Biochem IA2 (PDF)

Biochem
2
Carbohydrate Metabolism 174
El Glycolysis
Bass
Synonyms:
Embden Meyerhoff pathway or EMP. nom
Insulin
Definition :
Glycolysis is the breakdown of glucose to pyruvate (in aerobic conditions) or lactate (in
anaerobic conditions) for energy.
Site:
a) Tissue site:
All the tissues of the body.
It is the onJy pathway that is taking place in all the cells of the body.
b) Intracellular site:
Cytosol.
Starting Material :
Glucose
End Products :
a) In aerobic condition:
Pyruvate
b) In anaerobic condition:
Lactate
Conditions for anaerobic glycolysis are,

a) In tissue under hypoxic conditions
b) In vigorously contracting skeletaJ muscles
c) In RBC's, Lens etc., which lack mitochondria
NADH automatically
causesreach
For queries and suggestions, contact the author at prasad_text@yahoo.com or 9986449575
Glucose
I
Reaction Pathway II ATP
Mg·' Hexokinase in all tissues. Glucokinase in liver
A DP
Glucose 6•Phosphate
t Phosphohexose isomerase
Fructose 6·phosphate
ATP
2
ADP ~ i
Phosphofructokinase (PFK)
Fructose 1,6-bisphosphate
PGAIGASP
EE
DHAP
Glyceraldehyde 3·phosphate Dihydroxyacetone phosphate
p~ Phosphotrobe isomerase
NAD+ Glyceraldehyde 3-Phosphate dehydrogenase
24 NADH+H+ BPGA
1.3
1, 3-bisphosphoglycerate
A DP ~
Mg•2 Phosphoglycerate kinase
ATP
3 PGA
t
3-pbosphoglycerate
Phosphoglycerate mutase
2PGA
2-phosphoglycerate
Mg·2 j Enolase
H20 Y!
PEP
Phosphoenolpyruvate
ADP:i
Mg·2 Pyruvate kinase
ATP
Pyruvate
In aerobic conditions, pyruvate is the end product of glycolysis, but in anaerobic
condition, pyruvate is further converted to lactate by lacta te dehydrogenase (LDH).
ADH• + tt• NAD•
Pyru vate •1-----'-----L,._____ •~Lactate
4 -i
Lactate dehydrogenase
LDH utilizes NADH formed in glyceraldehyde

G lyceraldeh yde -3-phosphale
]-phosphate dehydrogenase & gives NAD+.
Importance: Glycolysis requires a conrinuous
Pit==: :::;l.:.----7
gain
supply of NA D+ (for the glyceraldehyde ]-
phosphate dehydrogenase step). In aerobic
glycolysis, reconversion of NA DH to NA D•
1,3-Bisphosphoglycerale
i
t ... I
NADH• + H• NAO·
takes place during oxidative phosphorylation.
During anaerobic glycolysis, LDH ensures a
Pyrut ate "-, . / • Lactate
Lactate deh ydrogenase
co111i11uous supply of NAD•.

TCA
Inhibitors of Glycolysis:
I • Iodoacetate is an inhibitor of enzyme glyceraldehyde 3-phosphate dehydrogenase
p
• Arsenate is also an inhibitor of enzyme glyceraldehyde 3-phosphate dehydrogenase
• Fluoride is an inhibitor of enolase enzyme. Fluoride is an inhibitor of enolase enzyme.
So, fluoride is added to the blood to prevent glycolysis during blood glucose estimation.
Energetics of glycolysis :
Aerobic glycolysis gives 7
8 ATP and anaerobic glycolysis gives 2 ATP.
Note that in aerobic conditions, NADH produced in glycolysis enters ETC and gives 2.5
3
ATP by oxidative phosphorylation. ADH does not give ATP under anerobic conditions.
Step <Enzym e) Source ISQ. of A T P fQrmeg

i) Hexokinase / Glucokinase -1 ATP
Aerobic ii) Phosphofructokinase -1 ATP
glycolysis iii) Glyceraldehyde-3-phosphate dehydrogenase NADH 6ATP(3ATPx2)
65 85 I
iv) Phosphoglycerate kinase ATP 2ATP
v) Pyruvate kinase ATP 2ATP
Net Gain = SATP
Step <En zym e) Source N12, of ATP fQrrn eg

i) Hexokinase / Glucokinase -1 ATP
Anaerobic ii) Phosphofructokinase -1 ATP
glycolysis iii) Glyceraldehyde-3-phosphate dehydrogenase AOH 0ATP
iv) Phosphoglyccrate kinase ATP 2ATP
v) Pyrnvate kinase ATP 2ATP
Net Gain = 2ATP
N ew concept: According to the current concept, NADH gives 2.5 ATPs in ETC. So, in
glycolysis, glyceraldehyde-3-phosphate dehydrogenase step gives 5 ATPs (2.5 X 2).
Accordingly, aerobic glycolysis gives 7 ATP's (Instead of 8 ATPS'of earlier calculation.
Significance of glycolysis :
Importance
1. Provision of energy: Glycolysis is the only pathway that takes place in all the cells of
the body to provide energy. Almost all cells use glucose as principle source of energy.
2. In muscle cells, glycolysis provides energy even under oxygen deficient conditions.
3. In erythrocytes and lens, anaerobic glycolysis is the only pathway that provides
energy as these cells lack mitochondria (Because, TCA cycle and !}-oxidation, two
major pathways that provide energy take place in mitochondria).
4. Glycolysis provides the precursors for the synthesis of some compounds. For e.g,
Dihydroxy acetone phosphate for the formation of glycerol 3-phospha te, which in turn
required for the triacylglycerol synthesis & pyruvate for the transamjnation to alanine.
5. Glycolysis provides the intermediate 1,3 BPG for the production of 2,3 BPG, which is
required for the release of oxygen from oxyhemoglobin in tissues.

Regulation of glycolysis :
The energy requirement of the cell regulates the rate of glycolysis. When the cell
requires energy, the glycolysis operates in a faster rate and when the cell has sufficient
energy, glycolysis runs slowly. Hexokinase I Glucokinase, Phosphofructokinase
(most important enzyme), Pyruvate kinase are the regulatory enzymes of glycolysis.
nnninasesexceptpnospuogyceraten.name
Indu cer Re pressor Activator Inhibitor
SIRAI
Hexol<lnase Glucose 6-phosphate
- - Glucose
Pdt
Glucokinase Insulin Glucagon
- -o nosmaee
-
Phosphofructokinase Insulin Glucagon AMP,
I
Fructose 6-phosphate
fi
ATP, Citrate
Fructose 2,6-bis phosphate
Pyruvate kinase Insulin Glucagon Fructose 1,6-bis phosphate ATP
brapat
Pasteur Effect: Under aerobic conditions, glycolysis is inhibited. The inhibitory effect
of oxygen on glycolysis is known as Pasteur effect.
Difference between Hexokinase and Glucokinase

Hexokinase G lucokinase
D istribution All tissues Only in Liver
Action Acts on glucose, fructose, Acts only on glucose
Mannose
Km Low (0.01 mmol/L) High (20 mmol/L)
Affinity to glucose High Low
Inducer Not inducible Inducible by Insulin
Even when blood glucose Acts on when blood glucose level is high
Significance level is low, glucose can i.e.> 100 mg/ di; then glucose is taken
be utilized by body cells up by liver cells to synthesize glvcogen
Liver converts glucose to glycogen in fed state, mainly due to action of glucokinase.
Glucokinase is an inducible enzyme (by insulin), present only in liver. It has absolu te
specificity for glucose & has a higher Km for glucose than hexokinase. So, glucokinase
acts on only when glucose concentration is high i.e. fed state. It phosphorylates glucose,
which then converted to glycogen by glycogenesis (due to high insulin).
Significance of lactate dehydrogenase (LDH) reaction:

Glycolysis requires an urzi11terrupted supply of NAO· for glyceraldehyde 3-phosphate dehydrogenase
step of glycolysis, which utilizes NAD • and produces NADH, which is reconverted to NAO• by
oxidative phosphorylation. But in anaerobic conditions, this is not possible.
The formation of lactate by lactate dehydrogenase reconverts NADH to NAO+ and ensures an
uninterrupted supply of NAD for glycolysis.
This is very essential in skeletal muscle during strenuous exercise, where 02 supply is limited.

EZ Carbohydrate Metabolism 195
Gluconeogenesis
Definition :
Synthesis of new glucose molecules from non-carbohydrate sources is termed as
Gluconeogenesis or neoglucogenesis.
Sites:
Kidney
a) Tissue site: Liver (90%) and renal cortex (10%)
b) Intracellular site: Partly in cytosol and partly in mitochondria.
Starting Compounds (Non carbohydrate sources of gluconeogenesis):

a) Lactate: From contracting muscles and RBC.
b) Glycerol: From fats
AAG
c) Glutamate, Aspartate and Alanine etc (Glucogenic Amino acids)
d) Propionyl CoA: From ~- oxidation of odd chain fatty acids.
e Pyruvate
Reaction pathway:
Gluconeogenesis (formation of new glucose) & glycolysis (breakdown of glucose) are
opposing pathways, but, gluconeogenesis is not complete reversal of glycolysis. Seven
reversible reactions of glycolysis are common for both gluconeogenesis & glycolysis.
Three irreversible steps of glycolysis are bypassed by the new en zymes, called the key
oxaeocn iperac.ir
gluconeogenic enzymes, namely, Pyruvate carboxylase, Phosphoenolpyruvate
s.uastep
carboxykinase (PEPCK), Fructose-1, 6-Bisphosphatase and Glucose sn
6-phosphatase.
Note that starting compounds of gluconeogenesis enters into glycolysis or TCA cycle,
and from that point, the process goes in the reversible order to produce glucose.
Thus, the gluconeogenic pathway involves,
a) Reactions of TCA cycle.
b) Reversible reactions of glycolysis.
c) 4 key gluconeogenetic enzymes to bypass the irreversible steps of glycolysis.
1) Pyruvate carboxylase
2) Phosphoenolpyruvate carboxykinase (PEPCK)
3) Fructose-1,6-Bisphosphatase
4) Glucose 6-phosphatase
Irreversible steps of g l yco l ysis Correspond ing key g lu coneogenic e n zy mes
a) Pyruvate kinase 1) Pyruvate carboxylase

2) Phosphoenolpyruvate ca rbo xykinase
b) Phosphofructokinase (PFK) 3) Fructosc -1, 6-Bisphosphatase
c) Hcxokinase / Glucokinase 4) G l ucose 6-phosphatase .

Ketogenic Glucogenic a a
t Gostinto Carbohydrate Metabolism 196
intolipidsynth
Goes
Tsacycle
Reaction pathway: GLUCOSE

p·1
IGlucose 6-phosphatase I H20
tutiniina Glucose 6-phosphate
only
Cntinmusug Note:
t
Fructose 6-phosphate
1. Key enzymes are written
inside the rectangle boxes
Pi 2. Substrates are written
Fructose 1,6-bisphosphatase IV H20
inside the ellipsoidal circles
Fructose 1,6 -bis pho phate

.,,
·;;;
Q,I
C:
Q,I
00
0
Q,I
•I 1
Glyceraldehyde 3-phosphate
Pi ..._ t:,NAO"
s
l
Dihydroxyacetone phosphate
=
0
V I ·~
correspenrymes
NADH+H•
NADH+H+
Glycerol 3 -phosphate
:;s
ch I , 3-Bisphosphoglycerate dehydrogenase
.....
0 I I ?.ADP NAD + mallee
E
C:
r>l ATP
..0 I
0
'.tl Glycerol 3-phosphate pose
V
Q,I 3--Phosphoglyceratc ADP
I t lycerol kinase
2- Phosphoglycerate
Cvtoplasm
t
Phosphoenolpyruvate
ATP
GDP +C0 2 13C V ADP
Phosphoenolpyruvate
carbox kinase
r-.. +------------
Pyruvate
ATP LOH
GTP
Oxaloacetate Pyruvate +------------ ~ A
as ALT
k!::iotin
alanineaminotransferase
!Pyruvatecarboxylase I Acetyl CoA

Malatedehydrogenase ~ ADP + P/
tanen
JI"Oxaloacetate A ST
Malate g
Malate / Malate Citrat
suttee
G OH ~
Fumarate MITOCHONDRIA a -Ketoglutarate .-- ~
Succinyl CoA ------- ~
Answer hint for gluconeogensis from diffrent individual sources
(like alanine, lactate, glycerol or propinyl CoA) questions:
This is a all-in-one representation . Start from the individual source that
is asked (for e.g. lactate), draw gluconeogenesis from that point.

Significance of Gluconeogenesis:
1) Maintains blood glucose level during starvation :
Gluconeogenesis provides glucose when carbohydrate is not available in sufficient
amounts from the diet or from glycogen reserves (mainly during starvation ).
A continual supply of glucose is necessary for brain, nervous system, RBC, skeletal
muscles, lens etc. Liver glycogen stores can meet this need for only up to 12-18 hours
of fasting. During starvation, gluconeogenesis ensures the continual supply of glucose
to these tissues. As the glycogen stores start depleting, gluconeogenesis progressively
takes over, which ensure a continuous supply of glucose to brain and other tissues.
So, gluconeogenesis is critical for maintenance of the blood glucose level during starvation
conditions, especially for the proper functioning of brain tissues (Even though brain can
utilize ketone bodies during starvation, they prefer glucose to ketone bodies).
During starvation, body tissues can derive energtJ from alternative sources like proteins and fats.
But brain, RBC's, skeletal tissus, lens etc have an obligatory requirement of glucose.
2) Gluconeogenesis used to clear the w aste products of metabolism:

Certain metabolites produced in the tissues accumulate in the blood. These waste
metabolites are cleared from blood by gluconeogenesis.
E.g.: a) Lactate produced from muscle and RBC's
b) Glycerol from adipose tissues fat.
c) Alanine from muscle
d) Propionyl CoA produced from ~-oxidation of odd chain fatty acids.
These compounds will be taken up by liver & converted to glucose by gluconeogenesis.
Regulation of Gluconeogenesis:
The four key gl uconeogenetic enzymes are the regulatory enzymes of gluconeogenesis.
•Induction / repression: Glucagon & cortisol increase the rate of gluconeogenesis by
inducing them. Insulin decreases the rate of gluconeogenesis by repressing them.
• Allosteric regulation: Pyruvate carboxylase is activated by acetyl CoA & inhibited by
ADP; Fructose 1,6-bisphosph atase is activated by citrate & inhibited by fructose 2,6-
bisphosphate and AMP. Fructose 1,6-bisphosphate also inhibits this enzyme.
Regulatory enzymes Inducer TripsRepressor emulate
Activator
In hibitor
Glucagon, Cortisol Acetyl ADP

Pyruvate carboxylase Adrenalin Insulin CoA
fees soacetat.ro
Phosphoenolpyruvate
carboxy kinase Same Same
-- --
Fructose 1,6- AMP, Fructose 2,6-
bis phosphataseOf Same Same Citrate bisphosphate
ipospite
fructose
Glucose 6-phosphatase Same Same -- --

E5 Carbohydrate Metabolism 180
Citric acid cycle
Synonyms:
5 common
9 acid cycle, Final
TCA I cycle, Tricarboxylic2 acid Cycle, Krebs3cycle, Citric
metabolic pathway.
Definition:
TCA cycle is final common metabolic pathway for the oxida tion of Acetyl-CoA
obtained from carbohydrates, lipids and proteins (amino acids) to CO2, H 2 0 & energy.
Glucogenic Ketogenic
A min o acids Amino acid s
G lucose - --I ••
i
Pyru vate --- •
i
Acetyl-CoA ____. TCA cycle
•
Fatty Acids
TCA cycle is the central pathway connecting almost all the individual pathways
(either directly or indirectly).
Site:
a) Tissue Site:
All the tissues of the body except RBC's & lens (as these cells lack mitochondria).
b) Intracellular Site:
Mitochondrial matrix
Starting Material
Acetyl-CoA
End products:
CO2, H 20 , Energy

Reaction pathway:
~-
AcetylCoA
""~::....
- - - ~ Oxaloacetate
fe i
,-J'' -• -,.,• _,, Cl:~-
times
Simon
water
Co~H
coenzyme A
F
te H:!O H2<> Cis-aconitate
Socclnate
dehydrogenase
FAOHz F c H2<>
Fe•2 1Aconitase
FAD
Citric acid cycle was cb lsocitrate
Succinate
CoASH
NAO' ~ lsocitrata
Gll'
Succlnate NADH + H+ dehydrogenase
thloldnase
GDP+PI [Oxalosuccinate]
Succinyf-CoA
5C
4C 2 COz Mn+2 ~ t rate
3 =ydrogenase
--~- o- ketoglutarate
5C
Inhibitors of TCA cycle: Eca Gylolysis

• Fluoroacetate is an inhibitor of aconitase
• Arsenite is an inhibitor of a-KG dehydrogenase
• Malonate is an inhibitor of succinate dehydrogenase
Energetics:
STEP Coenzvme ATP formed
a) Isocitrate dehydrogenase NADH 3
b) a -Ketoglutarate dehydrogenase NADH 3
c) Succinate thiokinase GTP 1
d) Succinate dehydrogenase FADH2 2
e) Malate dehydrogenase NADH 3
TOTAL 12
One molecule of Acetyl CoA gives 12 ATPs in TCA cycle. According to the current
concept, NADH g ives 2.5 ATPs & FADH2 gives 1.5 ATPs in ETC. In TCA, 3 NADH
give 7.5 ATPs (2.5 X 3) & 1 FAD8z gives 1.5 ATPs. So, according to the new concept,
TCA cycle gives 10 ATPs (Instead of 12 ATPs as per earlier calculation).

Functions of TCA Cycle :

1) Primary function of TCA cycle is the provision of energy.
2) TCA cycle has amphibolic (both Anabolic and Catabolic) nature.

a) Catabolic role:
TCA cycle is the final common metabolic pathway for the production energy from
acetyl CoA obtained from carbohydrates, lipids and proteins / amino Acids.
f
Amino acids Amino acids
Glucose -
i
__. Pyruvate "
I
Acetyl-CoA • TCA cycle
•
FatJ Acids
b) Anabolic role:
Starting from intermediates of TCA cycle, several compounds can be produced .
i) Heme synthesis:
Succinyl-CoA is used as a starting material in heme synthesis.
ii) Synthesis of aspartate and glutamate: 21699Eur
31 the orof
Oxaloaceta te and a-ketoglutarate can be converted to amino acids aspartate and
gluconeogenesis
glutamate respectively by transamination process.

iii) Fatty acid synthesis:
Citrate is transported out of mitochondria into cytoplasm where it can give back acetyl-
CoA & oxaloacetate. Acetyl-CoA can be used for the synthesis of fatty acids.
iv) Glucose synthesis:
12 prash
who
Intermediates of TCA Cycle can be sources for gluconeogenesis. Varun
• Since TCA cycle has both anabolic and catabolic role, TCA cycle said to be amphibolic
(both Anabolic and Catabolic) nature.
Regulation: is by CIKenzyme
Hueb's Cycle Regulator
Citrate synthase, Isocitrate dehydrogenase and a -ketoglutarate dehydrogenase
enzymes are the regulatory enzymes of TCA cycle.
Regulatory enzyme Inducer Repressor Activator Inhibitor
IRA
C itrate synthase Insulin Glucagon - rainy Citrate, NADH, ATP,frat's
annote
snawn antconservat fa ttv acvl CoA
lsocitrate dehydrogenase lrsulin Glucagon ADP,NAD• NADH,ATP
Reagents Pdt's
a -keto g lutarate dehydrogenasc Irsulin Glucagon ADP, NAD• NADH,ATP

The energy (ATP) requirement of the cell regulates the rate of TCA cycle. When the cell
requires energy, TCA cycle runs in a faster rate & when the cell has sufficient energy,
TCA cycle runs slowly. So, ATP is the inhibitor and ADP is activator of TCA cycle.
Anaplerotic reactions of TCA cycle (Greek: Ana = fill up) :

In its anabolic role, several intermediates of TCA cycle are used up for biosynthesis
of many compounds. These intermediates should be replenished. The reactions, which
replenish these intermediates, are called anaplerotic reactions or anaplerosis.
E.g. 1: Pyruvate carboxylase reaction:
Pyruvate carboxylase
Pyruvate + CO2 ----=--:::-------+• Oxaloaceta te
;;:-:-=-;iot~
ATP Mn2+ ADP+ Pi
This is a very important anaplerotic reaction. Since a large entry of acetyl-CoA to the
TCA cycle depletes the supply of oxaloacetate required for the first reaction of TCA
cycle (citrate synthase).
E.g. 2: Malic enzyme:

Malic enzyme
Pyruvate + CO2 Malate + H20
NADPH + H· NADP•
Other examples of anaplerotic reactions are Glutamate dehydrogenase and

transaminases (ALT and AST).
Oxidative decarboxylation reactions:

Several keto acids undergo oxidative decarboxylation reactions by keto acid
dehydrogenases. These are multi-enzyme complexes with 3 enzymes and 5 Coenzyrnes.
These reactions require 3 B-complex vitamins (Thiamine, Riboflavin and iacin).
E.g.: a-keto glutarate dehydrogenase and Pyruvate dehydrogenase (PDH).
) a-keto glutarate dehydrogenase enzyme complex (of TCA cycle):

a,.ketoglutarat e dehydrogenase
cx-ketoglutarate 77'" • Succiny l CoA
CoASH - / FAD, TPP ~-co.
NAD Lipoic acid NADH•+H •
3 enzymes are a-ketoglutarate dehydrogenase. Dihydrolipoyl tmnsacetylase, Dillydrolipoyl dehydrogenas
5 Coenzymes are TPP, CoASH, NAO•, FAD, Lipoic acid.
) PDH is already exaplained before.

Carbohy drate Metaboli sm 185
Glyco gen metab olism (Glycogenesis & Glyco genol ysis)

Glycogen is an animal storage polysaccharide. It is synthesi zed (Glycoge nesis) during
fed (energy rich) conditio ns and broken down (Glycoge nolysis) during fasting (energy
deficient) conditio ns.
Homo
of a DGlucose
polysaccharide Branched
Glyco genesi s (Glycogen synthesis)

Definition:
Formatio n of glycogen from glucose is called glycogenesis.
Site:
a) Tissue site : Liver and muscle
b) Intracell ular site : Cytosol
Starting compound:
Glucose
End product:
Glycogen
Reaction pathwa y:
It takes place in two phases.
a) Activation glucose [Formation of UDP glucose or UDPG]:

triphosphate
uridine
ATP ADP UTP PPi
~g+2Jf \. 1'
Glucose Glucose 6-phosph ate • ., Glucose lpho p a h t ~ UDPGlc
Hexokinase/ Phosphog luco UDPGlc
Glucokina se mutase pyophosph or}'lase
r
esomeneat
asreverse is
phosphorylate
UDP glucose is the donor of glucose in glycogen synthesis. of

uppalacou
b) Length ening of chain & formation of glycogen:

It requires two enzymes i) Glycogen synthase ii) Branchin g enzyme.
Glycogen synthesizesGlycogen
primer
i) Glycoge n synthase: By the action of glycogen synthase enzyme, UDP glucose adds
its glucose to a pre-existing glycoge n primer & lengthen s it by one glucose molecule
to form glycogen amylase (l iberating free UDP). The new glucose is added in a(l • 4)
direction. (Glycogen primer is synthesi zed by glycoge nin, a glycopro tei n).
UDP gluco~e UDP

Glycogen primer Glvcogcn amyla5€
{Gl UCO'>C) n Glycogen syn thase {Glu co<,l') n 1
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ii) Branching enzyme:

When the chain has been lengthened to a minim um of 11 glucose residues, a secon d
enzym e, the branch ing enzym e transfers a part of a(l • 4) chain (minim um length
of 6 glucose residues) to a neighb oring chain, produ cing a new a(1• 6) linkage, thus
forming a new branch point.
Then the branc hes grow by furthe r addition of glucos e in a(1 • 4) direct ion by
glycogen synthase enzym e and then furthe r branch ing by branch ing enzyme.
it
VD P
i
;1')
Diagramatic representation of glyco genes is
Signi fican ce:

Glycogen is an animal storage polysaccharide. It is stored in liver and muscle. When
the blood glucose level increases, excess glucose is conve rted to glycogen.
Regu lation of glyco gen synth esis:

Glycogen syntha se is the regulatory enzym e of glycogenesis. It is regula ted by covalent
modification . (Refer enzym e chapter).The phosp horyla ted form of glycogen syntha se
is inactiv e, where as depho sphor ylate d form of g lycoge n synthase is active .
by insuli n.
Glyco gen syntha se activit y is decrea sed by adren aline and enhan cedtesamiose
Fad
FFF
iesamc.se
p.ly
Phosphy
Phosphorylated
ATP ~ n as:.-- ----::: : DP form
Glycog en syntha se a Glycog en syntha se b
(De phosph o ry la ted fo rm) (Phosp ho ry la ted fo rm )
(Active form)
:::-------
Pi
--
Ph osphat ase H 20
(Inacti ve form)
Rever sible covale nt modif icatio n of glycog en syntha se enzym e
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Glyc ogen olys is (Gly coge n brea kdow n)

Defin ition:
GlycogenoJysis means the breakd own of glycogen.
Site: Glycogenesis Glycogensynth

GY.EEDesp1 Efaeed
a) Tissue site: Liver and Muscle
b) Intracellular site: Cytosol Glycogenolysis Glycogenbledwn Glycogenphosphorylase
ActivePhosphorylated
Staiting material:
Glycogen
End product:
i) Glucose in liver and
ii) Gluco se-6-p hosph ate in muscl es (as muscl e lacks glucose-6-phosphatase).
Reaction pathway:
Glyco gen is broken down by combi ned action of 3 enzym es, namely,
a) glycog en phosp horyla se b) glucan transferase c) debra nchin g enzyme.
Glycogensynthase
Branching enzyme
a) Glyco gen phosp horyla se:
It acts on glycogen & breaks the termin al a(1 • 4) glycosidic bonds of glycog en and
releases the termin al glucose molecule as glucos e ]-phos phate.
Pi
Glyco gen
(Gluco se) n
---- =-- ---- --+ Glycogen
Glycog
+ Gluco se 1-phos phate
en phosphorylase (Gluco se) n-1
Action of glycogen phosp horyla se contin ues till about 4 glucose residu es remain on
either sides of the branch point.
Muscle glycogen phosp horyla se is a PLP depen dent enzym e.
b) Gluca n transferase: It transfe rs 3 glucose residu es from one chain to anothe

r chain
and thus expos ing the a (1 • 6} glycos~dic bond (branc h point).
c) Glyco gen debra nchin g enzyme: It splits the a(1 • 6) bond at the branch point and
releases free glucose. After the remov al of branch , action of glycogen phosp horyla se
continues.
on chainelongated
by
Glucan transferase
Thus the action of these 3 enzym es leads to the compl ete break down of glycog en
to give mainl y glucos e 1-pho sphate (90%) and few free glucos e molec ules (10%).
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Carbo hydrat e Metab olism 188
Diag rama tic repre senta tion of glyco genol ysis
by
Released glycogenphosphorylase
Fate of gluco se 1-pho sphat e:
a) In liver: Gluco se 1-pho sphate is conve rted to gluco se in liver.

Mg•2 H20 Pi
Glucose 1-phosphate - - - -+ Glucose 6-phosphate ':::- 2! •
Glucose
Phospho gluco mutase Glucose -6-phosp hatase
b) In musc le: Muscle lacks glucos e 6-pho sphata se enzym e . So, in muscl e
glycogenolysis, the end p roduc t is glucose 6-pho sphate.
4 iMusanglycogencan'tmaintain bloodglucoselevel
Signi fican ce of glyco genol ysis:

a) Hepatic glycogenolysis takes place in order to maintain blood glucose level.
b) Muscle glycogenolysis takes place for the provision of energy.
Regulation of glyco genol ysis :

Glycogen phosp horylase is the regula tory enzym e of glycogenolysis. It is regulated by
covale nt modification. (Refer enzym e chapter). The phosp horylated form of glycogen
phosp horyla se is active, wh ereas depho sphor ylated form is inactive. Glyco gen
phosp horyla se activity is activated by glucag on and adrenalin; inhibi ted by insuli n.
4am.net
ATP ADP
Glycogen phosph orylase b • Glycogen phosph orylase a Prospect
(Depho sphory lated form) (Phosp horylat ed form) Phosphorylat

form
4~ -----
(Inactive form) Pi Phosph atase HiO (Active form)
Rever sible covale nt modif icatio n of glycog en phosp horyla se enzym e
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Vitamins 339
Vitamin D:
Chemistry:
There are two forms of vitamin D, Vitamin 0 2 (Ergocalciferol), Vitamin 0 3
(CholecaJciferol). Both have equal activity. Plant sources
Animalsources
Sources:
a) Exogenous sources:
• Ergocalciferol (Vitamin 0 2) is found in plants.
• Cholecalciferol (Vitamin 0 3 ) is obtained from fish liver oils (like cod liver oil,
shark liver oil), shrimps, milk, eggs etc.
b) Endogenous sources:
Vitamin 0 3 (Cholecalciferol) can be synthesized from 7-dehydrocholesterol (which is
a derivative of cholesterol) present in the skin, by the action of UV rays of sunlight. So,
vitamin Dis also referred to as sunshine vitamin.
(Skin) 1280 -3 1 on )
m
7- dehydrocholesterol • • • • • Cholecalciferol (vitamin D:J

• •
Ultraviolet rays of sunlight
Ergosterol Ergocalciferol vitaminDa
*** Similarly ergosterol is converted to vitamin 0 2 (Ergocalciferol).
Darkerskinvegmoresunexposure
Provitamin D :
7-dehydrocholesterol and ergosterol are caJled provitarnins of Vitamin D.
RDA:vitamin D
The RDA for adults is 10 µg (400 I.U.)of vitamin 0 3 •
During pregnancy and lactation, RDA increases to 15 µg.
-
duodenum
& Jejunum
it's lipid soluble
Absorption, Transportation and p Storage: → Minette formation to transport V it D
occurs as
• Vitamin D is absorbed in small intestine along with fats with the help of bile salts.
• In intestine, vitamin Dis incorporated into the chylomicrons & transported through
the lymph and enters the circulation. In the circulation, vitamin D binds to the vitamin
D binding protein (DBP, a plasma a2- globulin) and transported to different tissues.
S• Vitamin D is stored in liver and other tissues (kidney, adrenal glands etc) to a
significant extent.
Biochemical functions of vitamin D:

Vitamin D is not itself biologically active. First, vitmain D has to get converted to the
active form calcitriol (or 1, 25 dihydroxy cholecalciferol) by hydroxylase enzymes.
This activation steps involves the partcipation of organs like liver, kidney and bones.

Vitamins 340 C
Activation of vitamin D (formation of calcitriol):

Vitamin D3 (and D2) are not biologically active, but are converted to active form
1,25-dihydroxy cholecalciferol or calcitriol
Cholecalciferol (vitamin D3)
PON l 25-hydroxylase (liver)
2S-hydroxy choral:::::roxylase (kidney, bone etc)

PON
,
1, 25 -dihydroxy cholecalciferol (Calcitriol) , , , maman
,.,.,. Both the enzymes require cytochrome P 4501 molecular 0 2, NADPH PON
Calcitriol plays an important role in bone formation and calcium metabolism.

Funds
1) Bone mineralization and formation:
Vitamin D (more appropriately Calcitriol) is required for the proper mineralization
of bone. They increase the number and activity of osteoblasts, the bone forming cells.
In the osteoblasts of bone, cakitriol stimu.Jates calcium uptake and deposition. Bone
consists of matrix of collagen with crystals of calcium and phosphate.
2) Calcium and phosphate homeostasis:

Vitamin D plays an important role in calcium and phosphate homeostasis. Calcitriol
is a hypercakemic hormone. The sites of action are intestine, kidney and bones.
andhyperphosphatic
a) Intestine: Calcitriol increases the intestinal absorption of calcium and phosphates
and thus increases blood calcium and phosphate level. man.mn
Action of calcitriol is similar to steroid hormones. It binds to a receptor in the cytosol
and this complex is then transported to the nucleus, where it acts on DNA to stimulate
the synthesis of calcium binding protein (CBP), which increases calcium absorption.
Hyperphosphatic
b) Kidney: Cakitriol promotes the reabsorption of calcium and phosphate by renal

tubules of kidney and thus reducing excretion of calcium and phosphate.
c) Bone: Calcitriol also promote bone resorption and calcium mobilization to raise the
serum calcium and phosphate levels (During calcium deficiency sta te.)
Note: But no net loss ofbone calcium results when both vitamin D & calcium intakes are adequate in the diet.

Vitamins 341
I Deficiency of vitamin D:
Vitamin D deficiency causes Rickets in children and osteomalacia in adults.
Rickets:
Rickets is caused due to the deficiency of vitamin Din children.
Symptoms:
Rickets is a disease of growing bone. There is insufficient mineralization of new bones.
Bones become soft and pliable (easily bent). Rickets is characterized by,
• Bowlegs, Genuvamm
of • Knock knees, Genuralgumcaneesin
• Pigeon-chest, antpurtunen sternum
if of
• Rickety-rosary (beaded appearance) of ribs prominentknobsatcostochondratian
Basis: Rickets is a disease of growing bones. ln vitamin D deficiency during the stage of bone
growth, the depositions of minerals (calcification) fail to occur in the newly formed matrix, but
matrix formation continues. This results in the soft, pliable (easily bent) bones. Deformities occur
because cartilaginous structure cannot withstand the weight of the growing body. This results in
bowlegs, knock-knees, and pigeon-breast and rachitic-rosary a beaded appearance of the ribs.
Osteomalacia:
Osteomalacia is caused due to the d eficiency of vitamin Din adults.
Symptoms:
Osteomalacia is characterized by
• Insufficient mineralization of bones
• Softness of bones Pelvic isparticularlyaffected
• Bone pain and aches
gist• Easy fracture of bones
Renal rickets (Vitamin D resistant rickets):
This is seen in patient with chronic renal failure. Kidney is required for the formation
of calcitriol (active form of vitamin D) that is required for calcium absorption and bone
mineralization. So, chronic renal disorders lead to poor bone mineraJization and rickets,
which is referred to as renal rickets. These do not respond to vitamin D provision, but
respond to provision of calcitriol. So, some regards this as vitamin D resistant rickets.
2 Toxicity of vitamin D (Hypervitaminosis D):

Excess consumption of vitamin D leads to a toxicity condition - hypervitaminosis D.
Symptoms:
hypercalcemia
2
• Demineralization of bones, hypercalcemia 3
are main findings. Calcification of soft
tissues, especially renal tissues leading to renal stone is one of the main features.
Is
• Loss of weight, weakness, polyuria, increased thirst are the other symptoms.

Minerals 365
• Calcium (Ca)
Distribution:
Calcium is the most abundant mineral in the body. The total content of calci um in the
body is about 1 -1.5 kg. 99% of which is present in bones and teeth and rest in extra
cellular fluid (mainly blood).
Normal blood level of Ca+2 is 9 to 11 mg per 100 ml of blood.
Dietary sources:
Milk and milk products are rich sources of calcium.
Egg, fish, mutton, dates and vegetables are good sources.
i
Cereals (except rice) and millets are good sources of calcium. (Particularly, millets
like ragi and bajra are good sources of calcium).
Note: Phytates (present in cereals) and oxalates (present in certain green leafy
vegetables like spinach, amaranth etc) inhibit calcium absorption. So, bioavailability
of calcium from cereals and green leafy vegetables depends on their phytate and
oxalate content.
RDA:
Adult: 0.8 gm per day
Children: 0.8 to 1.2 gm per day
Pregnancy and lactation: 1.5 gm per day
Absorption of calcium:
Calcium is absorbed in duodenum against concentration gradient. Absorption requires
calcium binding protein (CBP, a carrier protein), which requires energy.
Factors facilitating calcium absorption;

• Calcitriol (active form of vitamin D): Facilitates the absorption of calcium by inducing
the synthesis of CBP in the intestinal mucosal cell.
• PTH: It indirectly stimulates calcium absorption by promoting the synthesis of
calcitriol.
• Lactose
• Amino acids lysine and arginine also facilitate calcium absorption.
• Gastric acidity (low pH) favors calcium absorption.

Minerals 366
Factors decreasing calcium absorption:

• Phytates (present in cereals, tea, coffee)
• Oxalates (present in green leafy vegetables like spinach, amaranth etc)
• Fatty acids
Phytates, oxalates, fatty acids form insoluble salts with calcium and reduce calc ium
absorption.
• High phosphate content in the food causes the precipitation of calcium as insoluble
calcium phosphate and interferes w ith calcium absorption. The optimum ratio of calcium
to phosph ate for maximum absorption is 1:2 to 2:1.
• Alkaline condition is unfavorable for calcium absorption.
Chronic renal failure / Nephrosis cause decreas ed calcium absorption: Kidney is required
for the formation of calcitriol that is required for formation of calcium binding protein
(CBP) required for calcium absorption. So, chronic renal disorders / nephrosis lead to
impaired calcium absorption. It can be treated by administration of calcitriol.
Steatorrhea (as in malabsorption syndrome) decreases calcium absorption: Steatorrhea
occurs due to defective digestion and absorption of fat and fatty acids (in malabsorption
syndrome). These unabsorbed fatty acids inhibit fat absorption.
Calcium : Phosphate ratio in diet: The optimum ratio of calcium to phosphate for maximum
absorption is 1:2 to 2:1 (as present in milk). If the amount of either calcium or phosphate
exceeds double the amount of the other, then insoluble complex will be formed which will
not be absorbed. Ideal ratio is 1:1.
Storage:
Bones, teeth and muscles s tores calcium. But, teeth calcium is not used to maintain
blood calcium level.
Excretion:
About 500 mg of calcium is excreted in the urine p er day.
Functions of calcium:
BigBlackMantainmasrighagayamadaechod
I
1) Bone and teeth formation: Calcium is required for the forma tion of bone and teeth,
2
as a structural component. Calcium gives h ardness and strength to these tissues.
3 Bones act as reservoir of calcium .
2) Blood coagulation: Calcium is required for blood coagulation process. FactorIV
3) Muscle contraction: Calcium is required for the excitation and contraction of m uscle
fibres.
4) Transmission of nerve impulse: Calcium is required for the transmission of nerve
impulses in the synapses (pre-synaptic to post-synaptic region).
Explain
5) Neuromuscular excitability: Calcium decreases neuromuscular excitability.

Minerals 367
6) Hormone action: Calcium acts as a secondary messenger to some hormones.
l
E.g.: glucogon, adrenalin, vasopressin etc.
7) Release of hormones: Calcium is required for the release of some hormones such as
insulin, PTH, calcitonin from their storage vesicles.
8) Activation of enzymes: Calcium is requ ired for activation of some enzymes.
i) As calcium calmodulin complex: Calmodulin is a calcium binding protein. It can
bind with four calcium ions. Calcium-calmodulin complex activates many enzymes.
E.g.: Adenylate cyclase, calcium dependant protein kinase etc.
ii) Direct action: Some enzymes are directly activated by calcium like pancreatic lipase,
rennin etc (where calcium acts as a metal activator).
9) Membrane integrity and cell permeability:
Calcium promotes or controls the permeability of cell membrane.
10) Cell to cell contact:
Calcium has an important role in cell to cell contact.
Dietary deficiency of calcium:

a) Children: Pure dietary deficiency of calcium in children may lead to impa ired (bone)
growth leading to rickets like symptoms, negative calcium balance, early occurrence of
osteoporosis etc.
b ) Adults: In adults, pure deficiency of calcium in diet may lead to osteoporosis. It also
result in hyperexitable state of nerves and muscles.
ote: Pure dietary deficiency of calcium is very rare and it manifests only after years
of insufficient intake. If intake of vitamin Dis adequate, the problems of osteomalacia
and rickets do not occur even if dietary calcium is low. During vitamin D deficiency
and hypoparathyrodism, deficiency of calcium can occur (because vitamin D and
PTH are required for calcium absorption).
Dietary deficiency of calcium mainly affects the bones (osteoporosis).
Osteoporosis: Osteoporosis is a condition where bones become porous due to decalcification.

Dietary deficiency of calcium in diet is one of the risk factors for osteoporosis. In osteoporosis,
serum calcium levels are normal but body stores are reduced. But, in vitamin D deficiency &
hypoparathyrodism, along with reduction of body stores, serum calcium level is also reduced.
Overconsumption and toxicity:

Dietary excess is unlikely to reach toxic amounts. However, individuals receiving high
calcium supplements over a long period of time may develop toxicity. Too much calcium
is associated with an increased risk of kidney stones and decreased absorption of other
minerals (like iron, zinc, phosphorous, magnesium etc).

Minerals 368
Calcium homeostasis (Blood calcium level and factors regulating it):

Normal blood calcium level is 9 to 11 mg / dl.
Blood calcium level is maintained within the normal level by various factors. They are,
1) Vitamin D
Hypercalcemia
2) PTH
3) Calcitonin Hyoracemic
1. Vitamin D:
It has hypercalcemic effect. It increases the blood calcium level. It has three major
independent sites of action - intestine, kidney and bones.
i) Intestine: Ca lcitriol induces the syn thesis of a carrier protein (Calcium binding protein,
f
CBP) in the intestinal mucosa, which increases absorption of calcium and blood
calcium level.
Eient ii) Kidney: PTH activity on kidney is enhanced by vitamin D, which increases
Act reabsorption of calcium.
Festive
iii) Bones: Vitamin D enhances osteoblastic activity of bones and thus promotes
calcification of bones mainly in growing children, but it causes bone resorption
during hypocalcemia in order to maintain the blood calcium level.
2. Parathyroid hormone (PTH):

PTH is a hypercalcemic hormone. It has three major independent sites of action -
intestine, kidney and bones.
i) Intestine: PTH stimulates production of calcitriol which increases intestinal absorption
of calcium.
ii) Kidney: PTH increases calcium reabsorption by kidney tubules.
iii) Bones: PTH causes demineralization (resorption) of bones, resulting in increasing
blood calcium level. But this activity is mainly seen during calcium deficiency.
anemia
ng
por
3. Calcitonin:
Calcitonin is secreted by parafollicular cells of thyroid gland. Calcitonin is a hypocalcemic
hormone, which decreases the blood ca lcium level.
GIT ut asTuycasetowin
i) Kidney: Calcitonin
can'taffectdigestive
inhibits calcium reabsorption by lodneys
ii) Bones: Calcitonin inhibits bone resorption by increasing activity of osteoblasts and
decreasing activity of osteoclasts.
Phosphate (PO/) level also has an effect on calcium homeostasis:

Hypophosphatemia (Decreased serum PO/ level) increases the serum calcium level.
Hypophosphatemia enhances the hydroxylation (activation) of vitamin D in kidnet;s to form
calcitriol, which has hypercalcemic effect.

Minerals 369
Clinical aspects:
Normal blood/ plasma calcium level is 9 to 11 mg/ dl.
H ypocalcaemia:
Definition:
When the plasma calcium level is below 9 mg/ dl, condition is known as hypocalcaemia.
Causes:
• Vitamin D deficiency
• Hypoparathyrodism fry
racemic
f
I
• Accidental surgical removal of parathyroid glands (generally during thyroidectomy
due to its close proximity to thyroid gland),
• Dieta ry deficiency of calcium,
• Steatorrhea (due to accumulation of fatty acids, which inhibit calcium absorption),
• Chronic renal diseases (due to impaired formation of calcitriol which is required for
calcium absorption).
Manifestations:
Calcium decreases neuromuscular excitability. Deficiency of calcium increases
neuromuscular excitability. When serum calcium level is less than 8.5 mgIde %, mild
tremors (hyper-excitable state of the nerve and muscle) are seen. If it is lower than 7
mg % a life threatening condition called tetany will occur. Symptoms include numbness
of extremities, emotional irritability, tightness and spasm of m uscle, muscle cramps,
convulsions etc. I
I
Two clinical signs, Chvostek's sign and Trousseau's sign will be positive.
Yitiis
Low levels of calcium also lead to bone deformities.
Hypercalcemia:
Definition:
When blood calcium level increases above 11 mg/ dl, the condition is known as
hypercalcemia.
Causes:
Hypervitaminosis D and Hyperparathyroidism. salami
Hyper
Symptoms:
I
An increased excretion of urinary calcium leading to renal calculi. Bone2deformities
(due to increased bone resorption), ectopic calcification of urinary bladder, renal tissues,
In
pancreas etc, anorexia, depression and muscle weakness are other symptoms.

Electron Transport Chain and Oxidative Phosphorylation 294
The process of mitochondrial respiration can be discussed under two headings;

I. Electron transport chain (ETC), where oxidation of NADH/FAD8i in ETC.
II. Oxidative Phosphorylation of ADP to form ATP.
I. Electron transport chain:

Electrons from NADH and FADH2 are transferred to 0 2 through a series of electron
carriers known as Electron transport chain (ETC). ETC is loca ted in inner
mitochondrial membrane Sznthat fl AT Pdependent -
>
synthase → HTPindependent
Components and organization of ETC:
Electron carriers in ETC are organized in to 4 complexes and 2 mobile carriers.
These complexes are arranged i n increasing o rde r of redox potentials. This
arrangement ensures the continuous flow of electrons from NADH I /for
affinity es
FAD8i to 0 2•
(As electrons flow from lower to higher redox potential
systems.) Each electron carrier in ETC
can receive electrons from an electron donor and subsequently dona te electrons to
the next carrier in the chain. Molecular oxygen (02) acts as the final electron acceptor,
which is eventually converted to water.
Components of electron transport ch ain (ETC)

NADH
½ Ch+2H•
Complex I Complex III Complex IV (

NADH dehydrogenase Cytochrome c reductase Cytochrome oxidase
E last
orgasm comes
FM ·, Fe-S Q + Cyt b, Fe-S, Cyt c1 - • Cyt c - • Cyt a, Cu, Cyt a3 /4
NAO• l H20
Complex II Fumarate
S ucc inate dchy droge n ase
FAD, Fe-S Succinate
Complex I (NADH dehy drogenase or NADH Q reductase complex):

1 • The electrons of NADH enter complex I. It contains 2 types of prosthetic groups.
FMN (which exists as a flavoprotein) and Fe-S cluster (which exist as Fe-S proteins,
Eth the iron ion of which alternate between Fe+2 and Fe+3 states).
• The reducing equivalents (2 electrons and2H +) from NADH are first accepted by
FMN to form FMNH2 •
• The electrons from FMNH 2 are then transferred to Fe-S clusters.
• The electrons are finally transferred to mobile carrier Q (CoQ or Coenzyme Q).

Complex II (Succinate dehydrogenase complex or Succinate Q reductase):
I• Complex II is a part of TCA cycle enzyme, succinate dehydrogenase, where FADH2

is formed with the oxidation of succinate to fumarate.
2• The electrons from FADH 2 a re transferred to Fe-S centers and then to Q.
CoQ (Coenzyme Q or CoQ or Ubiquinone):
I• Q is the first mobile electron carrier in ETC.

2• Q can accept pair of electrons from both complex I and complex II. Q receives a pair
of electrons and 2 H+(from matrix) to form QJ\.
3• QH2 then transfer a single electron to Complex JII.
Complex III (Cytochrome reductase or Cytochrome c oxidoreductase):
I • Complex JU h as Cytochrome band cytochrome c1 and Fe-S clusters.

2 • Cytochromes are conjugated proteins with heme as prosthetic group. The iron ion of
cytochromes alternate between Fe+ & Fe+ states during electron transport.
2 3
• CoQ transports a single electron to Cyt b of complex III. The electron from CoQ is
3first recieved by Cyt b, which then transfers it to FeS. From FeS electrons are transferred
to cyt c1 . Electrons from cyt c1 are then transferred to mobile carrier cytochrome C.
Cytochrome c:
I • Cytochrome c also is a mobile electron carrier. Cytoch rome c is a h eme protein, which
contain iron ion, which alternates between Fe+2 & Fe+3 states.
2 • Cytochrome c then transfers the electron to cytochrome oxidase (complex IV).
Complex IV (Cytochrome c oxidase):
• Cytochrome oxidase contain cytochrome a, cytochrome a 3 and copper ions.

• The electrons from Cyt c is first recieved by Cyt a, which then transfers it to copper.
From copper electrons are transferred to cyt ~-
• cyt a 3 (of Cytochrome oxidase) then finally delivers the electrons to oxygen, w hich
forms water. Oxygen is the final acceptor of electrons.
4 cyt C (fe+2) + 4 H + + 0 2 4 cyt C (Fe+3 ) + 2H20

Inhibitors of ETC
There are a number of site specific substances that inhibit Electron Transport Chain.
Th ese inhibitors have contributed immensely to the knowledge of m itochond rial
respiration.
Site of inhibition Inhibitors
Complex I Ritters Rotenone (Fish poison, which is used as an insecticide)

e
Boys Barbiturates such as amobarbitol, amytal (sedative)

-
Prison's Piericidin A (antibiotic)
Complex II Mad Malonate
Complex III Antimycin A (antibiotic)

BAL (British Anti Lewisite, an antidote used against war-gas)
Dimerca prol
Complex IV Cyanide
Criminals
CO (competes with 0 2 for binding with cytochrome c oxidase

Eies ~Sand Azides (N - containing compounds)
3
II. Oxidative Phosphorylation:
Definition of Oxidative phosphorylation:

Oxidative phosphorylation can be defined as the process of coupling of oxidation of
NADH/FADH2 in ETC to the phosphorylation of ADP to form ATP in ETC.
• When electrons from NADH/FADH2 flow through the ETC (oxidation), they release
free energy; a part of this energy is utili zed to generate ATP from ADP and Pi
(phosphorylation). Since, thjs process couples the oxidation of NADH/FAD~ in ETC
with the ATP synthesis, it is called oxidative phosphorylation.
• Remaining free energy which is not trapped as ATP is used to generate heat, which
maintains the body temperature.
Mechanism of Oxidative phosphorylation:

Flow of electrons from NADH / FAD~ in ETC is not directly linked with ATP synthesis.
It is a complex process. Several h ypotheses have been put forward to explain this
process. Among them, chemjosmotic hypothesis explained by Mitchell is the most
accepted one.

Inhibitors of ETC
There are a number of site specific substances that inhibit Electron Transport Chain.
Th ese inhibitors have contributed immensely to the knowledge of m itochond rial
respiration.
Site of inhibition Inhibitors
Complex I Rotenone (Fish poison, which is used as an insecticide)

e
Barbiturates such as amobarbitol, amytal (sedative)

-
Piericidin A (antibiotic)
Complex II Malonate
Complex III Antimycin A (antibiotic)

BAL (British Anti Lewisite, an antidote used against war-gas)
Dimerca prol
Complex IV Cyanide
CO (competes with 0 2 for binding with cytochrome c oxidase
~Sand Azides (N3- containing compounds)
II. Oxidative Phosphorylation:
Definition of Oxidative phosphorylation:

Oxidative phosphorylation can be defined as the process of coupling of oxidation of
Def NADH/FADH2 in ETC to the phosphorylation of ADP to form ATP in ETC.
• When electrons from NADH/FADH2 flow through the ETC (oxidation), they release
free energy; a part of this energy is utili zed to generate ATP from ADP and Pi
eness(phosphorylation). Since, thjs process couples the oxidation of NADH/FAD~ in ETC
with the ATP synthesis, it is called oxidative phosphorylation.
• Remaining free energy which is not trapped as ATP is used to generate heat, which
maintains the body temperature.
Mechanism of Oxidative phosphorylation:

Flow of electrons from NADH / FAD~ in ETC is not directly linked with ATP synthesis.
It is a complex process. Several h ypotheses have been put forward to explain this
process. Among them, chemjosmotic hypothesis explained by Mitchell is the most
accepted one.

Chemiosmotic hypothesis (also known as Mitchell hypothesis):

• According to this hypothesis, free energy that is released when electrons flow through
plumping
the ETC causes the pumping of protons from mitochondrial matrix to the intermembrane
space across the inner mitochondrial membrane. Complex I, III & N of ETC act as
proton pumps.
2 • These protons cannot diffuse back into the matrix because the inner mitochondrial
membrane is impermeable to protons (and other ions). This causes the accumulation
of protons in the intermernbrane space resulting in the generation of proton gradient
across the inner mitochondrial membrane. This creates a proton motive force, which
tends to force the protons back into the mitochondrial matrix.
3• mitochondrial
The protons that accumulate in the inter membrane space can flow back to the
matrix only through the ATP synthase complex present in the inner
mitochondrial membrane. The proton motive force generated drives the synthesis
of ATP from ADP and Pi by ATP synthase comp lex.
4 • ATP synthase complex (also referred to as Complex V):
ATP synthase complex is embedded in the inner mitochondrial membrane. It consists
polypeptide chairs
→ it →a
of 2 functional subunits, F0 & Fl' which are attached with each other. F0 spans the
membrane and forms a proton channel. F1 projects into matrix and has phosphorylation
mechanism.
The flow of protons through the proton channel F0 subunit causes the rotation of FO'
mescu
driving the formation of ATP from ADP and Pi in the F1 subunit. The energy for this
endergonic reaction of ATP synthesis is derived from the proton motive force.
Proton pumps
y 0 circle Channel
ATP synthetase
mituin.net 2mm
items
Chemiosmotic hypothesis of Oxidative phosphorylation

Digestion and Absorption 161
Abnormalities of carbohydrate digestion:
1) Lactose intolerance:
I Deficiency of lactase enzyme leads to lactose intolerance.2Lactase is present in brush
7 hydrolyzes lactose to glucose and galactose.t In lactose
border of intestine. This enzyme
intolerance, lactose (hence milk) cannot be digested by the affected person.
Types: There are three types of lactose intolerance,

i) Inherited lactase deficiency (Congenital): Rare, seen in infants.
ii) Primary low lactase activity (Congenital): Most common, manifests in adults. its
presumed to represent a gradual decline in lactase activity in susceptible individuals
over the years
iii) Secondary low lactase activity: Due to intestinal diseases like gastroenteritis, sprue
(tropical & nontropical), kwashiorkor, colitis, etc, where lactase production is decreased.
Clinical manifestations:
a) Osmotic diarrhea: Since lactose is not digested due to lactase defect, it accumulates
in the intestine. Lactose takes up water into the bowel by osmotic effect and lumen
will be filled with water (endosmosis) leading to osmotic diarrhea.
b) Flatulence: Accumulated lactose is acted upon by intestinal bacteria to form CO2
which leads to abdominal cramps, flatulence and diarrhea. These manifestations are
together called intolerance.
c) Lactosuria: Lactose also appears in urine, the condition known as lactosuria.
Diagnosis:
Lactose intolerance is tested by giving a test dose about 50g lactose and observe the
prevalence of diarrhea and rise in blood glucose level after test dose. Defect in lactase
immediately causes gastric irritation & they will not show any significant rise in glucose.
Treatment:
Elimination of milk and milk products from diet. Curd is an effective treatment as
lactobacilli present in curd contains the enzyme lactase.
2) Sucrase deficiency:
The deficiency of sucrase enzyme causes intolerance to sucrose. It is a very rare disease.
This deficiency is generally seen in Eskimos.
Clinical manifestations are similar to lactose intolerance (diarrhea, flatulence etc).

aFuranesIncauer if
6 at
Diagnosis of diabetes mellitus:

1. Blood glucose estimation:
Normal fasting plasma glucose level is 70 to 110 mg%.
Main diagnostic criterion for diabetes is fasting plasma glucose level above 126 mg%.
3884
Glucoseoxidaseperoxidase
Glucose oxidase method is best for blood glucose estimation as it measures only glucose.
2. Urine test for glucose:
Urine is tested for the presence of glucose by Benedict's qualitative test. Urine of normal
individuals does not show the presence of any glucose. Presence of glucose in urine is
a possible indicator of diabetes mellitus. The normal renal threshold for glucose is 160
to 180 mg/ dl. Blood glucose level must exceed this value for glycosuria to occur.
Benedict's test is answered by all other sugars & reducing substances in urine, giving a
false positive test for diabetes. So, urinary glucose should be interpreted with care.
In symptomatic cases, diabetes can be confirmed by estimating plasma glucose level and urine
testing for glucose. But, in asymptomatic and suspected coses, the diabetes is diagnosed by GIT.
Management of diabetes mellitus:

Diet, exercise, drugs & insulin are the options for the management of diabetes mellitus.
a) Insulin injections: Insulin is the drug of choice for type 1 diabetes. It is also used in
type 2 diabetes, where oral drugs are not sufficient.
b) Diet and exercise: Diet and exercise can control about 50% of type 2 diabetes. A
diabetic patient is advised to take a diet low in carbohydrates and fat and rich in protein
and fibers. Refined sugars and saturated fat should be avoided.
c) Oral hypoglycemic drugs: Sulfonylurea and biguanides are the two types of oral
hypoglycemic drugs, mainly used in type 2 diabetes (NIDDM).
Hypoglycemia
When blood glucose level falls below 50 mg/di the condition is known as hypoglycemia.
Causes:
1. Overdose of insulin during the treatment of diabetes.
2. Insulinoma: Due to insulin secreting tumors of p -cells of pancreas.
3. Decreased secretion of hyperglycemic hormones like glucagon, pituitary, thyroid etc
4. Hyperactivity of pancreas of newborn infants born to diabetic mothers.
5. von Gierke's disease, leading to failure in hepatic glycogenolysis.
6. Defective P-oxidation of fatty acids (camitine deficiency, inherited CPT deficiency, defects
in P-oxidation enzymes or by poisons like hypoglycin), lead to nonketotic hypoglycemia.
Defective P-oxidation leads to low acetyl CoA formation, which is the activator of pyruvate
carboxylase, a key gluconeogenic enzyme. This leads to decreased gluconeogenesis. Also,
since fatty acids cannot give energy, glucose utilization increases leading to hypoglycemia.
8. Liver failure by poisons, alcoholism etc are other reasons.
Symptoms and manifestations:
Headache, anxiety, confusion, sweating, seizures, fatty liver and coma, if not treated, death.

3 Glucose tolerance test (GTT)

I Definition: Glucose tolerance test or GTT is a test to measure the capacity of the body
to dispose off an additional load of glucose entering into the body.
2 Oral GTT refers to measureent of tolerance after a oral dose of glucose.
3GTT is usually performed in diagnosis of doubtful cases of diabetes mellitus. belie
4 Dowe inpregnantwomentocheckforgestationaldiabetes no nomydypredia
Procedure:
• The patient is instructed to have a good carbohydrate diet for 3 days prior to the
test and the patient is starved overnight (12 hours fasting condition).
• In the morning a sample of blood and urine is collected in fasting condition.
• Then the patient is given glucose load (75 g of glucose in a glass of water).
• Then the blood and urine samples are collected at an interval of half an hour for the
next 2½ hours (30, 60, 90, 120, 150 minutes).
• Plasma glucose level is estimated quantitatively in all the blood samples. A graph is
plotted with plasma glucose va lues (in mg/ dl) on y-axis and time (in minutes) on
x-axis. Urine samples are tested for sugar by benedict's qualitative test.
Response: Three types of responses are seen.

1) Normal GTT: Fasting plasma glucose level is less than 110 mg %, which rises to peak
within 1 hour and is not above 160 mg%. Postprandial plasma glucose level (i.e. 2 hours
after the load or food) is not above 140 mg%. Sugar is absent in all the urine samples.
(Renal glycosuria shows similar GTT graph, but sugar is present in most urine samples).
2) Diabetic GTT: A decreased glucose tolerance is seen in diabetic patients.
Fasting plasma blood glucose level will be above 126 mg%; a very high increase in peak
value and postprandial glucose level (2hr glucose level) will be above 200 mg%.
Urine sugar is detected in at least one of the urine samples.
In severe diabetics, all the urine samples show positive benedicts test.
3) Impaired Glucose Tolerance UGn: IGT is a condition, when plasma glucose level is
in between normal and diabetic levels, i.e. fasting plasma glucose level is 110-126 mg%
& postprandial plasma glucose level is 140-200 mg%. In GTT, urine sugar may be present
in some urine samples. These IGT individuals may progress to diabetic patients.
Response Fasting plasma Postprandial pl asma

glucose level glucose )eve I
ormal GTf < 110 mg•• < 140mg~.

-
Diabetic GTI > 126 mg¾ > 200mg%
-
Impaired glucose tolerance (IGT) 110 - 126 mg •• 140 - 200 mg
punaianetic

250
Blood glucose 200

(mg/di) Diabetic
150
IGT
100
Normal
50
½ 1 1½ 2
Time (Hours)
Importance of GTT:
1) GIT has a great value in investigation of mild diabetes and symptomless glycosuria
(i.e. Suspected cases of diabetes).
2) GIT may also provide useful information in some endocrine disorders.
4 • Glycated hemoglobin (HbA1):

Glycated hemoglobin refers to any sugar (mainly glucose) added to hemoglobin (Represented
as HbA1), Among them, HbA1c is the most abundant form. The rate of addition is directly
proportional to blood glucose level. So, diabetics have higher percentage of HbA1c• Normal
level of HbA1c is 4 to 7%. But in diabetics the level goes up to 8-15% (depending on severity).
Significance:
Glycated hemoglobin measurenet is an index of long term control of blood glucose. This is
·because glucose once attached, cannot be removed from glycated hemoglobin. So, glycated
hemoglobin once formed, remains inside RBC throughout the life span of RBCs (120 days).
Therefore, glycated hemoglobin reflects the blood glucose level over a period (about 60 days,
which is the half life period of RBCs).
ote that the measurement of glycated hemoglobin is not for the diagnosis of diabetes, but
for monitoring the response of treatment in diabetes mellitus as it reveals the mean glucose
level over the previous 10 to 12 weeks. An elevated HbA1c indicates the poor control of
diabetes in the previous 2 to 3 months. In diabetics, if the level of HbA1c concentration is less
than 7%, the diabetic patient is considered to be in good control in the previous 2 to 3 months.
• Fructosamine:
G lycosylated serum proteins (mainly albumin) are termed as fructosamine.
Normal serum level = 1.6-2.7 mrnol / 1
Significance: Half life of albumin is only about 2-3 weeks; So, fructosamine reflects the glucose
control for only the preceding 2-3 weeks. Thus, measurement of serum fructosamine is an
index of short term control of glucose level in diabetes mellitus (whereas, HbA 1c reflects
long term control). Estimation of fructosamin.e is useful in gestational diabetes.
• Microalbuminuria:
Microalbuminuria is defined as the presence of 30-300 mg of albumin in a 24-hour collection.
It serves as an early & independent predictor of progressive renal damage in diabetic patients.
(In contrast to macroalbuminuria, whcih serves as definite indicator of severe renal failure).

Glucose tolerance test (GTT)

I Definition: Glucose tolerance test or GTT is a test to measure the capacity of the body
to dispose off an additional load of glucose entering into the body.
2 Oral GTT refers to measureent of tolerance after a oral dose of glucose.
3GTT is usually performed in diagnosis of doubtful cases of diabetes mellitus.
Procedure:
• IThe patient is instructed to have a good carbohydrate diet for 3 days prior to the
2 test and the patient is starved overnight (12 hours fasting condition).
•3In the morning a sample of blood and urine is collected in fasting condition.
•4Then the patient is given glucose load (75 g of glucose in a glass of water).
•sThen the blood and urine samples are collected at an interval of half an hour for the
next 2½ hours (30, 60, 90, 120, 150 minutes).
•6Plasma glucose level is estimated quantitatively in all the blood samples. A graph is
plotted with plasma glucose va lues (in mg/ dl) on y-axis and time (in minutes) on
x-axis. Urine samples are tested for sugar by benedict's qualitative test.
andgraphplotted
Response: Three types of responses are seen.
1) Normal GTT: Fasting plasma glucose level is less than 110 mg %, which rises to peak
within 1 hour and is not above 160 mg%. Postprandial plasma glucose level (i.e. 2 hours
after the load or food) is not above 140 mg%. Sugar is absent in all the urine samples.
(Renal glycosuria shows similar GTT graph, but sugar is present in most urine samples).
2) Diabetic GTT: A decreased glucose tolerance is seen in diabetic patients.
Fasting plasma blood glucose level will be above 126 mg%; a very high increase in peak
value and postprandial glucose level (2hr glucose level) will be above 200 mg%.
Urine sugar is detected in at least one of the urine samples.
In severe diabetics, all the urine samples show positive benedicts test.
3) Impaired Glucose Tolerance UGn: IGT is a condition, when plasma glucose level is
in between normal and diabetic levels, i.e. fasting plasma glucose level is 110-126 mg%
& postprandial plasma glucose level is 140-200 mg%. In GTT, urine sugar may be present
in some urine samples. These IGT individuals may progress to diabetic patients.
Response Fasting plasma Postprandial pl asma

glucose level glucose )eve I
ormal GTf < 110 mg•• < 140mg~.

-
Diabetic GTI > 126 mg¾ > 200mg%
-
Impaired glucose tolerance (IGT) 110 - 126 mg •• 140 - 200 mg

250 so
Blood glucose 200 soo

(mg/di) Diabetic
too so
150
IGT
no
100
Normal
no
50
½ 1 1½ 2
Time (Hours)
Importance of GTT:
1) GIT has a great value in investigation of mild diabetes and symptomless glycosuria
(i.e. Suspected cases of diabetes).
2) GIT may also provide useful information in some endocrine disorders.
• Glycated hemoglobin (HbA1):

Glycated hemoglobin refers to any sugar (mainly glucose) added to hemoglobin (Represented
as HbA1), Among them, HbA1c is the most abundant form. The rate of addition is directly
proportional to blood glucose level. So, diabetics have higher percentage of HbA1c• Normal
level of HbA1c is 4 to 7%. But in diabetics the level goes up to 8-15% (depending on severity).
Significance:
Glycated hemoglobin measurenet is an index of long term control of blood glucose. This is
·because glucose once attached, cannot be removed from glycated hemoglobin. So, glycated
hemoglobin once formed, remains inside RBC throughout the life span of RBCs (120 days).
Therefore, glycated hemoglobin reflects the blood glucose level over a period (about 60 days,
which is the half life period of RBCs).
ote that the measurement of glycated hemoglobin is not for the diagnosis of diabetes, but
for monitoring the response of treatment in diabetes mellitus as it reveals the mean glucose
level over the previous 10 to 12 weeks. An elevated HbA1c indicates the poor control of
diabetes in the previous 2 to 3 months. In diabetics, if the level of HbA1c concentration is less
than 7%, the diabetic patient is considered to be in good control in the previous 2 to 3 months.
• Fructosamine:
G lycosylated serum proteins (mainly albumin) are termed as fructosamine.
Normal serum level = 1.6-2.7 mrnol / 1
Significance: Half life of albumin is only about 2-3 weeks; So, fructosamine reflects the glucose
control for only the preceding 2-3 weeks. Thus, measurement of serum fructosamine is an
index of short term control of glucose level in diabetes mellitus (whereas, HbA 1c reflects
long term control). Estimation of fructosamin.e is useful in gestational diabetes.
• Microalbuminuria:
Microalbuminuria is defined as the presence of 30-300 mg of albumin in a 24-hour collection.
It serves as an early & independent predictor of progressive renal damage in diabetic patients.
(In contrast to macroalbuminuria, whcih serves as definite indicator of severe renal failure).

Clinical Biochemistry 585
Thyroid Function Tests (TFT)

Definition:
Thyroid Function Tests (TFf) are the group of tests that are performed to assess the
function of thyroid gland.
Significance:
Thyroid Function Tests are performed to diagnose various thyroid diseases. They are
also useful in monitoring the progress of treatment.
1) Measurement of T3, T4 and TSH in serum:

Free T3' Free T4_ Total T4 (Total = Bound + Free) and TSH are measured in serum by
RIA or ELISA method.
Normal serum values:

a) Free T3 : 80 - 220 ng / dl, b) Free T4 : 0.8 - 2.4 ng / dl
c) TSH : < 10 µ U / ml, c) d) (Total T4 : 5 - 12 µg / dl)
Significance:
a) Hyperthyro idism:
• In primary hyperthyroidism (Due to primary thyroid decrease),
isease T3 and T4 level
increased and TSH level decreased.
• In secondary hyperthyroidism (Due to Pituitary cause), All Ty T4 and TSH level
increased.
b) Hypothyroidism:
• In primary hypothyroidism (Due to primary thyroid decrease),
isease T3 and T4 decreased
and TSH increased.
• In secondary hypothyroidism (due to hypothalamic / pituitary defect), T:v T4 and
TSH all decreased.
c)T3 toxicosis:
• T4 level normal, T3 level increase. TSH level decreased.
Condition T3 T4 TSH
Primary hypothyroidism
SecCJ1dary hypothyroidism "' "'"'
'V
1'
'V
Primary hyperthyroidism 1' -t 'V
Secoodary hypcrth yroidisrn -1' -t t
""
T3 th yrotoxkosis 1' Norma l

Clinical Biochemistry 586
2) Thyroid uptake studies:

Hyperthyroidism Hypothyroidism
Radio active iodine uptake (RAIU) by thyroid gland is used to detect function
derangements of thyroid gland. 131I is used for this study.
Technique:
About 15µ Ci of 1311 is given intravenously. After few hour, the patient' s neck is
monitored by movable gamma camera (gamma ray counters), which will pick up
the radiation emitted by the thyroid gland.
Normal value:
25 % Uptake within 2 hours; 50 % Uptake within 24 hours
Significance:
• Hyperthyroidism: Uptake increases
• Hypothyroidism: Uptake decreases
3) Thyroid scanning:
Technique:
131
1 is given intravenously. After 24 hours, patient is placed under the scanner to detect
the radioactive emission from the neck.
Significance:
Approximate size and shape of thyroid gland and actual distribution of radioactivity
will be known by thyroid scanning. In hyperthyroidism, increased radioactivity is
shown as darkly shaded areas. Defective distribution of 131I in some specific areas
such as silent nodules is suggestive of thyroid cancer.
4) D e tection of thyroid antibodies:

In some thyroid diseases, specific antibodies (anti-tpo antibodies) are detected in
plasma.
• In Grave's diseases, TSig (thyroid stimulating immunoglobulins), alias LATs (long

acting thyroid stimu lator), which mimic TSH action are seen in circulation.
• In Hashimoto thyroiditis, anti thyroglobulin antibodies, anti microsomal antibodies

and anti nuclear antibodies are seen in circulation.

Significance of HMP Shunt pathway:
1) Provision of NADPH: NADPH SoRudely Problematic

HMP shunt pathway generates NADPH, which is required for
2 bodies, cholesterol,
i) NADPH is required for the synthesis of fatty 1acids, ketone 3
acids / bile4salts, steroid
S 0
hormones.
bile
HMP shunt is very active in testis, ovary, adrenal cortex (as these tissues are the sites ofsteroid
hormone synthesis, which requires NADPH generated by HMP shunt pathway).
ii) In RBC's NADPH protects the RBC membrane against hemolysis and maintains
O
integrity of RBC membrane:
Explanation: .
In RBC's, NADPH is required to maintain the availability of reduced glutathione (GSH).
RBC has a high concentration of reduced glutathione (GSH), which removes H 2O2 by
glutathione peroxidase enzyme (H20 2 is a reactive oxygen species, which destroys the
RBC membrane and causes haemolysis). During this reaction, reduced glutathione
(GSH) converted to oxidised glutathione (GS-SG).
This oxidised glutathione (GS-SG) is reduced back to reduced glutathione (GSH) by
glutathione reductase enzyme, which requires NADPH formed in HMP shunt pathway.
In other words, In RBC's, NADPH produced in HMP shunt pathway required to keep
glutathione in reduced state (GSH), which is required to destroy removes H 2O 2 and
protect the RBC membrane against hemolysis and maintains integrity of RBC membrane.
(Harmful) (Reduced glutathione)

8i02 2GSH NADP+
HMP shunt
GS-SG NADPH+H+ ~• -- pathway
(Oxidized glutathione)
iii) NADPH is also needed for the phagocytosis.
2) Provision Ribose 5-phosphate (pentose sugar-phosphate):

HMP shunt produces ribose 5 - phosphate, which is required for nucleotide synthesis.

Clinical significance of HMP shunt pathway:

Glucose 6-phosphate dehydrogenase (G6PD) deficiency:
• Glucose 6-phosphate dehydrogenase deficiency (G6PD) is an inherited x- linked
disorder. It is one of the most common genetic defects in this world, w ith more than
400 million people having this genetic defect, most of them are asymptomatic.
• G6PD deficiency usually does not exhibit clinical symptoms.
• There is a relationship between glucose 6-phosphate dehydrogenase deficiency and
increased hemolysis & hemolytic anemia (as NADPH synthesised by G6PD is required
for intergrity of RBC).
Drug induced hemolysis:

Persons with G6PD deficiency develop hemolytic anemia, when they are administered drugs
such as primaquin (antimalarial), sulphanamide (antibiotic), acetanilide (antipyretic).
This called drug induced hemolysis.
Reason:
• Decreased activity of G6PD impairs the synthesis of NADPH. NADPH is required for
keeping glutathione in reduced state (GSH), which is required to destroy free radicals and
protect the cell membrane. Although G6PD deficiency occurs in all the cells, the effect is more
severe in erythrocytes because HMP shunt is the only pathway to produce NADPH in
erythrocytes. However, G6PD deficiency usually does not exhibit clinical symptoms.
• Some drugs like primaquine (antimalarial), sulphanamide (antibiotic), acetanilide (antipyretic),
etc require GSH for their metabolism. These drugs use up GSH (reduced glutathione) and
convert them to oxidized glutathione. Thus, the use of such drugs accentuates the G6PD
deficiency conditions. NADPH produced by HMP shunt converts oxidized glutathione back
to GSH. In G6PD deficiency, HMP shunt does not operate properly to produce NADPH, and
glutathione remains in oxidized state. This lead to accumulation of free radicals in cells,
especially in RBCs. Accumulatedfree radicals damage the cell membrane and lead to hemolysis
(drug induced hemolysis).
• Decreased NADPH also results in the accumulation of methemoglobin in erythrocytes,
which also can cause hemolysis.
Note: G6PD confers some protection against malaria caused by Plasmodium falciparum. This
is because the parasite requires GSH for its survival and also their life cycle is obstructed by
early destruction oferythrocytes.
Favism:
Ingestion of uncooked Java beans (Vicia Java), may also cause hemolysis in G6PD deficiency
patients. This anemic condition as a result of eating Java beans is called Favism.
This is due to the presence of vivin (a toxic glycoside).
Cooking & decanting removes this toxin.

Clinical significance of HMP shunt pathway:

Glucose 6-phosphate dehydrogenase (G6PD) deficiency:
• Glucose 6-phosphate dehydrogenase deficiency (G6PD) is an inherited x- linked
disorder. It is one of the most common genetic defects in this world, w ith more than
400 million people having this genetic defect, most of them are asymptomatic.
• G6PD deficiency usually does not exhibit clinical symptoms.
• There is a relationship between glucose 6-phosphate dehydrogenase deficiency and
increased hemolysis & hemolytic anemia (as NADPH synthesised by G6PD is required
for intergrity of RBC).
GGPD NADPH GSH
Drug induced hemolysis: PAS
Persons with G6PD deficiency develop hemolytic anemia, when they are administered drugs
such as primaquin (antimalarial), sulphanamide (antibiotic), acetanilide (antipyretic).
This called drug induced hemolysis.
Reason:
• Decreased activity of G6PD impairs the synthesis of NADPH. NADPH is required for
keeping glutathione in reduced state (GSH), which is required to destroy free radicals and
protect the cell membrane. Although G6PD deficiency occurs in all the cells, the effect is more
severe in erythrocytes because HMP shunt is the only pathway to produce NADPH in
erythrocytes. However, G6PD deficiency usually does not exhibit clinical symptoms.
• Some drugs like primaquine (antimalarial), sulphanamide (antibiotic), acetanilide (antipyretic),
etc require GSH for their metabolism. These drugs use up GSH (reduced glutathione) and
convert them to oxidized glutathione. Thus, the use of such drugs accentuates the G6PD
deficiency conditions. NADPH produced by HMP shunt converts oxidized glutathione back
to GSH. In G6PD deficiency, HMP shunt does not operate properly to produce NADPH, and
FR
Glutathione accumulateof
glutathione remains in oxidized state. This lead to accumulation of free radicals in cells,
especially in RBCs. Accumulatedfree radicals damage the cell membrane and lead to hemolysis
(drug induced hemolysis).
• Decreased NADPH also results in the accumulation of methemoglobin in erythrocytes,
which also can cause hemolysis.
Note: G6PD confers some protection against malaria caused by Plasmodium falciparum. This
is because the parasite requires GSH for its survival and also their life cycle is obstructed by
early destruction oferythrocytes.
Favism:
Ingestion of uncooked Java beans (Vicia Java), may also cause hemolysis in G6PD deficiency
patients. This anemic condition as a result of eating Java beans is called Favism.
This is due to the presence of vivin (a toxic glycoside).
Cooking & decanting removes this toxin.

Clinical significance of glycogen metabolism
Glycogenosis (Glycogen storage diseases)

The inherited genetic defects related to the glycogen metabolism (Synthesis and
degradation) are called glycogenosis or glycogen s torage diseases.
These genetic diseases are characterized by deposition of abnormal type or abnormal
quantity of glycogen in the tissues.
Glycogenosis are the group of genetic diseases that result due to enzyme defects of
glycogen metabolism. There are several types of glycogen storage diseases. In each
type there is deficiency or low activity of a particular enzyme.
Types: There are 6 major types of glycogen storage diseases.

UP CAMEHer
Type Name Enzyme defect Characteristics
Hepatomegaly (Due to
Von-Gierke's disease GI ucose-6-phospha tase liver cells loaded with
I in liver glycogen, hypoglycemia,
Lactic acidosis, Ketosis.
Fatal, accumulation of
II Lysosomal acid maltase
Pompe's disease glycogen in lysosomes,
(a-1 • 4 glucosidase)
heart failure.
Limit dextrinosis/ Accumulation of a

m Cori's disease/ Debranching enzyme branched polysaccharide
Forbes diseaseBidi (limit dextrin)
Accumulation of
Andersen's disease/ polysaccharide having
IV Branching enzyme few branch points. Death
Amy lopectinosis
due to cardiac or liver
Banyan
Y
failure in } st year of life.
nobrown
Diminished exercise
tolerance; Muscles have
V McArdle' s disease Muscle phosphorylase
abnormally high
glycogen content.
High glycogen content in

VI Hers' disease Liver phosphorylase liver; tendency towards
Hepatic hypoglycemia.

Type I glycogen storage disease or von Gierke's disease:

von Gierke's disease results from absence of glucose-6-phosphatase in liver.
Prevalence 000livebirths
in hypoglycemia, familyc.ge Yaiogeiiys
Features include hepatomegaly, lactic acidosis, ketosis, hyperuricemia.
• Hypoglycemia: Blood glucose is maintained by hepatic glycogenolysis in the
interdigestive periods. In von Gierke's disease, there's no con version of glucose 6
phosphate to glucose, leading to hypoglycemia.
• Hepatomegaly: There is an abnormal accumulation of glycogen in liver due to inability
of breakdown of glycogen, leading to hepatomegaly. Thus, this accumulated glycogen
in liver is not useful.
• Lactic acidosis and ketosis: Glucose 6-phosphatase is also required for gluconeogenesis
for the conversion of lactate to glucose in liver. Failure of lactate to glucose leads to
accumulation of lactate, causing lactic acidosis and subsequent ke tosis.
• Hyperuricemia and Gout:von Gierke's disease is associated w ith other secondary
manifestations like hyperuricemia and gout. Glucose 6-phosphatase deficiency blocks
the conversion of glucose-6-phosphate to glucose. Accumulated glucose-6-phosphate
is then diverted to HMP shunt pathway resulting in elevated levels of ribose-5-phosphate
and hence purine n ucleotides and nucleic acids synthesis. Breakdown of excess purine
nucleotides & nucleic acids causes hyperuricemia (Refer nucleotide metabolism chapter).
piiipiesifiidiagiieik.siaeEgijofjiiiitiiqieneiu.ua
Type II or Pompe's disease:
Lysosomal a. 1, 4-glucosidase (also called acid maltase) defect. Normally this enzyme
is involved in the degradation of glycogen in lysosomes. In its absence glycogen
accumulates in lysosomes, maninly in heart, which lead to cardiac failure & early death.
Type III or Cori's disease (limit dextrinosis):

Debranching enzyme defect. There is accumulation of a polysaccharide of the "limit
dextrin" type.
Type IV or Anderson's disease (amylopectinosis):

Branching enzyme defect. There is accumulation of a polysaccharide with few branch
points. Death occurs due to cardiac or liver failure in first year of life.
Type V or McArdle's syndrome:

Muscle phosphorylase defect. These patients exhibit a markedly diminished tolerance
to exercise as glycogen cannot be degraded to provide energy to con tracting muscle,
causing cramps (not the lactic acidosis cramps, as very little lactate formed after exercise).
These are normoglycemic, even in fasting state, as liver glycogenolysis is not affected.
Type VI or Her's syndrome:

Liver phosphorylase defect. Glycogen accumulates in the liver. Failure to glycogenolysis
leads to hypoglycemia.

Functions of TCA Cycle :

1) Primary function of TCA cycle is the provision of energy.
2) TCA cycle has amphibolic (both Anabolic and Catabolic) nature.

a) Catabolic role:
TCA cycle is the final common metabolic pathway for the production energy from
acetyl CoA obtained from carbohydrates, lipids and proteins / amino Acids.
Amino acids Amino acids
i
g
I
Glucose - __. Pyruvate "
Acetyl-CoA • TCA cycle
•
FatJ Acids
b) Anabolic role:
Starting from intermediates of TCA cycle, several compounds can be produced .
i) Heme synthesis:
Succinyl-CoA is used as a starting material in heme synthesis.
oxaloacetate Aspartate OPP
ii) Synthesis of aspartate and glutamate: ofgluconeogenesis
a ketoglutarate Glutamate
Oxaloaceta te and a-ketoglutarate can be converted to amino acids aspartate and
glutamate respectively by transamination process.
iii) Fatty acid synthesis:
Citrate is transported out of mitochondria into cytoplasm where it can give back acetyl-
CoA & oxaloacetate. Acetyl-CoA can be used for the synthesis of fatty acids.
iv) Glucose synthesis:
Intermediates of TCA Cycle can be sources for gluconeogenesis.
• Since TCA cycle has both anabolic and catabolic role, TCA cycle said to be amphibolic
(both Anabolic and Catabolic) nature.
Regulation:
Citrate synthase, Isocitrate dehydrogenase and a -ketoglutarate dehydrogenase
enzymes are the regulatory enzymes of TCA cycle.
Regulatory enzyme Inducer Repressor Activator Inhibitor
C itrate synthase Insulin Glucagon - Citrate, NADH, ATP,

fa ttv acvl CoA
lsocitrate dehydrogenase lrsulin Glucagon ADP,NAD• NADH,ATP
a -keto g lutarate dehydrogenasc Irsulin Glucagon ADP, NAD• NADH,ATP

The energy (ATP) requirement of the cell regulates the rate of TCA cycle. When the cell
requires energy, TCA cycle runs in a faster rate & when the cell has sufficient energy,
TCA cycle runs slowly. So, ATP is the inhibitor and ADP is activator of TCA cycle.
Anaplerotic reactions of TCA cycle (Greek: Ana = fill up) :

In its anabolic role, several intermediates of TCA cycle are used up for biosynthesis
of many compounds. These intermediates should be replenished. The reactions, which
replenish these intermediates, are called anaplerotic reactions or anaplerosis.
StepofTCA
E.g. 1: Pyruvate carboxylase reaction:
Pyruvate carboxylase
Pyruvate + CO2 ----=--:::-------+• Oxaloaceta te
;;:-:-=-;iot~
ATP Mn2+ ADP+ Pi
This is a very important anaplerotic reaction. Since a large entry of acetyl-CoA to the
TCA cycle depletes the supply of oxaloacetate required for the first reaction of TCA
cycle (citrate synthase).
E.g. 2: Malic enzyme:

Malic enzyme
Pyruvate + CO2 Malate + H20
NADPH + H· NADP•
ADH
Other examples of anaplerotic reactions are Glutamate dehydrogenase and
transaminases (ALT and AST).
Oxidative decarboxylation reactions:

Several keto acids undergo oxidative decarboxylation reactions by keto acid
dehydrogenases. These are multi-enzyme complexes with 3 enzymes and 5 Coenzyrnes.
These reactions require 3 B-complex vitamins (Thiamine, Riboflavin and iacin).
E.g.: a-keto glutarate dehydrogenase and Pyruvate dehydrogenase (PDH).
) a-keto glutarate dehydrogenase enzyme complex (of TCA cycle):

a,.ketoglutarat e dehydrogenase
cx-ketoglutarate 77'" • Succiny l CoA
CoASH - / FAD, TPP ~-co.
NAD Lipoic acid NADH•+H •
3 enzymes are a-ketoglutarate dehydrogenase. Dihydrolipoyl tmnsacetylase, Dillydrolipoyl dehydrogenas
5 Coenzymes are TPP, CoASH, NAO•, FAD, Lipoic acid.
) PDH is already exaplained before.

Lactic acidosis:
Definition: Elevated level of lactate and subsequent decrease in plasma pH is termed
as lactic acidosis (a type of metabolic acidosis).
Normally, lactate produced by anaerobic glycolysis is taken up by liver and converted
to glucose by gluconeogenesis. Lactic acidosis results from either overproduction or
underutilization of lactic acid. Different causes of lactic acidosis are,
• Pulmonary embolism, myocardial infarction, uncontrolled hemorrhage, severe shock
etc. causes decreased oxygen supply to tissues, resulting in decreased oxidative
phosphorylation to produce ATP. To survive, the cells rely on anaerobic glycolysis and
overproduction of lactic acid.
• Strenuous exercise and under hypoxic conditions, there is increased rate of anaerobic
glycolysis (as oxygen supply is not enough to sustain aerobic glycolysis) and
overproduction of lactic acid. Lactic acid accumulates and causes muscle cramps.
• Inherited pyruvate dehydrogenase (that converts pyruvate to acetyl CoA) & pyruvate
carboxylase (that converts pyruvate to oxaloacetate) deficiency, lead to an accumulation
of pyruvate and subsequent conversion to lactic acid, resulting in lactic acidosis.
• Dietary deficiency of thiamine (thiamine forms TPP, coenzyme of PDH) can cause
lactic acidosis. Arsenite & mercury, whcih inhibit PDH also can cause lactic acidosis.
• Alcoholism: Oxidation of alcohol in the body generates NADH which favours
conversion of pyruvate to lactate. Also alcohol inhibits thiamin absorption.
• von Gierke's disease: It is the defect of glucose-6-phospatase in liver, (a gluconeogenic
enzyme which is required to convert lactate to glucose). Failure of conversion of lactate
to glucose leads to accumulation of lactate, causing lactic acidosis.
Acetyl CoAac
Pyruvate dehydrogenase (PDH) reaction (Oxidative decarboxylation of pyruvate):
Pyruvate is oxidatively decarboxylated to acetyl CoA by pyruvate dehydrogenase (PDH)
enzyme complex. Acetyl CoA then enters TCA cycle. PDH is a muJtienzyme complex.
Pyruvate dehydrogenase
complex
c sina.ca
Pyruvate Acetyl CoA
CoASH FAD, TPP CO, Transacetylase
Lipoic acid
NAO• ADH+H•
Pyruvate dehydrogenase enzyme is a multie11zyme complex. ft contains 3 enzymes & 5 coenzymes.

5 Coenzymes are;
3 enzymes are; • TPP (Thiami11e pyrophosphate)
• Pyruvate dehydrogenase • Coenzyme A (CoASH)
• Dil,ydrolipoyl transacetylase • NAD+
• Dihydrofipoyl dehydrogenase • FAD
• Lipoic acid
Pyruvate dehydrogenase requires TPP
• Dihydrolipoyl transacetylase requires lipoic acid & Coenzyme A
• Dih drolipo l deli dro enase requires FAD and NA O•

Rapaport leubering cycle (2, 3 -BPG shunt or Diphosphoglycerate shunt)
• There is a high concentration of 2, 3-Bisphosphoglycerate (2, 3-BPG) in erythrocytes.

This is formed from 1, 3-Bisphosphoglycerate (1, 3 BPG) by Phosphoglycerate mutase
enzyme. The phosphoglycerate kinase step is bypassed.
• Next, 2, 3-BPG is degraded by a phosphatase enzyme to form 3-phosphoglycerate.

The bypass around phosphoglycerate kinase step is known as Rapaport leubering
cycle or 2, 3-BPG shunt. In this shunt, no ATP is generated.
Glucose
( Rapaport leubering cy cle )
l
I, 3-Bisphosphoglycerate
ADP Phosphoglycerate mutase
2, 3-Bisphosphoglycerate
ATP ~ H 20
J phosphatase
3-phosphoglycerate Pi
l
Pyruvate
1 02transport Faycerate
Importance of 2,3 -BPG: 2 Hyoxia
3 Paakinasestep
I • 2, 3 - BPG is a regulator of oxygen transport in erythrocytes. It combines with Hb and
reduces the affinity of hemoglobin to 0 2. So, in presence of 2, 3 - BPG, oxyhemoglobin
will unload 0 2 more easily in tissues.
2• Rapaport leubering cycle and the concentration of 2, 3-BPG in erythrocytes increases

in hypoxic conditions. This favors the release of 0 2 to tissues even when p0 2 is low.
leabring cycle
Rapaport
3• The energy requirement in erythrocytes is minimal. In 2, 3 -BPG shunt, the energy
yielding phosphoglycerate kinase step is bypassed and hence no energy is formed.
This allows the glycolysis to proceed without the synthesis of extra energy.

Transport of reducing equivalents from cytosol to mitochondrial matrix:

ETC is present in mitochondria. So, NADH is produced in the cytosol by the glycolytic
enzyme glyceraldehyde 3-phosphate dehydrogenase (and other cytosolic enzymes)
should be transported to mitochondria. But, the inner mitochondrial membrane is
impermeable to NADH, so they cannot enter mitochondria. This problem is overcome
by two shuttle systems, which transport the reducing equivalents from cytosol to
mitochondria. These are,
1) Malate shuttle 2.5ATPfrom
2) Glycerophosphate shuttleftp.AHayp from NADHdueto producer FADH inwitoch
of
1) Malate shuttle:
In the cytosol, oxaloacetate accepts the reducing equivalents (NADH) and becomes
malate. Malate then enters into mitochondria where it is oxidized by mitochondrial
malate dehydrogenase. In this reaction, NADH and oxaloacetate are regenerated.
NADH enters ETC and produces 3 ATP. This is in contrast to glycerophosphate
shuttle where only 2 ATP are produced.
In the mitochondria, oxaloacetate participates in transamination reaction with
glutamate to produce aspartate and a - ketoglutarate. The aspartate enters the cytosol
and transaminates with a-ketoglutarate to give oxaloacetate and glutamate.
Oxa loa ceta te Glutamate

NAOH+H+
tree
Malate Aspartate a-KG Cytosol
! l
Malate Aspartate
l
a-KG
Mitoch membrane
Mitochondrial Matrix
NAO+
Mitochondrial
Malate dehydrogenase
NADH+H+
Oxaloacetate Glutamate
1
ETC

2. Glycerophosphate shuttle:
Cytosolic glycerol 3-phosphate dehydrogenase oxidizes NADH to NAD+. The reducing
equivalents are transported through glycerol 3- phosphate into the mitochondria.
Glycerol 3- phosphate dehydrogenase present on outer surface of inner mitochondrial
membrane reduces glycerol 3-phosphate back to dihydroxy acetone phosphate, which
also converts FAD to FADH2 • Dihydroxyacetone phosphate escapes into the cytosol
and the shuttling continues. FADHz gets oxidized in ETC to generate 2 ATP.
NADH+H+ NAO+
Dihydroxy acetone ---~-"""--L""'---- •ll Glycerol

Phosphate Cytosolic glycerol 3-phosphate
3-phosphate dehydrogenase
Cytosol
Mitochmembrane
Mitochondrial Matrix
Mitochondrial glycerol
Dihydroxy acetone 3-phsphate dehydrogenase Glycerol
7'\
4
Phosphate 3-phospahte
FADH2 FAD
l
ETC
Note:
Reducing equivalents transported through glycerophosphate shuttle yields only 2
ATP, because FADH2 is produced in the mitochondrial matrix, whereas reducing
equivalents transported through malate shuttle can give 3 ATP as NADH is produced
in the cytosol.
Transport of ATP and AD P (ATP-ADP tran sporter):

1n oxidative phosphorylation, ATP is produced in the mitochondria. Many energy
requiring reactions in the cytosol need ATP (ATP is converted to ADP in the process).
Therefore, ATP needs to be transported from mitochondria into cytosol, but
mitochondrial membrane is impermeable to ATP. This is accomplished by ADP-ATP
exchange transporter present in the inner mitochondrial membrane. It transports ATP
and ADP in the opposite direction, ATP from mitochondrial matrix to cytosol and ADP
from cytosol to mitochondrial matrix.

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Disa If I traumas
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Biochem

Conditions for anaerobic glycolysis are,

NAD+ Glyceraldehyde 3-Phosphate dehydrogenase

LDH utilizes NADH formed in glyceraldehyde

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Step <Enzym e) Source ISQ. of A T P fQrmeg

Step <En zym e) Source N12, of ATP fQrrn eg

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Difference between Hexokinase and Glucokinase

Significance of lactate dehydrogenase (LDH) reaction:

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Starting Compounds (Non carbohydrate sources of gluconeogenesis):

Irreversible steps of g l yco l ysis Correspond ing key g lu coneogenic e n zy mes

a) Pyruvate kinase 1) Pyruvate carboxylase

b) Phosphofructokinase (PFK) 3) Fructosc -1, 6-Bisphosphatase

c) Hcxokinase / Glucokinase 4) G l ucose 6-phosphatase .

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Reaction pathway: GLUCOSE

Fructose 1,6 -bis pho phate

GDP +C0 2 13C V ADP

!Pyruvatecarboxylase I Acetyl CoA

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2) Gluconeogenesis used to clear the w aste products of metabolism:

Glucagon, Cortisol Acetyl ADP

Glucose 6-phosphatase Same Same -- --

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Citric acid cycle

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Inhibitors of TCA cycle: Eca Gylolysis

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Functions of TCA Cycle :

2) TCA cycle has amphibolic (both Anabolic and Catabolic) nature.

glutamate respectively by transamination process.

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Anaplerotic reactions of TCA cycle (Greek: Ana = fill up) :

E.g. 2: Malic enzyme:

Other examples of anaplerotic reactions are Glutamate dehydrogenase and

Oxidative decarboxylation reactions:

) a-keto glutarate dehydrogenase enzyme complex (of TCA cycle):

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Glyco gen metab olism (Glycogenesis & Glyco genol ysis)

Glyco genesi s (Glycogen synthesis)

a) Activation glucose [Formation of UDP glucose or UDPG]:

UDP glucose is the donor of glucose in glycogen synthesis. of

b) Length ening of chain & formation of glycogen:

UDP gluco~e UDP

ii) Branching enzyme:

Diagramatic representation of glyco genes is

Signi fican ce:

Regu lation of glyco gen synth esis:

Rever sible covale nt modif icatio n of glycog en syntha se enzym e

Glyc ogen olys is (Gly coge n brea kdow n)

Site: Glycogenesis Glycogensynth

b) Gluca n transferase: It transfe rs 3 glucose residu es from one chain to anothe

Diag rama tic repre senta tion of glyco genol ysis

a) In liver: Gluco se 1-pho sphate is conve rted to gluco se in liver.

Signi fican ce of glyco genol ysis:

Regulation of glyco genol ysis :

(Depho sphory lated form) (Phosp horylat ed form) Phosphorylat

Rever sible covale nt modif icatio n of glycog en phosp horyla se enzym e

7- dehydrocholesterol • • • • • Cholecalciferol (vitamin D:J

Biochemical functions of vitamin D:

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Activation of vitamin D (formation of calcitriol):