rate is observed in which 35-23. What are the salient features of Kwashiorkor?
clinical condition? Hypoalbuminemia, hypomagnesemia, growth retarda-
Grave's disease (Hyperthyroidism). tion, lethargy, loss of appetite.
35-10. What are the beneficial effects of dietary fiber? 35-24. How much energy is required for a 60 kg person,
Increased motil ity of intestine; prevents constipation; with sedentary work?
decreased absorption of cholesterol; increased glu- 2000 kcal.
cose tolerance. 35-25. A balanced diet should have calories for carbohy-
35-11 . Which food stuffs contain polyunsaturated fatty drate, proteins and fats in which ratio?
acids (PUFA)? 60:20:20.
Vegetable oils, such as sunflower oil, groundnut oil. 35-26. What is the optimum ratio for cereals an d pulses in
35-12. Name polyunsaturated fatty acids. a balanced diet?
Linoleic acid; Linolenic acid; Arachidonic acid. Cereals:pulses ratio= 5: 1.
35-13. What is the dietary advice for a patient with hyper- 35-27. What is the recommended ratio of saturated, mono-
cholesterolemia? unsaturated and polyunsaturated fatty acids in a
They should reduce intake of food containing cho- balanced diet?
lesterol and increase intake of polyunsaturated fatty SFA:MUFA:PUFA = 1:1 :1 ratio.
acids. 35-28. What is the recommended fat intake?
35-14. What foodstuffs contain high cholesterol? The ideal fat intake is about 20% of total calories, out
Egg, butter, animal fat, meat (Table 35.5). of which about 30% be of PUFA.
_ _ _ _ _Chapter 36
Detoxification and
Biotransformation of
Xenobiotics
Chapter at a Glance
• Biotransformation is the process whereby a substance more toxic than the parent substance. This is known
is changed from one chemical to another by a chemi- as bioactivation or toxication. An example is the bio-
cal reaction within the body. For some drugs, it is not transformation of vinyl chloride to vinyl chloride epoxide,
the absorbed drug, but a metabolite that has therapeutic which covalently binds to DNA and RNA, a step leading
action . For example, phenoxybenzamine, a drug given to cancer of the liver. The compounds that are detoxified
to relieve hypertension , is biotransformed into a metabo- include:
lite, which is the active agent. a. Compounds accidentally ingested like preservati-
Biotransformation also serves as an important ves, food additives and adulterants.
defense mechanism in that toxic xenobiotics and meta- b. Drugs taken for therapeutic purposes.
bolites are converted into less harmful substances that c. Compounds produced in the body which are to be
can be excreted from the body. eliminated, e.g. bilirubin and steroids. Bilirubin is
In general, biotransformation reactions generate toxic to the brain. Biotransformation of the lipophilic
more polar metabolites, that are readily excreted from bilirubin molecule in the liver results in the pro-
the body. The liver plays the most important role in the duction of water-soluble (hydrophilic) metabolites
biotransformation reactions. excreted into bile.
The biochemical processes whereby the noxious d. Compounds produced by bacterial metabolism,
substances are rendered less harmful and more water e.g. amines produced by decarboxylation of amino
soluble, are known as detoxification. Lipophilic toxi- acids:
cants are hard for the body to eliminate and can accu- Histidine -> Histamine
mulate to hazardous levels. Lysine -> Cadaverine
Xenobiotics are compounds which may be acciden- Ornithine --> Putrescine
tally ingested or taken as drugs or compounds produced Tyrosine --> Tyramine
in the body by bacterial metabolism (Greek, xenos = Tryptophan -> Tryptamine
strange). Molecules to be eliminated/detoxified are called The transformation of a specific xenobiotic can be
xenobiotics. either beneficial or harmful, and perhaps both depending
Biotransformation is not exactly synonymous with on the dose. A good example is the biotransformation of
detoxification, since in many cases, the metabolites are acetaminophen, a commonly used drug to reduce pain
526 Section D: Nutrition l
OH
O cH
,T) O cH,-oH + 02
0 - P
_ha_se 1_
6
Toluene NADPH NADP• (Benzyl alcohol)
Benzene Phenol Phenyl sulfate
Fig. 36.1: Phase one; oxidative reaction Fig. 36.2: Sometimes both phase one and two reactions are
needed to detoxify a compound
and fever. It normally undergoes rapid biotransformation (detoxification). Sometimes this may result in increased
with the metabolites quickly eliminated in the urine and toxicity (entoxification), e.g. methanol to formic acid.
feces; hence no toxicity is observed . The phase one reactions include hydroxylation, oxida-
But at high doses, the normal level of enzymes tion, reduction , hydrolysis, dealkylation, epoxidation, etc.
may be saturated . The excess acetaminophen under- The products of metabolic transformations are
goes additional biosynthetic pathway, which produces a either excreted directly or undergo further meta-
metabolite that is toxic to the liver. bolism by phase two reactions. They involve con-
jugation with a conjugating agent, thus converting
I CYTOCHROME P450 lipophilic compounds into water soluble, easily excre-
table forms. Phase two reactions are sulfation , acetyla-
The cytochrome P450 enzymes are involved in the bio-
tion, methylation and conjugation with glucuronic acid ,
transformation reactions. They are heme-containing
glutathione or glycine.
..
enzymes, localized in the endoplasmic reticulum of
In some instances, products of phase two reactions
liver.
may further be metabolized by phase three reactions.
NADPH (and not NADH) is the coenzyme for all the
P450 enzymes. One atom of oxygen is inserted into the
substrate. IPHASE ONE REACTIONS
Almost all common drugs are metabolized by the
Oxidative Reactions
P450 system. They are inducible enzymes. Phenobar-
bital causes increased activity of P450. The anticoagu- It may be either aromatic or aliphatic hydroxylation.
lant Warfarin is metabolized by CYP2C9. If the same The reactions also include sulfoxidation, N-oxidation
patient is taking phenobarbitone, the enzyme is induced, and epoxidation. For example, toluene is hydroxylated
the level of the enzyme is increased, warfarin is broken to benzyl alcohol by mixed function oxidase system
down quickly, and the dose of warfarin becomes inade- (Fig. 36.1). Sometimes both phases one and two reactions
quate. are necessary. The biotransformation of benzene req-
Some of the isoforms of the enzyme exhibit low uires both phase one and phase two reactions. Ben-
catalytic activity (polymorphism). This explains the vari- zene is biotransformed initially to phenol by a phase one
ation in drug responses among different persons. For reaction (oxidation). Phenol has the functional hydroxyl
example, there are 3 alleles for the enzyme which cata- group that is then conjugated by a phase two reaction
lyze nicotine of tobacco. The person with the inactive (sulfation) to phenyl sulfate (Fig. 36.2).
allele is protected against addiction to tobacco. The oxidation and detoxification of alcohol is also
an important function of the liver. Two enzymes are
Phases of Detoxification Processes involved in this process: alcohol dehydrogenase oxidizes
Biotransformation reactions are usually classified as alcohol to aldehyde; and aldehyde dehydrogenase oxi-
phase one and phase two reactions. dizes aldehyde to acid (Fig. 36.3).
Phase one is the alteration of the foreign mole- Oxidation of some compounds may result in produc-
cule, so as to add a functional group, which can be tion of substances which are more toxic, e.g.
conjugated in phase two. Phase one reactions result in Methanol ---+ Formic acid
the formation of compounds with decreased toxicity Ethylene glycol _. Oxalic acid
Chapter 36: Detoxification and Biotransformation of Xenobiotics 527
0 6
CH3-CH2-0H - ---T"-:-=
""""'----+• CH3-CHO
Ethyl alcohol ( + Acetaldehyde
NAO+ NAOH+H+
•
CH 3- CHO
Acetaldehyde n
- - -------::-=------+•
NAO+ NADH+H+
CH3-COOH
Acetic acid
Nitrobenzene Amlnobenzene (Aniline)
(5i
CONJUGATIONS
Acetanlllde Aniline + acetic acid
H 3C-CO A xenobiotic that has undergone a phase one reac-
6
I CH,-COOH tion is now a new metabolite that contains a reactive
chemical group, e .g . hydroxyl (-OH), amino (-NH 2),
and carboxyl (-COOH ). These metabolites must un-
TABLE 36.1: Phase two con1ugat1ng agents TABLE 36.2: Con1ugat1on with glucuronic acid
Steroids
acid side chain
Ester with OH group
22)
Glucuronide of steroid
'
GSH transferase Amines Amide N-glucuronides
R-X + Glutathione - - -- - - G-S· R + X-H
GS-R + Acceptor GGT Cysteinyl glycine-R +
gamma glutamyl acceptor
lsoniazld - N-acetyl
- +---=------
transferase
Acetylated isoniazld
6NH-NH, 6H-NH-<:O-CH,
Peptidase acetyl-CoA
Cysteinyt glycine-R _ _ _ _.,. R-cysteine + Glycine
N-acetyl transferase
R-cysteine + - - ~ -- - - Mercapturic acid
Acetyl-CoA +CoA
N N
Fig. 36.6: Phase two; glutathione as detoxifying agent
(GGT = Gamma glutamyl transferase)
Sulfanilamide
N-acetyl transferase
o~CH,
Acetyl sulfanllamide
6~oA
+ acetyl-CoA
0
Benzoyl-CoA + Glycine --+ Hippuric acid + CoA
CO-NH
( )H,-COOH
Fig. 36.7: Phase three reaction; acetylalion
CH2-COOH !Hz
6 ?H- COOH
CH 2
I
CH2
I
Cysteine and Glutathione
The cysteine is derived from glutathione, which is the
active conjugating agent. The reaction is given in detail
in Figure 36.6. Alkyl or aryl halides, and epoxides are
CO-NH
Phenylacetic- + Glutamine - -- -+ Phenylacetyl•
detoxified in this manner.
acid glutamine
Environmental Pollution
'
and Heavy Metal Poisons
Chapter at a Glance
The learner will be able to answer questions on the following topics:
D Corrosives D Pesticides and insecticides
D Irritants D Occupational or industrial hazardous agents
D Heavy metal poisons D Air pollutants
D Lead, Mercury, Aluminium D Toxic substances in foodstuffs
•
Man has tried to change the environment in different oxidase. Poisoning may be due to suicidal attempts.
ways from the days when he was a cave dweller. Any Industrial exposure may occur in the persons working
substance present in the environment, which may pro- with hydrocyanic (prussic) acid or with potassium cyanide.
duce abnormality in metabolism or alter the wellbeing of Ingestion of amygdalin, present in kernels of certain
an organism, is called an environmental pollutant. fruits (apricots, almonds, peaches) is also a common
A poison is a substance which causes death or cause.
harm if introduced in the living body or brought into con- Dicobalt edetate (kelocyanor) is the antidote, which
tact with parts of the body. chelates the cyanide. Another method is to give sodium
or rat poisons. Symptoms resemble acute liver disease. vi. One pack of cigarette contains 15 micrograms of
The poison is oxidized in the body. lead and chronic smokers have higher blood levels
of lead.
Neurotoxins vii. Lead chromate is commonly used as adulterant in
curcumin.
These may act at cerebral level, e.g. opium, alcohol, ether,
chloroform. datura, belladona, cannabis, etc. Those act-
ing at spinal level are aconite, quinine and oleander.
viii. Battery repair, radiator repair, soldering, painting
and printing are occupations prone to get lead poi- •
soning.
prevented. Diagnosis depends on the estimation of cho- The poisonous mixture of smoke, fog, air and other
line esterase in serum and RBC. The antidote is atro- chemicals is called smog. The chemically reducing
pine sulfate and cholinesterase reactivators (diacetyl smog is derived from the combustion of coal and oil,
monoxime or pralidoxime ). and contains sulfur dioxide (SO2 ), sulfur trioxide (SO3 ),
mixed with soot. SO2 and SO3 in the presence of atmos-
I
OCCUPATIONAL AND pheric water vapor, become sulfurous and sulfuric acids,
INDUSTRIAL HAZARDS respectively. This is the precursor of acid rain that may •
be carried by wind to long distances.
Methanol Chronic respiratory symptoms are associated '
.1,
It is the organic solvent widely used in paints and anti- with sulfur oxide or particulates in air. Exacerbations of
freezes. It may be consumed in place of ethanol as a bronchitis were associated with high concentrations of
substitute. Alcohol dehydrogenase converts methanol to smoke and sulfur oxide. Children living in polluted areas
formaldehyde and then formic acid. It is more toxic show diminished ventillatory function when compared
than ethanol. Optic neuritis and blindness is the cha- with their counterparts living in less polluted areas.
racteristic toxicity. The treatment is to give large doses Heart diseases are also related to pollutants such
of ethanol, which is preferentially oxidized in the body so as ozone, sulfur dioxide, sulfates and cadmium in the air.
that formaldehyde generation is reduced.
Industrial Pollution
I AIR POLLUTANTS Dramatic and disastrous episodes of air pollution have
The atmosphere contains mostly nitrogen (78.09%) and been documented in many industrialized centers in the
oxygen (20.94%), carbon dioxide (0.03%), and water world. An example was the London Fog of 1952, in which
vapor. The permissible level of total suspended particles approximately 4,000 deaths occurred over a period of
(TSPs) is 230 mg/cu.m. 2 weeks, following 5 days of severe cold and dehse fog.
A chemical other than those conventionally accep- Another such event was the Bhopal gas tragedy in
ted in the composition of clean air is called a Contami- December 1984 which claimed thousands of lives due
nant. A contaminant that occurs in the atmosphere in to methyl isocyanate (MIC) poisoning.
sufficiently high concentrations to cause an adverse
effect, is called a Pollutant.
Passive Cigarette Smoking
The major artificial sources of pollution arise due to The particulate load in a household is directly propor-
emissions from automobiles, industry and power plants. tional to the number of cigarette smokers living at home.
These are carbon dioxide, carbon monoxide, hydro- Increased preval ence of respiratory illnesses and redu-
carbons, oxides of nitrogen, oxides of sulfur and lead. ced levels of pulmonary function measurements have
Chapter 37: Environmental Pollution and Heavy Metal Poisons 535
been found in children of smoking parents. Studies have ii. Ergot (C/aviceps purpurea): It is the fungus that
also concluded that lung cancer risk is higher in non- usually grows in moist food grains (rye, millet,
smokers, who live under the same roof with smokers. wheat, barley, bajra). Ergotamine, ergotoxin and
ergometrine are present in this fungus (Ergometrin
Other Obnoxious Indoor Agents is clinically used to prevent postpartum hemor-
Common industrial pollutants and their effects are listed rhage). The toxins may produce peripheral vascular
in Table 37.1. contraction, causing painful cramps, gangrene in
extremities and convulsions. The disease is called
Toxic Substances in Foodstuffs ergotism.
These may be considered under the following headings:
Adulterants
Toxins Normally Present in Plants
i. Lathyrism: It is characterized by paralysis of lower
i. Goitrogens: They prevent iodine uptake or utiliza- limbs. It is seen in persons consuming large quanti-
tion by thyroid gland. Thio-oxazolidone is present in ties of Lathyrus sativus (Khesari dal). Khesari dal
cabbage, radish, turnip and brussel sprouts. Thio- is widely used to adulterate ordinary dal, and hence
cyanates and isothiocyanates are seen in mustard the disease may be seen sporadically all over India.
and other oilseeds. Polyphenolic glycosides are Neurotoxins present in lathyrus cause damage of
present in the red skins of groundnut and almonds. upper motor neurons. There is exaggerated knee
All these compounds have goitrogenic effect. jerk, ankle clonus, scissor gait and spastic paraly-
ii. Antivitamins: Orange peel, used in making orange
sis. The toxic principle from lathyrus sativus is
marmalade, contains citral, which inhibits vitamin A
• activity. Linseed oil, which contains linetin, inter-
identified as beta oxalyl amino alanine (BOAA).
Thorough cooking and decanting the supernatant
feres with pyridoxine utilization. Black berries and
! two or three times will remove these toxins (leach-
"- red cabbage contain thiaminase, which destroys
ing out the toxin by hot water). Ironically, the protein
vitamin 8 1 . Raw eggs containing avidin can dec-
content in khesari dal is of very good quality.
rease available biotin.
ii. Argemone oil: Mustard oil may be adulterated with
iii. Cyanogenic glycosides: Legumes (lima beans)
argemone oil. This is from a wild plant, Argemone
and tubers (tapioca or cassava) contain cyano-
mexicana. Argemone seeds are similar to mustard
genic compounds, which on hydrolysis produce
seeds, and oil from both seeds are similar in con-
hydrocyanic acid. Hence, they are highly toxic
sistency. Hence, adulteration is easy. Argemone oil
when taken raw. Cattle and sheep eating tapioca
contains the alkaloid, Sanguinarine which causes
leaves often get acute fatty degeneration of liver
vomiting, diarrhea, congestive cardiac failure and
with fatal outcome. The toxins can be removed by
cooking and decanting the supernatant water. edema. It is then called epidemic dropsy.
iv. Favism: Ingestion of uncooked broad bean (vicia
fava) may cause hemolytic anemia in susceptible Toxins Entering during Food
persons with glucose-6-phosphate dehydrogenase Preparation
(GPO) deficiency (see Chapter 6). The toxic glyco- Mono sodium glutamate (Aginomoto): It is a com-
side is known as vicin. Cooking and decanting will
mon food additive. Packets of mono sodium glutamate
remove the toxins.
carry the statutory warning that it is unsuitable for child-
Storage Contamination ren below the age of 5. It produces transient symptoms
like numbness and palpitation. It may deteriorate mental
i. Fungal infections: During postharvest storage,
alertness in children.
contamination with fungus is very common. Asper-
gil/us ffavus produces aflatoxins, which are hepato-
Related Topics
toxic and carcinogenic. The fungus grows in moist
conditions in groundnut, coconut, rice, maize, Ethanol metabolism (see Chapter 12); Carbon mono-
wheat, etc. xide (see Chapter 23); Detoxification and xenobiotics
536 Section D: NutrWon
(see Chapter 36); Free radicals (see Chapter 30); 3. Corrosive substances like strong acids and alka-
Chemical carcinogens (see Chapter 48). lis have been long recognized as poisons, mainly
found in laboratories and chemical industries.
0 4. Cyanide, the most potent poison, will arrest cellular
• · Clinical Case Study 37.1
respiration by its effect on cytochrome oxidase.
A 3-year-old girl was brought to the emergency depart- 5. Organic irritants include camphor, castor, croton
ment. She was cold and clammy and was breathing and calotropis.
rapidly. She was obviously confused and lethargic. Her 6. Neurotoxins are opium, alcohol, ether, chloroform,
mother had indicated that she had accidentally ingested datura, cannabis, etc., which are ingested acciden-
automobile antifreeze while playing in the garage. Fol- tally or willfully or by substance abuse.
lowing GI lavage and activated charcoal administration, 7. The most common environmental poison in India is
a nasogastric tube for ethanol was administered. What lead. In spite of banning, lead in paints and ink still
is the likely cause? occurs.
8. Lead inhibits heme synthesis and anemia is a very
0 common finding. Permanent neurological damage
• · Clinical Case Study 37.1 Answer
like lowering of intelligence and optic atrophy may
This is a case of ethylene glycol poisoning. Ethylene be seen.
glycol is the major ingredient of radiator fluid products. It 9. Chelation of lead using penicillamine, dimercaprol,
is used to increase boiling point and decrease freezing dimercaptosuccinic acid are the lines of manage-
point of radiator fluid. Thus, overheating and freezing of ment.
the fluid is reduced, depending upon season. 10. Entry of organic mercury into fish from industrial
effluent can cause indirect poisoning as in Mina-
Ethylene glycol is sequentially converted to glyco-
aldehyde, glycolate, glyoxylic acid and finally to oxalate. mata disease. •
Glycolate can produce acidosis and hyperventilation. Cal- 11. Widespread use of aluminium in all fields of life,
cium oxalate crystals can form and accumulate in blood including cooking has posed the problem of poison-
and other tissues and hypocalcemia can occur. Thus, ing.
symptoms include severe anion gap metabolic acidosis, 12. Arsenic used in rat poison, pesticides and insect
tachypnea, confusion, convulsions, and coma, oxalate repellents can cause blood diseases.
crystalluria and renal failure. 13. Organophosphorus (ORP) compounds used as
Laboratory investigations are (1) Ethylene glycol pesticides and fungicides are potent poisons. ORP
levels in blood, (2) Serum osmolality, (3) Serum electro- Inhibits acetylcholine esterase.
lytes, (4) Serum calcium, (5)Arterial blood gas analysis, 14. Contamination of ethyl alcohol by methanol (illicit
and (6) Urinalysis for calcium oxalate crystals. liquor) has claimed a large number of lives. Metha-
nol gets oxidized to formic acid, a potent poison
I LEARNING POINTS, CHAPTER 37 which can cause blindness and even death,
15. Lathyrism and epidemic dropsy are due to intake of
1. Cyanide poisoning causes tissue anoxia by chelat- adulterated dal and mustard oil respectively.
ing the ferric ions of the enzyme cytochrome oxi- 16. The following also cause environmental pollution
dase. Death is instantaneous. (a) new building materials and internal carpet-
2. Lead is a cumulative poison. Acute toxicity can ing practices, (b) newly built/remodeled build-
cause encephalopathy, convulsion, mania, anemia ings are known to cause "sick building syndrome",
and renal damage. Blue line along the gums is a
characteristic feature of acute lead poisoning.
(c) releasing of "off gas" toxicants and (d) carpets
are notorious for trapping toxicants.
'
PART-1 : SHORT NOTE QUESTIONS
37-1. What is most common environmental poison? 37-4. What are the manifestations of chronic lead poi-
Lead. soning?
37-2. What are common causes of lead poisoning? In children, mental retardation, learning disabilities,
,. Paint, lead containing petrol, lead pipes, newspapers behavioral problems are seen. Anemia and loss of
appetite are very common.
and cigarette smoke are important contaminants.
37-5. What is the cause of anemia in lead poisoning?
37-3. What are the occupations in which persons are
Lead inhibits delta amino levulinic acid (ALA) synthase,
prone to get lead poisoning?
ALA-dehydratase. So. heme synthesis is blocked.
Battery repair, radiator repair, soldering, painting and 37-6. What ls Mlnamata diseases?
printing. Organic mercury poisoning .
•
r
,
<.
'
SECTION E
Molecular Biology
Chapter at a Glance
Th e learner wi ll be able to answer questions on the following topics:
A Purines and pyrimidines
E Uric acid and gout
B Nucleosides and nucleotides
0 De novo synthesis of pyrimidines
C De novo synthesis of purine nucleotides
"' 0 Disord ers of pyrimidine metabolism
j) Degradation of purine nucleotides
• ~·
,'
" i·._.
t- )
•
~4
Kassel described the 4..bases in nucleic
·J
Friedrich Albrecht Adolph Frederick
Miescher Kosse/ Strecker Hopkins
a s eticJnfo:m:mnon. The universal currency of 1844-1895 NP 1910 1822-1871 NP 1929
energy, namely AIE. is ~t1ele~e derivative. Nucleo- 1853-1927 1861-1947
Hypoxanthine Xanthine
N
9 N N H2N
Purine nng
f
Adenine
3No~5 4
N~
¾
_j [ _
-
HO N r---
11 Pyrimidi ne Lactam or keto form of uric acid
La or enol form of unfaod
2 ".,..:;::: 6
N
Fig. 38.2: Minor bases seen in nucleic acids
2.~
~WlcP.~ ~
V·
+o."'....,,
Uracil ,, hymine THYMINE is the bas_g present in DNA.
I
elN
(~O~ THIAMINE is a emb r \dtamin13 complex.
Adenosine Deoxythymidine
0 0
NH2 0
'"~).
2
O
~N)
aN ~ C H ,
I
~ - -~ N 6
OH OH
9
D-ribose 2-deoxy-D-ribose
s· 0 s· I
O
H-";Vo":•·
Fig. 38.4: Sugar groups in nucleic acids 0
2
OH-H C
~
4' 1·
TABLE 38.1: Base + sugar are nucleosides
Ribonuc/eosides
IHJ 3' 2· H
OH OH OH H
Adenine + Ribose Adenosine
Guanine+ Ribose Guanosine Fig. 38.5 : Numbering in base and sugar groups. Atoms in sugar
are denoted with prime numbers
Uracil + Ribose Uridine
Cytosine + Ribose Cytidine
- TABLE 38.2: Base + sugar + phosphate = nucleotide
Hypoxanthine + Ribose lnosine Ribonuc/eotides
Xant hine + Ribose Xanthosine Adenosine + Pi monophosphate
Deoxyribonucleosides (AMP) (Adenylic acid)
Guanosine + Pi monophosphate
Adeni ne + Deoxy ribose Deoxy adenosine (d-adenosine)
(GMP) (Guanyiic acid)
Guanine + Deoxy ribose d -guanosine
Cytidine + Pi monophosphate
Cytosine + Deoxy ribose d-cytidine (CMP) (Cytidylic acid)
-
Deoxyribonucleotides
Nucleoside d-ad enosine + Pi (d-adenylic acid)
Nuc/eoside monophosphote
d-guanosine + Pi (d-guanyllc acid)
Ribonuc/eoside phosphates
d -cytidine + Pl (d-cytid yiic acid)
Adenosine Adenosine Adenoslne Adenosine
d-thymidine + Pi (d-thymidylic acid)
.;;--)
mono phosphate diphosphate triphosp hate
(AMP) (ADP) (ATP)
Guanosine GMP GDP GTP
lnosine IMP IDP ITP
Cytidine CMP CDP CTP
Uridine UMP UDP UTP
Deoxyribonucleoside phosphates ' 0 0 0
~ -~ N
I
I
&
N
d-adenosine d-AMP d -ADP d-ATP II II o
d-guanosine d-GMP d-GDP d -GTP HO- i~O-, -O-, -O-H2C
~ • O
d -cytidine d-CMP d-CDP d-CTP
OH OH OH
d-thymidine d -TMP d-TDP ·d-TTP
H
OH OH
@ Nucle Tri phosphates
Fig. 38.6 : Adenosine triphosphate (ATP)
Corresponding nucleoside di- and tri-phosphates are
formed by esterification of further phosphate groups to diphosphate contains one high energy bond and
the existing ones. In general, any nucleoside triphospt)ate triphosphates have 2 high energy bonds. ATP is the
is abbreviated as t:JTP or d-NTP (Table 38.3). Nucleoside universal energy currency (Fig. 38.6). It is formed
Cr)
5'
N N
•
o- - -
1
•
HO-P=O
I
(1~3 '~ "",;;;-57
Fig. 38.8: The assembly of purine ring is from various sources.
-
sized from j Since the ~ ma~ ..i,
being can synthesize the~ rine and yrimidine ba~ s~ J
-
nova, they are said to be
;,;;:.:.;.;..;;.;;;.;.;..,;;;.;;,;.;.;;.;;...;.;.;:..-~difl~
he contribution of
~ for the formation of
the purine ring is shown in the Figure 38.8.
During de nova' synthesis, purine ring is built up on
a ,.......,_.... molecule. He.n ce~ teotide-s-are-
mediating the action of several hormones. th~ products""tlf the de-.ro~ t~ . There are
Deoxy ribonucleotides are used for synthesis of . . in the de novo synthesis pathway. The enzymes
DNA and ribonucleotides for RNA. In pse11daurjdyjic a£!? catalyzing these reactions are existin as a mu ·e me
(found in tRNA) uridine is attached to ribose phosphate
in a bond instead of C-N bond in UMP. High energy
compound"'s are listed in Table 21.2. Please note that
s I
~1o®.1
I • ¥ N
©JI ClN:rru
~ tt,yi c.~
-4 -
Formyl
c) . ,1 ADP
,.
ATP
ormyl glycinamide
methenyl-
THFA THFA
0
I /
)
5'-.,-----~
Q N
glycinamide
ribonucleotide fA'I
ribonucleotide
(FGADI
-------,........
If:\ R p
I (FGAI'\) 0; IV 01 5
J
~"'
JCI
ATP ADP+ Pi N-succlnyl-5-amino-i:::-'"
N~ Amino imidazole (+) \ 1m1dazole
ribonuteotide (AIR) as \
2
. . 5-amin~carboxtamino- ->-----~-+ carboxamide
1 \.V 1m1daz
~ole nbonucleollde (ACAIR) - (?)
H2 N :i- ~ 0 - N> (+)~ C,
s rtic acid O N
R 5P £ 't cw"\:tu:a t ~ '\
\'v.P>
, 1""'1 cu-f
\,-.J.M £._
H2 N I Asp/
N
I / @
- ~- R5P N
H2N I
').{t'-1~\e. ref_
(§1\\
¥
R5P
C - - ...H c.a8 °-formyl-
v Fumaric acid
GDP + Pl GTP .:y THFA
Adenylic ~cid
(Adenosine
lnosinic acid
(lnosine
N-f?r'.1'1yl amino J
5-amino-imidazole-
1m1dazole +-~~.,e;.___ 4-carboxamlde
N>
monophosphate) • monophosphate) @' 4-carboxamide
4
®
ribonucle.otide tr~
(AMP) @ (IMP) 1-1 rlbonucleotide (AICAR) 1\;.f CU..,.O,..,.,
I
Fumaric Aspartic H20 (FAICAR) AICAR
o 0
@)[)
ac~ cid 0
R,P I ~ ) "'")I)
'lNJ-, @D---N I R5 P R5P
H2N I
R5P
Fig. 38.10: First eight steps of purine synthesis
~ .,0 h\~-p @ ~ ~Q . y _ -4 h"TQ."t 2...btl'lc.~e.s?.'~ 'S ~esrt~~c:a9..')
Ll 8 ::l:1"'1"1---.\bt-ro~
~be · C>"'~
TABLE 38.4: Summary of steps of purine synthesis of purine nucleotides; it is called a Jprelirninary or pre-
Added atom • ........ 1111. paratory stea .....,
1 Glutamine N9 (Rate limiting) PRA
2 Glycine C4,s'®ATP required) GAR F ormation of AMP
3 Methenyl-THFA ca FGAR
Steps 1 to 10 are summarized in Table 38.4. Flow dia-
4 Glutamine @ ATP required) FGAM
grams of these steps are shown in Figures 38.10 and
5 Ring closure (ATP) AIR
6 Carbondioxide C6 ACAIR
38.11.
7 Aspartic acid ®ATP required) SAICAR .. ('";!Fr;:ir:::-
s.-t s::.t:::-
e=-p"is~ ra:r
te=---r.
tir,,;,n·;.
;t...
,~- Step 4 is inhibited by aza~
8 Fumarate removed AICAR serine, an anticancer drug. 6-mercaptopurine inhibits
9 Formyl-THFA C2 FAICAR amination of IMP to AMf, and therefore it is used as an
10 - Ring closure IMP anticancer drug. C1.'1.. "-~I
546 Section E: Molecular Biology
Amidotransferase
IMP • XM P • GMP
(inosinic acid) { \ (xanthylic acid) ( '\ { \ (guanylic acid)
11~..)LNN
>9 NAO NADH •HN:x~;m,oe GMamate
0 + + 0 .
ATP
AMP.PP
;N~N>
N I
@AN N
I
~-)LNI
N
.'.3 Ribose-5-phosphate Ribose-5-phosphate RsP
Most common
Common
Less common
URICACID @_ ~
.,~
...-,<.;iJjlllll":...
---
Uric acid
1--- - - 4 - - Uricase Lesch-N~n Syndrome
breaks down uric acid
Probe. + ~ ( It is an X-linked inherited disorder of purine metabolism.
1-+- - + - - - Probenecid
Increases excretion of
uric acid in urine
I Incidence is 1:10,000 males. There is deficiency of
~PRTase. So, the rate of salvage pathway is decreased
Crystals deposit
in tissue ( ~n ~ t~ ) resulting in accumulation of PRPP and decreased level,,
of inhibitory puf.l!l,e nucleotides. e disease : ~::~:; &l'\dl ,1
i-+- - - i - - - NSAIDs, colchicine, terized by self tff6r tion, m t rdation, . moIW
corticosteroids
reduce inflammation ~ d and· nep litbiasis Gout develops in later life.
The neurological manifestations suggest that the brain is
dependent on the salvage pathway for the requirements
of IMP and GMP. -
Fig. 38.15: Action of medicines in gout o
• •
Jt Hypour1cem1a
n ,otil ~Y\~~-
r· ( pq ,9(\-)
Adenosine Deaminase (ADA) Deficiencr,,
2:n.o~,"e_ __. KlobPS11'.;!-0uni• ,-_;Q
It is associated with severe immunodeficien[y where~ V
iCD.9. ~ e . .
4
C HC0 3- A •~' \
( .>t( +
1
r '\+•
'
NH2-C0-0-P03
~\001
Carbamoyl
,J
~C 5 Glutamine GM amate 2ATP 2ADP phosphate
From
From
carbamoyl
phosphate
~
I
C6
aspartic
acid
3 2
NH2-CO-NH-CH--COOH
1 6
I
7
2
Aspartate
CH:2- COOH ~ Pi
N 5 4
1 0
0
COOH
7
"2 :;"t:
/'
Dlhydro orotic acid (OHO)
NAO+
moyl phosphate synthetase II (CPS-II). The differences
4
between CPS-I and CPS-II are described in Table 17.2. N d;6,1'.°'4 NADH
Uridine monophosphate (UMP) is the first pyrimidine I
f......, 'D~ Orotic acid (OA)
that is synthesised (step 6). T zyme' is OMP-
0 N COOH
decarboxylase (OMPOC). 6-aza-uridin inhibits this 0 <!)O"M'.&.
N~ - 'Vt-""""'"'-' PPi O
Regulation of Pyrimidine S
O~ N ) lCOOH Oroti dylic acid (Orotidine
i. In eukaryotes the ~ ~ e zy , viz, CPS lL, I ~, _A\...., ,.?lmonophosphate) (OMP)
ATC and OHOase are pre~ ~ myltjenzvme R5P IV\ ~
boxylase are also present as a s.ua_le functjg~I _____ Uridylic acid (U ridine
tp
0 N ..,,-" monophosphate) (UMP)
complex. ecause of this clustering of enzymes,
C)~l..4 f'-'~ ATP ~
the synthesis is well co-ordinated. Both complexes \-\me. Ot\"'- 7
are cytosolic
ADP
ATP ~
iv. ~1~ ,c:k
@
8
d,.::,r,<J1-:..J-- - - - - - , ADP
NH.,
(, J
~ - 7\Uridine triphosphate (UTP)
V.
<l\~ ATP * Glutamine
'-T'• =~~i~~~~=
-~-;.--:.
- •-
- - l.~2
carbamoylase (ATC ; . . ••r = asparty1 trans-
&AWi • t. - dihydro
orotate dehydrogen:ase {Dt10DH); = orota e p osplio ribosyl
transferase enzyme. transferase (OPRTase); 6 = OMP-<lecarboxylase (OMPDC); 7 =
Cytosine + PRPP CMP UMP kinase; 8 =nucleoside diphosphate kinase; 9 =CTP synthetase
Thus, both purines and pyrimidines can be salvaged. Fig. 38.17: Synthesis of pyrimidine nucleotides
-~ t& o.a \1"''h\~m
Chapter 38: Nucleotides: Chemistry and Metabolism 551
• The condition results from absence of either or both of Orotic aciduria OPRTase, OMP decarboxylase
the enzymes, OP~[ase and OMP decarboxylase. It Orotic aciduria Ornithine Transcarbamoylase
is an autosmti~~cessiv disease. There is r~larded Drug induced Orotic OMP decarboxyla se
growth and The rapidly growing aciduria
cells are more affected and hence the anemia. J3 amino isobutyric Transamina,sJ, affects urea cycle
are _excreted in urioe which may ca~se o c~ ry tea!? aciduria function, deamination of a amino acids
to a keto acids
obstruction. Due to lack of feedback inhiWon orotic
acid production is excessive. The.t q_~ ition can be
successfully treateo by feeding~ ihe or aine. They Cytosine
may be c ~ UIP which £aD acl_j.s..feedback
~ r. See Table 38.6 for summary of disorders of
1•0 Nf' 1
Deoxyribonucleotide Formation ,
t NADP H+
t:ADPH;H'
C..'PP d c..'"DP Cl Tf NA! + ADP+
Deoxyribonucleotides (both P. nes and pyrimidine
Dihydrouracil Dihydrothymine
uctiort at the( g: carbon
. hospha ':9 (NDP l l
ribonucleotide reductase Beta ureido
propionate
Beta ureidoc ~B)
1sobutyrate I
'T·~:ae)
complex, which contains thioredoxin. Thus, UDP is
1~0,
(carbamoyl (carbamoyl
first converted to dUDP, and then to dUTP. beta amino isobutyrate)
)
-1\1
ucleotides
0
• · Clinical Case Study 38.1
Anticancer Agents Acting on Pyrimidines A40-year-old male presented with severe pain, redness
and swelling of the base of the first metatarsophalangeal
joint in the night after a bout of alcohol consumption.
The patient was in usual state of health until early in
the morning when he woke up with severe pain in his
right big toe. The patient denies any trauma to the toe
and no previous history of such pain in other joints. On
examination, he had mild fever 38.2°C. The right big toe
was swollen , warm, red, and exquisitely tender. Serum
uric acid was 9.7 mg/dl. What is the likely diagnosis?
What is the pathogenesis of the condition? How would
you make a definite diagnosis?
552 Section E: Molecular Biology
14. De nova synthesis of purine nucleotides uses 21. Lesch Nyhan syndrome is an X-linked trait cha-
from amino acids and racterized by nephrolithiasis, mental retardation ,
aggressive and self-mutilation.
15. The first purine nu~leotide formed is- which is 22. De nova synthesis of pyrimidine nucleotides use
then converted to AMP and GMP. amide group of glutamine and aspartic acid.
16. Requirement of GTP for the formation of AMP from 23. There is a reciprocal relation between the synthesis
IMP and ATP for the formation of GMP ensures of purine and pyrimidine nucleotide synthesis which
balanced synthesis of both the nucleotides. ensures a balanced supply of both.
17. Amidotransferase is subject to feedback inhibition 24. dUMP can be converted to dTMP by thymidylate
by the products AMP and GMP. synthase enzyme using one-carbon group carried
18. Catabolism of purine nucleotides produces uric acid. byTHFA.
19. Normal serum uric acid level varies from 2-5 mg/di 25. Inhibition of nucleic acid synthesis by nucleotide
in women and 3-7 mg/di in men. and nucleoside analogues is used in cancer
20. A high level of uric acid {hyperuricemia) can lead chemotherapy (6-mercapto purine, 6-thioguanine,
to deposition of urate crystals in joints producing 5-flourouracil etc). Methotrexate which inhibits thy-
arthritis. Treatment of hyperuricemia is by giving allo- midylate synthase is also an anticancer drug.
purinol a competitive inhibitor of Xanthine oxidase. 26. Some antiviral drugs are also nucleotide analogues.
38-1 . All the following amino acids are used for synthesis C. Hypoxanthine
of purine nucleotides, except: D. Beta aminoisobutyric acid
.. A. Glycine
C . Aspartate
8 . Alanine
D. Glutamine
38-5. Hyperuricemia is observed in all the following con-
ditions, except:
38-2. N-3 of purine ring is donated by:
A. Lesch-Nyhan syndrome
A. Ammonia 8 . Glutamine
B. von Gierke's disease
C. Aspartate D. Glycine
C. Glutathione reductase deficiency
38-3. Wh ich amino acid is required for both purine and
pyrimidine synthesis? D. Xanthine oxidase deficiency
A. Glycine B. Aspartate 38-6. Hyperuricemla can result from deficiency of all the
C. Alanine D. Glutamate following enzymes, except
38-4. The chief product of catabolism of purines in A. HGPRTase
human beings is: B. APRTase
A. Urea C. Glucose-6-phosphatase
B. Uric acid D. Adenosine deaminase
554 Section E: Molecular Biology
38-7. The reduction of nucleotide to deoxyribonucleotide: C. Methylation is taking place on dUMP and not on
A. Takes place at the level of nucleoside diphosphate UMP
B. Involves the cleavage of glycosidic bond D. 1st nitrogen is donated by glutamine
C. d-ATP will enhance the reaction 38-16. Regarding gout, all are true, except:
D. Needs hydrolysis of ATP A. It is seen in HGPRT deficiency
38-8. Orotic aciduria is a feature of deficiency of all the B. There is deposition of urate in jjoints
following enzymes except: C. Can be treated using allopurinol
A. OMP decarboxylase D. It is seen in PRPP synthetase ,deficiency
B. Ornithine transcarbamoylase 38-17. If a cell is unable to synthesise PRPP, synthesis of
C. OPRTase which is likely to be directly affected?
D. Aspartate transcarbamoylase A Pyridoxal B. OMP
38-9. Pyrimidine nucleotide synthesis and purine nucle- C. CoASH D. FAD
otide synthesis are similar in which aspects? 38-18. During de novo synthesis of Ipyrimidine, which
A. Enzymes are present as multienzyme complexes nucleotide is first formed?
B. Ring is assembled and ribose-5-phosphate added A. TMP B. OMP
C. Ribose-5-phosphate is donated by PRPP C. UMP D. CMP
D. Aspartic acid is fully incorporated into the ring 38-19. The drug of choice for primary gIout is:
38-10. All the following molecules have regulatory effect A. Allopurinol B. Aspirin
on synthesis of purine nucleotides, except C. Colchicine D. Probenecid
A. PRPP 38-20. All manifestations are seen i n ILesch-Nyhan syn-
drome, except:
B. AMP
A. Self mutilation B. lmmun,odeficiency
C. UMP
C. Hyperuricemia D. X-linke,d inheritance
D. GTP
38-21. All are true regarding pyrimidine nucleotide syn-
38-11. Which property of uric acid is responsible for the
thesis, except:
manifestations of gout?
A. The reactions occur in the cytoplasm
A. Ketoenol tautomerism :.
B. CTP is a negative modifier
B. Acidic nature
C. Glutamine and aspartic acid are used
C. Reducing action
D. Pyrimidine ring is built up on a ribose-5-phosphate
D. Solubility constant
38-22. Formation of dTMP requires:
38-12. Which compound is not derived from purine nuc-
leotides? A. S-adenosylmethionine
A. Uric acid B. Beta aminoisobutyric acid B. NADPH
C. Allantoin D. Xanthic acid C. Adrenodoxin
38-13. Uric acid is the end product of breakdown of all the D. Molybdenum
following, except 38-23. Orotic aciduria is characterized by all the findings,
A. AMP B. IMP except
C. Xanthosine E. CMP A. Megaloblastic anemia
38-14. Secondary hyperuricemia is seen in all the follow- B. Response to oral uridine
ing conditions, except: C. Failure to thrive
A. Leukemia B. Psoriasis D. Deficiency of HGPRTase
C. Hepatitis D. Cytotoxic drug therapy 38-24. Which of the following conditions is not associated
38-15. Which is correct regarding thymine synthesis? with hyperuricemia?
A. 4th carbon is donated by carbamoyl phosphate A. Multiple Myeloma B. Xanthi1nuria
B. 3rd nitrogen is donated by aspartate C. Psoriasis D. Polycythemia vera
38-1. What is a nucleotide? 38-20. What is the normal uric acid level in blood?
Nitrogenous base+ sugar+ phosphate. 2-5 mg/dl in females and 3-7 mg/dl in males.
38-2. What is a nucleoside? 38-21 . What is gout?
Nitrogenous base + sugar. It is a disease associated with hyperuricemia, deposit
38-3. What are the sugars in nucleic acids? of urate crystals in joints and excruciating joint pain.
Ribose in RNA and deoxyribose in DNA. 38-22. Which property of uric acid is responsible for the
38-4. What are the bases present in nucleotides? manifestations of gout?
Purines and pyrimidines. Uric acid is sparing soluble in water.
38-5. Name the common purines. 38-23. Increased uric acid level is seen when?
Adenine and guanine. Gout, pre-eclampsia, Hodgkin's lymphoma, leukemia.
38-6. Name the common pyrimidines. 38-24. Hyperuricemia is observed in which conditions?
Cytosine, uracil, thymine. Gout, Lesch Nyhan syndrome, van Gierke's disease
38-7. What are the bases present in DNA? 38-25. What is the mechanism of Allopurinol?
Adenine, guanine, cytosine, thymine. It is an analogue of hypoxanthine. It inhibits xanthine
38-8. Which base is absent in DNA? oxidase. thereby decreasing the formation of uric acid.
Uracil. 38-26. It is what type of inhibition?
38-9. Which base is found exclusively in DNA and not in Xanthine oxidase converts allopurinol to alloxanthine.
RNA?
It is a more effective inhibitor of xanthine oxidase. This
Thymine.
is a good example of 'suicide inhibition' .
38-10. Which base is found exclusively in RNA?
38-27. What is the reason for severe combined immuno-
Uracil.
deficiency?
38-11 . Which amino acid is required for both purine and
Adenosine deaminase deficiency.
pyrimidine synthesis?
Aspartic acid and Glutamine. 38-28. What are starting materials for pyrimidine synthe-
38-12. NJ of purine ring is donated by what? sis?
Glutamine. Carbamoyl phosphate and aspartic acid.
38-13. Glycine donates what part of the purine ring? 38-29. What is the rate limiting step in pyrimidine synthe-
C4, CS, N7 atoms. sis?
38-14. What is the key enzyme of de novo synthesis Aspartate transcarbamoylase.
pathway of purines? 38-30. How is pyrimidine synthesis pathway regulated in
Phosphoribosyl amidotransferase (step 1). mammals?
38-15. How is de novo synthesis of purine regulated? CPS II is inhibited by CTP.
Amidotransferase enzyme is inhibited by AMP and 38-31 . Orotic aciduria is a feature of deficiency of which
GMP. enzymes?
38-16. Give a few examples of purinP. analogues, used as OMP decarboxylase; OPRTase; Ornithine transcarba-
anticancer drugs. moylase.
6-mercapto-purine; Cytosine arabinoside; Methotre- 38-32. What are the characteristic features of Orotic aci-
xate; Azaserine. duria?
38-17. What is the end product of catabolism of purines Megaloblastic anemia.
in human beings? 38-33. What is the mechanism of Methotrexate?
Uric acid. II inhibits dihydrofolate reductase and thereby reduces
38-18. What is xanthine oxidase? the regeneration of THFA; it is a powerful anticancer
It is the enzyme catalysing the reactions, hypoxanthine agent.
to xanthine and xanthine to uric acid. 38-34. What is the mechanism of 5-fluorouracil?
38-19. Name an enzyme containing molybdenum. It inhibits conversion of dUMP to dTTP; and acts as an
Xanthine oxidase contains FAD, molybdenum and iron. antimetabolite.
_ _ _ _ _ Chapter 39
Deoxyribonucleic Acid:
Structure and Replication
Chapter at a Glance
The learner will be able t o answer questions on the following topics:
D Watson-Crick model of DNA stru cture D DNA polymerase
D Chromosomes D Okazaki pieces
D Replication of DNA D DNA repair mechanisms
Thomas Morgan (1866-1945), the founder of modern genetics, showed that chromosomes contain genes in a sequential
manner in Drosophila (Nobel Prize, 1933). In 1931 , Barbara McClintock showed the rearrangement of genes or mobile genes
in chromosomes in corn (Nobel Prize, 1983). George Beadle, working with mutant strains of Neurospora suggested •one
enzyme one gene" hypothesis in 1941 (Nobel Prize, 1958). Avery in 1944 demonstrated that DNA is the genetic material. In
1952, Hershey (Nobel Prize, 1946) showed that only DNA of virus and not the proteins will enter into the host before infection.
Erwin Chargaff elicited the base pairing rule of DNA in 1950. X-ray crystallographic studies on DNA by Maurice Wilkins (Nobel
Prize, 1962) showed the details of structure of DNA. Rosalind Franklin worked out the helical structure of DNA. (She died of
ovarian cancer at the age of 37, probably due to irradiation during her work on X-ray crystallography). Based on these data,
James Watson and Francis Crick in 1953 deduced the double helical structure of DNA (Nobel Prize, 1962).
OH
I 5' phosphate end S'end
Phosphate bonds
O=P-0- form the rail of
1 staircase
O-H2C Thymine
0 Base pair
0 H
0
I
I
0 HC Cytosine
2
0
Francis Crick
NP 1962
3' end 5' end
1916-2004
H
Fig. 39.2: Watson-Crick model of double helical structure of DNA.
0 Adjacent bases are separated by 0.34 nm. The diameter or width
of the helix is 2 nanometers
Adenine
0
Common abbreviations
in DNA chemistry:
A= Adenine, G = Guanine
C = Cytosi ne, T = Thymine
OH H
Red boxes show Erwin Chargaff
Phosphodiester linkages 3' OH end
1905-2002
Maurice Wilkins
Polarity of DNA Molecule Nucleotide NP 1962, 1916-2004
In the case of DNA, the base sequence is always written F ig . 39.3: Base pairing rule. Base pairing of A with T and G with
from the 5' end to the 3' end. This is called the polarity C. Hydrogen bonds between bases
of the DNA chain.
Right Handed Double Helix
Watson-Crick Model of DNA Structure DNA consists of two polydeoxyribonucleotide chains
The salient features of Watson-Crick model of DNA are twisted around one another in a right handed double helix
given in Figures 39.2 and 39.3. similar to a spiral stair case. The sugar and phosphate
558 Section E: Molecular Biology
Fig . 39.4: DNA wraps twice around histone octamer to form one
groups comprise the handrail and the bases jutting nucleosome
inside represent the steps of the staircase. The bases
are located perpendicular to the helix axis, whereas the Denaturation of DNA Strands
sugars are nearly at right angles to the axis.
The double stranded DNA may be denatured and
Base Pairing Rule separated by heat. This is called as melting of DNA. At
lower temperature, the melted strands are re-associated;
Always the two strands are complementary to each
this is called annealing.
other. So, the adenine of one strand will pair with
thymine of the opposite strand, while guanine will pair
Higher Organization of DNA
with cytosine. The base pairing (A with T; G with C) is
called Chargaff's rule, which states that the number of In higher organisms, DNA is organized inside the
purines is equal to the number of pyrimidines. nucleus. Double stranded DNA is wound round histones
to form nucleosomes (Fig. 39.4). Chromatin is a loose
Hydrogen Bonding term employed for a long stretch of DNA in association
with histones. Chromatin is then further and furthe r
The DNA strands are held together mainly by hydrogen
condensed to form chromosomes (Fig. 39.5).
bonds between the purine and pyrimidine bases. There
are two hydrogen bonds between A and T while there are
Histones
three hydrogen bonds between C and G. The GC bond
is therefore stronger than the AT bond. A mispairing will These are proteins containing unusually higher con-
disturb the stable double helical structure. centration of basic amino acids. Histones synthesized
in the cytoplasm migrate to the nucleus. Acetylation of
Antiparallel histones leads to activation of transcription, whereas
deacetylation causes depression of transcription. Meth-
The two strands in a DNA molecule run antiparallel,
ylation generally occurs when the gene is repressed.
which means that one strand runs in the 5' to 3' direction,
while the other is in the 3' to 5' direction (Fig. 39.2). This
DNA is a Very Long Molecule
is similar to a road divided into two, each half carrying
traffic in the opposite direction. Human diploid genome consists of about 7 x 109 base
pairs. So when placed end to end it will be about 2 meters
Other Features long, If one nucleotide is added per second, it will take
In the DNA, each strand acts as a template for the 250 years to synthesize the whole DNA of a human cell.
synthesis of the opposite strand during replication The length of a DNA molecule is compressed to 8,000
process. The spiral has a pitch of 3.4 nanometers per to 10,000 fold to generate the chromosomes (Fig. 39.5).
turn. Within a single turn, 10 base pairs are seen. Thus,
Inactivation of DNA during Differentiation
adjacent bases are separated by 0.34 nm. The diameter
or width of the helix is 1.9 to 2.0 nm. A major groove All human cells are derived from a single cell, the zygote.
(1.2 nm) and a minor groove (0.6 nm) wind along the Therefore, all cells contain the same genetic information.
molecule, parallel to the phosphodiester backbone. In But, a cell from the gastrointestinal epithelium is different
these grooves, proteins interact with the exposed bases. from a cell of central nervous system, by structure and
DNA is the storehouse of genetic information (Box 39.1). function. How such a differentiation is made possible?
Chapter 39: Deoxyribonucleic Acid: Structure and Replication 559
i
Nucleosomes
about 5,00,000 times, and accounts for about 5% of total
human DNA.
10 nm
i I REPLICATION OF DNA
During cell division, each daughter cell gets an exact
Chromatin fibril
30 nm copy of the genetic information of the mother cell.
!
This is called semi-conservative type of DNA replication.
,'I, I , I ,
Each strand serves as a template or mold, over which a
I ' • I
Chromosome new complementary strand is synthesized (Fig. 39.7).
I 11 ,• \ .. ·, ' 1 , l 1400 nm
(\ Absorbance
I \ at 260 nm
I
N1•
Matthew Meselson
(b .1930)
and Franklin W Stahl
(b.1929)
Template strand
--G
- T - C - C - G - A - C -T- C -
P
jpJJP
JJO
H OH
S'end New strand 3' end
Synthesis from 5' to 3' direction
Parent cell
First generation
(15N + 14N) x 2
Fig. 39.8: New strand is synthesized from 5' lo 3' direction. Base
pairing rule is always maintained
II 11 Second
generation
Old
(15N + 14N) x 2
11 1111 11 (14N + 14N) x 2 Old strands
about 100 nucleotides per second . (Bacterial enzyme and repair enzyme, where as DNAP gamma is concerned
has 10 times more speed). It is the major enzyme which with mitochondrial DNA replication.
synthesizes Okazaki fragments. DNAP delta completes
lagging strand synthesis and DNAP epsilon is used for RNA Primer is Required for DNA Synthesis
leading strand synthesis. DNAP beta is a proofreading
An RNA primer, about 100-200 nucleotides long, is
synthesized by the RNA primase. Then the RNA
Origin of replication primer is removed by DNAP, using exonuclease activity
r7
l
and is replaced with deoxyribonucleotides by DNAP
:::::::·:::::::::~:::~::!·n·:::::::l::im::::::::::. (Fig. 39. 11).
Opening of
,. , ., )~..,.... .................
l ...
DNA helix
JI''"'' Elongation of DNA Strand
........... ..............
.,.
ftfft HtHtlftUOtttlO ..,,,- - , ,.. ...... H tmu 1ottttH
l
YllOflWf{llfllffflWl..,pli' "'""""""""
IIINHINll llffllllUIJ~-IIIJt= ---~
pyrophosphate is released from the deoxynucleoside
Synthesis of triphosphate (Figs. 39.8 and 39.9). This newly added
leading strand
nucleotide would now polymerise with another one,
1"'1"""""'"''""41c.
"'llWll'"""'""
.Mhllllll1h-..,
,=:Ioo,,
, ....,_tllllllJII
~ IIUlllllQWIIIIM
Primase
RNA primer
"~
! - - - --+ DNAP synthesizes the new strand of DNA
5'
DNA polymerase
ONA polymerase
5' ·•••111...•••11111...
llaal!• 3'
. , -,;•
Then ligase joins the ends
Daugher strand is now complete
3' ~
Fig. 39.11: RNA primer is needed for the DNA synthesis; blue is the parent strand ; green is the new strand ; red is the primer.
562 Section E: Molecular Biology
Exonucleolytic Proofreading
The DNA polymerase has 3' to 5' exonuclease activity.
Hence any mispaired nucleotide added is immediately
removed (Fig. 39. 13).
Mismatch Repair
The original template DNA contains methylated residues
(N6-methyladenine and 5-methylcytosine). The newly
Aziz Sancar, Paul Modrich, Tomas Lindahl,
NP 2015, NP2015, NP 2015, synthesized strand will not have methylated bases.
b 1946 b 1946 b 1938 So enzymes can recognize the original (correct) DNA
strand. The mismatched base is identified and removed
BOX 39.2 : Summary of DNA rephcalion
along with a few bases around that area. The wrong
1. Origin of replication is identified. Then unwinding of parental
base is removed by the endonuclease activity. A small
DNA to form a replication fork. segment of DNA with correct base sequence is then
2. RNA primer complementary to the DNA template is synthesized by DNA polymerase beta. Then the gap or
synthesized by RNA primase. nick is sealed by DNA ligase (Fig. 39.14).
3. DNA synthesis is continuous in the leading strand (toward
replication fork) by DNA polymerase.
4. DNA synthesis is discont inuous in t he lagging strand (away
Nucleotide Excision Repair (NER)
from t he fork), as Okazaki fragments. This mechanism repairs damage of DNA and replaces
5. Elongation: In both stran ds, the synthesis is from 5' to 3'
a segment up to 30 nucleotides in length. Pyrimidine
direction.
6. Then the RNA pieces are removed; the gaps filled by
dimers caused by UV light, benzopyrene (guanine
deoxynucleotides by DNAP and the pieces are ligated by adducts formed by smoking), other modifications
DNA ligase. caused by chemical agents, cancer chemotherapy etc.
7. Proofreading is done by the DNA polymerase. are thus repaired. Initially two phosphodiester bonds on
8. Finally organized into chromatin.
the damaged strand are hydrolysed by endonucleolytic
9. Main enzym es involved in replication are: DNA polymerases;
helicases; topoisomerases; DNA primase; single strand cleavage and the activity is referred to as excinuclease.
binding proteins; and DNA ligase. A fragment of DNA, 25-30 nucleotides in length is
Single strand binding Co-operative binding to the SS DNA 55 DNA binding pro teins bind at the replication fork.
proteins
RNA primer Required for synthesis of both strands DNAP alpha has primase activity and initiates synthesis of both lagging
and leading strands
DNA polymerases Major polymerizing enzyme is DNAP- 111 Major polymerizing enzymes are DNAP delta and DNAP epsilon.
The DNAP delta completes polymerization of the Okazaki fragments
(lagging st rand) and DNAP epsilon com pletes polymerization of
leading strand
Proofreading DNAP Ill al so has 3' to 5' exonuclease DNAP delta and epsilon both have 3'-> 5' exonuclease activity and
Activity so that any wrong base is removed therefore serve the proofreading function
and the correct one added
Gapfilling The RNA primer is removed and gap filled RNA primer is removed by RN Ase H and FEN1. Polymerase beta is
byDNAP involved in gap filling and DNA repair. Mitochondrial DNA replicat ion
is by DNAP gamma
Inhibitors Ciprofloxacin and Novobiocin inhibit Etoposide, Adriamycin and Camptothedn inhibitTopoisomerase
topoisomerase (DNA gyrase)
564 Section E: Molecular Biology
Remo,~ :"+!Q
incorrectly added; this
blocks further elongation. sequences by DNA
polymerase
3 = 3'-5'-exonuclease nucleotides
activity of DNA polymerase
removes the unwanted base. CH3 TGC CH 3 DNA ligase seals
the nicks
!
Fanconi's Anemia: Defect in DNA cross link repair; increased occur- lagging strand
Telomerase
rence of cancer
•
Bloom's Syndrome: Defect in DNA ligase or helicase; lymphoreticu- New telomere repeat
lar malignancies
Cockayne Syndrome: Defect in NER mechanism; transcription factor
coupled repair, transcription factor II H is defective; stunted growth
and mental retardation.
Hereditary Polyposis Colon Cancer (Lynch syndrome): Mismatch
repair is defective.
! RNA template
inside telomerase
3. The two strands run antiparallel to each other. 11 . DNA synthesis is continuous on the leading strand
4. Supercoiling of DNA is mediated by the enzymes and discontinuous on the lagging strand. This
topoisomerases and gyrases. results in the formation of the replication fork.
5. DNA is assembled into nucleosomes. 12. The small DNA molecules attached to their own
6. Histones are unusually rich in basic amino acids. RNA primers are called Okazaki fragments.
7. Transcriptionally active chromatin is called "euchro-
13. Xeroderma pigmentosum is an autosomal reces-
matin" and stains less densely as compared to
"heterochromatin" which is the inactive region of sive condition caused due to a defective nucleotide
the chromatin. excision repair mechanism.
8. DNA replication in vivo is semiconservative. 14. In every replication the 3' end of the parent strands
9. In mammals, the DNA polymerase is called alpha cannot be replicated. This end piece of the chromo-
polymerase. There are five DNA polymerases. some is called telomere. This shortening is prevented
(alpha, beta, gamma, delta and epsilon) by telomerases.
10. The DNA polymerase requires a RNA primer syn- 15. Telomerases have been implicated in aging pro-
thesized by RNA polymerase for initiation of its
cess and cancers.
activity. This forms the first step in DNA synthesis.
39-1. Describe the structure of DNA. What are the differences between DNA and RNA?
39-2. What are the salient features of Watson-Crick model of DNA?
39-3. Describe the process of DNA replication. Name two inhibitors of replication.
39-10. All the following statements are true with regard to C . Reverse transcriptase
Watson-Crick model of DNA, except D. Deoxyribonuclease
A. Double stranded helix 39-17. All cause fidelity in replication, except
B. DNA strands running in opposite directions A. There is an inherent repair mechanism
C. Adenine bonds with thymine B. DNA polymerase beta has an editing function
D. Guanine bonds with uracil C. DNA polymerase also has exonuclease activity
39-11 . All are true with regard to histones, except D. Replication stops if a wrong base is added
A. They are basic proteins 39-1 8. Regarding DNA structure which is not correct?
B. Nucleosomes contain histones and DNA A. Two strands are covalently bonded
C. Phosphorylation increases transcriptional activity
B. Two strands are antiparallel
D. Histone synthesis is coupled with DNA synthesis
C. Strands have polarity
39-12. Histones are modified by all, except:
D. Strands are complementary to each other
A. ADP ribosylation B. Acetylation
39-19. Which of the following is not a feature of mitochon•
C. Glycosylation D. Phosphorylation
drial myopathies?
39-13. Transcriptionally active chromatin is:
A . Cytoplasmic inheritance
A. Resistant to digestion by deoxyribonuclease
B. Heteroplasmy
B. Methylated at cytosine residues
C. Genomic imprinting
C. Densely packed
D. Low energy threshold
D. Called euchromatin
39-20. All are regulatory sequences on DNA, except
39-14. DNA replication requires all, except
A. Attenuator sequences
A. Elongation factor
B. Deoxynucleotide phosphates B. Enhancersequences
C. DNA polymerase C. Consensus sequences
D. RNA primer D. Promoter sequences
39-15. Which enzyme is not required for replication? 39-21. Denaturation of DNA is accompanied by all the
A. DNA ligase B. Topoisomerase changes,except
C. Helicase D. Reverse Transcriptase A. Increase in viscosity
39-16. All are involved in synthesis of DNA, except B. Hyperchromicity
A. DNA polymerase C. Unstacking of bases
B. Topoisomerase D. Destabilization of DNA helix
39-1. What are the salient features of Watson-Crick 39-3. What are histones?
model of DNA? They are nuclear proteins found in nucleus. They bind
(i) Right-handed double helix; (ii) Each turn of helix has with DNA and stabilizes DNA structure.
10 base pairs; (iii) Hydrogen bonds between purines 39-4. What is a nucleosome?
and pyrimidines; (iv) DNA strands running in opposite DNA wrapped around histones.
directions (anti parallel). 39-5. What is meant by DNA replication?
39-2. What is base pairing rule (Chargaff's rule)? During cell division, each daughter cell gets an exact
Pairing (hydrogen bonding) of adenine with thymine copy of the genetic information of the mother cell.
and guanine with cytosine. Purine is paired with This process of copying the DNA is known as DNA
pyrimidine. A+ T = G+C. replication.
568 Section E: Molecular Biology
39-6. What is semi-conservative replication? 39-13. What is meant by the lagging strand?
Each one of the newly synthesised DNA has one-half The strand which is discontinuously synthesized is
parental strand (old) and one-half new DNA strand. referred to as the "lagging strand" and the one conti-
Since half of the original DNA is conserved in daughter nuously polymerized as the "leading strand"
DNA, it is called semi-conservative replication. 39-14. What are Okazaki fragments?
39-7. What are the enzymes required for ONA replication?
The small DNA molecules attached to its own primer
DNA polymerase, Topoisomerase, DNA ligase. RNA in the lagging strand are called Okazaki
39-8. What is a replisome? fragments.
DNA replication needs more than 20 enzymes and 39-15. Xeroderma pigmentosum is due to deficiency of
proteins, collectively called DNA replicase system or what process?
replisomes. Defect in DNA repair mechanism (nucleotide excision
39-9. What is a replication bubble? repair).
Helicases move on both directions, separating the 39-16. In prokaryotes, DNA replication is inhibited by
strands in advance of the replication. This forms a what drugs?
replication bubble. Ciprofloxacine, nalidix acid, novobiocin.
39-10. Replication is in which direction? 39-17. DNA replication in eukaryotes is inhibited by what
Polymerization of the new strand of DNA is taking place drugs?
from 5' to 3' direction. This means that the template is 5-fluorouracil; 6-mercaptopurine; Cytosinearabinoside;
read in the 3' to 5' direction. Etoposide.
39-11. How replication starts? 39-18. What is telomere?
An RNA primer, about 100-200 nucleotides long, is The end piece of the chromosome is called telomere.
synthesised by the RNA primase. 39-19. What is telomerase?
39-12. What is meant by semi discontinuous nature of It replicates the end piece of chromosomes.
replication? 39-20. What is the clinical significance of telomerase?
DNA synthesis is always in the 5' to 3' direction in both In old age, the telomerase activity is lost; leading to
strands. In one strand, the replication is taking place chromosome instability and cell death. As cancer cells
continuously; but in the other strand replication is in have increased and persistent activity of telomerase,
small pieces. the cancer cells become immortal.
t - - -_ __ _ Chapter 40
Transcription
Chapter at a Glance
The learner will be able to answer questions on the following topics:
0 Ribonucleic acid 0 Post-transcriptional processing
0 Messenger RNA 0 Reverse transcriptase
0 Transcription
I RIBONUCLEIC ACID
Ribonucleic acid (RNA) is also a polymer of purine
and pyrimidine nucleotides linked by phosphodiester
bonds. However, RNA differs from DNA as shown in Table DNA
40.1 and Figure 40.1. Cellular RNAs are of five types:
a. Messenger RNA (mRNA): The genetic information
present in DNA is transcribed into mRNA, which is
the coding RNA . They have a short half life and are
generally degraded quickly. mRNA
! l
RNA polymerase attaches to DNA RNAP enzyme
l. Start site
!
Promoter site
~ .lqV""'/&.."'-'JJ.:J~ , DNA
Template 3' C A T T GA T G
DNA - - -- - - - - - - -- - - 5'
strand 5' G U A 3'
New RNA strand - 11111111"'"""
Roger Kornberg, F ig . 40.6 : Initiation of transcription
NP 2006
(b. 1947)
the starting point is numbered as -1 . Further upstream,
these negative numbers are increased. The DNA helix
partially unwinds, and the RNAP binds with the promoter
site on DNA with the help of sigma factor (Fig. 40.7).
When it reaches the appropriate site on the gene, the
first nucleotide of the mRNA attaches to the initiation site
Fig. 40.5: Transcription process on the beta subunit of RNAP. This becomes the 5' end of
the mRNA. It will be complementary to the base present
Signals for Initiation of Transcription in the DNA at that site. This is the initiation of transcrip-
tion. The next nucleotide attaches to the RNAP. A phos-
Promoters
phodiester bond is formed. Then the enzyme moves to
There are certain consensus sequences on DNA which the next base on the template DNA (Fig. 40.6).
act as start signals which may be located upstream or
downstream from the start site. The RNAP attaches at Mammalian System
the promoter site on the template DNA strand. In eukaryotes, the situation is more complex. There are
atleast 7 transcription factors, collectively called as
TATA Box and Golberg-Hogness Box Tf-11. First, the TATA box is recognized by TSP (TATA
In the case of bacteria, about 10 bp upstream, there is binding protein). In humans about 105 transcription ini-
sequence 5'-TATAAT-3'. This is referred to as TATA box tiation sites are available.
or Pribnow box. The TATA box is not on template strand,
but on coding strand. In mammals, the exact sequence Elongation Process of Transcription
in TATA box is slightly different {TATAAA) and is known The RNAP moves along the DNA template. New nucleo-
as Goldberg-Hogness box. This signal sequence located tides are incorporated in the nascent mRNA, one by
at -25 to -30 position indicates the start site. one, according to the base pairing rule {Fig. 40.8). Thus,
A in DNA is transcribed to U in mRNA; T to A; G to C and C
Other Regulatory Signals to G. The synthesis of mRNA is from 5' to 3' end. That means
Enhancers increase the rate of transcription and silencers the reading of template DNA is from 3' to 5' (Fig. 40.3).
decrease the rate. Other regulatory signals for transcrip- This is analogous to the polarity in DNA synthesis.
tion are hormone response elements (HRE) {see Chap- As the RNAP moves along the DNA template, rewin-
ter 45), repressors, inducers and derepressors {see ding of the double helix occurs at the upstream areas. RNAP
Chapter 42). has DNA unwinding property. Topo-isomerase will also
help in this unwinding process. A transcription bubble
Initiation of Transcription containing RNAP, DNA and nascent RNA is formed
{Fig. 40.7). This bubble is about 20 bp length. RNAP
Bacterial System has no nuclease activity; so there is no proof reading.
The starting point of transcription corresponds to the 5' Hence , fidelity is less; mistake rate in mRNA transcrip-
nucleotide of the DNA, designated as +1. Then num- tion is 104 or 105 times more than DNA replication. But it
bering is done 2, 3, 4, etc. to the downstream region of is less serious, since these mistakes are not transmitted
the DNA. The nucleotide adjacent to downstream of to the daughter cells or to the next generation .
572 Section E: Molecular Biology
l l
RNAP enzyme
Start site
Pro=""''
Template
DNA
strand
3
, l C A T TG A TG
5' G U A A C U A C 3'
5'
Fig. 40.7: DNA unwinds for transcription process Fi g . 40.8: Elongation process of transcription
-
Exons lntrons
s·
DNA I
•
:, <> em 8 ... a I
- -
e Cl I Iii CC- :::J
Rho factor
identifies stop Primary RNA transcript ! Transcription
signal II I CS 0 AAAAA
•
I Adding 5' cap and
5' cap poly A tail
c::j CSI I Cll Q AAAAA
Sma I Nuclear RNAs (snRNAs) a ribozyme. The amino acid binding sequence CCA is
Their ,ize ranges from 90-300 nucleotides. They take added to the 3' end. Modification of bases is another
part ir the formation of spliceosomes. All of them are change during processing.
locate j in the nucleus. They complex with specific pro-
teins, to form small nuclear ribonucleoprotein particles Ribozymes
(SnRI\ PsJ. It is pronounced as "Snurps". Production Enzymes made up of RNA are called ribozymes. Ribo-
of aut ::>antibodies against "Snurps" is implicated in the zymes or RNA enzymes are catalytic RNA molecules
patho ienesis of systemic lupus erythematosis (SLE), with sequence specific cleavage activity. Thomas Cech
a fata auto-immune disease. and Sidney Altman discovered the ribozymes, for which
they were awarded Nobel Prize in 1989. Spliceosomes
Splii~eosomes contain ribozymes as well as protein components which
SnRN 0 s associated witn hnRNA at the exon-intron junc- serve to stabilize the structure of ribozymes. RNAse-P is
tion fc rm spliceosomes. This is taking place inside the another ribozyme, which generates the ends of tRNAs.
nucle JS . Cuts are made at both ends of intron; it is Peptidyl transferase present in ribosomes (used for
remO\ ed and exon-exon ends are Iigated at G-G residues. protein biosynthesis is another exam ple of ribozy me.
For elpcidation of spliceosome activity, Richard Roberts
and Rhillip Sharp were awarded Nobel Prize in 1993. Reverse Transcriptase
essing of rRNA Generally speaking, the genes are made up of DNA.
mal RNA is transcribed by RNAP-I and different Usually, DNA dependent RNA polymerase transfers the
rRN molecules are transcribed as single precursor. information of DNA to mRNA. However genetic material
Then it is cleaved into three different molecules; 28s of some animal and plant viruses is made up of RNA.
RNA, 5.8s RNA & 18s RNA. The rRNA genes are
locat d in the nucleolar region .
1
Virus RNA Inhibitor Source Mode ofaction
Primer RNA 5' AAAAA 3' Polymerization by Actino- Antibiotics from Insertion of phenoxa-zone
reverse transcriptase mycin-D streptomyces ring between two G-C bp
of DNA
3' AI\I\.I\/\AI\I\I\I\I\I\I\AAI\I\I\AAl\lt 5•
Rifampicin Synthetic derivative of Binds to beta subunit of
RNA-DNA hybrid 5' AMA- - - - - - 3'
l
rifamycin RNA polymerase which is
inactivated
RNAse H hydrolyzes RNA
!
Alpha Toxin from mushroom Inactivates RNA
DNA copy of RNA amanitin polymerase II
DNA polymerase Ill
3' -deoxy Synthetic analog Incorrect entry into chain
Double stranded DNA adenosine causing chain termination
Howard Temin
NP 1975
David Baltimore
NP 1975
Robert Holley
NP 1968 ILEARNING POINTS, CHAPTER 40
1934-1994 b. 1938 1922- 1993
1. Template strand of the DNA is transcribed to mRNA.
Chapter 40: Transcription 575
2. R AP type II or B is the enzyme synthesizing RNA 11 . Both rRNA and tRNA are non coding RNAs, but play
in mammals. It is susceptible to amanitin. crucial roles in translation.
3. A transcription bubble contains RNA polymerase, 12. The enzyme which brings about transcription is
D A and nascent RNA. DNA dependent RNA polymerase.
4. li rmination of transcription can be Rho factor 13. Bacterial RNA polymerases are different from mam-
d pendent or independent. malian enzymes; so antibiotics which inhibit bacte-
5. P st-transcriptional processing of the primary RNA rial RNAP will not affect human beings.
tr nscript includes RNAse-P tailing at 3' end, cap- 14. The reverse transcription process is catalyzed by
pi g at 5' end , methylation and intron splicing. RNA-dependent DNA polymerase (RT) which was
6. R bozymes are enzymes made up of RNA. Exam- first reported in tumor viruses.
p s are RNAse-P, peptidyl transferase and spliceo- 15. The HIV is the most clinically relevant retrovirus
s mes. now.
7. R verse transcriptases are RNA dependent DNA 16. The inhibitor of RNA synthesis (in both eukaryotes
p lymerases. They synthesize a DNA strand using and prokaryotes) is actinomycin D and mitomycin
A as their template. Retroviruses, such as HIV which intercalate with DNA, blocking transcription.
a d tumor viruses, possess this enzyme. 17. The widely used anti-tuberculous drug rifampicin
8. D A replicates before cell division, so that each (Rifamycin) inhibits prokaryotic RNA polymerase.
d ughter DNA molecule gets an exact copy of the 18. The small interfering RNA (si-RNA) protects the
p rent cell. genome from bacteriophages and viruses in lower
9. T e genetic information present in the mRNA is
organisms.
tr nscribed to mRNA and then translated into the
19. In eukaryotic cells, small dsRNA molecules will
a ino acid sequence of the polypeptide chain, as
silence specific genes, and this is a normal regula-
t e gene product.
10. T e strand of DNA which is transcribed is called tory mechanism.
t e templet strand, which is complementary to the 20. Use of synthetic antisense strands (RNA mole-
RNA. The anti-template strand has the same cules having complementary sequence of cellular
!
s quence as the mRNA transcript, and is called the mRNA) to manipulate genes are now being used as
ding strand. therapeutic tools.
4 -1. All the bases are found in mRNA, except B. A subunit of RNA polymerase
A. Adenine B. Guanine C. A subunit of 50 S ribosome
C. Uracil D. Thymine D. Responsible for initiation of replication
4 -2. The nitrogenous base absent in DNA Is: 40-4. The processing of mRNA involves:
A. Uracil B. Cytosine A. Capping at 5' terminus
C. Thymine D. Guanine B. Poly A tail attachment
4 -3. Sigma factor is: C. Removal of intrans
A. A subunit of DNA polymerase D. Translocation of mRNA into nucleus
576 Section E: Molecular Biology
40•5. lntron is the portion of: 40-1 6. In an organism, only DNA, but not RNA is
A. DNA that is cleaved off during replication labelled when grown in a medium co ntaining
B. mRNA that is removed after transcription rad ioactive nucleotide of:
C. tRNA that is added on after its synthesis A. Adenine B. Guanine
D. Protein that is removed after translation C. Thymine D. Cytosine
40-6. Which is an inhibitor of transcription in 40-17. The RNA present in largest amounts in a cell at
eukaryotes? any time is
A. Rifampicin 8 . Chloramphenicol A. mRNA B. tRNA
C. Alpha amanitin D. Streptomycin C. rRNA D. hnRNA
40-7. Mechanism of action of mitomycin is: 40-1 8. All the bases are found in mRNA, except
A. Inactivation of EF-2; blocks elongation of proteins A. Adenine B. Guanine
C. Uracil D Thymine
B. Intercalation with DNA strands; blocks transcription
40-19. The nitrogenous base absent in DNA is:
C. Abrupt termination of protein synthesis
A. Uracil B. Cytosine
D. It is a structural analogue of amino acyl tRNA
C. Thymine D. Guanine
40-8. Reverse transcriptase differs from DNAP in
40-20. The TATA box:
that
A. Present on the template strand
A. Synthesizes DNA in 5' to 3' direction
B. Present about 70 bp away from transcription start
B. Has 3' to 5 ' exonuclease activity
site
C. Follows the base pairing rule
C. Serves as the signal for attachment of RNAP-11
D. Can insert IMP in the growing strand
D. Acts as a silencer of the gene
40-9. RNAP is inhibited by
40-21 . The processing of mRNA involves
A. Streptomycin B. Chloramphenicol
A. Capping at 5' terminus
C. Ciprofloxacin D. Rifampicin
B. Poly A tail attachment
40-10. Which of the following nucleotides is not
C. Removal of intrans
present in mRNA?
D. Translation of mRNA into nucleus
A. GMP B. TMP
40-22. All are post-transcriptional modifications in
C. CMP D. UMP
mRNA, except
40-11 . Which of the enzymes listed below is involved
A. Modification of bases
in processing of RNA?
B. Splicing of intrans
A. RNA polymerase II
C. Trimming of 3' end to CCA sequence
B. Ligase
D. Attachment of poly A tail.
C. Helicase 40-23. Transcription is terminated when:
D. Ribonuclease-P A. The codon on the mRNA is AUG
40-12. All the following are inhibitors of eukaryotic B. Rho factor binds to mRNA
transcription, except: C. The codon on the mRNA is UGA
A. Rifampicin B. Puromycin D. The ribosome reaches the poly-A tail on mRNA
C. Actinomycin D. Amanitin 40-24. Which is caused by autoantibodies to Snurps?
40-13. The coding unit of the DNA is called as: A. Xeroderma pigmentosum
A. lntron B. Exon B. Mushroom toxicity
C. Cistron D. Prion C. Systemic lupus erythernatosus
40-14. Which is not a transcription start signal? D. Thalassernia
A. CAAT box B. Hogness box 40-25. lntron is the portion of:
C. Pribnow box D. Homeo box A. DNA that is cleaved off during replication
40-15. All are true regarding transcription, except: B. rnRNA that is removed after transcription
A. RNA polymerase has proofreading capacity C. tRNA that is added on after its synthesis
B. RNA strand is complementary to template strand D. Protein that is removed after translation
of DNA 40-26. All are correct with retroviruses, except:
C. Is inhibited by rifampicin A. Contains a DNA dependent RNA polymerase
D. RNA is synthesized in the 5' to 3' direction B. Transcription occurs in the reverse direction
Chapter 40: Transcription 577
C. Viral RNA is copied into host cell DNA 8. Intercalation of DNA strands and blocks trans-
D. New DNA is incorporated into host cell genome. cription
40- 7. Mechanism of action of mitomycin is: C. Abrupt termination of protein synthesis
A. Inactivation of EF-2, and so blocks elongation D. It is a structural analogue of amino acyl tRNA
Chapter at a Glance
The learne r will be able to answer question s on the following topics:
Translation
Anti odon Arm of tRNA Ribosomes provide necessary infrastructure for the
mRNA, tRNA and amino acids to interact with each other
At th opposite side of the acceptor arm is the anticodon for the translation process. Thus ribosomal assembly is
arm ( ig. 41 .3). It recognizes the triplet nucleotide codon the protein synthesizing machinery. Nucleolus is the
prese t in mRNA. The specificity of tRNA resides in area where rRNA is synthesized and ribosomal assem-
the a ticodon site, which has base sequences comple- bly is produced. For studies on structure and function
ment ry to that of mRNA codon. of ribosomes, Nobel Prize was awarded in 2009 to
F r example, if the mRNA has a codon with the Venkataraman Ramakrishnan (born in India), Ada Yonath
sequ nee UUU, the anticodon sequence of the tRNA and Thomas Steitz. Ribosomal RNA is synthesized by
will b AAA, by which it base pairs with mRNA codon. RNAPI .
So, t e specific tRNA can bind correctly to the mRNA
codo s. In this case, the UUU codon is translated as
Components of rRNA
phen !alanine. Recognition of codon by the tRNA anti-
codo is illus~~ated in see Figure 41 .3. The mammalian ribosome has a sedimentation constant
T e tRNA molecule will show specificity in both of 80S unit. It has a larger 60S subunit and another
smaller 40S subunit. They contain different rRNAs and
aspe ts; in recognizing the mRNA codon as well as in
specific proteins. Ribosomal RNA has catalytic activity.
acce ting the specific amino acid coded by that codon.
Peptidyl transferase activity is carried out by 28 S RNA
The t NAs act as adapter molecules between mRNA
which acts as a ribozyme.
and t e amino acids coded by it. Methionine-tRNA was
isolat d by Paul Berg in 1956 (Nobel Prize, 1980). The
compete sequencing of alanine-tRNA was done by
Robe Holley in 1963 (Nobel Prize, 1968). Khorana
(lndi n born US scientist) got Nobel Prize in 1968, for
synth sizing the gene for alanine tRNA.
Initiator Codon
Thomas A Steitz Ada E Yonath Venkatraman
NP2009 NP 2009 Ramakrishnan In most of the cases, AUG acts as the initiator codon.
b. 1940 b. 1939 NP2009 AUG also acts as the codon for methionine. In a few
b. 1952 proteins, GUG may be the initiator codon.
Chapter 41 : Genetic Code and Translation 581
Mite chondria have Different Codons ATP. Then the carboxyl group of the amino acid is esteri-
fied with 3' hydroxyl group of tRNA.
The Iirotein synthesizing machinery of mitochondria is
Amino acyl tRNA
differ mt from that in the cytoplasm. There are only about Amino acid synthetase ) Aminoacyl tRNA
22 tF NAs in mitochondria; but there are 31 tRNA spe- +tRNA+ ATP +AMP
cies in cytoplasm. Therefore, some of the mitochondrial
codo ~s are different from nuclear codons. In this reaction, ATP is hydrolyzed to AMP level, and
so two high energy phosphate bonds are consumed.
I TI 'ANSLATION PROCESS
Initiation of Protein Synthesis
Tran• lation is a cytoplasmic process. The mRNA is
Initiation can be studied as 4 steps: 1. Recognition steps.
trans ated from 5' to 3' end. In the polypeptide chain
2. Formation of preinitiation complex. 3. Binding of
synt~ esized, the first amino acid is the amino terminal
mRNA to the preinitiation complex. 4. Formation of
one I see Fig. 41 .1). The chain growth is from amino ter-
ribosomal complex.
mina to carboxyl terminal. The process of translation
can I e conveniently divided into the phases of:
Recognition Steps
A. !\ctivation of amino acid
B. nitiation In eukaryotes, the first amino acid incorporated is methio-
C. =longation nine (AUG codon). But in prokaryotes, the same codon
D. rermination stands for N-formyl methionine, which is the first amino
E. ~Ost-translational processing. acid.
The first AUG triplet after the marker sequence is
Act vation of Amino Acid identified by the ribosome as the start codon . In mam-
(Charging Reaction) malian cells, the marker is the "Kozak" sequence; in
bacteria, it is the "Shine-Dalgarno" sequence. For the
The enzymes aminoacyl tRNA synthetases activate
process, initiation factors (elF) are required. Poly-A
the c mino acids. The enzyme is highly selective in the
tail of the mRNA is also important for the recruitment of
reco 1nition of both the amino acid and the transfer RNA
40S ribosomal subunit to the mRNA.
acceptor. There is at least one tRNA for each of the
20 amino acids. The D arm of tRNA is very important
Formation of Preinitiation Complex
for ti e recognition by the enzyme. The CCA 3' terminus
of t~e acceptor arm carries amino acid (see Figs. 41 .2 GTP, IF-2, met-tRNA (tRNA carrying methionine)
and 41.3). Amino acid is first activated with the help of and 40S ribosomal subunit are complexed to form
582 Section E: Molecular Biology
Met-tRNA
+GTP + IF-5 + IF-2
+ elF-2 + factor 4
(\
ATP ADP+Pi
{+)605 ' \
GDP+Pi
UAC AC
+ elF-3
AUG ACC
+ elF-1 /\AAA/\ AAAAAAAAA AA
pre-initiation complex (Fig. 41.4 ). Met-tRNA has the The anti-codon of met-tRNA is correctly base pairing
anticodon UAC. with the AUG codon on mRNA (Figs. 41.4 ahd 41 .5).
-----• l~:.: :
.... •····· •• free protein
Protein synthesis
Polypeptide half nearing to completion
length synthesized NH2
I
Synthesis started NH 2
NH 2
I I
RF+GTP
AMAMMMA free mRNA
mRNA
energy supply for the purpose of peptide bond forma- peptide chain from the tRNA at the P site. This needs
tion. Now, the growing peptide chain is occupying the hydrolysis of GTP to GDP. The completed peptide chain
"A" site (Fig. 41.5). is now released.Finally, SOS ribosome dissociates into
its component units of 60S and 40S (Fig. 41 .6). The
Translocation Process differences between mammalian and bacterial transla-
At this time, the tRNA fixed at the "P" site does not carry tion are given in Table 41.3.
any amino acid and is therefore released from the ribo-
some. Then the whole ribosome moves over the mRNA Polyribosomes
through the distance of one codon (3 bases). The pep- One eukaryotic ribosome can synthesize 5 to 6 peptide
tidyl tRNA is translocated to the "P" site. Now, the "A" bonds per second . Many ribosomes can work on the
site is ready to receive another aminoacyl tRNA bear- same mRNA molecule simultaneously and these aggre-
ing the appropriate anticodon. The new aminoacyl tRNA gates are called polyribosomes or polysomes (Fig. 41 .7).
is fixed to the "A" site, by base pairing with the mRNA Polyribosomes may be attached on the walls of the
codon. Translocation requires hydrolysis of GTP to GDP endoplasmic reticulum to form the rough ER (see
(Fig. 41 .5). The elongation reactions are repeated till Chapter 2). The proteins are then transported through
the polypeptide chain synthesis is completed. cisternal space to Golgi apparatus, where they are tem-
porarily stored. Cytoplasmic proteins are synthesized by
Energy Requirements ribosomes that exist free in cytoplasm.
For each peptide bond formation, 4 high energy phos-
phate bonds are used; two for the initial activation and Protein Targeting
one for binding of amino acyl tRNA to A site (GTP to Proteins for External Secretion
GDP) and one for translocation step (GTP to GDP).
The process is also called as "protein sorting" or "protein
Actual peptide bond formation (peptidyl transferase
localization". The secreted proteins, plasma membrane
step) does not require any energy, because the amino integral proteins, lysosomal enzymes and membrane
acids are already activated. Further, 1 ATP is used for proteins of ER are synthesized on rough endoplasmic
initiation complex formation ; 1 GTP for SOS ribosome reticulum by membrane bound polyribosomes. The
formation and 1 GTP for termination. newly synthesized protein is then delivered to the des-
tined compartment. Blobel and Sabatini proposed the
Termination Process of Translation
signal hypothesis to explain the different destination
After successive addition of amino acids, ribosome rea- of proteins. Guenter Blobel was awardec Nobel Prize in
ches the terminator codon sequence (UM, UAG or UGA) 1999.
on the mRNA. Since, there is no tRNA bearing the The nascent protein is passed through the mem-
corresponding anticodon sequence, the "A" site remains brane into the channels of ER. Then protein synthesis
free. The release factor (RF) enters this site. The RF binds is completed, and the protein molecule is now inside
with RF3 and GTP. This complex along hydrolyzes the the endoplasmic membrane. As the nascent protein is
584 Section E: Molecular Biology
TABLE 41 .2: Post-translaltonal mod1ficat1ons TABLE 41 .3: Comparison of translation In eukaryotes and
prokaryotes. M1tochondna are similar to prokaryotes
Reversible Irreversible
Eukaryotes
Disulfide bridge Proteolysis
(mammalian Prokaryotes
Glycosylation Ubiquitination Feature cells) (bacteria) Mitochondria
Phosphorylation Lysine hydroxylation DNA Open Circular Circular
Acylation Proline hydroxylation Ribosomes 80S 70S 70S
N-acetylation Methyl at ion tRNA (No.) 31 22 22
Initiating Methionine Formyl Formyl
amino acid methionine methionine
BOX 41 .2: Protein targeltng Inhibited
Effect of tetracycline Not affected Inhibited
Zellweger syndrome is due to defective oxidation of very long chain
fatty acids (VLCFA). Here the correct "address• is not printed on
the protein packet; so that it could not be delivered to the correct
location. Peroxisomal enzymes are produced; but their entry into
peroxisome is denied. This leads to insufficient oxidation of VLCFA. NH 2 V---
------
-----
- ----•~-
Pre-proinsulin
====~ - - - • • • COOH
Accumulation of VLCFA in CNS causes neurological impairment and
death in childhood.
Another example is primary hyperoxaluria, which causes kidney
stones at an early age. The defect is due to protein targeting defect Proinsulin
and the enzyme alanine glyoxylate aminotransferase (see Fig. 18.7)
is seen in mitochondria, inst ead of its normal peroxisomal location.
Inclusion cell disease is due to non-entry of normal enzymes into
lysosomes. Mannose-6-phosphate is the marker to target enzymes
to lysosomes; this is absent.
Mature insulin
bacteria and are nontoxic to human beings. This is Mitochondrial DNA and RNA
because mammalian cells have 80S ribosomes, while
1. There is a dichotomy on the mitochondrial metabo-
bacteria have 70S ribosomes.
lism. Some of the mitochondrial protein synthe-
Reversible Inhibitors in Bacteria sis is under the control of mitochondrial DNA; but
important proteins of the outer membrane of the
These antibiotics are bacteriostatic. Tetracyclines
mitochondria are synthesized under the influence of
bind to the 30S subunit of bacterial ribosome and so
inhibit attachment of aminoacyl tRNA to the A site of nuclear DNA. Table 41.4 shows that mitochondria
ribosomes. Chloramphenicol inhibits the peptidyl trans- are similar to bacteria more than mammalian cells.
ferase activity of bacterial ribosomes. Erythromycin This fact supports the theory that mitochondria are
(macrolides) and clindamycin prevent the translocation derived from prokaryotes symbiotically adapted to
process. multicellular organisms.
2. Maternal inheritance: Since, the mitochondria are
Irreversible Inhibitors in Bacteria inherited cytoplasmically, the mtDNA is inherited
These antibiotics are bactericidal. Streptomycin and from the mother. Mother transmits mtDNA through
all other aminoglycoside antibiotics bind to 30S subunit oocyte.
of bacterial ribosomes. They cause misreading of mRNA 3. There are hundreds of copies of mtDNA in each
and at high concentrations, they completely inhibit the cell (nuclear DNA has only 2 copies). During cell
initiation complex formation and totally inhibit protein division, mtDNA replicates and they segregate to
synthesis. the daughter cells. If a mutation occurs in mtDNA,
the daughter cells may inherit the mutant or normal
Inhibitors of Protein Synthesis in Mammals mtDNA. Heteroplasmy is defined as the presence
They are not suitable for clinical use; but they are used of normal and mutant mtDNA in different propor-
as research tools. Puromycin is structurally similar to tions in different cells.
tyrosine-tRNA and gets attached to the "A" site of the 4. Defects in mitochondrial genome will lead to mito-
ribosome. So, the incomplete peptide is released. It acts chondrial myopathies. Leber's hereditary optic
both in bacterial and mammalian cells. Cycloheximide neuropathy is caused by a single base mutation
inhibits peptidyl transferase in 60S subunit. It acts only which alters one arginine to histidine in the NADH
on eukaryotic cells. Inhibitors of transcription (Chapter 40) Coenzyme Q reductase. OXPHOS (oxidative phos-
will also in turn inhibit translation process. phorylation) diseases are shown in Table 41 .5.
586 Section E: Molecular Biology
TABLE 41 .5: OXPHOS diseases iv. Proteomics: It directly addresses the protein com-
Syndrome Features
plement of the genome. The study of all proteins by
a cell type or an organism is called 'proteomics'.
Leber's Hereditary Complex I defect; blindness, cardiac
Neuropathy (LHON)
Myoclonic epilepsy ragged
conduction defects
Myoclonic epilepsy, myopathy,
I LEARNING POINTS, CHAPTER 41
red fiber disease (MERRF) dementia
1. Transfer RNA (tRNA) or soluble RNA (sRNA) is the
Leigh's syndrome Complex I defect; movement adapter molecule between transcription and trans-
disorders
lation. Each amino acid has a specific tRNA.
2. The triplet sequence on the anticodon arm of the
Genomics and Proteomics tRNA is complementary to the codon triplet on the
mRNA.
i. Genome means all the DNA contained in an orga- 3. Six important characteristics of the genetic code
nism or a cell, which includes both the chromosomes are that it is triplet, universal, degenerate, non-over-
within the nucleus and the DNA in mitochondria. lapping, nonpunctuated and exhibit wobbling.
Thus the genome of an organism is the totality of 4. Three terminator codons are UAA, UAG and UGA.
5. Four high energy phosphate bonds are required
genes making up its hereditary constitution.
for the formation of one peptide bond, two for initial
ii. Genomics is the study of the genome and its activation, one for EF-1 step and one for EF-2 step.
actions. 6. Post-translational processing of proteins includes
iii. Proteome is the sum of all proteins expressed removal of the signal sequences, gamma carboxy-
by the genome of an organism, thus involving the lations, methylations, acylation, subunit aggrega-
identification of the proteins in the body and deter- tion and phosphorylations.
mination of their role in physiological and pathologi- 7. Clinically useful protein synthesis inhibitors are
streptomycin, chloramphenicol, tetracyclines, erythro-
cal functions. While the genome remains largely
mycin.
unchanged, the proteins of a particular cell change 8. Chaperones are proteins that govern protein fold-
dramatically as genes are turned on and off in ing. Improper protein folding may lead to Prion
response to the environment. diseases.
41-3. Which is not true regarding the genetic code: A. Amino terminal modification
A. Degenerate 8 . Ambiguous 8 . Protein folding
C. Nonoverlapping D. Universal C. Phosphorylation of serine residues
41-4. During protein biosynthesis, high energy bonds D. Glycosylation of asparagine
are utilized in all the following steps, except 41-15. In eukaryotes, the initiation of translation requires
A. Formation of amino acyl tRNA all the following, except:
8. Binding of amino acyl tRNA to A site of mRNA A. Methionyl tRNA
ribosome complex 8 . 70 S ribosome
C. Formation of peptide bond (peptidyl transferase C. Kozaksequence
step) D. mRNA codon AUG
D. Translocation 41-16. Elongation of polypeptide chain requires all the
41 -5. Post-translational modifications include all the fol- following proteins, except:
lowing, except A. Peptidyl transferase 8 . Rho factor
A. Glycosylation 8 . Hydroxylation C. Translocase D. GTPase
C. Decarboxylation D. Phosphorylation 41-17. Which complex is involved in translation?
41 -6. All are diseases resulting from failure of post- A. Polysome 8. Nucleosome
translational modifications, except C. Spliceosome D. Lysosome
A. Lathyrism 41-18. Signal peptide (SP) region is necessary for all the
8. Ehler-Danlos syndrome following, except
C. Osteogenesis imperfecta A. Attachment of ribosomes to mRNA
D. Scurvy 8 . Anchorage of ribosomes to endoplasmic reticulum
41-7. Failure of post-translational modification occurs in C. Release of newly synthesized proteins from
deficiency of which vitamin? ribosomes
A. Ascorbic acid 8. Retinol D. Digestion of signal recognition particle (SRP)
C. Vitamin 812 D. Vitamin D 41-19. Which is not a post-translational modification?
41-8. The mechanism of action of tetracycline is: A. Gamma carboxylation of prothrombin
A. Inhibits tRNA binding to ribosome 8. Hydroxylation of proline in collagen
8. Decreases binding of ribosomes to mRNA C. Methylation of histones
C. Causes misreading of codes D. Hydroxylation of benzene ring of phenyl alanine
D. Inhibits translocation 41-20. Which is an inhibitor of translation in eukaryotic
41-9. All are true regarding tRNA, except: cells?
A. Amino acid is attached to 3' end. A. Erythromycin 8 . Puromycin
8. Anticodon sequence is at 5' end. C. Chloramphenicol D. Tetracycline
C. Contains several modified bases. 41-21 . Mechanism of action of streptomycin is that it:
D. Has a clover leaf pattern due to hydrogen bonding. A. Inhibits tRNA binding to ribosome
41-10. Unusual nucleotide bases are found in significant 8 . Decreases binding of ribosomes to mRNA
quantities in: C. Causes misreading of codes
A. mRNA B. tRNA D. Inhibits translocation
C. rRNA D. snRNA 41-22. All the following statements are true with regard to
41-11 . All are inhibitors of RNA synthesis, except: mitochondrial DNA, except
A. Rifampicin B. Puromycin A. All mitochondrial proteins are encoded by mito-
C. Amanitin D. Actinomycin D chondrial DNA
41-12. All are structural features of tRNA, except: 8 . Mitochondrial DNA is inherited by cytoplasmic
A. Has clover leaf appearance inheritance.
8 . Has amino acid binding site at 5'end C. Inheritance is predominantly from mother
C. Contains unusual bases D. DNA in mitochondria is circular
D. Has a pseudouridine arm 41 -23. The disease resulting from mutation in mitochon-
41-13. All the following are inhibitors of translation in drial DNA is:
prokaryotic cells, except: A. Zellweger syndrome
A. Clindamycin 8. Streptomycin 8 . Jakob-Creutzfeldt disease
C. Mitomycin D. Erythromycin C. Xeroderma pigmentosum
46-14. Which of the following post-translational events is D. Leber's hereditary optic neuropathy
defective in mad cow disease?
588 Section E: Molecular Biology
41-1 . What are structural features of tRNA molecule? 41 -8. What is meant by the term wobbling?
It has clover shape appearance. Amino acid binding is Anticodons pair with codons that differ at the third
at 3' end. The opposite part has anticodon arm. base.
41-2. What is a codon? 41-9. Where is protein biosynthesis is taking place?
Codon is a triplet of bases, present in mRNA. Collec- Ribosomal assembly either attached to endoplasmic
lion of these codons make up the dictionary of genetic reticulum, or in cytoplasm.
code. 41-10. Give examples for post-translational modifications.
41-3. During replication, DNA is synthesised in wh ich Gamma carboxylation of prothrombin; Hydroxylation
direction? of praline in collagen; Methylation of histones; Glyco-
From 5' to 3' direction. sylation of proteins.
41-4. During transcription, mRNA is synthesized in 41-11 . Give examples of inhibitors of translation in
which direction? eukaryotic cells?
From 5' to 3' direction. Puromycin; Cycloheximide; Diphtheria toxin; Ricin.
41-5. During translation, protein is synthesized in which 41 -12. Give examples of reversible inhibitors of protein
direction? synthesis in bacteria.
From amino terminal end to carboxy terminal end. These antibiotics are bacteriostatic. Tetracyclines;
41-6. What are the salient features of genetic code? Chloramphenicol; Erythromycin and clindamycin are
i. They are consecutive three bases pairs in mRNA. examples.
ii. Codons are nonoverlapping; iii. degenerate; iv. but 41-13. Give an example of irreversible inhibitor of protein
unambiguous and v) universal. synthesis in bacteria.
41-7. What is meant by degeneracy of genetic code? These antibiotics are bactericidal. Streptomycin is an
One amino acid is represented by multiple codons. example.
-----------Chapter 42
Control of Gene Expression
Chapter at a Glance
The learner will be able to answer questions on the following topics:
0 Principles of heredity 0 0peron concept
0 Dominant and recessive inheritance 0 Repression and derepression
0 Mutations 0 Transcription regulation in eukaryotes
..
It is estimated that more than 6% of all infants born alive suffer from genetic diseases and 1% from chromosomal aberrs -
tions. The former conditions involve minor alterations in DNA make up, e.g. phenylketonuria. The latter onies are due to major
changes in chromosomes; e.g. Down's syndrome, Turner's syndrome. Gregor Johann Mendel (1822-H4}, who was Abbot
I
of Bron, described the principles of heredity in 1866. As it was printed in an obscure journal, it remained unnoticed for many
years. In 1900, Hugo de Vries and C Correns, rediscovered and confirmed Mendel's theory. Walther Flemming demonstrated
chromosomes in 1882. In 1902, Walter Sutton showed that chr mosomes are in pairs and are the carriers of Mendel's Unit of
heredity. The word 'Gene' was coined by Wilhelm Johannsen in 1909.
-
father
® d
9 d N n
•
D Dd Dd N NN Nn
Dd dd
Nn ~ Nn
cf
INN Nn Nn nn l
I
n
d dd dd
® 0 0 Nn nn
•I
lod dd Dd ddi
®
1111
affected normal
I
affected
®
II]
normal
D = abnormal allele
(dominant)
d= normal allele
[I
normal carrier
Result: offsprings
25% are affected
[]
carrier affected
n = abnormal allele
(recessive)
N = normal allele
Result: 50% offsprings 50% are carriers
are affected 25% normal
Fig. 42.1: Autosomal dominant inheritance Fig. 42.2: Autosomal recessive inheritance
chains will be abnormal. He is homozygous for sickle X chromosome carries the abnormal gene (Fig. 42.3).
cell disease. In certain cases, the carrier state may be When a normal male marries a carrier female (unaffected
identified biochemically, then it is referred to as the trait parent), the children can be affected male (25%), female
of the disease. For example, in sickle cell trait, one beta- carrier (25%), normal male (25%) and normal female
globin gene (allele) is normal; while the other one is (25%). All male children of an affected male and normal
abnormal (carrier state). Such an individual is hetero- female will be normal; but all female children will be car-
zygous for that character. Therefore, normal gene pro- riers since they inherit the abnormal X from their father.
duces normal Hb and abnormal gene produces HbS. There is no male-to-male transmission, but male-to-
Thus inside the RBC, 50% of hemoglobin molecules are female and female-to-male transmission of the affected
X can occur. X-linked traits are expressed in males
abnormal. This can be identified by electrophoresis.
who are hemizygous (XY) for the condition, but not in
When both father and mother are carriers, one-
females who may be heterozygous (XX).Hemophilia, glu-
quarter of siblings express the disease (both alleles
cose-6-phosphate dehydrogenase deficiency, pseudo-
abnormal), another one-quarter of siblings are normal, and
hypertrophic muscular dystrophy (Duchenne type), and
half of the children are carriers (Fig. 42.2). This chance
red-green color blindness are examples of sex-linked
factor is acting on each progeny. If only one parent is
recessive inheritance.
carrier and the other is normal; then there will be no
affected child, but 50% children are carriers. Most of the Marriages with Close-Cousins
inborn errors of metabolism are recessively transmitted. are Inadvisable
A few examples are phenylketonuria, albinism, galacto-
The probability of two carriers getting married is increa-
semia and sickle cell anemia.
sed in consanguineous marriages (Latin, con = with;
sanguis = blood). So, there is increased frequency of
Sex-linked (X-linked)
genetic diseases in their children. For example, phenyl-
Recessive Inheritance
ketonuria has an incidence of 1 in 25,000 in general
In the autosomal conditions, the disease occurs in both population; but it is 13/ 25,000 in children of first cousin
sexes with equal frequency. But in sex-linked conditions, marriages.
Chapter 42: Control of Gene Expression 591
X V Carrier
female
gametes
X V Carrier
female
gametes
X V
Normal
X xx xv
Carrier Normal X xx xv
Carrier Affected X xx xv
Affected Affected
xx xv xx xv xx xv
daughter son daughter son daughter son
female
gametes
useless protein is produced. Frameshift mutations can a mutation of a single gene resulted only in a single
also lead to thalassemia, premature chain termination chemical reaction. which gave evidence to the concept
and run-on-polypeptide. of "one gene, one enzyme·.
Frameshift mutations can result from aberrant splic-
ing of primary transcript due to mutations at splice sites.
Lethal Mutations
Alternative splicing can give rise to different isoforms of The alteration is incompatible with life of the cell or the
the same protein in different tissues e.g. Tropomyosin organism. For example, a mutation which does not pro-
in skeletal and cardiac tissue. mRNA editing can also pro- duce alpha chains (4 gene deletion) will result in intra-
duce two different proteins: e.g. Apo-B-100 and Apo-B-48. uterine death of embryo.
but also how healthy one is during one's lifetime is de- 5 different cyclin dependent kinases (CDK 1, 2, 4, 5
termined at least partially be genes. Molecular variations and 6) control the cycle. Cyclins are so named because
or single nucleotide polymorphisms (SNPs) in numerous they are synthesized throughout the cell cycle, and are
genes are responsible for this phenomenon. abruptly destroyed during mitosis.
There are 3 gene systems which determine lifespan,
namely. , insulin signaling, free radicals and antioxidants Retinoblastoma (RB)
as well as DNA repair mechanisms. Oncosuppressor Protein
Insulin greatly influences the body's ability to grow
RB protein is the product of an oncosuppressor gene (see
and utilize energy. Elderly people can live longer if they
Chapter 48). It is so named, because it was isolated from
consume fewer calories.
patients of retinoblastoma (cancer arising from retina).
Antioxidants mitigate the dangerous cell-killing
Rb inhibits cell cycle at G1 phase. But in controlled cell
activities of free radicals and might prolong life.
cycles, cyclin D levels rise in the late G 1 phase. The
DNA repair systems help the body's fight against
cyclin D inactivates Rb, which is separated from E2F.
harmful agents in the environment and help to sustain This is the normal mechanism to overcome the G1 arrest
healthy lifespan. by Rb. Certain tumor antigens derived from viruses such
Specific variations in certain genes are also found to as SV40, HSV, HPV may combine with Rb. Then, Rb
influence healthy lifespan. Some of these genes include cannot inhibit cell cycle, leading to continuous cell divi-
telomerase, Foxo3, inflammatory cytokines, and mito- sion and cancer (Fig. 42.5).
chondrial DNA.
The p53 Oncosuppressor Protein
So named because it is a protein with 53kD size, hav-
LCELLCYCLE_ _ __ ing 393 amino acids. The half-life is only 5-10 minutes.
The term 'cell cycle' refers to the events occurring p53 inhibits cell division, allowing time for any damage
during the period between two mitotic divisions. It is to DNA to be repaired. If damage is extensive and repair
divided into G1 (gap-1 ), S (synthesis), G2 (gap-2), and is not possible, the p53 directs the cell to apoptosis. In
M (mitosis) phases. The cell division is taking place in most cancer cells, the p53 is mutated or nonfunctional.
M phase. It is the shortest phase, lasting about 1 hour.
The daughter cells then either enter into GO (undivid-
ing or dormant) phase or re-enter the cell cycle when UV
there is necessity for growth and repair. In a normal cell
population, most of the cells are in GO phase. Inter- ! released
ATM
phase is the period between the end of M phase and
the beginning of the next mitosis. In G1 phase, protein !
and RNA increase. Duration of G1 phase is about 12 CHK 1 kinase activated
hours. In the S phase, DNA is synthesized, but only !
CDC25 inactivated
once. DNA content doubles, nucleus becomes tetra-
ploid (4n). The entire diploid genome is replicated into !
Cyclin CDK2 active
a tetraploid genome. S phase lasts about 6 to 8 hours.
!
In the G2 phase, there is cytoplasmic enlargement. G1-S checkpoint released
DNA repair is also taking place in the G2 phase. It !
lasts for about 4 to 5 hours. The total cell cycle is about
20-22 hours duration in mammalian cells (Fig. 42.4 ). G1 f:: s
Cell Cycle
Cell Cycle Controls or Checkpoints
Hartwell, Hunt and Nurse were awarded Nobel Prize in
2001 for their contributions in elucidating the cell cycle
regulation. Four types of cyclins (A, B, D and E) and Fig. 42.4: Cell cycle phases (total 20-22 hours)
Chapter 42: Control of Gene Expression 595
·- ·
Rb is removed bl( tumor antigen;
G2 checkpoint ---+ No inhibition
•••
••••
Rb maximum phosphorylated
Rb phosphorylated
•-
.' No inhibition
Rb inhibits G1 checkpoint
G1 to Sentry
GO (resting)
Fig. 42.5: Cell cycle controls or Checkpoints. Retinoblastoma protein inhibits cell cycle at G1 checkpoint. Body ,circumvents this block
by phosphorylation of Rb protein. This is done normally by cyclin D-CDK. Tumor antigens will attach with Rb protein, so Rb inhibition is
lost; there will be uncontrolled cell division, leading to cancer
I REGULATION OF
GENE EXPRESSION
Induction and Repression
called inducers. Induction is turning "on" the switch of the
gene. Repression is turning "off" the f)ene expression.
Francois Jacques
Sydney Robert JohnE BruceN
Jacob Monad
Brenner Horvitz Sulston Ames
NP 1965 NP 1965
1920-2013 1910-1976 NP2002 NP2002 NP 2002 b. 1928
b. 1927 b. 1947 b. 1942
The Lac Operon
operator region. So there is no repressor molecule at
Operon is a unit of gene expression; it includes struc- the operator site. Now, RNAP can transcribe the struc-
tural genes, control elements, regulator/inhibitor gene, tural genes, which are then translated (Fig. 42.6B). Thus
promoter and operator areas. In the bacterial cell, the lactose switches the genes "on". Lactose induces the
z gene encodes beta-galactosidase, the enzyme which synthesis of lactose utilizing enzymes. Hence, lactose is
hydrolyzes lactose to galactose and glucose. The Y
an inducer of these genes and the mechanism is said
gene is responsible for production of a permease which
to be derepression of the gene.
transports lactose and galactose into the cell. The A
gene codes for thiogalactoside transacetylase. Since Z, Clinical Applications
y and A code for the structure of the proteins, they are
Lactase in human intestine is an inducible enzyme.
called structural genes. These three genes are present
Clinical manifestations of lactase deficiency and lactose
as contiguous segments of DNA (Figs 42.6). The trans-
intolerance are described in Chapter 10. In humans,
cription of these genes start from a common promoter
(P), located close to the Z gene. The RNA polymerase examples of derepression include induction of trypto-
binds to the promoter and transcribes these three struc- phan pyrrolase, and transaminases by glucocorticoids;
tural genes as a single mRNA. as well as ALA synthase by barbiturates.
Operon
Inducer •
•• • •
=t=
Inducer + repressor
!
Promoter Operator Structural (Lactose) now repressor
cannot bind
I Iz y A
RNAP binds -+
- I
' "ii, !
RNAP
Repressor bound
to operator site RNAP moves on
a Structural genes
Not transcribed
Structural genes
now transcribed
Figs. 42.6A and B: (A) Repression of Lac operon. When lactose is absent, repressor molecules bind to the operator site. So RNAP
cannot work, and genes are in "off' position. (B) Induction or Derepression of Laci operon. Lactose attaches to repressor; so repressor
cannot bind to operator site which is free; genes are in ·on" position; protein is synthesized
l
Ai.A synthase
l
~:::~/ \ • • Co-repressor (Heme)
VIRUSES
•U:- Clinical Case Study 42.1
Viruses are absolute parasites on living cells. They con-
tain only the bare minimum of genetic information for A healthy 10-year-old boy interested in sports started
survival and replication. Salvador Luria in 1942 showed complaining of muscle cramps and weakness of lower
that viruses are visible only by electron microscopy. limbs after the sports. On examination, he had mild
Alfred Hershey proved that nucleic acids, but not pro- wasting of the lower limb muscles. History revealed that
teins, are the genetic material in viruses. Both got Nobel his mother had a younger brother, who had a similar ill-
Prize in 1969. Viruses generally bind to specific recep- ness to which he succumbed around the age of 20.
tors on the host cell surface. For example, binding of HIV A. What is the most probable cause?
to CD4 receptor occurs in lipid raft areas through GP120 B. What are the investigations to be done?
(glycoprotein of the virus). Influenza virus binds NANA C. How does history give a clue in this case?
(N-acetyl neuraminic acid) residues on glycoprotein D. What is the prognosis?
receptors on cell surface. After entry into the host cell, E. Explain the nature of inheritance in this case.
the viruses utilize the host cell machinery for growth and 0
replication. • · Clinical Case Study 42.1 Answer
X-linked muscular dystrophy. Creatinine in urine and CK
Epigenetic Regulation in blood will be increased.
Yet another mechanism for gene regulation occurring in
both prokaryotes and eukaryotes is by the phenomenon
(b_EARNING POINTS, CHAPTER 42
known as epigenetic regulation. The important epige- 1. Mutations can be acceptable (Hb Bristol), partially
netic mechanisms are DNA methylation and histone acceptable (HbS) or unacceptable (HbM).
modification. External or environmental factors causes 2. Cell cycles have 5 phases: GO, M, G1 , Sand G2.
genes to switch on or off. Such changes may or may 3. Operon is a unit of gene expression. It includes
not be heritable. This is especially important during the structural genes, control elements, regulator/inhibitor
phenomenon of cellular differentiation. gene, promoter and operator areas, e.g. Lac operon.
42-1 . All the following traits are inherited through the X B. 25 % of children will be carriers
chromosome, except: C. 25 % of children will be normal
A. Sickle cell anemia D. All the children will be genotypically abnormal
B. Hemophilia 42-3. All the following lead to cancer, except
C. Glucose-6-phosphate dehydrogenase deficiency A. Promoter insertion
D. Duchenne type muscular dystrophy B. Chromosomal translocation
42-2. In autosomal dominant inheritance, if only one C. Point mutation
parent is affected: D. Trinucleotide expansion
A. 50% of children will be sufferers 42-4. Point mutations may be silent because:
Chapter 42: Control of Gene Expression 599
A. A change in third base of the codon may produce 42-11. In the process of transduction:
a synonym A. Bacteria takes up DNA from the surrounding
B. Change in base sequence is corrected during medium
transcription 8 . New genes are produced by accumulation of
C. Non-specific base pairing between mRNA and DNA mutations
D. High fidelity of transcription of mRNA C. Normal cell is transformed into a malignant cell
42-5. HbS (Sickle cell anemia) is a result of D. Virus carries some part of the genetic information
A. Transversion mutation from one host to another host
B. Transition mutation 42-12. All are correct with retroviruses, except
C. Nonsense mutation A. Can use their RNA as template
D. Frameshift mutation B. Have reverse transcriptase
42-6. Mutagenicity of a compound is checked by: C. Can multiply in host cells
A. Hay's test B. Guthrie test D. Are inactivated after the first multiplication cycle
C. Ames' test D. FISH test
42-13. Frameshift mutation results from
42-7. In which phase of the cell cycle synthesis of deoxy-
A. Substitution of a single base
ribonucleotide triphosphate is highest?
8 . Deletion of a single base
A. GO B. G1
C. Addition of a codon
C. S D. M
D. Deletion of a codon
42-8. Which hormone does not act at the level of trans-
42-14. Abnormal genotype and normal phenotype is seen
cription?
in mutation of
A. Cortisol B. Calcitriol
A. Splice site B. Intervening sequences
C. Aldosterone D. Calcitonin
42-9. Viruses may have all the following fates within the C. Terminator codon D. Transcription factors
host cells, except 42-15. When GAC is replaced by GAA, the result is:
A. Virus particles may multiply within host cells A. Back mutation B. Deletion mutation
B. Viral DNA may be incorporated into host cell DNA C. Silent mutation D. Conservative mutation
C. Viral DNA may be transcribed 42-16. The cell cycle is regulated by
D. Viruses may suppress oncogenes A. Availability of nucleotides
42-10. All the following are antiviral drugs, except B. Presence of inducers in cells
A. Cytosine arabinoside B. Acyclovir C. Cyclin dependent kinase
C. Ribavirin D. Azidothymidine D. DNA dependent kinases
42-1. Give examples of X chromosome linked trans- 42-6. What is cell cycle?
mission. Cell cycle refers to the sequence of events that occur
Hemophilia; Glucose-6-phsophate dehydrogenase during the growth and cell division.
deficiency; Duchenne type muscular dystrophy. 42-7. In which phase of the cell cycle, DNA synthesis is
42-2. What is a mutation? maximum?
An alteration in the genetic material results in a muta- During S phase.
tion. 42-8. What is the p53?
42-3. Give an example. It is an oncosuppressor gene product.
HbS or sickle-cell hemoglobin is produced by a mu- 42-9. What is an operon?
tation of the beta chain in which the 6th position is One or more structural genes, together with their
changed to valine, instead of the normal glutamate. operator gene.
42-4. Give examples for mutagens. 42-10. Give examples of enzyme induction.
Acridine orange: X-rays; gamma rays; Methyl cholan- Beta galactosidase by lactose; Tryptophan pyrrolase
threne by glucocorticoid and ALA synthase by barbiturates.
42-5. What is apoptosis? 42-11 . Give an example of repression.
Prog rammed cell death. ALA synthase by heme.
_ _ _ _ _ Chapter 43
Recombinant
DNA Technology and
Gene Therapy
Chapter at a Glance
The learner will be able to answer questions on the following topics:
Recombinant DNA technology Molecular cloning
Restriction endonucleases Gene therapy
Vectors
C ECOMBINANT DNA
ECHNOLOGY
-----------
8 io tech noIog y may be defined as "the method by which
Risk of Contamination is Eliminated
It is now possible to produce a biological substance with-
out any contamination. Hepatitis, caused by the hepa-
titis B virus (HBV), is highly contagious. A vaccine is
a living organism or its parts are used to change or to
prepared from the hepatitis B virus surface proteins,
incorporate a particular character to another living
which will give protection from infection. Originally the
organism". Biotechnology involves the application of
virus was isolated from pooled blood of patients, and the
scientific principles to the processing of materials by
specific protein was isolated. However, blood of hepatitis
biological agents. The use of new varieties of micro-
patients is known to be highly infective. It is absolutely
organisms to breakdown pollutants in soil or water to harm-
essential to make sure that the preparations of vaccines
less end products is known as bioremediation. Genetic
or clotting factors are free from contaminants such as
recombination is the exchange of information between
hepatitis B particles. Recombinant DNA technology pro-
two DNA segments. This is a common occurrence within
the same species. But by artificial means, when a gene vides the answer to produce safe antigens for vaccine
of one species is transferred to another living organism, production.
it is called recombinant DNA technology. In common
Specific Probes for Diagnosis of Diseases
parlance, this is known as genetic engineering.
Specific probes are useful for:
Applications of i. Antenatal diagnosis of genetic diseases. For example,
Recombinant Technology many of the single gene defects (e.g. cystic fibrosis,
phenylketonuria, etc.) could be identified by taking
Quantitative Preparation of Biomolecules
cell samples from fetus.
If molecules are isolated from higher organisms, the ii. To identify viral particles or bacterial DNA in sus-
availability will be greatly limited. For example, to get pected blood and tissue samples.
1 unit of growth hormone, more than 1000 pituitaries iii. To demonstrate virus integration in transformed
from cadavers are required . By means of recombinant cells.
technology, large scale availability is now assured. iv. To detect activation of oncogenes in cancer.
Chapter 43: Recombinant DNA Technology and Gene Therapy 601
.Yi-rn-
C TTAA G
Hindlll Haemophilus influenzae Rd A 1 AGCT l T
T TCGA A
Taql Thermus aquaticus T~A
A GC T - C- T- C- G- A- T- G Sticky ends G- T- G- G-
GTT j AAC
Hpal Haemophilus
parainfluenzae CAA ! TTG 111 11 11 I
-G-A- G- C- T-A-ef T-T-A-AI
v. To pinpoint the location of a gene in a chromosome. Fig. 43.1 : EcoRI enzyme cuts the bonds marked with red arrow.
This results in the sticky ends
vi. To identify mutations in genes and for pedigree
analysis: point mutations, deletions, insertions and
rearrangements of DNA could be identified. Sickle
cell disease is an example of point mutation (see
Chapter 23). The substitution of T for A in the tem-
plate strand of DNA in the beta globin gene changes
the Mstl l restriction site. Thus normal, heterozygous
and homozygous individuals in the family could be Paul Hamilton Wemer Daniel
Berg Smith Arber Nathans
identified.
NP 1980 NP 1978 NP 1978 NP 1978
b. 1926 b. 1931 b. 1929 1928-1999
Gene Therapy
An important application of recombinant technology is Restriction Sites
in gene therapy. Normal genes could be introduced into
Restriction endonucleases have specific recognition
the patient so that genetic diseases can be cured. These
sites where they cut the DNA. (Table 43.1 ). There are
techniques are described later in this chapter.
more than 800 such enzymes now available commer-
Restriction Endonucleases (RE) cially. These enzymes recognize specific sequence with
In order to transfer a gene, it is to be fi rst selectively split palindrome arrangement. Palindrome in Greek means
from the parent DNA. This is usually achieved by restric- "to run backwards". It is similar to a word that reads back-
tion endonucleases which are referred to as "molecular wards or forwards similarly, e.g. "madam". These are
scissors". also called inverted repeat sequences, which means
Werner Arber showed that certain enzymes of bac- the nucleotide sequence in 5' to 3' direction is the same in
teria restrict the entry of phages into host bacteria. both strands. The resultant DNA fragments will generally
Hence, the name restriction endonucleases. Hamilton have overlapping sticky ends (Fig.43.1).
Smith in 1970 isolated the first restriction enzyme beta
Hind-I. Daniel Nathans in 1971 for the first time applied I VECTORS
the enzyme to cut the DNA. All the three got Nobel Prize
in 1978. Paul Berg (Nobel Prize 1980) developed the In order to introduce the human gene into bacteria, at
cutting technique for recombinant DNA. The restriction first, the gene is transferred to a carrier, known as a
endonucleases are named after the species and strains vector. Most commonly used vectors are plasmids.
of bacteria and the order of discovery. For example, the Plasmids are circular double-stranded DNA molecules
enzyme EcoRI is isolated from Escherichia coli RY13 seen inside bacteria. In nature, plasmids confer antibiotic
strain. The Roman numeral "one" indicates the order of resistance to host bacteria. This feature has profound
discovery of an enzyme from that species. Restriction significance in clinical practice because, antibiotic resis-
enzymes are isolated from bacteria. tance property is exchanged between bacteria. Plasmids
602 Section E: Molecular Biology
l
meric DNA or hybrid DNA or Recombinant DNA. A sum-
5' dTTT• • • • • • • • • • • • • • • • • • - 3' DNA mary of the procedure is given in Figure 43.3.
3' AAAMAMMAMMMMMMMMM 5' RNA
A circular plasmid vector DNA is cut with a specific
Extend 3' end of DNA by terminal deoxy nucleotidyl
transferase + dCTP
restriction endonuclease (RE). If EcoRI is used, sticky
l
ends are produced with TTAA sequence on one DNA
5' dTTT· • • • • • • • • • • • • • • • - • CCCC 3' DNA
3' AAAAI\I\AI\I\I\I\AI\I\I\AAI\AA/+,I\I\AI\AAl\1\1\ 51 RNA
strand, and AATT sequence on the other strand (Table
l
43.1 and Fig. 43.3). The human DNA is also treated with
RNA hydrolyzed by RNAase; making single stranded DNA
the same RE, so that the same sequences are gene-
rated at the sticky ends of the cut piece. Then the vector
s· ,m-------------------•cccc ,, DNA
DNA and human cut-piece DNA are incubated together
DNA double strand is primed by a piece of oligo dG
so that annealing takes place. The sticky ends of both
vector and human DNA have complementary sequences,
5' dTTT • · • • • • • • • • • • • • • • • • • • CCCC 3' DNA and therefore they come into contact with each other.
1 GGGG 5'
Human mRNA
!
cDNA copy (see Fig. 43.2)
!
Plasmid vector cleaved by
specific restriction
. • endonuclease;
y
Plasmid cut
by ECoRI
0 _'
, Donor DNA
cut by ECoRI
vector carrying human
gene (see Fig. 43.3)
i
Selection of bacteria carrying
1• 0 • 00
The human proteins can be harvested from the bacterial 1. Recombinant human Insulin
2. Recombinant human growth hormone (HGH, somatotropin)
cu lture. A summary of the DNA recombinan t technology and other human hormones (e.g. FSH)
is shown in Figure. 43.4. Depending on the length of the 3. Recombinant blood clotting factor VIII and other clotting
factors (Factor IX, tPA, hirudin)
DNA insert, different vectors like plasmids, cosmids and 4. Recombinant hepatitis B vaccine, HPV vaccine, etc.
artificial chromosomes are used. 5. Cytokines and growth factors (interferon, interleukins, etc.)
6. Monoclonal antibodies and other related products (rituximab,
trastuzumab, etc.)
Human Recombinant Proteins 7. Recombinant enzymes (acid a glucosidase [myozyme),
alpha-L-iduronidase [aldurazymel)
Hundreds of human proteins are now being synthe- 8. Recombinant HIV protein for HIV ELISA testing
9. Herbicide and insect resistant crops
sized by the recombinant technology. A list of important 1O. Other products: Bone morphogenic protein (BMP), albumin,
proteins produced by recombinant technology is given fibrinolytic and thrombolytic agents, etc.
in Box 43.1.
set of human DNA contains about 3 billion base pairs
Human Genome Project (HGP) (one cell contains 2 sets) and about 10,000 genes. The
"Book of Human Life" contains "23 Chapters", as the
The US Department of Energy together w ith the US
23 chromosomes.
National Institutes of Health started this project in 1990.
The impact of HGP will be on all branches of medi-
James Watson (co-discoverer of the structure of DNA,
cine and related health sciences. It is now possible to
Nobel laureate, 1962) was the first head o f the project;
isolate any human g ene of interest. Many previously
later Francis Collins succeeded him. The project included unknown genes have been identified.
scientists from 16 centers all over the world, mostly By December 1998, human chromosome 5 (about
from USA, with coordination from laboratories of Britain , 6% of human genome) was sequenced completely.
France, Japan and Germany. So, the Project was The final version of the sequence of the entire human
named as International Human Genome Sequencing genome was completed in 2003.
Consortium. Pharmacogenomics is a recently emerged science
It is one of the greatest achievements of humanity. from the genome project; it is the use of genetic infor-
The ambitious project was to decode the whole human mation towards the development of new drugs and their
genome and to sequence the whole human DNA. One targets of action.
604 Section E: Molecular Biology
GENE THERAPY
Gene therapy was once considered a fantasy. However, Take cells
from patient
thousands of individuals have already undergone human
clinical trials. A great leap in medical science has taken
place on 14th September, 1990, when a girl suffering
from adenosine deaminase deficiency (severe immuno-
©
Patient·s~II
now cont ns
normal
deficiency) was treated by transferring the normal gene gene
for adenosine deaminase.
Summary of the Procedure deleted from the wild type retrovirus, rendering it inca-
pable of replication inside human body. Then the human
1. Isolate the healthy gene along with the sequence
gene is inserted into the virus. This is introduced into a
controlling its expression.
culture containing packaging cells having gag, pol and
2. Incorporate this gene into a carrier or vector as an
env genes (Fig . 43.6). These cells provide the neces-
expression cassette.
3. Finally deliver the vector to the target cells. sary proteins to pack the virus. The replication-
deficient, but infective, retrovirus vector carrying the
How the Genes are Introduced? human gene, now comes out of the cultured cells. These
There are three ways of applying gene carrying vectors: are introduced into the patient. The virus enters into the
a. Ex vivo strategy: Where the patients' cells are cul- target cell via specific receptor. In the cytoplasm of
tured in the laboratory, the new genes are infused the human cells, the reverse transcriptase carried by
into the cells; and modified cells are administered the vector converts the RNA to proviral DNA, which is
back to the patient (Fig. 43.5). integrated into the target cell DNA. The normal human
b. In situ strategy: When the expression cassette is gene can now express (Fig. 43.6).
injected to the patient either intravenously or direc- Advantages of retroviruses: The virus is modified,
tly to the tissue. and replication deficient. So infection with viral particle
c. In vivo strategy: Where the vector is administered is limited to one cycle, and is very safe. They can infect
directly to the cell, e.g. CF (cystic fibrosis ) gene to a wide variety of human cells. This strategy is very suit-
the respiratory tract cells. able for treatment of all diseases produced by single
gene mutations.
The Vectors Adenoviruses, Liposomes and Gene gun are some
other commonly employed carriers for transfer of foreign
Different vector (carrier) systems used for gene delivery
genes to host cells.
are: retroviruses, adenoviruses, adeno associated viral
vectors and herpes simplex viruses. Nonvirus systems
Accomplishments
include liposomes, plasmids and physical methods.
Gene therapy is effective in inherited disorders caused
Retroviruses by single genes. Several clinical trials have been
Retroviruses are RNA viruses that replicate through conducted. Success stories are few. The best developed
a DNA intermediate. Moloney murine leukemia virus and most successful cell therapy is hematopoietic stem
(MMLV) is commonly used. The gag, pol, env genes are cell transplantation (HSCT). Another established gene
Chapter 43: Recombinant DNA Technology and Gene Therapy 605
l
(SCIO) lymphocytes; by retrovirus
Delete gag, pol, anv genes; In that place add
Cassette containing human gene 2, Duchenne muscular Oystrophin gene on short arm of
dystrophy (OMO) X chromosome; by retrovirus
r-"""-~
LTR I '
3. Cystic fibrosis (CF) CFTR gene on chromosome 7 to
bronchial epithelium; adenovirus
4. Familial hyper- LDL receptor gene on chrom 19 to
cholesterolemia hepatocytes; retrovirus
5, Hemophilia A_a nd 8 genes for factor VIII and IX into
Naked viral RNA has to get fibroblasts; retrovirus
protein coat to enable them to
enter into human host cell. 6. Cancer Activation of p53 (tumor suppressor
So, defective virus is put gene) by liposome
into packaging cells, which
provide the viral coat 7. Leber's hereditary Introducing the gene for the enzyme
optic neuropathy (isomerohydrolase) using an adenoviral
vector directly to the retina
Receptor mediated
entry of retrovirus into
STEM CELLS
human host cell
Stem cells are defined as cells with the capacity for
self-renewal and having potential to differentiate into
Reverse transcriptase
progenitors of different lineages which ultimately give
of virus copies RNA rise to mature tissues. Mario R. Capecchi, Sir Martin J
into virus ONA
Evans and Oliver Smithies were awarded Nobel Prize
in 2007 for their discoveries of principles for introduc-
Integrates with host ing specific gene modifications in mice by the use of
DNA inside nucleus
embryonic stem cells. Stem cells have the ability to divide
for an indefinite period. They can give rise to a variety gene of one species is transferred into another under
of specialized cell types. This phenomenon is known as laboratory conditions, the technique is called
developmental plasticity. Stem cells can be isolated from recombinant DNA technology or genetic engineering.
embryos, umbilical cord as well as from adult bone marrow. 2. Restriction endonucleases (RE), also known as
Plasticity is more for embryonic stem cells. Plasticity is 'molecular scissors' cut at sequences which are
defined as the ability of stem cells from one germinal palindromes. Each RE is characterized by a spe-
layer to give rise to tissues of another germinal layer. cific 'restriction site'.
Stem cells have the unique capacity to produce 3. Plasmids are commonly used vectors. They pro-
unaltered daughter cells (renewal) and also to generate vide antibiotic resistance to their host bacteria. This
specialized cells (potency). Stem cells may be capable property is used as a marker in genetic engineering.
of producing all types of cells of the organism (totipo- 4. A vector carrying a foreign DNA is called 'Chimeric
tent), or able to generate cells of the three germ layers DNA'.
(pleuripotent). Active research is being done to utilize
5. The process of introducing a plasmid into a host is
stem cells in the treatment of the following diseases:
called transfection.
stroke, brain injury, Alzheimer's disease, Parkinsonism,
6. Gene therapy involves the delivering genes to gen-
wound healing, myocardial infarction, muscular dystro-
erate a therapeutic effect by correcting an existing
phy, spinal cord injury, diabetes, cancers.
abnormality.
General belief was that only stem cells are plastic,
Introducing genes involves three ways of applying
and the differentiated matured cells will lose their plas-
gene carrying vectors, ex vivo, in situ and in vivo.
ticity. But Sir John Gurdon and Shinya Yamanaka have
7. Retroviruses, adenoviruses and herpes simplex
independently discovered that mature cells can be
reprogrammed to become pluripotent; they were awar- viruses have been used carrier systems in human
ded Nobel Prize in 2012. gene studies.
8. Diseases for which gene therapy has been attemp-
·LEARNING POINTS, CHAPTER 43 ted are severe combined immunodeficiency (SCI O),
Duchenne muscular dystrophy (DMD), cystic fibro-
1. Genetic recombination involves the exchange of
sis, hemophilia.
information between two segments of DNA. When a
C. They replicate independent of bacterial DNA replication 43-9. The c DNA is prepared by using the enzyme:
D. They confer antibiotic resistance to host bacteria A. RNA polymerase B. DNA polymerase
43-5. All the following are nucleic acids, except C. Reverse transcriptase D. Restriction endonuclease
A. Plasmids B. Prions 48-10. Which of the following is not used as a vector in
C. Cosmids D. Virions molecular cloning?
43-6. Synthesis of recombinant DNA (rDNA) requires all A. Plasmid B. Cosmid
the following, except: C. Artificial chromosome D. Hapten
A. Restriction endonuclease 43-11. Restriction endonucleases
B. RNA primer
A. Cleaves DNA at splice sites
C. Plasmid vector
B. Protects bacterial genome
D. DNA ligase
C. Hydrolyses DNA from 5' end
43-7. Which is not amenable to gene therapy?
D. Inhibits human DNAP
A. Hemophilia
43-12. The enzyme deficiency which was first corrected
B. Severe combined immunodeficiency
by gene therapy is:
C. Cystic fibrosis
A. Adenosine deaminase
D. Infective hepatitis
43-8. All are vectors for gene therapy, except: B. HGPRTase
A. Proteasomes B. Liposomes C. Glucose-6-phosphatase
C. Adenoviruses D. Retroviruses D. APRTase
43-1. What are restriction endonucleases? They are small circular, extra chromosomal DNA
They act as "molecular scissors". These enzymes recog- present in bacteria. They are used as vectors in DNA
nise specific sequences in the DNA, and then cleave recombinant technology.
at those sites. They are useful in recombinant DNA 43-4. What are the vectors used for gene therapy?
technology. Retrovirus; Adenovirus; Plasmid, liposome complex.
43-2. What are required for preparing a recombinant 43-5. Name some diseases in which gene therapy is
DNA molecule? used successfully?
Restriction endonuclease; Plasmid vector; DNA ligase. Severe combined immunodeficiency; Duchenne mus-
43-3. What are plasmids? cular dystrophy; cystic fibrosis; hemophilia
_ _ _ _ _Chapter 44
Molecular Diagnostics and
Genetic Techniques
Chapter at a Glance
The learner will be able to answer questions on the following topics:
D DNA hybridization techniques Restriction fragment length polymorphism
D Southern, Northern and Western blots D Polymerase chai n reaction (PCR)
D Animal cloning D Monoclonal antibodies
D Molecular cloning DNA sequencing
Molecular (DNA based) diagnostics is rapidly becoming Nick-translation. Non-radioactive probes are also
a standard laboratory procedure for a large number of available and fluorescent probes are easier to detect.
disorders. Many DNA based molecular techniques are
being used in clinical practice. In addition to the diag-
Southern Blot Technique
nosis of a particular patient, molecular diagnostics can It is based on the specific base pairing properties of
be used for diagnosis of index cases, prenatal diagnosis complementary nucleic acid strands. This technique is
and screening. In many cases, the diagnosis is based therefore based on DNA hybridization (Fig. 44. 1). The
on detecting mutations in genes involved, but in some blot technique was developed by EM Southern in 1975.
cases it may be to detect specific OMA sequences as This is used to detect a specific segment of DNA in the
in infectious diseases. In this chapter, a brief review of whole genome.
major molecular techniques employed in clinical diagno- DNA is isolated from the tissue. It is then fragmented
sis is given. by restriction endonucleases. The cut pieces are
I HYBRIDIZATION AND
b LOT TECHNIQUES 3'
Double stranded DNA
the radioactivity is tagged into the gene. This is called Fig. 44.1: DNA-DNA hybridization
Chapter 44: Molecular Diagnostics and Genetic Techniques 609
--
1. DNA cut with
(for DNA) (for RNA) (for protein)
II
restriction enzymes;
DNA cutpieces or
Electrophoresis
--+ on agar gel
RNA or proteins
placed in the well
and electrophoresed
2. DNA fragments
3
blotted on nitrocellulose
membrane
--
4. Bands visualized DNA* cDNA * Antibody* Radioactive ( • )
by autoradiography • • • probe added
~--$
(b) diagnosis of B-cell lymphoma by demonstration of Prepare cDNA probe Prepare microarray chip
reduced light-chain mRNA (c) determination of amplifi- RNA from patient
cation of HER2/neu in breast cancer and (d) diagnosis of
various types of lymphomas.
l l te;~:ue
Microarray Technique
A microarray is a series of thousands of microscopic
spots of DNA oligonucleotides (probes) on a slide that
~ =--
are used to hybridize a particular cDNA or cRNA sample Label~
fluorescent
(target). Microarray generally contains 5,000 to 20,000
genes in a gene chip. Each gene (DNA probe) solution
dye +
Hybndae +----
is prepared, a nanoliter sized drop is added to one well
in the plate; the droplet dries in a few seconds, leav- Fig. 44.4: Microarray technology
ing the DNA in the well; thousands of such wells, each
containing a different gene is prepared. The DNA from A single adult cell contains all the genetic material
the clinical sample (or PCR amplified DNA) is tagged for making the entire animal. In the case of Dolly, it was
with a fluorescent dye. These tagged DNA fragments a cell from the udder (mammary cell).
are then incubated with the chip. After the DNA-DNA Next, the mammary cell and denucleated oocyte
hybridization is over, unbound DNA is washed away. were fused. The fused cell was then implanted into the
The surfa ce of the microarray is then scanned with a uterus of a surrogate mother, which delivered the baby
laser beam. Color intensity indicates the extent of hybridi- in course of time.
zation (Fig. 44.4). In a similar manner, specific proteins
can be identified using monoclonal antibody microarray. Applications of Cloning of
Microarray tests are useful in diagnosis of (a) Cancer; Animals and Plants
(b) Infectious diseases and (c) Allergy. 1. Animals with genetically desirable traits could be
bred more efficiently, e.g. cows yielding more milk.
Animal Cloning 2. Biopharmaceuticals: By November 1998, the first
goats were born , who were genetically engineered
The term cloning has two broad meanings. When a gene
to produce milk containing antithrombin Ill. Any
of higher organism is introduced into a bacterial DNA, it
human protein could be introduced into the make up
is called "cloning of the gene" or "molecular cloning";
of goat or cow and get the desired protein cheaply
details of which are described in Chapter 43. When a
through milk. Eggs have been genetically manipu-
cell from an animal is grown to an exact duplicate of
lated to produce interferon and insulin in the egg-
that animal, it is known as "cloning of an animal" or
white.
"somatic cloning".
3. Cloning is successfully employed in agriculture, to
It made big news when Ian Wilmut and Keith Camp-
propagate plants such as rubber, banana, orchids,
bell of Scotland cloned a sheep named "Dolly" in July
etc. If a good yielding rubber is available, it is cloned
1996. (Dolly died naturally in 2002, not due to any com-
so that, thousands of progenies of the same quality
plications of cloning). Today the sheep, tomorrow it could
could be produced within a short time.
be the shepherd. So this raised a number of moral, ethi-
cal and legal issues.
Disadvantages of Cloning
Cloning will never replace selective breeding. Cloning
Dolly and Bonnie. Roslln Institute, Scotland halts any further progress. Cloning can produce the ani-
reported that the first cloned sheep "Dolly" mals/plants with the same characteristics; new charac-
was born in 1996. Her first lamb, Bonnie,
teristics could not be developed. The cloned animal and
was born in 1999. Dolly died in 2002.
parent need not be exactly identical. First, mitochondrial
DNA invariably comes from the egg. Second, DNA in
Chapter 44: Molecular Diagnostics and Genetic Techniques 611
Cloning in Medicine
Diagnosis of Genetic Diseases .__ __________ _,1
1.15 kbp fragment
AA AS ss Genotype
Various genetic diseases can be identified by using Normal HbS trait HbS disease Phenotype
appropriate probes from defective genes. A point muta-
Fig. 44.5: Southern blot analysis of DNA from normal. HbS trait
tion may destroy or create a restriction enzyme cleavage and s ickle cell anemia disease
site. Then the fragment size produced from normal gene
and mutated gene will be different. This can be easily or individuals in which the disease has not yet been
identified by Southern blot. manifested.
For example, sickle cell anemia is caused by a point Such a test is possible for prenatal diagnosis also.
mutation. In the beta chain of the hemoglobin, the 6th DNA from cells collected from amniotic fluid can be used
amino acid normally is glutamate. In sickle-cell anemia, for Southern blot analysis.
this is altered to valine. In the DNA, this is seen as a Duchenne muscular dystrophy (DMD) is a degen-
change from normal T replaced by abnormal A nucleo- erative disease of muscle affecting only male children.
tide (T to A substitution). The normal gene for HbA has The gene is in the X chromosome. This gene produces
the following nucleotide sequence. a protein called dystrophin with 3700 amino acids. It
CC t TGA GG is one of the largest human genes known. In DMD pa-
Coding strand tients, the gene for dystrophin is mutated. This could be
GG AC(!) t CC identified by using a cDNA probe for dystrophin. In the
Template strand Southern blot analysis, the fragment corresponding to
The arrows indicate the cleaving site for the restric- this gene will be absent.
tion enzyme Mst-I1. In the sickle cell anemia gene, the
underlined T is replaced by A, and the DNA sequence of DNA Finger Printing in
the HbS gene is as follows: Forensic Medicine
CC TGT GG There are tandem repeats (TR) in chromosomes. These
Coding strand are short sequences of DNA, located at scattered sites.
GG AC@ CC The number of these repeat units varies from person to
Template strand person, but is unique for a particular person. Therefore,
This alteration in base sequence abolishes the recog- it serves as a molecular fingerprint. It is also known as
nition site for this RE. But other Mst-II cleavage sites are ONA profile. Probability of similarity between two per-
preserved. sons is only 1 in 3 x 1010 persons. The technique is used
T herefore, analysis of DNA from AA (normal HbA), to pinpoint the culprit of the crime, and also in disputes
heterozygous AS (Sickle-cell trait) and homozygous SS of parenthood. DNA can be isolated from stains on
(Sickle-cell disease) individuals fall into 3 different pat- clothing made of blood.
terns in Southern blot technique (Fig. 44.5). The muta-
Restriction Fragment Length
tion eliminates one restriction site for Mst 11 enzyme, and
hence a larger fragment is present in sickle cell anemia.
Polymorphism
This becomes useful as a diagnostic test for the The human genome contains hundreds of variations in
presence of the disease allele in heterozygotes (carriers) base sequences that do not affect the phenotype. The
612 Section E: Molecular Biology
alleles are common in more than 1% of the total popula- Old Strand
- -
ID
tion. The existence of two or more types of restriction c1::c:
1 ===!-~ ~> New Strand
New Strand
fragment patterns is called restriction fragment length < I I I
Old Strand
polymorphism (RFLP). This can be used as a genetic Cycle 2 =
I Add dNTPs and
marker. +Taq polymerase at n •c 8 strands
i
restriction fragments. to '>O rt' nQ Ado primers dNTPs, Cycle 3 =
l m, I l, 16 strands
Karry Mullis invented this ingenious method in 1989, who Step 2: Priming (Annealing): The primers are
was awarded Nobel Prize in 1993. Polymerase chain re- annealed by cooling to 50°C for 0.5 to 2 minutes. The
action (PCR) is an in vitro DNA amplification procedure primers hybridize with their complementary single stran-
in which millions of copies of a particular sequence of ded DNA produced in the first step.
DNA can be produced within a few hours. It is like xerox Step 3: Extension: New DNA strands are synthe-
machine for gene copying. sized by Taq polymerase. This enzyme is derived from
The flanking sequences of the gene of interest bacteria Thermus aquaticus that are found in hot springs.
should be known. Two DNA primers of about 20- 30 Therefore the enzyme is not denatured at high tempera-
nucleotides with complementary sequence of the flank- ture. The polymerase reaction is allowed to take place
ing region can be synthesized. The reaction cycle has at ?2°C for 30 seconds in presence of dNTPs (all four
the following steps: deoxy ribonucleotide triphosphates). Both strands of
Step 1: Separation (Denaturation): DNA strands DNA are now duplicated (Fig. 44.6).
are separated (melted} by heating at 95°C for 15 sec- Step 4: The steps of 1, 2 and 3 are repeated. In each
onds to 2 minutes (Fig. 44.6). cycle, the DNA strands are doubled. Thus 20 cycles
Chapter 44: Molecular Diagnostics and Genetic Techniques 613
provide for 1 million times amplifications. These cycles BOX 44.1: Apphcat1ons of PCR
are generally repeated by automated instrument, called Detection of infectious diseases
Thermal cycler. AIDS, tuberculosis, CMV, H1 N1 , hepatitis C, etc.
Step 5: After the amplification procedure, DNA Detect 3 sexually transmitted diseases in one swab- herpes,
papilloma virus, Chlamydia.
hybridization technique or Southern blot analysis with a
PCR can diagnosis even one bacteria or virus present in the
suitable probe, shows the presence of the DNA in the specimen.
sample tissue. Latent viruses can also be diagnosed.
Detection of variations and mutations in genes
Detects people with inherited disorders and carriers
. Clinical Applications of PCR Track presence o r absence of DNA abnormalities characteristic of
cancer
1. Diagnosis of bacterial and viral diseases: In early
Prenatal diagnosis of genetic disorders.
phases of tuberculosis, the sputum may contain PCR combined with RE cleavage and Southern blot t ing is used for
only very few tubercle bacilli, so that usual acid mutation detection.
fast staining may be negative. But PCR can detect PCR and the law
DNA fingerprinting-can multiply small amounts of DNA found in
even one bacillus present in the specimen. Any other blood samples, hair, semen, and other body fluids
bacterial infection can also be detected similarly.
The specific nucleotide sequences of the bacilli are
cancer (oncogenes and oncosuppressor genes are
amplified by PCR and then detected by Southern
described in Chapter 48). See also Box 44.1.
blot analysis. If reverse PCR is done, living organ-
isms can be detected. This technique is widely used Reverse Transcriptase PCR (RT-PCR)
in the diagnosis of viral infections like hepatitis C, It is the method used to amplify, isolate or identify a
cytomegalovirus and HIV. known sequence from a cell or tissue RNA library. The
2. Medicolegal cases: PCR allows the DNA from PCR is preceded by reverse transcription (to convert the
a hair follicle or a blood cell to be analyzed.The RNA to cDNA). This is widely used to determine when
restriction analysis of DNA from the hair follicle from and where certain genes are expressed. Instead of Taq
the crime scene is studied after PCR amplification. polymerase described above, Tth polymerase from Ther-
This pattern is then compared with the restriction mus thermophilus may be used. This enzyme has both
analysis of DNA samples obtained from various DNA polymerase and reverse transcriptase activities at
suspects; the culprit's sample will perfectly match high temperature. This allows both cDNA synthesis from
with that of PCR amplified sample. The restriction mRNA followed by PCR amplification . In ordinary PCR,
analysis pattern of DNA of one individual will be very DNA is detected; that DNA could be from a living or non-
specific (DNA fingerprinting); but the pattern will be living organism. But in reverse PCR, mRNA is detected;
different from person to person. This is highly useful that means, it is derived from a living organism. Pres-
in forensic medicine to identify the criminal. ence of HIV RNA in blood can be detected as early as 4
3. Diagnosis of genetic disorders: The PCR techno- weeks after infection.
logy has been widely used to amplify the gene seg-
ments that contain known mutations for diagnosis Real-time PCR
of inherited diseases such as sickle cell anemia, By this method , quantitation of the number of virus pre-
beta thalassemia, cystic fibrosis, etc. sent in a sample can be calculated, e.g. viral load in HIV
4. PCR is especially useful for prenatal diagnosis of or HBV. So, the treatment modalities can be planned
inherited diseases, where cells obtained from fetus and th e response to treatment can be assessed.
by amniocentesis are very few.
5. Cancer detection: PCR is widely used to monitor Multiplex-PCR
residual abnormal cells present in treated patients. By targeting multiple genes at once, additional informa-
Similarly identification of mutations in oncosup- tion may be elicited from a single test run that otherwise
pressor genes such as p53, retinoblastoma gene, would require several times the reagents and time to
etc. can help to identify individuals at high risk of perform.
614 Section E: Molecular Biology
Spleen cells
Aot q"
HGPRT +ve
HAT resistant
lg secretion +ve • Myeloma cell
HGPRT-ve
1• • 1 1 • 1 HAT sensitive
....
lg secretion -ve
/ Proliferation +ve
unwanted.
In the laboratory, monoclonal antibody can be myeloma cells are defective in the enzyme HGPRTase
generated. In this case, only a particular type of antibody and so they lack the salvage pathway for DNA synthesis.
against a specific epitope of the antigen is produced In the culture, HAT medium is used containing
(Fig. 44.7). Monoclonal antibodies were fi rst produced hypoxanthine, aminopterin and thymidine. The amino-
by Georges Kohler and Cesar Milstein in 1975. They pterin, a folic acid antagonist, will inhibit the de novo
were awarded Nobel Prize in 1984. synthesis of DNA. Since both pathways are blocked, the
non-fused myeloma cells also die in the special medium
Production of Hybridoma provided. The only cells that survive are the cells where
The antigen is injected into mice. Spleen cells from the fusion has taken place between normal spleen cells with
immunized mice are fused with mice myeloma cells, so myeloma cells. In this case, normal cells provide the
as to produce a hybrid cell. The hybrid cells now con- HGPRTase enzyme and so DNA synthesis is possible
tain the gene of normal mice as well as the myeloma from the hypoxanthine and thymidine provided in the
cells. (Fig. 44.8). However, hybridization might have medium. The normal cellular genes also provide the
occurred between two normal cells. Normal cells lack the information for specific antibody synthesis. The myeloma
multiplication potential. So all the hybridized normal cells cancer genes provide the endless multiplication drive,
die in the usual culture conditions within 5-6 days. The so that hybrid cells are immortalized (Fig. 44.8).
.:t.
,,.. ,.~ , Chapter 44: Molecular Diagnostics and Genetic Techniques 615
'ti
·-r Table 44.1: Common therapeutic uses of monoclonal antibodies
• • 1
,a,-
-
•~
~ Indication
Transplantation rejection
Non-Hodgkin's lymphoma
Target
CD3
CD20
Georges Cesar
&.~
Walter
- · -
Frederick
Rheumatoid arthritis
Metastatic breast cancer
TNF -a
HER-2
Kohler Milstein Gilbert Sanger
NP 1984 NP 1984 NP 1980 NP1958 and
When growth hormone gene is introduced, the ani-
1946-1995 1927-2002 b.1932 1980
1918-2013 mals became twice as large as their normal counterparts.
The potential applications of these findings are many.
Applications of Monoclonal Antibody Attempts are already under way to increase milk pro-
Monoclonal antibodies are used for immunosuppres- duction in cows by transgenic method . Production of
sion (Basiliximab and Daclizumab), for treatment of biopharmaceuticals, described under the heading appli-
autoimmune diseases (Etanercept, lnfliximab, Adali- cation of cloning of animals, is another example of
mumab), and as anticancer drugs. These are shown transgenesis.
in Chapters 46 and 48. Common therapeutic uses of mono-
DNA Sequencing
clonal antibodies are shown in Table 44.1.
In 1977, two sequencing techniques have been develo-
Advantages of Monoclonal Antibody ped. Sanger's technique is called "controlled termination
In a monoclonal preparation, all the antibody molecules of synthesis"; it uses chain terminating agents. Gilbert
are specific against a particular antigen. Therefore, developed the base-specific chemical cleavage method.
more and more monoclonal antibodies are now com- Both were awarded Nobel Prize in 1980.
mercially produced. These are highly useful to detect (a) Sanger's technique is summarized below: Suppose
serum proteins, (b) enzymes, (c) hormones, (e) drugs, the sequence of the polynucleotide is 3'AACTCGAG-
(f) bacterial antigens, (g) viral antigens, (h) cell surface TA5'. This DNA sample is taken in 4 different test tubes
receptors, (i) HLA antigens and U) cancer antigens. (Fig. 44.9). In all tubes, the Kienow enzyme (DNA poly-
merase without exonuclease activity) and radiolabeled
Single Nucleotide Polymorphism (SNP) TT as the primer are added. In all tubes radioactive
SNP is a change in a single nucleotide, which can pro- dNTPs (all the 4 nucleotides which are labeled with 32P)
are added. Synthesis of a new strand of DNA having a
duce change in one amino acid in a protein by altering
sequence of 5' TTGAGCTCAT3' is started. But in the
single codon. The SNP may not produce clinical symp-
first test tube, ddTTP (2',3'-di deoxy TTP) is also added.
toms. The person will show only an abnormal genotype
The ddTTP will add the T, but it cannot form the next
which is detected by DNA analysis. SNPs are detected
phosphodiester link, and so further chain lengthening is
by single strand conformation polymorphism.
stopped. In other words, ddTTP will stop chains at T.
There are many techniques now available for detec-
Instead of ddTTP, the polymerase might add a normal
tion of mutations. Some of the most commonly employed
dTTP, in which case chain growth will continue till the
methods are Single Strand Conformation Polymor-
next T. Thus in first test tube, TTGAGCT (7 nucleotides)
phism and Conformation Sensitive Gel Electrophoresis and TTGAGCTCAT (10 nucleotides) are produced.
(Table 44.1 ).
In the second test tube, ddATP is added, so chain
length is stopped at A. So in the 2nd tube, DNA strands
Transgenesis
having sequences of TTGA (4 nucleotides) and TT-
It is a form of germ cell gene therapy. A recombinant GAGCTCA (9 nucleotides) are produced. Similarly, in
DNA segment, containing the desired gene from another the 3rd test tube, ddCTP is added, which will contain
species, is introduced into the fertilized ova. The embryos TTGAGC (6 nucleotides) and TTGAGCTC (8 nucleo-
are allowed to develop in the uterus of another tides). In the 4th tube containing ddGTP will have TTG
animal. The animals born are called transgenic animals. (3 nucleotide) and TTGAG (5 nucleotides).
616 Section E: Molecular Biology
-- -
1st tube 2nd tube 3rd tube 4th tube Length Sequence of Table 44.2: Techniques and their apphcat1ons
ddTTP ddATP ddCTP ddGTP of new new strand
strand Technique Applications
10 Southern blot Specific DNA segment (sequence) detection
----
ti)
~g
9
8 li1 a ro Northern blot Detection of a specific gene expression
-
m ~--
6 ti) 0 In situ hybridi- Location of a gene on a specific chromosome
5 zation
m
4 DNA microarray Presence and expression of various genes
3
Polymerase chain In vitro amplicat ion of a DNA sequence; PCR
Fig. 44.9: DNA sequencing reaction can detect even a single bacterium or virus
Single strand Detection of mutations in small fragment s
conformation
Then the contents of each tube are simultaneously
polymorphism
examined on polyacrylamide electrophoresis. The posi-
Conformation Detection of single base mutations in
tion of these fragments will correspond to the chain sensitive gel longer fragments
length; e.g. chain with 10 nucleotides will move least, electrophoresis I
while with 3 nucleotide will move maximum and other
nucleotides will be arranged in their order of molecular the extracellular matrix. Dystrophin and its isoforms are
size. The gel is then autoradiographed. The radiation found in skeletal muscles, smooth muscles and brain.
from 32P labelled primer will be available in all pieces Patients usually present in the 3rd- 5th year of life
and will be seen as dark bands in the X-ray plate (Fig. with motor delay or abnormal gait, difficulty in running,
44.9). From the picture, it can be inferred that 7th and getting up from ground, frequent falls, etc. There may be
10th bases are T. Similarly the positions of other bases pseudohypertrophy of calf muscles (fibro-fatty replace-
can also be deciphered. Thus the sequence of the newly ment of degenerating muscle tissue). Cardiac defects
synthesized strand is known. The complementary include cardiomyopathy and rhythm changes. Respira-
sequence will be present in the original unknown DNA. tory involvement by 16-18 years include scoliosis (which
Applications of the above mentioned techniques are impairs pulmonary functions), respiratory failure (due to
summarized in Table 44.2. progressive failure of intercostals muscles), hypercap-
nia and severe respiratory infections.
·• · Clinical Case Study 44.1 Laboratory analysis reveals elevated serum CK
(10-100 times above normal), AST, ALT and LOH. Muscle
A 4-year-old boy was brought to the hospital. His mother
biopsy (shows deficient dystrophin), electromyography
was concerned that he was walking in an awkward
and PCR can be used for confirmatory diagnosis.
manner, fell over frequently, and had difficulty in climb-
ing stairs. Family history revealed a maternal uncle who
died due to muscular dystrophy at the age of 19. Clini-
ILEARNING POINTS, CHAPTER 4_!_
cal examination showed muscle weakness in shoulder 1. A probe is a single stranded piece of DNA, labeled
with a radioactive or a non-radioactive label, the
and pelvic girdles and enlargement of calf muscles.
sequence of which of complementary to the target
A tentative diagnosis of Duchenne muscular dystrophy
DNA.
was made. What is the defect in this condition?
2. Southern blot is a technique used to detect a speci-
fic segment of a DNA. It has diagnostic and forensic
•i i• Clinical Case Study 44.1 Answer applications.
3. Northern blot is used to detect a specific RNA.
Duchenne muscular dystrophy (DMD) is due to muta-
4. Western blot is used to detect a specific protein .
tions in DMD gene. The gene encodes a protein known
5. Molecular cloning in medicine can be used for diag-
as dystrophin. All muscular dystrophies are progressive nosis of genetic diseases such as Duchenne Mus-
in nature and many are genetically inherited. Dystrophin cular Dystrophy (OMO), Sickle cell anemia, etc.
acts as a link between actin in the cytoskeleton and the 6. Polymerase chain reaction (PCR) is an in vitro DNA
extracellular matrix and helps anchor the muscle fiber to amplification procedure in which millions of copies
Chapter 44: Molecular Diagnostics and Genetic Techniques 617
of a particular sequence of DNA can be produced 8. Hybridoma technique is used to generate mono-
within hours. clonal antibodies. They are used in immunological
7. The enzyme used in PCR is Taq polymerase Appli- research, ELISA, nephelometry, etc.
cations of PCR include diagnosis of bacterial and 9. DNA microarray is used for identification of the
viral diseases, genetic disorders, cancers, solving presence and expression of various genes.
medicolegal cases. 10. Polymerase chain reaction is used for in vitro ampli-
fication of a DNA sequence.
44-1. Specific DNA can be identified by all the following 44-7. All the following are requisites for PCR, except.
techniques, except A. DNA primer
44-1 . What is the use of Southern blotting? 44-6. What is the application of DNA fingerprinting?
To identify abnormal genes, to demonstrate virus inte- It has medicolegal application.
gration. A lso used for prenatal d iagnosis. 44-7. Which enzyme is required for PCR (polymerase
44-2. What Is Northern blotting? chain reaction)?
The Northern blot is used to demonstrate specific RNA . Taq polymerase.
44-3. What is Western blotting? 44-8. What is the use of PCR?
This is to identify proteins (not nucleic acids). (Fig. 44.3).
Diagnosis of bacterial and viral diseases; Medicolegal
44-4. What Is meant by DNA probes?
cases; Diagnosis of genetic disorders, especially pre-
They are single stranded DNA , labeled with radio-
natal diagnosis. Only very minute quantity of sample is
activity. They are used to identify specific bases seq-
required.
uences (genes, virus etc) in the cellular DNA material.
44-9. How are monoclonal antibodies produced in the
44-5. RFLP (restriction fragment length polymorphism)
laboratory?
is used for what?
Locating mutations in DNA. By means of hybridoma technology.
SECTION F
Advanced Biochemistry
Chapter at a Glance
The learner will be able to answer questions on the following topics:
Signal transduction Biological effects of glucocorticoids
Cyclic AMP and G-proteins Adrenal hyper and hypofunction
Cyclic GMP Ovarian hormones
Hormone response element Testicular hormones
Antidiuretic hormone Synthesis of thyroxine
Oxytocin Metabolic effect of thyroid hormones
Hypothalamic releasing factors Assessment of thyroid function
Growth hormone Hyperthyroidism
Adrenocorticotropic hormone Hypothyroidism
Thyroid stimulating hormone Gut hormones
Gonadotropins Growth factors
Synthesis of steroid hormones
...r.,..). -,.cu.C\J"y s~
The nervous system and endocrioe___system are the
major control mechanisms that integrate the functions of
the tissues in the body. The nervous system transmits
electrochemical signals between the brain and periphe-
ral tissues for coordinatin the diverse body functions.
The endocrine system releases chemical
-.::
me.Qialor.s_or
Earl Alfred Martin
hormones into the circulation. However, b th these sys-
Sutherland Gilman Rodbe/1
t; ms n ~errjf. t~am u~ 10n of endocrine NP 1971 NP 1994 NP 1994
glands is arfecterl. oreover, neurotransmitters have 1915-1974 b. 1941 1925-1998
RvJ'I r'1
sev~: r~;es in ~common 1th hormones.
· e signaling occurs when ame cell acts as mesengers secreted into circulation . Once they reach
sender and reci ie t, e.g. gro~ /1, diffe~ ntiation, immune the target cell, they bind to specific target cell receptors
and inflammatory response. P~rac ne signaling is effec- with high affinity.
ted by local mediators which have their effect o.e.ar.Jl!e The classical definition of a hormone is "substances
site of secretion without enterin the circulation. The released from ductless or endocrine glands directly to the
effect is rapid and transient. Pi signaling is blood". A more modem definition of a hormone is that it is
between cells which are located at a distance from each s nthesized by on(1Jxp__e_of...CEII an transported through
other and the signal may be hormones or chemical @ o act on angth.er fy.Jl!l. of ce 1s. There are hundreds
622 Section F: Advanced Biochemistry
Mechanism of action
Hormones that bind to
intracellular receptors
,
(:,
Robert Brian Kobilka
IIA Hormones bind with cell CTH, ADH, FSH, HCG, LH, Lefkowitz NP 2012
SH, PTH, CRH, Glucagon, NP 2012 b. 1955
Calcitonin, Catecholamines b. 1943
~-i\,
the excitation signal to adenylate cyclase (Fig. 45.4-2).
,~ yclic AMP (cAMP) was first discovered by Earl Suther-
The hormone is not assed throu h the Qliilllb.(ane;
Tand in 1961, who was awarded Nobel Prize in 1971. Signal
but only the si nal is Q.g_S_,§ed; hence this mechanism
transduction pathways are like a river flowing in one
is called signal transduction. The a5tepy.Lcycla§e is
directiop on~ ; ~ll1P.?b1~..ntscloser to the receptor are
emged~ in thr; plasma membrane (Fig. 45.4).-
~ ~called "ue~ ',.,anBcioser to the response are called
"(;' ~ "dmID.§tream" ~"6\".'.
Subunit Activation of G-Protein
Signal Transduction through G-Protein The inactive G-protein is a trimer with alpha, beta and
Action is through ~ protein coupled receptors (GPCRi)) gamma subunits. When activated, GTP binds and the
Binding of different types of signal molecules to G-pro- beta-gamma subunits dissociate from the alpha subunit.
tein coupled receptors is a ~ b , w i s m atsignijJ Adenylate cyclase is activated by G-alpha-GTP (Fig.
t~ansductiQ.!1 Action of several hormones is effected 45.4-2). The ~n_9:!.ng__of brumoneJo.Jru rece tor 1_[ggers
through this mechanism (Table 45.1). a confi U@tionalcilang · h - rotein which induces
Chapter 45: Hormones and Growth Factors 623
Fig. 45.1 : Synthesis, degradation of cyclic AMP Fig. 45.2: Inactive and active forms of, G-protein
- i
Adenyl cyclase C C
produces cAMP (2nd messenger)
R R
Fig. 45.3: Hormone binding activates G;_P_rotein R R
3. 4.
4 ,Jk....~~~
Protein kinase
..
the rel~ase o ~ s GJE....tQ..jjjnd.
The hormone has an amplified response, since several
t,.
molecules of G-alpha-GTP are formed.
Inactivation_.,
The ac~iv G-alpha-GTP is imme~ ~ ated by R= receptor: G= G-protein with a, 13. y subunits : AC= adenyl
- r ,, ·~ cyclase: H= hormone; C= catalytic unit; R= regulatory unit;
(2TPas . he G-alpha-GDP form is ive (Fig. 45.2).
cAMP = cyclic AMP.
Theacti'vation is switched off when the GTP is hydrolyzed
1= Receptor is attached to G-protein, which has a , 13, y subunits. It
to GDP by the GTPase activity of the alpha subunit (Fig. is bound with GDP, and is inactive. These are membrane bound.
45.2). This is a built-in mechanism for ~ tivation. Thus 2= When hormone attaches, er. subunit detaches, GTP is bound :
@ TPase acts as a molecular switch) 12S:.) Ga-GTP activates adenyl cyclase , cAMP is generated.
3= Protein kinase contains two catalytic units; but these are
Cyclic AMP attached to two regulatory units, and are inactive.
4= cAMP binds with regulatory units; now catalytic units are free;
Adenyl cyclase or adenylate cyclase converts ATP to kinase is now active.
cAMP (3' ,5'-cyclic AMP), and phosphodiesterase hydro- 5= Active protein kinase phosphorylates enzyme proteins.
- - - + Glycogen phosphorylase
(active; phosphorylated)
(tetramer)
Cholera toxin is present inside the bacteria Vibrio cholerae. The
enterotoxin contains A and B subunits. The B subunit binds to - - - - Glycogen (n- 1 units)
a ganglioside GMt. The A subunit then enters into the mucosal + Glucose-1-phosphate
cell membrane, the alpha subunit of Gs protein is activated.
This results in the irreversible activation of G protein. Therefore, Fig. 45.5: cAMP mediated cascade
t.mr~-e,ag C9VT > t..l\S'
I
adenyl cyclase remains continuously active and keeps cyclic
AMP levels high. This prevents absorption of salts from intestine CALCIUM-BASED SIGNAL
leading to watery diarrhea and loss of water from body. Hyper-
activity of these channels w ill result in loss of sodium chloride TRANSDUCTION '> c~~~~i%qitjJw
w ith watery diarrhea (liquid stools), that may have fatal resu lts.
The patient may lose as much as 1 L of water per hour. Calcium is an impoJSnt intracellular reg1~ ~tor of cell
Pertussis toxin ribosylates the alpha subunit of Gi-protein and
function like contr ;t1'on of muscles secrmlon o or-
prevents the Gi-GDP complex from interacting with the activa- manes and neurot-=-=--~nsmitters, cell J~fsi~n and re ula J~n
ted receptor. Hence, the action of hormones acting through Gi of=9ene regulation. Rapid but transient increase in cyto-
is inhibited.
solic calcium result from either ~ ng_<?.!,_~um_gian-
~ ~n~ nels.lrr the
Kinase Phosphorylates the Enzymes sfi. The released calcium can be rapidly taken-up by
The catalytic subunit then transfers a phosphate group ER to term)-nate the response.
from ATP to different enzyme proteins (Fig. 45.4-5). T~ h i tra ltGlar calcium coi'i"Cel ,tr-ation is1k,w (10·7)
Phosphorylation u uall akes place on the OH groups wherea r llular calcium concentration is very high
of serine, threoni e or rosine residues of the sub- (10·3 ), aintaining a 10,000 fold calcium gradient across
strates. Hence, ese kinases are called Ser/Thr kinases the membrane.
The calcium transporting ATPase transporter accu-
The enzymes may be ~ted o~ ctiyated this
mulates calcium within the lumen of ER (sarcoplasmic
p~ j ~ This is an example of covalent modifi-
reticulum) in muscle.
cation. A summary of the cascade activation of enzymes
Hormones can increase the C>!tosolic calcit~m level
by the hormone is shown in Figure 45.5. Gty_cogmi.
by the following mechanisrnJ . ~
~ hory~~ e (see Fig . 10.36) and t1on::nooase0sif. 1
A. By altering the permea 6llit e membrane.
Ii ase (see Fig.13.14) are @lr'olle~
hormones that are acting through cyclic AMP are
~ he
B. The action of ~ 2+- +_A ¥f which
extrudes calcium in exchange forW.
enumerated in Table 45.1. 1..bt., c\.)u :f,t ... C. By rel e . intracellular calcium stores.
There are Many G-proteins D. ~ lmnH_· the calcium dependent .cegulstQ....ry
p~ II has fo:W calcium bjnding
About 30 different G-proteins are identified, each being
sites. When calcium binds there Is a conformational
used for different signal transduction pathways. The
change ~ -J .~,~ ~lmodulin, which has a role in
G-protein, adenyl cyclase, is called
regulating various kinases. Examples of enzymes
Gs (G-stimuY5ito ry) opposite group is called Gi
or functional proteins regulated by calmodulin are:
(G-inhibitQ['{)~-pc~ also involved in toxic
Adenyl cyclase, calcium-dependent protein kinases,
manifestations of ~ raao.c:Lper.tussis (see Box 45.2). nitric oxide synthase and phosphorylase kinase.
There are Many Protein Kinases
HORMONES ACTING
More than thousand protein kinases are now known. All [ THROUGH PIP2 CASCADE
the known effects of cAMP in euka otic cells result from
---------
activation of rotein kinases, which are serine/threonine
kinases.
The major player in this type of signal transduction is
phospholipase C that hydrolyses phosphatidyl inositol in
Chapter 45: Hormones and Growth Factors 625
""
(tetramer) (R) dimer form is active
PKC
s s
- - - WW + -,c;,
-
---+
Inside cell
(e-, ; p) GTP
IP3 .
\
flfl
R R
.l.. ' RNAP
lcl~ RNAP
i= !
No proteins No proteins
1
action of(iWospbol~that produce hos.ehatidic is smooth muscle myosin, leading to relaxation and
acid which is h~rolY?ed to DAG. 1 ( ..._.- • vasodilatation. Cyclic GMP is also involved in the rho-
The binding of hormones like serotonin to cell sur- dopsin cycle. The role of cGMP in the light sensing cells
face receptor triggers the activation of the enz me hos- of retina and its interaction with the G-protein transducin
, J)holi ~se..:,.C which hydrolyzes the phosphatidyl inositol is described under visual cycle, Chapter 32.
to diacylglycerol. IP3 can release ca+• from intracellular NO (Nitric oxide) is the major activator of guanylate
stores, such as from endoplasmic reticulum and from cyclase. NO in turn is produced by the action of NOS
(Nitric oxide synthase) in tissues like vascular endothe-
sarcoplasmic reticulum (Fig. 45.6).(RAG activates PKf .
lial cells (Chapter 18). Increased level of cyclic GMP in
The elevated intracellular calcium then triggers proces-
ses like smooth m~cle contraction, glyco~i break- smooth muscle triggers rapid and sustained relaxation
of the smooth muscles. The vasodilatation resulting from
down and exocytosis. -
NO induced increase in cGMP has great physiological
and pharmacological significance. The drugs that act via
._ Role of Cyclic GMP
NO release are nitroprusside, nitrites (used in angina as
Cyclic GMP (cGMP) is another important second mes- coronary vasodilators) and sildenaphil citrate (Viagra).
senger involved in contractile function of smooth mus-
__
cles, visual signal transduction and maintenance of Hormones with Intracellular Receptors
blood volume. Cyclic GMP degradation is catalyzed by The hormones in this group include the steroid hormo-
membrane bound PD Es ..- t Q.....,~~ ..,.,,.,__
-----___./ ¼ ' f, -~ nes and thyroid hormones. They diffuse through the
It is formed from GTP by the action of(guan~I c clas plasma membrane and bind to the receptors in the cyto-
Several compounds have been found to increase the con- plasm. The hormone receptor (HR) complex is formed
centration of cGMP by activating guanyl cyclase. These in the cytoplasm. The complex is then translocated to
include drugs like nitroprusside, nitroglycerin , sodium the nucleus. Steroid hormone receptor monomer binds
nitrite. All these compounds act as potent vasodilators, to a single steroid molecule at a hydrophobic site, but on
by inhibiting the phosphodiesterase. binding to genes they dimerize (Fig. 45.7).
626 Section F: Advanced Biochemistry
In the nucleus, the HR binds to the hormone res- are long polypeptides. They are synthesized in hypotha-
ponse elements (HRE) or steroid response elements lamus. It is cleaved into the active molecule, and then
(SRE). The SRE acts as an enhancer element and when transported to posterior pituitary and stored there. They
stimulated by the hormone, would increase the transcrip- are released to bloodstream by exocytosis.
tional activity. The newly formed mRNA is translated
to specific protein, which brings about the metabolic IANTIDIURETIC HORMONE
effects. This would lead to induction of protein synthesis. It is also called vasopressin. It has 9 amino acids. If
Best examples of the effect of hormones on genes are: arginine is replaced by lysine, it is called lysine vaso-
a. The induction of synthesis of amino transferases by pressin (LVP). Its main action is to prevent diuresis. So
glucocorticoids. it reduces the urine output. The ADH acts on the distal
b. Synthesis of calcium binding protein by calcitriol convoluted tubules of the kidney, producing reabsorp-
(see Fig. 32.11 ). tion of water. The ADH binds to membrane receptor
and activates adenylate cyclase. The cyclic AMP thus
Insulin-Signaling Pathway produced will activate the protein kinase. This, in turn,
phosphorylates proteins of the microtubules and micro-
filaments. The net effect is the reabsorption of water.
The regulation of ADH secretion is through the osmolal-
ity of blood. Lowering of the osmolality (hemodilution)
suppresses ADH secretion. Conversely, an increase in
osmolality (hemoconcentration or dehydration) leads to
stimulation of the secretion of ADH.
Horylates insulin receptor sub- Deficiency of ADH results in diabetes insipidus. It
different IRS molecules, named is characterized by excretion of large volumes of dilute
urine. Hypernatremia and hypertonic contraction of extra-
Activation of IRS2 results in activation of the Pl-3 cellular fluid volume are also seen. It is a very rare
kinase, which eventually activates various protein kina- condition. Excess secretion of ADH often results from
ses, PKB, PKC, SGK (serum glucocorticoid regulated ectopic production of ADH by malignant tumors else-
kinase), etc. This leads to transcription of specific genes where, referred to as the "syndrome of inappropriate
for key enzymes of glycolysis, such as glucokinase. secretion of ADH" or SIADH. Here ADH is continuously
There are more than 100 enzymes influenced by insulin. secreted and is not subjected to any control mechanisms.
I HYPOTHALAMIC AND
PITUITARY HORMONES
Generally, there is hypotonic expansion of extracellular
volume, water intoxication (headache, confusion, ano-
rexia, nausea, vomiting, coma and convulsions).
!
The paraventricular nucleus (PVN), located in the
anterior medial area of hypothalamus, is involved in
Tropic hormones
oxytocin and vasopressin release from the pituitary. The (ACTH, TSH, LH, FSH, GH, Prolactin)
arcuate nucleus (ARC) is involved in secretion of vari-
ous pituitary releasing hormones. The overall function of
!
Target organs
the hypothalamus is to link the central nervous system (Thyroid, adrenal glands,
_. gonads, liver)
to the endocrine system via the pituitary gland (also
termed the hypophysis).
l
Target organ hormones
(T3, T 4, cortisol, estrogen,
TABLE 45.3: Hormones of anterior pItuItary pattern of secretion is reflected in cortisol also. Factors
Acronym Full name Chemical nature Amino acids that increase ACTH secretion include stresses such as
GH Growth hormone Polypeptide 191 pain, cold exposure, acute hypoglycemia, trauma, dep-
ACTH Adrenocorticotropic Polypeptide 39 ression, and surgery.
hormone
Luteinizing hormone
ACTH binds to specific receptors on the adrenal
LH Glycoprotein; a, ~ a=89
chains ~ = 115 gland, then activates adenylate cyclase and so, cAMP
F5H Follicle stimulating Glycoprotein; a, a = 89 level is raised. ACTH induces adrenocortical steroido-
hormone chains = 11 5 genesis through the melanocortin-2 receptor. Steroid
TSH Thyroid stimulating Glycoprotein; a, a = 96
hormone chains = 115
hormones in turn cause feedback inhibition of HPA
MSH Melanocyte Polypeptide a = 13 (hypothalamopituitary adrenal axis).
stimulating hormone ~ = 18 ACTH secreting tumors of pituitary will cause Cush-
v = 12
ing's disease. Deficiency of ACTH secretion may occur
PRL Prolactin Polypeptide 198
Polypeptides 31 as a part of panhypopituitarism.
Cosyntropin stimulation (Rapid ACTH) test: With
Excess secretion by GH secreting tumor, leads to rapid administration of ACTH , blood cortisol level will
gigantism in children and acromegaly in adults. Defi- be raised. If there is adrenal insufficiency, adrenal gland
ciency of GH secretion in early childhood results in pitui- cannot be stimulated by the administered ACTH and
tary dwarfism. Dwarfism may also result from congenital subnormal or low response of cortisol occurs.
deficiency of GH due to end organ resistance. It is Dexamethasone suppression test: Dexamethasone,
treated by giving GH produced by recombinant technol- an analog of cortisol suppresses ACTH hormone and
ogy. Inhibition of GH secretion by hyperglycemia (GTT) cortisol production in normal subjects; but not in patients
and stimulation by hypoglycemia (insulin infusion) are with Cushing's syndrome.
used to check the status of GH secretion in hyper as well
as hypopituitarism. Endorphins
THYROID-STIMULATING
HORMONE JHYROTROPIN _ .____
cAMP by binding with a receptor on thyroid cell surface. High FSH levels are an indication of subfertility and/
TSH secretion is stimulated by TRH. The TRH also sti- or infertility. Diminished secretion of FSH can result in
mulates prolactin secretion. The TSH secretion is also hypogonadism. This condition is typically manifested
controlled by the level of thyroid hormones. High levels in males as failure in production of normal numbers of
of TSH may occur due to primary hypothyroidism and sperm. In females, cessation of reproductive cycles is
lack of feedback control. commonly observed.
'
.., Increased serum TSH levels are seen in primary
hypothyroidism (3-100 times normal), Hashimoto's thy-
Serum level of FSH is raised in primary gonadal
failure, ovarian or testicular agenesis, castration, Kline-
roiditis, ectopic TSH secretion by tumors (lung, breast). felter's syndrome, and gonadotropin secreting pituitary
The TSH is elevated in euthyroid patients during treat- tumors. Serum level of FSH is decreased in anterior
.ment of hyperthyroidism but TSH is low for 4-6 weeks pituitary hypofunction, hypothalamic disorders, preg-
after achieving euthyroid state in treated hyperthyroid nancy, anorexia nervosa, polycystic ovary disease and
patients. in hyperprolactinemia.
Decreased levels are observed in primary hyper- Serum level of LH is raised in primary gonadal dys-
thyroidism, secondary hypothyroidism (pituitary origin), function, polycystic ovary syndrome, postmenopausal
tertiary hypothyroidism (hypothalamic), subclinical women and in pituitary adenoma. Serum level of LH is
hyperthyroidism (e.g . toxic multinodular goiter, exo- decreased in pituitary hypothalamic impairment, ano-
genous thyroid hormone administration). rexia nervosa and severe illness.
~~;;:~:oer•~ 21
I
10 I
CH3
C=O20
l
17.
(Progesterone)
21
CH3
HO I
C=O 20
17-hydroxylase
(NADPH) HO
A 21
CH3
10 I I
17.
l
C=O 20
D 17-hydroxy progesterone - - - - -. 10 I
17.- 0H
HO 1~:tr~i""'
Pregnenolone CHpH
l
1a I
11-beta- n:- OH
Prolactin stimulation test by chlorpromazine and
hydroxylase
Prolactin suppression test by L-dopa are used to assess (NADPH)
the function.
I ADRENAL CORTICAL
HORMONES
Cholesterol is first acted upon by desmolase and a 6-car-
bon unit is cleaved off, formi ng the 21 carbon steroid,
pregnenolone (Fig. 45.9). It is a common precursor for
all the steroid hormones. Adrenocorticotropic hormone
The adrenal cortex has three different zones each res- (ACTH) stimulates this step. This is the rate limiting step
ponsible for production of different classes of steroid hor- for synthesis of all steroid hormones.
mones (C21, C19 and C18). The smallest and outermost Progesterone is the fi rst steroid hormone formed
zona glomerulosa produces the C21 steroids, mine- from pregnenolone in two steps. Progesterone is fur-
ralocorticoids. They have effects on water and electro- ther converted into glucocorticoids (Fig. 45.10), miner-
lyte balance. The middle zone of the adrenal cortex, the alocorticoids and sex steroids (Fig. 45.11 ). The major
zona fascicularis produces the glucocorticoids mainly; adrenal glucocorticoids are cortisol, 11-deoxycortisol
and adrenal androgens and estrogens to a lesser extent. and corticosterone in that order. The major mineralo-
The innermost zona reticularis produces the androgens corticoid is aldosterone, but 11-deoxycorticosterone and
(C19) and estrogens (C18). Cortisone was isolated by corticosterone also have significant mineralocorticoid
Tadeus Reichstein; the structure was identified by activity. These reactions are effected by hydroxylation.
Edward Kendall, while Philip Hench in 1948 showed its These specific hydroxylases are NADPH dependent.
efficacy in rheumatoid arthritis. All the three were awar- These hydroxylation reactions are summarized in Figure
ded Nobel Prize in 1950. 45.12. The ACTH stimulates the synthesis of all steroid
Chapter 45: Hormones and Growth Factors 631
Cholesterol (C21)
!
Pregnenolone (C21 ) - - - - - - - - Progesterone (C21)
Progesterone (C21)
l l
~r~• T
17-hydroxylase 17-hydroxy
17-hydroxy-
0
17-hydroxyprogesterone Androstene-
dione (C19)
1
Cortisol Dehydro-
(C21) epiandro- Corticosterone
sterone
(DHEA)
(C19)
Testosterone
(C19)
!
l
l
Aldosterone (C21)
Androstenedione - . Estradiol
l
(C19) (C18)
0
!
17-reductase 17-hydroxy DHEA 17-keto-
(NADPH) steroids sulfates steroids
in urine in urine in urine
l
box; intermediaries in grey box; hormones in b lue box; excretory
Aromatase products in brown box
(NADPH)
adrenal cortex. The term 17-ketogenic steroids is used immunoassay (CUA). Maximum activity is at 9 AM and
to include all the compounds having a keto or hydroxyl minimum activity is at 9 PM. A loss of diurnal rhythm may
group at 17th carbon. be an early indication of disease.
Adrenal carcinoma Increased; diurnal rhythm is lost Increased Decreased No suppression even with high dose
Pituitary adenoma Increased; no diurnal rhythm Increased Increased Suppression with high dose
Ect opic ACTH production Increased; no diurnal rhythm Increased Increased No suppression
I
Cause ofadrenal insufficiency Plasma cortisol Urinary free cortisol Plasma ACTH ACTH stimulation CRH stimulation Na• and K' in blood
Primary Low Low Elevated No effect No effect Na• ; K•t
..
TABLE 45.7: Summary of steroid hormones
Hormone Tissue of origin Function
Estrogen (Estradiol) Ovary Maturation and function of female secondary sex organs
Estrogens Placenta Maintenance of pregnancy
Progestins (Progesterone) Ovary Implantation of ovum and maintenance of pregnancy
Progestins Placenta Mimic action of progesterone
Androgens (Testosterone) Testes Maturation and function of male secondary sex organs
Glucocorticoids (Cortisol and corticosterone) Adrenal cortex Diverse effects on inflammation and protein synthesis
Mineralocorticoids (Aldosterone) Adrenal cortex Maintenance of salt balance
•-----------~ .
...
Plasma Thyroid cell Acinar space
(Iodide) ( '-\ (active
2
,- iodine)
l
Iodide
!3 ( \ H2 0 2 H2 0
TgF
9
HMP shunt pathway - NADPH NADP•
DIT
F i g . 45.14: Step 2 of thyroxine synthesis
10
PBI
.,(f , r
T, ~ Tgb-Tc +--
6
Tgl T;
Stored colloid
Step 2: Oxidation of Iodine
The iodide taken up by the thyroid cell is oxidized to
active iodine (Step 2 in Fig. 45.13). The thyroid is the
Fig. 45.13: Metabolism of thyroid hormones only organ which can perform this oxidation step. This
is catalyzed by the enzyme thyroperoxidase. The reac-
parts of the world, iodine is a scarce component of the tion needs hydrogen peroxide, which is produced by an
soil. Upper regions of mountains generally contain less NADPH-dependent reaction (Fig. 45.14). The NADPH
_. is generated by the hexose monophosphate shunt path-
iodine. Such areas are called goitrous belts, e.g. Hima-
way. This second step is stimulated by TSH and inhibited
layan region.
by antithyroid drugs such as thiourea, thiouracil and
Commercial source of iodine is seaweeds. The pro-
methimazole. In patients with an inborn error of iodide
gram of iodination of common salt has resulted in inc-
oxidation defect, treatment is T4 administration.
reased availability of iodine. Ingredients in foodstuffs,
which prevent utilization of iodine are called goitrogens. Step 3: Iodination
Goitrogens are seen in cassava, maize, millet, bamboo
Then thyroglobulin (Tgb) is iodinated. Thyroglobulin is
shoots, sweet potatoes and beans. Cabbage and tapi-
synthesized by the thyroid follicular cells. Iodination of
oca contain thiocyanate, which inhibits iodine uptake
the tyrosine is taking place on the intact Tgb molecule
by thyroid. Mustard seed contains thiourea, which inhi-
in the follicular space. Thus 3-monoiodotyrosine (MIT)
bits iodination of thyroglobulin. and 3,5-diiodotyrosine (DIT) are produced (Fig. 45.15).
The thyroid hormones are thyroxine (T4) and triiodo-
thyronine (T3). Step 4: Coupling
Some of the tyrosine residues in the thyroglobulin are
Synthesis and Secretion of Thyroxine aligned opposite each other, and are coupled (Step 4,
Fig . 45.13). When two DIT molecules couple, one mol-
Step 1: Uptake of Iodine ecule of tetraiodothyronine (T4) is formed (Fig. 45.1 5).
Thyroid gland takes up and concentrates iodine (Step 1 Triiodothyronine (T3) may be formed by deiodination of
in Fig. 45.13). This step is inhibited by thiocyanate and outer ring of T4 by 5'-deiodinase. Under normal condi-
perchlorate, which compete for the carrier mechanism. tions, 99% of the hormone produced by the thyroid gland
This step is stimulated by TSH. The congenital defect, is T4. The T4 residues are now attached to the thyro-
iodine trapping defect, may be treated by large doses globulin molecule. The iodotyrosyl coupling defect, an
of iodine. inborn error, affects this 4th step. Treatment is to give T4.
636 Section F: Advanced Biochemistry
Step 8: Release
3-monoiodotyrosine (MIT)
The T4 thus generated is released into the bloodstream.
The T3 is produced by deiodination at 5' position, either
HO inside the thyroid cell or in the peripheral tissues.
-b-
3,5,3'-triiodothyronine (T3) deficiency is manifested. Treatment is to give iodine.
'
HO 0 Step 10: Transport of Thyroid Hormones
Thyroid hormones are transported in plasma by proteins
(Step 10 in Fig. 45.13). The bound form is biologically
inactive, but they can be rapidly released. Total protein
bound iodine (PBI ) is about 10 mg/dl . The thyroxine
binding globulin (TBG) (54 kD) carries about 80% of T4
and 60% of T3. The rest of thyroid hormones are loosely
HO 0 bound with Transthyretin (TTR}, prealbumin and albumin.
\oiJ
the T 3 and T 4 secr_£tions will b~ ased~ normal
iv. Loss of body weight is a prominent feature of
\ response is observed in:
yroidism.-,C 1,-ll: .
J
hyp~ a. Hyp ro·dism: The negative feedback effecY-ni.. "n">u
v. GI eogenesis and car fl drate oxidation are of hi;;verpowers the stimulant effect of TRH~
increased. Glucose tolerance test shows rapid Here the thyroid hormone levels are elevated.
absorption. fji,\ (1;;'\ b. Hv.po~rism: The pituitary could not respond
vi. Fatty acid metabolis~ increased. Ch~ es~ to TRH.nthese cases the plasma thyroid hormon(I)
degradation is increased and hence cholesterol levels are subnormal.
level in blood is decreased, which is another hall- c. An exaggerated response is observed in primary
mark of hyperthyroidism. \-\~ , ()).)"'ooh, hypothyroidism since the n~ ve feed back effect
\'.o.\-, \7~'\:. >~'ct - oLT4 is reduced.
Thyroid Function Tests
c<ffe21esterol
.@say of Hormones
In hypothyroidism, cholesterol level in blood is incre-
The plasma levels of T 4 and T3 levels may be meas-
ased. It is not diagnostic, because hypercholesterolemia
ured by any of the techniques enzyme-linked immu-
is seen not only in hypothyroidism, but also in diabetes
nosorbent assay (~ . chemiluminescent immu- mellitus, hypertension, obstructive jaundice and nephro-
noassay (CLIA) orfiuorescent immunoassay (FIA) for tic syndrome. However, cholesterol level is a useful
the diagnosis of thyroid diseases. In hyperthyro1aism, index in monitoring the effectiveness of the therapy
thyroid hormone levels are increased. Both T3 and in thyroid conditions. Cholesterol level is increased in
T4 levels are increased, while TSH is reduced due to hypothyroidism, because cholesterol carrying lipopro-
feedback inhibition. In hypothyroidism, T3 and T4 are tein degradation is decreased.
reduced; but TSH levels are increased due to (\ick ~f
®
feed6ack effecU But when hypothyroidism is due to Detection of Thyroid Antibodies
hypothalamic or pituitary defect, then TSH, T3 and T4,
In Gcave'.s, disease, the resence of thyroid stimulat-
all are decreased .
ing Tmrminoglobuli TSlg) also known as long acting
® thyroid stimulator ( is seen cjrcu!atjan. The
Free T3 (fT3) and fT4 LATS can bind to TSH receptors on thyroid gland and
The free hormones are the really active molecules. produce stimulation which is not under feedback con-
Nowadays, very sensitive ELISA techniques are avail- trol. The TS lg is an antibody generated against the TSH
able to quantitate this free fraction. The free T4 consti- receptor.
tutes only 0.03% of the total T4, whereas free T3 forms The measurement of anti-TPn tibodies is useful
.a=:==~~
0.3% of total T3. in autoimmune thyroiditis (~ s). Anti-
© thyroglobulin antibodies are measured in thyroid cancer
Plasma TSH ~e.e..\o.Q'le. as well.
In primary hypothyroidism, TSH level is "elevated due
to lack of feedback. But in secondary hypothyroidism,
Abnormalities of Thyroid Function
TSH, T3 and T4 levels are low; this could point to a In 1835, Robert James Graves and in 1840 Carl
pituitary or hypothalamic cause. Hyperthyroidism due Adolph Basedow described hyperthyroidism (Graves-
to primary thyroid disease has high T3 and T4 levels, Basedow disease). In 1915, Kendall (Nobel Prize,
but suppressed TSH levels. Hyperthyroidism due to 1950), isolated thyroxine. Emil Kocher was the first
pituitary cause is indicated by high TSH, T3 and T4 surgeon to excise thyroid gland to treat goiter in 1883.
levels. For his contributions in thyroid pathology he was
38 Section F: Advanced Biochemistry
.
'
TABLE 45.8: Laboratory findings in hyperthyroidism TABLE 45.9: Laboratory findings 1n hypothyro1d1sm
.. , , , Plasma Response T, and T,in
r,4 TSH toTRH blood TSHin blood Response to TRH
Grave's disease Increase Primary Decreased Increased Exaggerated
High increase Decrease Nil
hy pothyroidism response
Toxic goiter Increase High increase Decrease Nil Secondary Decreased Decreased No response
T3 toxicosis T3 increase Increase Decrease Sluggish hypothyroidism
T4 normal "
Excess intake Increase Mild increase Decrease Sluggish tumors, pituitary surgery or irradiation, infiltration, Shee-
of thyroxin
han's syndrome and isolated TSH deficiency. Hypotha-
=
lamic diseases causing secondary hypothyroidism are
awarded Nobel Prize in 1909. Diseases of the thyroid tumors, trauma and infiltration.
are the most com mon afflictions involving endocrine Most common cause is primary thyroid disease,
system. The most common types of thyroid diseases often seen in autoimmune thyroiditis, leading to myxe-
are hyperthyroidism (excess secretion ), hypothyroi- dema in adults. Women are more affected than males.
dism (decreased secretion) and goiter (enlargement Symptoms are lethargy, tolerance to heat, cold intole-
of thyroid gland). Goiter may or may not be associated rance, slow heart rate, weight gain, dry coarse skin, slow
with abnormal function, e.g. euthyroid goiter (diffuse responses and sluggishness. Table 45.9 shows the lab
enlargement); nodular goiter which may lead to hyper- findings in hypothyroidsm.
function , or iodine deficiency goiter which may result In ch~n.~itPQlll!Yroidism reduces mental and
in hypothyroidism. physical retardation, known retinis . The TBG may
be elevated due to maternal hypefestrogenism and
HYPERTHYROIDISM therefore total T4 and T3 may be normal. The lack of
Patients have an increased rate of metabolism, weight feedback will give elevated TSH level also. Prompt diag-
loss, tachycardia, fine tremors, sweating, diarrhea, nosis and treatment are important c ~ since
emotional disturbances, anxiety and sensitivity to heat. aoy_,dela): in starting replacement may lead to irrever,Sible
damage. Maternal hypothyroidism may also cause con-
=--
Table 45.8 summarizes the laboratory findings in com-
mon types of hyperthyroidism. genital hypothyroidism in the newborn.
lmmunochemistry
Chapter at a Glance
Historical Perspectives
Introduction of cowpox vaccination by Jenner in 1798 paved the foundation for immunization procedures. This does not mean
that he had discovered the procedure. For 10 years, he was in Punjab, India as a doctor, when he came to know about the
inoculation procedure available throughout India from time immemorial. Jenner has developed and simplified the procedure.
Rabies vaccine was discovered by Pasteur in 1881 . Emil von Behring (Nobel Prize, 1901) introduced therapy with antibodies,
especially against diphtheria. In 1908, Nobel Prize was awarded to Paul Ehrlich for explaining the antibody production and to
Metchnikov for discovery of phagocytosis. Life saving blood transfusion was made possible by the pioneering work on human
blood groups, the ABO and Rh system by Karl Landsteiner (Nobel Prize, 1930). By employing specific antibodies, nano-
gram or picogram quantities of biologically important substances can be estimated. This is termed radioimmunoassay. Radio-
immunoassay was first described by Rosalyn Yalow (Nobel Prize, 1977). Peter Doherty and Rolf Zinkernagel were awarded
Nobel Prize in 1996 for their work on the specificity of cell mediated immune response. Bruce Beutler and Jules Hoffman were
awarded Nobel Prize in 2011 for their discoveries concerning the activation of immune system. Nobel Prize was awarded to
Ralph Steinman in 2011 for his work on dendritic cells in immunity.
Edward Emil Adolf Paul Henry Karl Daniel Niels K Jerne Rosalyn
Jenner Behring Ehrlich HDale Landsteiner Bovet NP 1984 Yalow
1749-1823 NP 1901 NP 1908 NP 1936 NP 1930 NP 1957 1911- 1994 NP 1977
1854-1917 1854-1915 1875-1968 1868-1943 1907- 1992 1921- 2011
648 Section F: Advanced Biochemistry
mainly in the paracortical areas of lymph nodes and
periarteriolar sheaths in the spleen. In peripheral blood
80% lymphocytes are T-cells, and 15% are B-cells.
Certain other cells originated from bone marrow and
processed by the Bursa of Fabricius in avians, are called
B-cells. The Bursa equivalent organs in human beings
Peter Doherty Rolf Bruce Beutler Ralph
NP 1996 Zinkernagel NP 2011 Steinman are gut associated (including Peyer's patches) and lung
b .1940 NP 1996 b. 1957 NP 2011 associated lymphoid organs. lmmunoglobulins are
b. 1944 1943-2011 secreted by Plasma cells belonging to the 8-lympho-
cytes. The 8-cells govern the humoral immunity.
Immunology is one of the rapidly advancing branches of Clonal selection: lmmunoglobulins of different
medical science. Small pox has been completely eradi- specificity are available on the 8-cell surface. When an
cated from the world by 1985; this is a triumph of im- antigen is introduced, the antigen selects out that parti-
munology. Three salient features of immunological reac- cular cell carrying the specific antibody. This results in a
tions are: Recognition of self from nonself or foreign series of divisions of that cell and a clone of cells are pro-
substances; specificity of the reactions, and Memory
duced. These cells are fi nally differentiated into plasma
of the response.
cells. This is the antigen dependent clonal selection.
When injected with 100 diffe rent proteins, the ani-
A particular clone of cells secretes antibodies of the
mal will produce 100 different antibodies; this is called
same specificity.
specificity. If a person belongs to A group, antibodies
against B group are seen in circulation. There is an extra Effector Mechanisms
N-acetyl group in antigen A; this is the only molecular
The following are the immunological effector mecha-
difference between A and B antigens. Immune system nisms by which foreign cells are destroyed or particles
is exquisitely specific to recognize even this small dif- are removed:
ference at molecular level. If the same antigen is intro-
duced for a second time, body will react immediately; Cell Mediated Immunity
this memory is the basis of vaccination.
The following are the major activities of T-lymphocytes.
A. Immunity against infections: T-cells mediate effec-
Antigens
tive immunity against bacteria, such as mycobacte-
Certain components of the cell membranes act as ria, many viruses and almost all parasites.
specific antigens. They will be different from person to 8. Rejection of allograft: When an organ (heart, kidney)
person in chemical composition and three dimension- is transplanted from one person to another, it is
al structure. Hence, the immunocompetent cells could called al log raft. Body tries to reject such transplanted
recognize the self from nonself. Any substance which organs, mainly by T-cell mediated mechanism.
invokes an immunological response is an antigen or C. Tumor cell destruction: Although other mecha-
immunogen. Antibody response will usually be selective nisms are also involved in killing tumor cells, T-cell
against specific spatial configurations on the antigen, activity is the predominant one.
which are called antigenic determinant sites, known as D. Helper function: T helper (Th} cells are a sub-
epitopes. group of cells which carry CD4 determinants on the
cell surface (CD = cluster determinant). They are
Immune Response necessary for optimal antibody production by plasma
The lymphocytes generated from the bone marrow, cells and for generation of cytotoxic T-cells. They
passed through and processed by the thymus gland, are selectively destroyed in AIDS (see Chapter 47).
are then called T-lymphocytes. They can directly kill the E. Suppressor function: T suppressor (Ts) cells are
target cells and are the effector cells for the cell- CD8 positive cells. They downregulate the activities
mediated immunity (CMI). The T-lymphocytes are found of both T- and 8-cells.
Chapter 46: lmmunochemistry 649
Macrophages
A Phagocytosis is the nonspecific mechanism by which
body tries to eliminate invading organisms. Foreign mate-
rials are ingested by the phagocytes and later digested Light chain
intracellularly. The myeloperoxidase present inside the
phagocytes destroys the bacteria (see Chapter 30). When
Complement activation
a foreign particle enters the body, the macrophages
phagocytose it, and present the antigens to the lympho- Heavy chain Heavy chain
cytes.
Cell binding
It can combine with 5 antigens simultaneously, and so in liver, reaches the intestinal muco:sal cells, where it
lgM is very effective for agglutinating bacteria. Being a combines with lgA dimer to form the siecretory lgA which
large molecule, it cannot come out of vascular space. is then released.
lgM are the predominant class of antibodies in primary
response. Natural antibodies are lgM in nature . Thus, lmmunog/obulin E (lgE)
a person having blood group A antigen will have anti-B
antibodies in his circulation (isohemagglutinins). These They are cytophilic antibodies. They mediate allergy
are produced without any known antigenic stimulation , (Greek, allo =other; ergon =work), hJtpersensitivity and
and hence called natural antibodies. These lgM anti- anaphylaxis. They have the property to fix on mast cells
bodies cannot cross placenta, and therefore the fetus, and basophils. When certain antigens such as penicillin
even though it carries an incompatible antigen, is protec- are injected a few times, lgE class antibodies are produced
ted from natural antibodies of the mother. which anchor on mast cells. When tlrle same chemical
is injected next time, the antigens fi:x on such antibo-
lmmunog/obulin A (lgA) dies, causing mast cell degranulatio n, and release of
lgA usually are dimers (total 4 heavy chains and 4 light histamine and slow reacting substance. This leads to
chains). The J chain connects the dimers (see Fig.46.2). vasodilatation, hypotension and bron chiolar constric-
They are the secretory antibodies seen in seromu- tion. This is the basis of penicillin anaphylaxis, hay fever
cous secretions of gastrointestinal tract, nasopharyn- caused by fungus, asthma by pollen and urticaria by
geal tract, urogenital tract, tears, saliva, sweat, etc. The absorbed food elements.
dimers are stabilized against proteolytic enzymes by The role of histamine in anaphyla:1Cis was elucidated
the secretory piece. The secretory piece is produced by Sir Henry Dale (Nobel Prize, 19~16). The first anti-
histaminic drug, mepyramine was prepared in 1944 by
Daniel Bovet who was awarded Nobel Prize in 1957.
•
fgG lgE lgD
IE_ARAPROTEINEMIAS •
Multiple Myeloma (Plasmac:ytoma)
lgM lgA When lg-secreting cells are transformed into malignant
cells, one clone alone is enormously proliferated. Thus,
lg molecules of the very same type are produced in
large quantities. This is seen on electrophoresis as the
myeloma band or monoclonal band or M band with a
sharp narrow spike (see Fig . 26.2). Multiple myeloma
is characterized by paraproteinemia , anemia, lytic bone
lesions and proteinuria. Bone marrow examination
Fig. 46.2: lgG, lgE and lgD have one basic unit each, lgM has
5 basic units and lgA has 2 basic units. Red circles represent J reveals large number of malignant plasma cells. Spon-
pieces. Green squares are secretory pieces taneous pathological fracture of weight bearing bones,
Primary
response;
Hypogammaglobulinemia
_g mainly
°E lgM antibody Decrease or absence of immunoglobulin levels may be
<(
seen in congenital or acquired conditions. Deficiency can
also occur due to loss of proteins as in nephrotic syn-
drome. A primary failure in production may occur as a con-
0 10 20 0 3 10 20 30 genital X-linked disorder (Bruton's disease). Decreased
Days after immunization production may also be secondary to diseases like
myeloma, leukemia or drug induced.
Fig. 46.3: Primary and secondary immune responses
(IL: Interleukin; IFN: Interferon; TNF: Tumor necrosis factor; G-CSF: Granulocyte colony stimulating factor, GM-CSF: Granulocyte macrophage
colony stimulating factor; MIF: Macrophage migration inhibition factor).
transplantation in 1956; both of them received Nobel Prize on their function (Table 46.2). The six major types are
in 1990. But Christian Bernard, who did the first heart receptors for hematopoetins, interferons, interleukins,
transplantation in 1964, was omitted from the Nobel Prize chemokines, tumor growth factors and lg-stimulating
list. The genes of major histocompatibility complex (MHC) factors.
are involved in the recognition between self and non-self Cytokine is the generic name of a group of polypep-
antigens. In human beings, the MHC genes are present tides that may be called the hormones of the immune
on chromosome 6. There are A, C, B, D and DR loci. All system.
these loci together contain more than 150 alleles. Permu-
tation and combination of them could produce an astro- Interleukins (IL)
nomical number of variations. Hence, the antigenic Interleukins are growth factors targeted to cells of hema-
constitution of one person will be entirely different from topoietic origin. More than 30 interleukins are known, some
another one. These are main transplantation antigens, important ones are listed in Table 46.2. They are a group
responsible for rejection of allograft. Frank Macfarlane of cytokines (signaling molecules) that were first seen
Burnet and Peter Brian Medawar were awarded Nobel to be expressed by white blood cells (leukocytes).
Prize in 1960 for elucidation of basic rules in transplan- Recombinant interferons and interleukins are used
tation immunity and acquired immunological tolerance. in immunomodulatory therapy and in viral infections.
Soluble Factors
Cytokines are a diverse group of low molecular weight
¥ Clinical Case Study 46.1
proteins, that are soluble mediators of inflammatory and A 45-year-old man presented with severe back pain and
immune response. They instruct the receiving cells to weakness. He had lost 7 kg in the last 3 months. Loss
proliferate, differentiate and secrete additional cytokines of appetite is present. No history of fever. He reports
and communicate signals from one cell type to another extreme fatigue, body pain and complains that he is
to effectively regulate growth and differentiation. unable to do any work. X-ray of skull revealed punched
Since cytokines may belong to different classes out lesions. Bone marrow biopsy was done and it
based on their structure, they are classified depending showed plasma cells in excess. Serum electrophoresis
Chapter 46: lmmunochemistry 653
Fig. 46.4: Protein electrophoresis of concentrated urine sample; Fig. 46.5: Serum protein electrophoresis showed a spike in the
presence of a spike in the beta region beta region
was ordered on the basis of clinical features. It showed a narrow spike (Fig. 46.5). What is the most probable
an abnormal band between 13 globulin and y globulin. diagnosis? What are the potential causes of increased
Urine was positive for Bence-Jones Proteins (BJP). 13-globulins? What investigations shoiuld be performed to
What is the probable diagnosis? What is the significance characterize the protein responsible for the spike in the
of laboratory findings in this patient? 13 region of the urine?
common confounding factors leading to apparent mono- bridges. Base on heavy chains, lgs are of 5 classes;
clonal peaks in the 13-globulin region of an electrophore- lgG, lgM, lgA, lgD, lgE.
sis analysis. Further, serum immunofixation electropho- 3. The light chains are either lambda or kappa.
resis revealed the presence of a monoclonal gamma 4. The antigen-binding region of the lg is the Fab por-
tion (2 in number). The rest of the molecule is the
band with no corresponding light chain. This finding is
Fe portion. lgG, the major antibody of secondary
consistent with the diagnosis of gamma-heavy chain
immune response forms about 75-80% of the total
disease (GHCD). HCD is a proliferative B-cell disorder
lgs. They can cross the placenta and is responsible
characterized by the production of monoclonal proteins for Rh isoimmunization.
with incomplete heavy chain components and without 5. lgM are macroglobulins; a pentameric structure
associated light chains. Fatigue, weakness, and lym- joined by J chains and are the predominant class
phadenopathy are the most frequent initial symptoms, of antibody in primary response. Natural antibodies
and hepatosplenomegaly are the most common physi- are of lgM type.
cal findings, which are often accompanied by anemia. 6. lgA are secretory dimeric antibodies seen in mucus
GHCD can also be associated with other lymphoprolif- secretions in the body.
erative diseases or autoimmune disorders. Patient's hip 7. lgE are cytophilic antibodies responsible for medi-
pain, subsequent fractured femur neck and associated ating allergic reaction, hypersensitivity and anaphy-
proteinuria and anemia suggested a gammopathy. laxis.
A monoclonal peak with 13 mobility was present in both 8. Malignancy of lg secreting plasma cells leads to
serum and urine electrophoresis. enormous proliferation of one clone and result in
multiple myeloma where monoclonal M band is
46-1 . lmmunoglob ulin structure consists of: 46-6. M band is seen in which condition?
A. 2 light chains A. Cirrhosis
B. 2 heavy chain B. Chronic infections
C. 1 light chain and 1 heavy chai n C. Multiple myeloma
D. Heavy chain disease
D. 2 light chains and 2 heavy chains
46-7. Which has anti-viral effects?
46-2. Which is the largest immunoglob ulin?
A. TNF alpha B. lnterfeirons
A. lgM B. lgG C. IL-1 D. IL-2
C. lgD D. lgE 46-8. Which is involved in primary immune response?
46-3. Hinge region is seen in which segment? A. lgG B. lgM
A. VL C. lgA D. lgD
B. VH
46-9. Which is involved in secon d ary immune res-
C. CL D. CH
ponse?
46-4. Which immunoglob ulin has the highest serum
A. lgG B. lgM
concentration? C. lgA D. lgE
A. lgG B. lgM 46-10. Which is involved in anaphylaxis?
C. lgA D. lgD A. lgG B. lgE
46-5. Which immunoglobulln exists as a dimer? C. lgG D. lgD
A. lgG B. lgM
C. lgA D. lgE
Chapter at a Glance
The learner will be able to answer questions on the following topics:
Acquired immunodeficiency syndrome (AIDS) 0 Immunology of AIDS
Human immunodeficiency virus (HIV) Laboratory diagnosis of AIDS
IEPIDEMIC OF AIDS at the National Institute of Health, USA and Luc Montagnier
at the Pasteur Institute, Paris. The latter was awarded
Toward the end of the 20th century, medical science
Nobel Prize in 2008. The virus was originally designated
was able to control and even conquer many of the pre-
as HTLV-11I (human T cell leukemia viirus). In 1986, it was
viously incurable diseases. But AIDS has now become
redesignated as HIV (human immunodeficiency virus). By
a disease of pandemic proportions.
retrospective analysis, it was inferred that the human pan-
In 1981, a cluster of 5 cases of Pneumocystis carinii
demic started in Africa by around 1970, and that a simian
pneumonia were reported in USA. These protozoa can
virus transformed into HIV by around HOO.
produce pneumonia only in immunodeficient individuals.
Based on the clinical manifestations, the disease was
Indian Scenario
named as acquired immunodeficiency syndrome with
acronym of AIDS. In 1983, Frarn;oise Barre-Sinoussi The virus entered in India in 1980. Fmm 1986 onwards,
(Nobel Prize, 2008) showed that the disease is due to a the Indian Council of Medical Research has started the
retrovirus (virus having reverse transcriptase enzyme). serosurveillance against HIV. The first seropositive indi-
The isolation of a virus from the lymphocytes of the AIDS viduals in India were identified in 1986. The Government
patients was done in 1984 independently by Robert Gallo of India has established the National AIDS Control
Organization (NACO) in July 1992 for the prevention
of AIDS. The adult HIV prevalence in India is now in a
declining phase.
Transmission
1. Eighty percent of the cases are sexually transmitted.
2. In about 15% of patients, the dise,asewas transmitted
Rhoab'elerts Luc Fram;oise through blood. The drug abusers usually use the
C , Montagnier Barre-Sinoussi
Gallo NP 2008 NP 2008 same needle without any sterilization for intrave-
_ _ _ _ _ _ _b_._1_9..;.
3 7_ _ _ _ _.....;..
b .....1..;.
9..;..
32 ::........,_ _ __,;,;,
b.;..
• .;..
19 ;..4;.;,7_ _ _ _ _..:.:
n..: o..:
u.:.
s...:.i:..::
nj:.::
e..:
ct=ioQ,,,,,,JJ;,-<u.~"' · 's..b.i...._.._...._ __
Chapter 47: Biochemistry of AIDS and H/V 657
Fig . 47.2: Structure of HIV There are 3 structural genes (gag, pol and env), 3 regu-
latory genes (tat, rev and nef) and 5 accessory genes
(vif, vpr, vpu, vpt and tev/tnv) in between long terminal
less than 2 weeks. Viral load is monitored during
repeats (LTR).
treatment by RT-PCR.
Immunology of AIDS
HUMAN IMMUNODEFICIENCY VIRUS The CD4 (T-helper) lymphocytes are decreased in num-
Structure ber, leading to immunodeficiency. The gp120 surface
unit could specifically attach with CD4 molecule present
HIV belongs to the retrovirus group. They are RNA-con- on the surface of T-helper cells. Therefore, HIV prefer-
taining viruses that replicate with the help of the reverse
entially enters into the T-helper cells and they are lysed.
transcriptase (RT) or RNA dependent DNA polymerase.
Since T-helper cells play a pivotal role in the immuno-
A schematic representation of the structure of the virus
logical system, their deficiency will lead to suppression
is shown in Figure 47.2. The virus has two copies of
of almost all the immunological effectors. T-helper (CD4)
single stranded genomic RNA. The protein components
count is less than 400 cumm of blood. T-killer cytotoxic
are named after its molecular weight. For example, pro-
activity is reduced. When all the effector mechanisms
tein having molecular weight 32,000 Daltons is called
p32. Other details are shown in Figure 47.2. of immunity are thus paralyzed, opportunistic pathogens
get entry into the body.
Virus Entry
Genetic Heterogeneity of Virus
The binding of HIV with target cell is through a receptor
Two types, HIV-1 and HIV-2 are characterized; HIV-1 is
mechanism. The gp 120 of the virus envelope will spe-
more virulent, infective and predominant. It is seen all
cifically bind with CD4 molecule on the surface of target
over the world. In India, both types are reported. There
cells. Thus CD4 acts as a receptor for the virus. The
are many subtypes of HIV-1 . Different subtypes may be
CD4 molecules are present on the surface of T-helper
seen in the same patient. Moreover, each type exhibits
cells and therefore helper cells receive the maximum
remarkable microheterogeneity. There is high mutation
attack of HIV.
rate in the virus. Such mutations accumulate to produce
the various types, strains and microvariations. About
Replication of HIV
15% of amino acids in the envelope gp120 are highly
After entry, the viral RNA is acted upon by the reverse variable. Therefore, the antigenicity of the virus also
transcriptase (RT). Based on the RNA, a DNA strand varies, and virus can escape from the immune attack.
Chapter 47: Biochemistry of AIDS and HIV659
47-1 . AIDS is caused by what type of virus? 47-3. All the following are structural genes of HIV, except
A. Adenovirus B. Adeno associated virus A. ~t B. gag
C. Retrovirus D. Parvovirus C. pol D. env
47-2. What is the most definitive test for detecting HIV? 47-4. Which anti-HIV drugs is a protease inhibitor?
A. ELISA B. Western blot A. Zidovudine B. Nevirapine
C. RTPCR D. T-helper cell count C. lndinavir D. Didano:sine
47.1 . What is the full form of AIDS? 47-6. What is the normal T-helper cell ,count?
Acquired immunodeficiency syndrome. More than 400 per cumm of blood.
47-2. What is the cause for AIDS? 47-7. Which molecule binds to CD4 re;ceptor on HIV?
Human immunodeficiency virus (HIV). gp120.
47-3. What type of virus is HIV? 47-8. What are the structural genes of HIV?
HIV is a retrovirus, an RNA virus. gag, pol and env.
47-4. A patient is Infected with HIV, but is.asymptomatic; 47-9. Which is the commonest treatmEmt for HIV?
what is this called Combination of drugs, highly active anti retroviral therapy
The window period. (HAART).
47-5. Is the window period dangerous? 47.10. How to handle blood spills?
Yes, he can transmit the disease as he is a carrier. Use 1% sodium hypochlorite solution.
t - -_ _ __ Chapter 48
Biochemistry of Cancer
Chapter at a Glance
The term "cancer" is derived from Latin word "cancrum" have occurred. Thanks to the surveillance by the immune
-
or Greek "karkinoma", that is equivalent to Sanskrit term ~ tern, these aberrant cells are usually destroyed. As
"karkitakam", which means "crab". The disease is so nces, the number of mutations accumulate,
called because of swollen veins around the area, resem- hence the st · tical probability of the incidence of can-
ble a crab's limbs. Indian Medical Science had identified cer is increased. wonder, cancer is a disease of old
cancer, gave the name "arbuda", which literally means age, especially after years.
the number 108 , identifying the extreme cellularjty of the 'l. Cancer is the second most common cause for death
cancer tissue. The International Union Against Cancer in developed countries, second only to cardio~ascular
(UICC; Union Internationale Cantre le Cancer) has defi- diseases. When the average life-expectancy is less, as
ned cancer as a disturbance of growth characterized by in the case of India, the death due to cancer is also low.
excessive proliferation of cells without apparent relation
Mutagens
to the physiological demands of the organs involved.
Oncology deals with the etiology, diagnosis, treatment, Any substance which increases the rate of mutation can
prevention and research aspects of cancer. also enhance ~ t.e of incidence oif cancer. Therefore,
all carcinogen's ar~ mutagens. Examples are X-rays,
ETIOLOGY OF CANCER gamma-rays, ultraviolet rays. Some human cancers are
caused by chemicals. These may be introduced into the
All cancers are multifactorial in origin . They include body by means of (a) occupation (aniline, asbestos),
genetic, hormonal, metabolic, physical, chemical and (b) diet (aflatoxins) or (c) lifestyle (smoking). Chemical
environmental factors. Most human cancers are ~ - carcinogens act cumulatively. Tobacco, food additives,
taneous. coloring agents, and aflatoxins are common carcino-
All cancers originate usually from one aberrant cell, gens in our environment.
which goes on to multiply and produce a tumor mass. Thousands of chemicals are known mutagens and
One mutation occurs out of 106 cell divisions. By the time carcinogens. A selected small list of chemical carcino-
a person reaches adulthood , about 1026 cell divisions gens is given in Table 48.1. Methyllcholanthrene is a
( 1t'4'1 ~ )
662 Section F: Advanced Biochemistry
Aflatoxins Progression
They are a group of chemically related compounds The biological history of a tumor shows progression of
synthesized by the fungi, Aspergillus f/avus. The mould malignancy. Cells with faster rowth r:~te have a selec-
grows on r¥ , wh~t and grou9 dnut, when kept in tion advantage. Thus, c Is with increased malignant
damp conditions. The fungi may grow in cattlejodder, Ch3/.acti3 are pro ressive selected. Familial adeno-
which may enter into human body through the cow's matgifs polyposis is a typical exam[Ple for multistep
milk. Aflatoxins are powerful carcinogens, which pro- progression. Mutations in the APC gene are inherited
duce hepatomas. from parents. By the time the patient becomes adult,
there will be different dy_sglastic ~ 1rant crypts in the
Cigarette large intestine. Some of the cells will ~Jet somatic muta-
t_Lung canci, is associated with the habit of cigarette tions in the K-Ras gene; these will progress to form
smoking. Cigarette contains many carcinogens, the adenomas. Further mutation in TGF qene or p53 gene
or Bax gene will give the push for the development of 1
most important group being benzo(a)pyrenes. Other
~r-..e.. important deleterious substances in cigarette smoke malignancy.
Co are nicotine, carbon monoxide, nitrogen dioxide and
carbon soot. Statistically, it is estimated that one ciga- Action of Chemical Carcino,gens
N~,.
·c; rette reduces 10 minutes from the lifespan of the indi-
vidual. The incidence of lung cancer is increased to 15
Mechanisms of action of chemical carcinogens are: (a)
Carcinogens are generally elec!J:2Bllllits (molecules
times more in persons smoking 10 cfgifettes per day deficient in electrons); they r~adily attack nucleoghilic
and 40 times more when smoking 20 cig1~~es per day. (electron rich) groups of DNA, (b) Carcinogens may
Thus, WHO suggested the slogan "Cigarf e smoke is bind covalent!_y to cellular DNA. N2, 1\13, and N7 atoms
injurious to health". Moreover, non-smok~g spouse of a of guanine are highly prone to addition of carcinogen
heavy smoker will have 5 times more probability to get groups, (c) These changes will lead to DNA alterations,
lung cancer than a non-smoker. This effect of "passive in spite of DNA repair, with increased probability of
smoking" made the International Union against Cancer mutations.
(UICC) to change the slogan to "Your smoking is injuri-
ous to our health". Physical Carcinogens
loral cancer} s strongly associated with chewing of
X-ray, gamma-ray and UV-ray may cause: (a) formation
obacco Pr !3 of pyrimidine dimers, (b) af)urinic sites with consequent
\ Alcohol · take increases the risk of oral, pha n- break in DNA, and (c) formation of free radicals and
geal, esophageal and Ii.Per cancers. Di~ igh i otal fat superoxides which cause DNA break, leading to somatic
an)icholesterol, increases the risk of cnlbn, ast and mutations. Exposure of X-ray in fetal life will increase
p~ ate cancers. the risk of leukemia in childhood. In p,opulation studies,
An often asked question is, why only some smokers 1 @d per _y_ear will increase the cancer incidence by
are getting cancer and not all smokers? Glutathione 40/million peo,rle per year.
Chapter 48: Biochemistry of Cancer 663
Anti mutagens
i. These are substances which will interfere with tumor
promotion. Vitamin A and carotenoids are shown Papova virus groulP
ii. Vitamin E acts as an antioxidant, preventing the Papilloma DNA Rabbit Papilloma
damage made by fr~ radicals and sup~ roxides. Marek DNA Chicken Lymphoma
~ -myc oncogene, with consequent malignancy. Michael Bishop and Harold Varmus, pioneers in the
HPV (Human Papilloma Virus) is the most com- oncogen~ rch were awarded Nobel Prize in 1989.
mon sexua lly tra nsmitted infectio n in adults. It has a ci r- A defin~i:2of for an oncogene was first d emonstrated
0 cular double stranded DNA. More tha n 100 HPV types
are known. H,r" ti'.pes 16 a nd..__W are associated with in avians but a
--- defjcje
in Rous sarcoma virus. The full viru~rod uces sarcoma
~~--.....__
ntJQ_a pa rticular
human ute rine cervical c r; they cause 70% of all gene, cou ld not cause the disease. He nce, this g ene
cervical cancers. a rald zur Ha uzen (Nobel Prize, 2008) was named as ~ e ne...abbreviated as Src. How-
showed the HPV DNA in the cancer cells. HPV infects ever, the same DNA sequences are avail~ le in normal
e_e_!the lial cells in the cervical mucosa; the virus avian cells also. This reveals th at no rma l cells do contain
plies a nd lyses the host cells , causing a lesion., $ ~ DNA sequences similar to vi ra l oncogenes. To distin-
of such cases healing occurs w ithin 6 months to~ years. g uish these two genes, they a re denoted as V-src (viral
But in about 1 % c~ses, the HPV DNA is integrated into gene) and C-src (cellular g e_!l.e ). The onco_g_enes...eresenf
some of th e host ce lls. After about 10- 30 ys)ars, these i~ s ar~ proto-;,;cog;,:;es.
cells develop inv_asive cancer. Vac~s against high risk A few important human proto-oncoge nes are abl, e rb ,
HPV 16 and 18 types a re 110w developed that provide myc, sis , etc.
Chapter 48: Biochemistry of Cancer665
nctioning gene
! • •
•
Embryonic cell
Methotrexate Follc acid analogue Competitive inhibitor of dihydrofolate reductase. THFA is n!q uired for nucleotide
synthesis
TABLE 48.7: Some monoclonal ant1bod1es used as anticancer is described in Chapter 44. The mechanism of action of
agents monoclonals against cancer may be!:
Name Target Used against the cancer a. The antibody marks the cancer cell and makes it
Rituximab CD20on Bcells NHL, CLL, B cell leukemla easier for the immune system to attack. The drug
Trastuzu mab HER-2/neu Breast cancer
rituximab attaches to CD20 found only on B cells;
(Hercepti n) (EGFR2, Erb-B2)
makes the cells more visible to the immune system,
Bevacizumab VEGF Colorectal, solid tumors of
kidney, breast which can then attack.
Alemtuzumab CD52 on B cells CLL b. Block growth factors. Certain cancer cells make
Cetuximab KRas Colorectal, head and neck extra copies of the growth factor receptor. This
Panitumumab EGFR Colorectal makes them grow faster than the normal cells.
lmatinib Tyrosine kinase CML Monoclonal antibodies can block these receptors
and prevent the growth signal. For example, Cetuxi-
methotrexate, tetrahydrofolic acid is not produced, which mab attaches to epidermal growth factor receptors
is necessary for incorporation of C2 and C8 of purines (EGFR) on cancer cells. Blod;ing this signal from
and CS methyl group in thymidine. Thus, there is inhibi- reaching its target on the canoer cells may slow or
tion of DNA synthesis and consequently of cell division. stop the cancer from growing.
Methotrexate is commonly employed in the treatment of c. Stop new blood vessels from forming. To attract
choriocarcinoma, which is a curable cancer. It is also blood vessels, cancer cells send out growth signals.
useful in acute leukemia. Monoclonal antibodies that block these growth sig-
nals may help prevent a tumor from developing a
6-Mercaptopurine
blood supply, so that it remainis small. The mono-
It is a purine analogue which prevents amination of IMP clonal antibody bevacizumab intercepts vascular
to AMP, so that the availability of AMP is reduced (see
endothelial growth factor (VEGF) and stops them
Fig. 38.11 ). This leads to inhibition of synthesis of DNA, from connecting with their targets (Table 4 8. 7).
and in turn cell division. It is commonly employed in
treating acute lymphoblastic leukemia.
Related Topics
Monoclonal Antibody Anti-oxidants (see Chapter 30), telomerase (see Chap-
These drugs are a relatively new innovation in cancer ter 39), cell cycle (see Chapter 42),. and apoptosis (see
treatment. Technique for monoclonal antibody production Chapter 42).
670 Section F: Advanced Biochemistry
••
St Clinical Case Study 48.1
ovarian cancer. However, it should be emphasized that
the vast majority of breast cancers aire not genetically
A 40-year-old female presented to the clinic with lump based, but occurs sporadically. BRCA2 mutations are
in right breast. A mammogram performed revealed a also associated with ovarian cancers and male breast
breast mass measuring 3 cm with numerous microcal- cancers.
cifications suggestive of breast cancer. The family his-
tory revealed that she had a sister who was diagnosed LEARNING POINTS, CHAPTER 48
with breast cancer at the age of 35. She also recalls that 1. All cancers are multifactorial in ori gin. They include
one of her aunts had died long ago with great abdominal genetic, hormonal, metabolic, pl1ysical, chemical
distension (probably ovarian carcinoma). On physical and environmental factors. Examples of physical
examination, there was a fixed and nontender mass on carcinogens are X-ray, gamma-ray, ultraviolet ray.
right breast, measuring 3 cm. Axillary lymph nodes were Examples of chemical mutagens are Aflatoxins,
palpable on the right side. Skin was not involved . The Methylcholanthrene, Nicotine.
biopsy report was intraductal carcinoma. What cancer 2. Examples of antimutagens include Vitamin A, Vita-
gene might be associated with cancer breast? What is min E, Vitamin C and Curcumin.
the likely mechanism of activation of oncogene in this 3. Example of viruses producing turmors in animals
case? are Polyoma, SV 40, Gross, Rous, etc.
48-1. Antimetabolites. 48-7. Name the t umor marker most appropriate for t he
48-2. Oncogenes. following: (a) prostate carcinoma; (b) c horio car-
48-3. Oncosuppressor genes. cinoma; (c) colon cancer; (d) hepatoma; (e) pheo-
48-4. Tumor markers. c hromocytoma; (f) carcinoid synd rome; (g) bone
48-5. A lpha-fetoprotein. metastasis.
48-6. Carcinoembryonic antigen.
48-1 . Name anti-mutagens and anti-carcinogens. 48-5. Name s,ome commonly used tumor markers.
Vitamin A, Vitamin C, Vitamin E, Curcumin. Alpha-fe'loprotein; Carcinoembryonic antigen; Prostate
48-2. What are Oncogenes? specific antigen
Genes capable of causing cancer. Oncogenes are spe- 48-6. Alpha-f1~toprotein (AFP) level is used as a tumor
cific sequences in DNA which when expressed may marker for which cancer?
Hepatoma.
produce cancer.
48-7. What is the marker for colorectal cancers?
48-3. What are tumor markers?
Carcinoembryonic antigen.
They are factors released from the tumor cells, which
48-8. What is the significance of beta chain of human
could be detected in blood and therefore indicate the
' presence of the tumor in the body.
chorionic gonadotropin?
It is a tumor marker for choriocarcinoma.
48-4. What is clinical application of tumor markers? 48-9. What is the mechanism of action of mitomycin?
They are useful (1) to follow-up cancer and to monitor Intercalation with DNA strands
the effectiveness of the therapy, (2) to detect the recur- 48-10. What is the mechanism of action of Methotrexate?
rence of the tumor. It is a folic acid antagonist.
_ _ _ _ _Chapter 49
Tissue Proteins in
Health and Disease
Chapter at a Glance
The learner will be able to answer questions on the following topics:
D Collagen, structure and synthesis D Muscle proteins
D Abnormal collagens D Myosin, actin, troponins
D Elastin D Lens proteins and cataract
D Keratins D Prions and Alzheimer's disease
• w
Fig. 49.1 : Triple stranded collagen fiber
2
3
---===------
5 _ _ __
4
----
F ig. 49.2: Quarter staggered arrangement in collagen fiber; each
row moves one-fourth length over the last row; the 5th row repeats
the position of the first row
Deficiency of Ascorbic Acid Fig. 49.3: Sliding and shortening of actin and myosin is the basis
It is characterized by defective hydroxylation of collagen. of muscle contraction. Compare the distance between Z lines in
the upper and lower pictures
The collagen formed is weak, leading to frag ility of blood
vessels, poor wound healing, etc. (see Chapter 33).
Keratins
Hyperhomocysteinemia Keratins are proteins present in hair, s~<in and nails, horn,
The accumulated homocysteine in this condition, reacts hoof, etc. The fibers present are called alpha keratins
with lysyl aldehydes to block crosslinking. The skeletal and matrix as keratohyalin. They mainly have the alpha
deformities, vascular and ocular defects are thus pro- helical structure. Each fibril has 3 polypeptide chains
duced (see Chapter 18). and each bundle has about 10- 12 fiibrils. The kerato-
hyalin matrix has cysteine-rich polypeptide chains which
Lathyrism are held together by disulfide bonds. The more the num-
It is due to ingestion of lathyrus sativa or sweet pea. It is ber of disulfide bonds, the harder the !keratin is.
due to a toxic agent beta oxalyl amino alanine (see
Chapter 35). This compound has been found to inhibit [ MUSCLE PROTEINS
------------
lysyl oxidase. This would interfere with formation of lysyl Striated muscle is made up of multinucleated cells bound
crosslinking. by plasma membrane called sarcolemma. Sarcomere is
I ELASTIN
the functional unit of muscle. Each muscle cell contains
myofibrils about 1 mm in diameter. lrhe myofibrils are
Elastin is a protein found in connective tissue and is the immersed in a cytosol that is rich in glycogen, ATP,
major component of elastic fibers. The elastic fibers can creatine phosphate and glycolytic enzymes.
stretch and then resume their original length. They have The functional unit of a myofibril is a sarcomere.
high tensile strength. They are found in the ligaments as The dark A bands and light I bands alternate regularly
well as in the walls of the blood vessels, especially large (Fig. 49.3). The central H zone of A band is lighter, while
vessels like aorta. One-third of the residues are glycine. the dark M line is found in the middle of the H zone. The
Praline is present in large amounts, so also alanine. I band is bisected by a very dense narrow Z line.
Hydroxyproline is present in small amounts while hydroxy- These bands are formed by variable combination of
lysine and glycosylated hydroxylysine are absent. Triple thick and thin filaments. The thick filament is primarily
helix structure is also absent. When elastin matures, myosin and thin filament contains actin, tropomyosin,
desmosine cross links are formed from 4 lysine and troponin. The Z line contains 2 actin molecules and
residues. M protein is located in the M line (Fig. 49.3).
Pseudoxanthoma elasticum: It is an inherited Thick and thin fi laments slide past each other during
defect in the formation of elastin. Clinical manifestations the muscle contraction, so that the muscle shortens by
are similar to Ehlers-Danlos syndrome. as much as a third of its original len!;ith. However, the
Chapter 49: Tissue Proteins in Heialth and Disease 675
BOX 49 .1: Muscle contract1on-relax,it1on events heated and denatured. In diabetes mellitus, when the blood
1. Nerve impulse releases acetyl choline (ACh) at motor end-plate.
glucose level is increased, lysine resiclues of these pro-
This ACh binds with the receptors. teins are glycated. This leads to increased susceptibility
2. Sodium-potassium conductance in neuromuscular end-plate, for sulfhydryl oxidation and consequent aggregation of the
so that a potential is generated at the end plate. proteins, resulting in opalescence and cataract. Protein
3. This Is transmitted as the action potential to m uscle fibers.
aggregates with molecular weight more than 50 million
4. Depolarization; release of calcium ions from SR \
5. Binding of calcium ions to TnC. will produce scattering of light.
6. Crosslink formation between actin and myosin In lens, the enzyme aldose reductase reduces mono-
7. Sliding of thin filaments over thick filaments; muscle contracts. saccharides to corresponding sugar alcohols; glucose to
8. Calcium is pumped back into SR.
sorbitol and galactose to galactitol. Th1~se polyols do not
9. Release of calcium from troponin.
10. Actin and myosin are separated; muscle relaxed. readily cross cell membranes and hE~nce accumulate;
causing osmotic swelling, and cons1equent disruption
of cell architecture. Thus diabetes m,ellitus (increased
I LENS PROTEINS
__ glucose in blood) and galactosemia (hifJh galactose level)
cause cataract. Drugs that inhibit aldose reductase are
India has the maximum number of blind persons in the shown to retard cataract formation in diabetic rats.
world. Cataracts and opacities of cornea are the cause
for 70% of blindness. The eyes of older people and dia- lf_RIONS
betics are prone to cataract formation. Being avascular,
The central dogma in molecular biology postulated by
lens relies on the aqueous humor for the provision of
Watson and Crick in 1953 was that g,enetic information
oxygen and essential metabolites. Uppermost part of
passes from DNA to RNA and then to protein. In general,
the lens consists of a monolayer of epithelial cells, which
this rule still holds good. In 1970, Ternin and Baltimore •
divides and differentiates to form the long fibre cells, that
showed that DNA could be synthesized from RNA by
make up the lens. These normal lens cells (but not the old
cells) possess the usual protein synthesizing machinery. reverse transcriptase. This has partly shattered the
The proteolytic activity of the lens is quite low and is central dogma . But could proteins act as an information
due to the presence of endogenous protease inhibitors. molecule? Could proteins replicate 1themselves? This
Lens tissue has a very active HMP shunt pathway, and question was considered to be heretical till a few years
has the maximum concentration of NADPH. Lens also ago, but no longer so. There are a few diseases charac-
contains high quantity of ascorbic acid. They scavenge terized by very long incubation period of many years.
the free radicals and maintain the transparency of lens. These "slow disease agents" were originally thought to
be "unconventional viruses", but now they are proved as
Crystallins prion proteins.
Major lens proteins are alpha, beta and gamma crystal- Prion Proteins: Abnormal
lins. Small quantities of delta and epsilon varieties are Tertiary Structure
also described. They undergo no replacement through-
"Prions" is the acronym for "proteinaceous infective par-
out the life of the individual. There is no turnover of these
ticles". Stanley Prusiner has described prion proteins
proteins. The proteins at the center of the lens are as old
(PrP) in 1982, who was awarded NobeII Prize in 1997. PrP
as the individual. The orderly arrangements of the mole- is a normal protein of 253 amino acicls, found in leuko-
cules make the lens proteins transparent. Alpha crystallin cytes and nerve cells. The matured prion protein (PrP)
is present not only in lens, but is seen in almost all cells has 210 amino acids. It exists as a sialoglycoprotein,
of the body. anchored on the cell surface. PrP molecules can
undergo a change in structural conformation. The altered
Cataracts
molecule is resistant to heat and proteolytic enzymes.
When there is change in three dimensional structure of The abnormal protein is called PrPsc; "sc" stands for
lens proteins, the lens becomes opaque. This is similar scrapie, the disease in which it was first isolated. Thus,
to the clear albumin becoming white and opaque when prions are proteins with correct primary structure,
Chapter 49: Tissue Proteins in Heialth and Disease 677
49-1 . Hydoxylation occurs in which of the following ami- 49-3. Glycosyla11ion of collagen occurs i n which of the
no acids in collagen? following ,residues?
A. Glycine B. Lysine A. Hydrox:yproline B. Lysine
C. Tyrosine D. Threonine C. Proline D. Hyproxylysine
49-2. Wh ich is the most common amino acid present in 49-4. Random c:oil structure obtained by boiling colla-
collagen? gen is kno,w n as
A. Glycine A. Procollagen
B. Lysine B. Gelatin
C. Threonine C. Collagen degradation product
D. Proline D. Tropooollagen
Chapter 49: Tissue Proteins in He,alth and Disease 679
49-5. Which disease is due to the toxin beta oxalyl ami- 49-8. Which troponin binds to tropornyosin?
no alanine? A. Troponin I B. Troponin C
A. Ehlers-Danlos syndrome C. Troponin M D. Troponin T
B. Marfan's syndrome 49-9. Which is associated with Alzheimer's disease?
C. Menke's disease A. Tau protein B. Amylc1id protein
D. Lathyrism C. APO-E4 gene D. All of 11he above
49-6. Functional unit of myofibril is 49-10. Which statement regarding prions is incorrect?
A. Actin B. Myosin A. They are infectious particles
C. Sarcomere D. A band B. Normal prion proteins have important functions in
49-7. Which of the following is not a component of tro- the body
ponin complex? C. It is normally anchored on the, cell surface
A. Troponin I B. Troponin C D. They have normal primary structure, but abnormal
C. Troponin M D. Troponin T tertiary structure
Chapter at a Glance
The learner will be able to answer questions on the following topics:
0 Isotopes 0 Diagnostic applicati ons
0 Radioactive decay and half-life 0 Treatment applications
0 Units of radioactivity 0 Biological effects of radiation
0 Resea rch applications
2
Isobars are atoms having same mass number, but : Radium - - 2~ Radon+ ~He (a particle)
are having different atomic numbers, e.g. 14C and 14N.
The nucleus of Radium. being unstable, emits 2 protons
and 2 neutrons (one helium nucleus) to become Radon-
Atomic Number and Atomic Weight
222. The alpha particles will carry 2 positive charges and
The number of protons (or electrons) in an atom will produce maximum ionization in their path. Thus, they
determine the mass number or its place in the periodic are most damaging to tissues. Alpha particles emitted
table. The presence of neutrons will add on the mass of have a high mass and therefore a high momentum. They
the atom. The atomic weight or mass number is equal
do not travel far and can be stopped by a few layers of
to the number of protons plus neutrons in the atom.
paper. However, they collide with other molecules and
Percentage of
original radioactivity
8 16 24 days
1
'~ Iodine ;.!Xenon(metastable) Roentgen (R)
H3) (-v)
1
t Xenon It is the measurement of exposure dose. 1 R is the radia-
A comparison of d ifferent forms of radiation is given tion which will give rise to 2 x 109 ion pairs/cc of air.
in Table 50.1. One mCi source kept at a distance of 1 cm will produce
12.9 R/hour.
Half-life of Radioactivity
The radioactivity is halved within a fixed time. For exam-
Rad and Gray
ple, if 100 mCi of 131
1 is kept , after 8 days the activity is Rad is the absorbed dose by tissue. 1 Rad = 1.5 x 10 12 ion
seen to be 50 mCi. The half-life of 1311is 8 days (Fig. 50.1). pairs/g tissue. One Gray (Gy) = 1()7 ergs/ kg tissue
The half-life is the time taken for a radioactive = 100 rads.
isotope to become half of its original activity. The
rate of decay or the half-life is a constant for a particular Applications of Radioactivity
isotope. Commonly used isotopes are listed in Table 50.2. in Research
Isotopes of an element will have identical chemical reac-
UNITS OF RADIOACTIVITY tions. Hence, when a radiolabeled compound is admin-
Curie (Ci) istered, these molecules are metabolized by the body
similar to normal molecules. This is cailled Tracer tech-
One Curie, abbreviated as Ci, is equivalent to 3. 7 x 1010 nique. Almost all biochemical research will utilize such
disintegrations per second (dps) or 37 giga becquerals tracer methods. A few examples are given below.
(gBq). This unit is used to measure the radioactivity of i. Almost all the pathways described in earlier chap-
the source. ters were studied by using traceirs. For example,
14
C-labeled acetoacetic acid is st1own to be incor-
Becqueral (Bq)
porated into palmitic acid. Suppose labeled "A" is
Becqueral (Bq) is defined as decay per second, (dps). administered to an animal. After a few minutes, liver
1 Bq = 1 dps. It is often expressed as kilobecquerals (kBq). contains labeled "B" and after one, hour, labeled "C"
Chapter 50: Applications of Isotopes in Medicine 683
is seen in liver. Thus, we can say that the pathway emission of positrons and their combination with
is A to B to C. an electron resulting in the simultaneous emission
ii. 32 P is useful to trace the nucleic acid synthesis in of two gamma rays is detected by a PET camera.
t
vivo and in vitro. It is therefore employed in genetic Combination of PET and CT (PET/CT) improves
research. 3 H-labeled thymidine is incorporated the diagnostic accuracy. The abnormality may be
in the newly synthesized DNA and therefore used either lesser uptake of the isotope by the organ
in assessing cell division kinetics. (cold spot) or more uptake (hot spot). A series of
iii. The total body content of a particular substance images taken over a period of time will give specific
(also designated as the "pool" of the substance) patterns that indicate normal or malfunction of the
can be quantitated by the isotope dilution tech- organ concerned. An advantage over X-ray imaging
nique. To cite an example, 1 ml of 131 1-labeled is that both bone and soft tissues can be studied.
albumin is seen to have 1 million dps. This is injected
intravenously to a man. The radioactivity will be
Applications of
uniformly mixed in the total blood volume. After Radioactivity in Treatment
10-15 minutes, a blood sample is withdrawn. One ml Radioactivity is used for treatment of cancer. Radiations
of blood is shown to have a radioactivity with 200 dps. when absorbed by the tissues, produce ionization in the
The volume injected and removed is the same 1 ml, path. Nucleic acids in the cell are damaged, so that next
but the original count is now diluted 5,000 times. cell division is not possible. Radiotherapy mainly affects
Thus, the intravascular space is 5,000 times more cells in the divis.ion phase. Since cancer tissue contains
than the volume injected. Therefore, the blood more dividing ciells than normal tissue, cancer cells are
volume is 5,000 ml. preferentially affected by radiation. Radiotherapy may
iv. Carbon dating technique is an important tool in be classified as:
paleobiology, the techn ique was developed by Wil-
lard Libby who was awarded Nobel Prize in 1960. Unsealed Sources
These are radioactive substances kept in liquid form.
Applications of The beta rays are the main effective radiation in these
Radioactivity for Diagnosis sources. For thyroid cancer and secondaries of thy-
The branch of medicine that deals with the diagnostic roid cancer, 131 1 (dose 50-100 mCi) is administered.
applications of radioactivity is referred to as Nuclear Similarly, 32P is used to treat polycythemia vera.
Medicine. A quick and accurate diagnosis can be made
by radioimaging of organs like thyroid, liver bone, etc. Sealed Sources
Diagnostic uses of radioactive tracers are using gam- They utilize gamma irradiation. The source is applied on
ma ray emitters. These are short lived isotopes linked the cancer or sometimes implanted as a needle into the
to chemical compounds that permit the observation of tissue.
specific physiological processes. They may be given by Cs (cesium) with a half-life of 30 years, is the pre-
injection, inhalation or orally. ferred sealed source nowadays. Application of such
i. Thyroid uptake studies by 131 1 are used to detect sources directly on cancer tissue is called Brachy-
functional derangements of thyroid gland. About 15 therapy. lntracavitary applications (for cancer body of
mCi of 131 1 is given intravenously. After a few hours,
.. the patient is monitored at the neck region by a
uterus, cancer of cervix uteri, cancer of vagina) and
interstitial applications (buccal cancer, tongue cancer)
movable gamma-ray counter, which will pick up the
are common.
radiation emitted by the thyroid gland. The normal
values are about 25% uptake by thyroid within
Teletherap~,
2 hours and about 50% uptake within 24 hrs. In
hyperthyroidism there will be increased uptake and The term "tele" means distant (as in the case of tele-
hypothyroidism shows the reverse effect. scope, telephone, etc.). Here the source of radiation is
ii. Positron emission tomography (PET) scan is kept at a distance from the patient. Historically, telethe-
a more precise and sophisticated technique. The rapy started with deep X-ray. X-ray was discovered by
684 Section F: Advanced Biochemistry
Wilhelm Rontgen in 1895 (Nobel Prize, 1901 ). Due to There will be atrophy of skin , hypopigmentation, fibros is,
its inefficiency, deep X-ray is no more used for cancer loss of elasticity, etc.
treatment.
Linear accelerator (LINAC): Here electrons are Effects on Mucous Membran1e
accelerated to higher energy levels of 8-12 MV and The gastrointestinal mucosa is very sensitive to radi-
directed into the cancer tissue. It has more penetrating ation. These include nausea, vomitin!J, diarrhea and in
power and accurate beam focussing capabilities. As there severe cases ulceration and bleeding. Late sequelae
is no permanent radioactive source in the machine, the such as adhesions, fibrosis, stenosis and obstruction
radiation hazards are minimal. LINAC is used for exter- may appear many months after radiotherapy.
nal beam radiation treatments for patients with cancer.
Effects on Blood Cells
Radiosens itivity Bone marrow and lymphoid tissues are highly radio-
The effectiveness of radiotherapy varies with different sensitive because of the higher rate of cell division in
tumors. In general, lymphomas, Hodgkin's disease and these organs. Leukopenia and thrombocytopenia is an
neuroblastoma are highly radiosensitive. Epithelioma, accepted side effect of radiotherapy. If WBC count is
cancer of oral cavity, cancer cervix, cancer breast and below 2,000/cu mm and platelet count is below 80,000/cu
cancer lung are moderately radiosensitive. Poorly radio- mm, the therapy is temporarily stopped till recovery is
sensitive tumors are osteosarcoma, and malignant mel- effected .
anoma.
Effects on Reproductive Organs
I BIOLOGICAL EFFECTS OF Gonads (ovary and testis) are highly radiosensitive.
I..BAQJATION Complete sterility is effected at 1000 rads. Even low
doses of radiation, too low to have any obvious effect
Direct Effects on Cancer Tissues on mitosis, can still affect the genes, so as to produce
The radiation damages DNA molecules. No effects are genetic alterations in the offspring. This is especially
visible immediately. But the damage is observed during important when radiation is given in pelvic region.
the next mitosis. Since new DNA cannot be synthesized,
cells die at the attempt of the next division. Chromosome Carcinogenic Potential
breakage is often noticed. Radiation produces large
During the period 1900-1910, people were working with
quantities of free radicals in tissues. The catastrophic
X-rays without any precautions. This caused non-heal-
effects of free radicals on different biological compounds
ing ulcers in many of them. During 1910s and 20s, lip
(including DNA) are described in Chapter 30.
cancer was common among painters of watch dial with
Effects of Radiation on Normal Tissues radioactive stain. Gradually, along w ith the increasing
knowledge on radiation hazard, strin£1ent safeguard for
In 1904, Madam Curie went for a lecture-demonstration
radiation protection was introduced.
class, keeping a few mg of impure radium ore in her
breast pocket. Within 1 hour, this caused severe der-
matitis. That was the first indication of a health hazard
Radiation Protection
by radioactivity. Madam Curie succumbed to radiation- There is always some amount of background radiation,
induced leukemia in 1934. of about 150 m Rem/year. Out of this, ,about 50% is from
the cosmic rays, about 30% from terrestrial environ-
Effects on Skin ment and 20% from internal environment (e.g. decay
Radiation will produce epilation, however hair may grow of 4°K). Granite and brick walls will increase external
after 3 months. Sweat glands may be permanently dama- background. At higher elevation, cosmic rays are more.
ged. There may be erythema and sometimes blisters. At an altitude of 2000 m (e.g. Gangtok, Sikkim state),
This is called acute radiodermatitis. Chronic radio- the background irradiation is 20% more. In some coastal
dermatitis is seen after a few months of radiotherapy. areas (e.g. Kerala state) natural deposits of radioactive
Chapter 50: Applications of lsoiopes in Medicine 685
thorium is seen, where background is 20-30% high. 2. Spontaneous degradation of nucleus and trans-
One diagnostic X-ray exposure may cause 75 milliRem. mutation of one element to another with conse-
quent emission of rays or particles is known as
f
Maximum Permissible Dose
radioactivity.
The MPD of radiation for whole body among radiation
3. The total body content of a particular substance can
workers (doctors, technicians) is 5 mRem/year, and for
be quantitated by the isotope dilution technique.
general population is 0.5 mRem/year.
4. Applications of radioisotopes in medicine include
LEARNING POINT~ CHAPTER 50 thyroid uptake studies, radiotherapy in cancers.
5. Radiations affect mucous membrane, skin, blood
1. Isotopes are the same element having the same cells, reproductive organs and ,can damage DNA
atomic number (protons) but different mass number
preventing mitosis.
(varying number of neutrons).
. '
PART-3: VIVA VOCE QUESTIONS AND ANSWERS
50-1. What are the major types of radioactivity? 50-3. Which isotope is used for thyroid scanning?
Alpha particle, along with beta and gamma radiation . 131 1
50-2. Wh ich isotope is used for carbon dating? 50-4. What is meant by half-life of an isotope?
,.C The half-life is the time taken for a radioactive isotope
to become half of its original activiity.
;