MD Rafi Biochemistry

CHAPTER 1
Section I
Prelude – Molecular and
Functional Organisation of
Life
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Rafi MD: Textbook of Biochemistry (4th Edition) Universities Press
CHAPTER 1
Chapter 1
Learning Biochemistry

CHAPTER 1
What is Biochemistry?
Anatomy is the study of structure, Physiology is the

study of function. Biochemistry integrates both these
aspects—it describes the structure and function of living
things in molecular terms.

CHAPTER 1
Energy in Living Organisms
Every organism needs energy for its survival.
Energy flow in the environment

CHAPTER 1
Essential Elements in the Body

CHAPTER 1
Polar and Non- polar Molecules
• Water
• Hydrophilic, hydrophobic and amphipathic

molecules

CHAPTER 1
Major Biomolecules in the Body

CHAPTER 2
Section I
Prelude
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Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press
CHAPTER 2
Chapter 2
The Cell and Biological

Membranes:
Structure and Function

CHAPTER 2
Structure of a Human Cell

CHAPTER 2
Composition of Membranes
Mem branes are made of a double layer of lipid

molecules containing proteins which are embedded in
them.
Lipids

The m ajor mem brane lipids are phospholipids. These
are amphipathic m olecules containing both hydrophilic
or polar (water soluble) and hydrophobic or non- polar
(water insoluble) groups.

The cell mem brane also contains equal amounts of
cholesterol.

CHAPTER 2
Proteins
●
Proteins in m embranes occur in two different ways.
Some proteins are located in the interior of the
m embrane and are called integral m embrane
proteins.
●
Others are located in the outer surface of the
m embranes and are known as peripheral membrane
proteins.
●
Transm embrane proteins serve as:
a) Channels b) Carriers
c) Pumps d) Receptors

CHAPTER 2
Carbohydrates
The ex tracellular surface of the plasm a m embrane

contains small am ounts of carbohydrate structures
known as glycocalyx.

CHAPTER 2
Fluid Mosaic Structure
Mem branes are organised into a structure known as

the fluid mosaic model. It is called so because
m embrane proteins float in a sea of lipids. This
m odel of the cell m embrane was proposed by Singer
and Nicholson in 1972.

CHAPTER 2
Fluid- Mosaic Model of
Membrane Structure

CHAPTER 2
Membrane Function
Transport Across Biological Membranes
Passive Transport
Here, substances move across the cell m embrane
without any energy ex penditure by the cell. It includes
diffusion and osm osis.
1 . Diffusion
Simple Diffusion
Facilitated Diffusion
2 . Osmosis

CHAPTER 2
Active Transport

When substances are transported against their
chemical and electrical gradient requiring energy,
the process is called active transport. The main
types are
1. Primary active transport
2. Secondary active transport
3. Carrier transport
4. Vesicular transport

CHAPTER 2
1 . Primary Active Transport

i) Sodium–potassium (Na+ –K+ ) pum p
ii) Calcium (Ca2+ ) pump
iii) Potassium –hydrogen (K+ –H+ ) pump
2 . Secondary Active Transport

CHAPTER 2
3 . Carrier Type Transport


Uniporters

Symporters

Antiporters
Carrier type processes

CHAPTER 2
4 . Vesicular Transport or Transcytosis

Many substances are transported across the cell
mem brane by endocytosis and ex ocytosis.
i) Endocytosis: pinocytosis, phagocytosis
Sequence of events in endocytosis

CHAPTER 2
ii) Exocytosis
Sequence of events in ex ocytosis

CHAPTER 3
Section II
Molecules of Life
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CHAPTER 3
Chapter 3
Carbohydrates

CHAPTER 3
Carbohydrates

Carbohydrates are aldehyde or ketone compounds
with multiple hydroxyl groups.

Carbohydrates can be defined as aldehyde or ketone
derivatives of polyhydric alcohols. Most
carbohydrates taste sweet and hence they are
popularly known as sugars.

They are the chief source of energy for all
organisms including humans.

CHAPTER 3
Classification and Nomenclature
Carbohydrates are classified into three major classes based

on the number of sugar units.
1. Monosaccharides
2. Oligosaccharides
3. Polysaccharides

CHAPTER 3
Monosaccharides
They are categorised into two groups based on the

functional group they possess.
Aldoses: Monosaccharides with an aldehyde group
are known as aldoses. For ex am ple, ribose (5C), glucose

(6C).
Ketoses: Monosaccharides having a keto group
are called ketoses. For ex am ple, fructose (6C).

CHAPTER 3
Classification of Monosaccharides

CHAPTER 3
D and L Isomers
The spatial arrangement of –H and –OH groups on the

penultimate carbon atom in a m onosaccharide (C2 for
glyceraldehyde, C5 for glucose) determ ines whether it
is a D- isomer or its m irror im age L- isomer.
If the –OH group is on the right side, the sugar

belongs to the D- series, and if it is on the left side it
belongs to the L- series.

CHAPTER 3
D- and L- Isomerism

CHAPTER 3

A carbon atom which is attached to four different
groups is known as an 'asymmetric' or 'chiral' carbon.
Optical Isomerism

Optical isomerism is ex hibited by those compounds
which possess asymmetric carbon(s).

Optical isomers are capable of rotating the plane of
polarised light either to the right or to the left.

CHAPTER 3

The optical isomer which rotates the plane of
polarised light to the right (clockwise) is said to
be dextrorotatory [represented by ‘d’ or (+ )] and
the substance which rotates the plane of
polarised light to the same ex tent but to the left
(counter clockwise) is termed as levorotatory
[represented by ‘ l’ or (–)].

A solution having equal concentrations of ‘d ’ and ‘l ’
forms, known as racemic mixture, cannot rotate the
plane of polarised light and hence shows zero
optical rotation.

CHAPTER 3
EPIMERS

Monosaccharides that differ only in their
configuration around one carbon atom (other
than anom eric) are known as epimers of one
another.

Thus, D- glucose and D- mannose are epimers
with respect to C2 and D- glucose and D-
galactose are epimers with regard to C4 , that is,
they differ from each other in the arrangement of
–OH group around that particular carbon atom.

CHAPTER 3
Epimers
Epimers (glucose and mannose are C2 epimers, whereas glucose and
galactose are C4 epimers).

CHAPTER 3
Anomers

The pair of stereoisom ers that differ in
configuration around the carbonyl carbon are
called anomers.
The hemiacetal / hemiketal or carbonyl carbon is
called anomeric carbon.
In case of - anomer, the - OH group of the anomeric
carbon is on the opposite side of the terminal
primary alcohol group (C6 ) of the sugar ring.
If both groups are on the same side, it is a

- anomer.

CHAPTER 3
α and β Anomers of Glucose
(Fischer Projections)

CHAPTER 3
 &  Anomers
Pyranose and furanose forms of glucose (Haworth projections)

CHAPTER 3
 &  Anomers
Pyranose and furanose forms of fructose (Haworth projections)

CHAPTER 3
Mutarotation
Mutarotation is defined as the change in optical rotation

representing interconversion of  and  forms of glucose
to an equilibrium mixture.

CHAPTER 3
Properties and Reactions of
Monosaccharides
Formation of furfurals from monosaccharides upon dehydration

CHAPTER 3
Tautomerisation and Enediol formation
Formation of a common enediol from glucose, fructose and mannose. ‘R’ represents
t he common structure of all the three sugars C3 – C6 .

CHAPTER 3
Osazone Formation

Only the first two carbons are involved in osazone
form ation.
The sugars which differ in configuration around the
first two carbons give identical osazones, since the
difference is masked by binding with phenyl
hydrazine.
This is the reason why glucose, fructose and mannose
give similar needle- shaped osazones.

Differently shaped crystals (osazones) CHAPTER 3
from different carbohydrates

Glucosazone and fructosazone – long needle-
shaped or broom stick- shaped crystals.

Maltosazone – Yellow coloured ‘petals of sun
flower’- shaped crystals.

Lactosazone – ‘Hedgehog- shaped’ or powder- puff-
shaped crystals.

CHAPTER 3
Disaccharides
As the name im plies, disaccharides are

carbohydrates composed of two monosaccharides
linked together through a glycosidic linkage. They
are of two types.
1 . Reducing disaccharides: They possess a free

aldehyde or keto group, e.g., maltose, lactose.
2 . Non- reducing disaccharides: They do not have

a free aldehyde or keto group,
e.g., sucrose.

CHAPTER 3
Maltose
Maltose is com posed of two - D–glucose units held

together by  (1–4)- glycosidic linkage.

CHAPTER 3
Lactose
Lactose is popularly known as milk sugar as it is

present in milk. Lactose is composed of - D-
galactose linked to - D- glucose through a - (1, 4)-
glycosidic bond.

CHAPTER 3
Sucrose

Sucrose is mostly present in sugarcane and fruits.
Hence it is also known as cane sugar. Sucrose is
synthesised by plants through photosynthesis.

Sucrose is composed of glucose and fructose linked
together through glycosidic linkage (1 –2 ). The
aldehyde group of glucose and the keto group of
fructose are engaged in bond formation leaving no
free functional group thus making sucrose
essentially non- reducing.

CHAPTER 3
Formation of sucrose
Formation of sucrose [ α - D- Glucopyranosyl- (1- 2)- β - D- fructofuranose]

CHAPTER 3
Polysaccharides
Hundreds of monosaccharides are linked together

through glycosidic linkages to form polysaccharides.
Polysaccharides are of two types:

1 . Homopolysaccharides
They are made up of a single variety of
m onosaccharides. For ex am ple, starch is a polym er
of glucose.
2 . Heteropolysaccharides
They are composed of different types of
monosaccharides or their derivatives, e.g.,
glycosaminoglycans.
CHAPTER 3
Starch
●
Starch is the high energy nutrient synthesised from
carbon diox ide and water by photosynthesis in
plants.
●
It is the chief carbohydrate fuel source for higher
animals, including humans. The dietary sources of
starch include cereals, tubers, roots, legumes and
vegetables.
●
Starch is composed of two com ponents:
Amylose and Amylopectin.
●
Starch is digested by pancreatic amylase in the
intestine.

CHAPTER 3
Structure of Starch
Cont inues. . .

. . . cont inued CHAPTER 3
Structure of Starch

CHAPTER 3
Glycogen
Glycogen is the storage form of carbohydrates in

animals, including humans, and hence is known as
animal starch. It is stored in muscle and in the liver.
Structure: Glycogen is composed of glucose units

linked by - (1–4) and - (1–6) glycosidic bonds. It is
structurally sim ilar to amylopectin ex cept in that
glycogen is m ore branched and well organised into a
num ber of concentric layers (about twelve). Hence,
glycogen is m ore com pact than am ylopectin (starch).

CHAPTER 3
Structure of glycogen
Glucose residues taking part in α - 1- 6- glycosidic linkages are shown in pink colour.
Glycogen is the instant storage form of energy in animals, including humans.
CHAPTER 3
Glycogenin in the gylcogen structure
Glucose residues taking

part in α-1-6-glycosidic
linkages are shown in
orange colour. The green
ball represents glycogenin,
a protein that acts as
glycogen primer.

CHAPTER 3
Cellulose
●
Cellulose is the most abundant carbohydrate in
nature. To be m ore precise, it is the m ost
abundant organic substance on earth. Cellulose
m akes up most of the plant framework. It is absent
in animals.
●
Cellulose is the unbranched, linear polymer
com posed of - D- glucose units linked through -
(1–4)- glycosidic bonds.

CHAPTER 3
Heteropolysaccharides
Glycosaminoglycans
Glycosamino glyc ans (GAGs), also known as

mucopolysaccharides, are m ade up of repeating units
of disaccharides containing am ino sugars (glucosamine
or galactosam ine) and uronic acids (glucuronic or
iduronic acids). Am ino sugars are further acetylated and
sulphated.

CHAPTER 3
The m ajor GAGs include:
1. Hyaluronic acid
2. Chondroitin sulphates
3. Keratan sulphates
4. Heparin and heparan sulphate
5. Dermatan sulphate

CHAPTER 3
Structures of Glycosaminoglycans
Hyaluronic acid
Chondroitin 4 - sulphate
(Continues…

…Continued) CHAPTER 3
Keratan sulphate
Heparin
(Continues…

Dermatan sulphate

CHAPTER 4
Section II
Molecules of Life
Universities Press
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CHAPTER 4
Chapter 4
Lipids

CHAPTER 4
Lipids

Lipids can be defined as heterogeneous
substances grouped together under one class
based on their common property, of being
insoluble in water and soluble in organic solvents
(ether, benzene, chloroform ).

They are predom inantly composed of carbon and
hydrogen atoms. These atoms are linked together
through neutral covalent bonds making lipids
essentially non- polar.

CHAPTER 4
FUNCTIONS OF LIPIDS
1. Lipids are the structural components of biological

membranes (phospholipids and cholesterol) and
are responsible for the selective permeability of the
m embranes.
2. They are the storage form of energy. Energy is

stored in the form of lipids (triacylgycerol) in
adipose tissue.
3. They act as electrical insulators (cholesterol and

glycolipids) helping in the propagation of nerve
impulse.
(Continues…

CHAPTER 4
…Continued)
4. They act as thermal insulators (subcutaneous fat)

providing insulation against the changes in
ex ternal tem perature.

CHAPTER 4
Classification of Lipids
Lipids are classified based on 'Bloor's classification‘

(with a few modifications) into four groups.
1 . Simple Lipids
They are fatty acid esters of alcohols
(Fatty acid + alcohol). They are of
two types.
a) Fats and oils
b) Waxes

CHAPTER 4
2 . Compound (Complex) Lipids


They are esters of fatty acids with alcohols having
additional groups like carbohydrates, proteins,
phosphoric acid

They are further classified into various subgroups
based on the type of additional group present.

CHAPTER 4
a) Phospholipids
i) Glycerophospholipids
ii) Sphingophospholipids
b) Glycolipids
c) Lipoproteins
3 . Derived Lipids
4 . Miscellaneous Lipids

CHAPTER 4
Fatty Acids

Fatty acids are components of the majority of lipids.
They are carbox ylic acids with a hydrocarbon side
chain.

The hydrocarbon chain accounts for the non- polar
nature of fatty acids.

Free and esterified fatty acids

Length of hydrocarbon chain

Even- and odd- number carbon fatty acids

CHAPTER 4
Saturated and Unsaturated Fatty Acids

Fatty acids which possess double bonds in their
structure are called 'Unsaturated fatty acids' (UFA)
and fatty acids with no double bonds are 'Saturated
fatty acids' (SFA).

Unsaturated fatty acids are further classified into
two subgroups based on the num ber of double
bonds.
1. Monounsaturated fatty acids (MUFA) are

fatty acids with single double bond.
2. Polyunsaturated fatty acids (PUFA) are fatty
acids with two or m ore double bonds.

CHAPTER 4
Unsaturated Fatty Acids

CHAPTER 4
Omega () Classification o f Fatty Acids
In this type of classification, the positions of the

double bonds are counted from the omega carbon end.
According to - classification, the unsaturated fatty
acids are broadly divided into three groups:

CHAPTER 4
Properties of Fatty Acids
Properties of fatty acids depend on chain length and

degree of unsaturation.

Solubility

Melting point

Salt form ation

Ester Form ation

Hydrogenation

Halogenation

Soaps and detergents

CHAPTER 4
Essential Fatty Acids


The fatty acids that are required by hum ans, but
are not synthesised in the body and hence need to
be supplied in the diet are known as essential
fatty acids (EFA).
1. Linoleic acid (18: 2; 9, 12)
2. Linolenic acid (18: 3; 9, 12, 15)

Arachidonic acid (20: 4; 5, 8, 11, 14) is considered
semiessential since it can be synthesised from
linoleic acid (note that both belong to - 6 series).

CHAPTER 4

Only linoleic acid and linolenic acid are essential
because hum ans lack the enzym es that can
introduce double bonds beyond carbon no. 9.

CHAPTER 4
Dietary Sources of Fatty Acids
Vegetable oils are the richest source of fatty

acids.

Oils rich in SFA: Palm oil, coconut oil, ghee.

Oils rich in MUFA: Groundnut oil, gingily oil,
m ustard oil, sesam e oil.

Oils rich in PUFA: Sunflower oil, cotton seed oil,
safflower oil, rice bran oil, soyabean oil, linseed oil,
corn oil.

CHAPTER 4
Eicosanoids

Eicosanoids are a group of biologically active m olecules
synthesised from eicosaenoic acids (C20 PUFA).

Eicosanoids are a group of five types of molecules.
1. Prostaglandins (PG)
2. Prostacyclins (PI)
3. Thrombax anes (TX)
4. Leukotrienes (LT)
5. Lipox ins (LX)

Most eicosanoids are synthesised from arachidonate.

CHAPTER 4
Biological Role and Therapeutic Applications
of Eicosanoids
Eicosanoids act as local hormones.
1 . Inflammation
Eicosanoids are the natural mediators of
inflammation. This is the basis of the use of
corticosteroids and various non- steroidal
antiinflammatory drugs (NSAID) (which inhibit
the eicosanoid synthesis) in the treatment of
various inflammatory disorders (e.g., rheum atoid
arthritis).

CHAPTER 4
2 . Regulation of Blood Pressure

Prostaglandins act as vasodilators. Hence, they
are used in the treatm ent of hypertension.
3 . Effects on Platelet Aggregation

Thrombax anes cause platelet aggregation.
Prostacyclins inhibit platelet aggregation.

CHAPTER 4
4 . Effects on Reproduction
Prostaglandins are used in medical termination of
pregnancy (MTP).
5 . Effects on the Gastrointestinal System

PGs increase intestinal motility and inhibit gastric
acid secretion. They are used in the treatm ent of
gastric ulcers.
6 . Effects on the Respiratory System

PGEs are used in the treatm ent of bronchial asthma.

CHAPTER 4
Triacylglycerols
Energy is stored as lipid droplets in adipose cells.

The storage lipids are chem ically triacylglycerols.
They are insoluble in water and because of their
non- polar character.

CHAPTER 4
Properties and Reactions
Properties and reactions of tiracyl glycerols in relation

to nutritional value.
Saponification number
Saponification num ber is defined as the mg of KOH
required to saponify 1 g of fat or oil completely.
Saponification num ber is a m easure of the average
m olecular weight and chain length of the fatty acids
present.

CHAPTER 4
Iodine Number
It is defined as the num ber of gram s of iodine absorbed
by 100 g of fat or oil. Iodine num ber is used to assess
the degree of unsaturation of fat.
Reichert–Meissl Number (RM Number)

RM num ber is defined as the amount of 0.1 N KOH
required to neutralise the volatile fatty acids distilled
from 5 g of fat. It is employed to assess the purity of
fats having more volatile fatty acids.

CHAPTER 4
Phospholipids

They are the structural components of biological
membranes and are responsible for the selective
perm eability of the m embrane.

CHAPTER 4
General structure of
phospholipids

Phospholipids are divided into two classes based on
the type of alcohol present.
I. Glycerophospholipids: They contain alcohol glycerol
(C3 )
II. Sphingophospholipids: They contain alcohol
sphingosine (C18 )

CHAPTER 4
Cephalin
It is a phosphatidic acid with ethanolam ine as the

nitrogenous base. It is a component of mem branes
along with other phospholipids.
It plays a role in blood coagulation.
Cephalin (Phosphatidyl
ethanolamine)

CHAPTER 4
Lecithin

It is the phosphatidic acid with choline as the
base.
Lecithin (phosphatidyl choline)
• Lecithin is the predominant

glycerophospholipid in cell membranes.
CHAPTER 4
Dipalmitoyl Lecithin

Lung alveoli are coated with a surfactant which is
predom inantly made up of dipalmitoyl lecithin.

Prem ature babies without sufficient
surfactant lining the alveolar walls can have
respiratory difficulties. This results in alveolar
collapse causing respiratory distress syndrome.

CHAPTER 4
Phosphatidyl Inositol
Phosphatidyl inositol

CHAPTER 4
Cardiolipin
Cardiolipin [diphosphatidyl glycerol]

CHAPTER 4
Ether Lipids
Plasmalogens

Plasm alogens are predominantly phophatidal
ethanolamines:

The brain and m uscle tissue are rich in
plasm alogens.
Plasmalogen

CHAPTER 4
Sphingomyelin
The num bers in circles represent the

num ber of carbon atoms in alcohol- sphingosine.

CHAPTER 4
L/ S Ratio
●
Lecithin- Sphingom yelin ratio (L/ S ratio) in am niotic
fluid is an indicator frequently used to evaluate fetal
lung m aturity.
Prior to 34 weeks of gestation, the concentrations of

lecithin and sphingomyelin in am niotic fluid are
alm ost equal. Later, the concentration of lecithin
rises m arkedly and the L/ S ratio becom es 5 at term.
In preterm infants, the L/ S ratio is 1 or < 1 ,

resulting in respiratory distress.

CHAPTER 4
Glycolipids

Glycolipids are high in nervous tissues

Types of glycolipids:
1. Cerebrosides
2. Globosides
3. Gangliosides

CHAPTER 4
Structure Of Galactosyl Ceramide and
Sulphatide

CHAPTER 4
Cholesterol
Cholesterol is a sterol that is ex clusive to animals.
Structure of cholesterol

CHAPTER 4
Functions of Cholesterol
1. Cholesterol is an integral component of cell

membranes and hence influences m embrane
permeability.
2. A number of biologically important substances are

synthesised from cholesterol (e.g. vitamin D, bile
acids, mineralocorticoids, glucocorticoids and
sex hormones).
3. Cholesterol acts as an electrical insulator and

helps in the propagation of nerve impulses.

General structures of various lipids (non- CHAPTER 4
polar and amphipathic

CHAPTER 4
Examples of Lipid Bilayers
Amphipathic lipids such as

phospholipids and cholesterol are
organised into bilayers in biological
membranes. Hydrophilic groups
are oriented towards the watery
phase (both ECF and ICF), while
hydrophobic groups are oriented
towards the oily phase (central
portion of membranes).

CHAPTER 5
Section II
Molecules of Life
Universities Press
3-6-747/1/A & 3-6-754/1, Himayatnagar
Phone: 040-2766 5446/5447
,, marketing@universitiespress.com
Rafi M D: Textbook of Biochemistry (4 th Edition ) Universities
Press
CHAPTER 5
Chapter 5
Amino Acids and Proteins

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CHAPTER 5
Proteins

Proteins are the building material for body
structure.

Proteins are com posed of carbon, ox ygen,
hydrogen, nitrogen and sm all am ounts of other
elements, notably sulphur.

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CHAPTER 5

Proteins play the role of ‘ brick and mortar’ of the
body.

Proteins perform a number of roles including
biocatalysis, defence, hormonal function, transport,
storage and muscle contraction.

Proteins are polymers of amino acids

Standard amino acids

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CHAPTER 5
Amino Acids

Am ino acids are the structural units (monomers) of
proteins.

An amino acid is made up of two functional groups—
amino (–NH2 ), carbox yl (–COOH). They also contain a
hydrogen atom and a side chain (R) linked to the carbon
atom.

Am ino acids differ from each other in their side chains.

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CHAPTER 5
Classification of Amino Acids
I. Amino acids with aliphatic side chains
1. Glycine 2. Alanine
3. Valine 4. Leucine
5. Isoleucine

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CHAPTER 5
II. Amino acids containing hydroxyl (–OH)

groups
6. Serine
7. Threonine

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CHAPTER 5
III. Sulphur containing amino acids
8. Cysteine
9. Methionine

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CHAPTER 5
IV. Acidic amino acids and their amides
10. Aspartic acid
11. Asparagine
12. Glutamic acid
13. Gultamine

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CHAPTER 5
V. Basic amino acids
14. Lysine
15. Arginine
16. Histidine

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CHAPTER 5
VI. Aromatic amino acids
17. Phenylalanine
18. Tyrosine
19. Tryptophan

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CHAPTER 5
VII. Imino acid
20. Proline

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CHAPTER 5
Essential or Indispensable Amino Acids

The amino acids which cannot be synthesised by the
body and hence need to be supplied through diet are
called essential amino acids. Some are
conditionally essential.

The ten essential am ino acids in ascending order of
dietary requirement:
1. Arginine 6. Isoleucine
2. Tryptophan 7. Valine
3. Histidine 8. Phenylalanine
4. Methionine 9. Lysine
5. Threonine 10. Leucine

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CHAPTER 5
Classification Based on Metabolic Fate

Ketogenic amino acids
Leucine is the only ex clusive ketogenic am ino acid.

Ketogenic and glucogenic amino acids
Isoleucine, lysine, phenylalanine, tyrosine and
tryptophan.

Glucogenic amino acids
The rem aining 14 amino acids are used for the
synthesis of glucose or glycogen.

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Classification Of Amino Acids Based On CHAPTER 5
Their Solubility

Polar (hydrophilic) amino acids
glycine, serine, threonine, cysteine, asparagine,
glutam ine and tyrosine.

Non- polar (hydrophobic) amino acids
Phenyl alanine, tryptophan and proline.

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CHAPTER 5
Isoelectric pH
Various ionic forms of amino acid at different pH

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CHAPTER 5
Sorenson's titration curves of valine

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CHAPTER 5
Colour Reactions of Amino Acids

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CHAPTER 5
Non- standard Amino Acids

D- amino acids

Non- Protein am ino acids

Am ino acid derivatives

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CHAPTER 5
Precipitation Reactions of Proteins
1. Salting out
2. Isoelectric precipitation
3. Precipitation by heavy metal ions
4. Precipitation by alkaloidal reagents
5. Precipitation by alcohols
6. Heat coagulation

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CHAPTER 5
Classification of Proteins

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CHAPTER 5
Methods to Determine Protein Structure

Determ ination of amino acid com position in a
protein

Determ ination of the number of polypeptides in a
protein

Determ ination of amino acid sequence in a protein
Sanger's reagent
Edm an's reagent
●
Autom atic sequencing

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CHAPTER 5
Methods to Explore Higher Levels of
Protein Structure
1. Electron microscopy
2. X- ray crystallography
3. Nuclear m agnetic resonance (NMR) spectroscopy

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Organisation of protein structure at CHAPTER 5
various levels

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CHAPTER 5
Formation of Peptide Bond

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CHAPTER 5
Primary Structure of Protein
 (psi) and  (phi) angles in the peptide backbone

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CHAPTER 5
Forces Stabilising Protein Structure
Covalent bonds

Peptide and disulphide linkages
Non- covalent bonds

Hydrogen bonds

Hydrophobic interactions

Electrostatic interactions

Van der Waals interactions

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CHAPTER 5
Secondary Structure of Protein
The polypeptide chain folded into regular (- helix , -

sheet) and irregular forms (turns and loops)
characterises the secondary structure.

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CHAPTER 5
Features of - helix
It is the first and the most comm on pattern (hence

designated a) of secondary structure.
1. - helix is the m ost stable conformation formed

spontaneously with the lowest energy.
2. It is a coiled structure with the tightly coiled

polypeptide backbone forming the inner part of the
helix with the side chains ex tending outwards from
the central ax is.
(Continues…

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CHAPTER 5
…Continued)
3. - helix can be either right handed or left handed.

But all a- helices found in proteins are right handed
(clockwise) because of the less probability of steric
clashes between the side chains and the backbone.
4. Covalent and various non- covalent forces stabilise

the - helix .
5. Ex cept the first and last peptide bonds at both

ends of the chain all other NH and CO groups take
part in hydrogen bonding.
(Continues…

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6. Each turn of the helix is 5.4 Å and accom modates 3.6

amino acid residues per turn of helix .
7. Thus, the amino acids spaced three to four residues

apart in sequence are spatially closer to one another
and each amino acid form s a hydrogen bond with the
fourth amino acid in linear sequence. In contrast,
amino acids which occur closer in linear sequence are
situated on opposite sides of the helix and hence
cannot m ake contact.
8. Proline fits only in the first turn of an - helix ;

otherwise it is never found in an - helix .

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CHAPTER 5
- Helix
- helix of a polypeptide chain

shown in a coiled structure
(right handed).
Only a few hydrogen bonds
are shown for clarity (dotted
lines).
Note that the side chains of
amino acids are not shown for
sake of clarity.

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CHAPTER 5
Features of - Pleated Sheet
1. It is the second type of regular repetitive pattern

(thus the name ) occurring in the secondary
structure of a protein.
2. Unlike the coiled - helix , the peptide backbone of a

- sheet is partly ex tended with a 'pleated
appearance'.
3. The distance between adjacent amino acids along a

- strand is 3.5 Å, in contrast to a distance of 1.5 Å
along an - helix . The side chains of adjacent am ino
acids orient in opposite directions to avoid steric
clashes.
(Continues…

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CHAPTER 5
…Continued)
4. - Pleated sheet is stabilised by ex tensive

hydrogen bonding. Hydrogen bonds can be
form ed between neighbouring polypeptide chains
or within a single polypeptide chain folded into
segm ents.
5. About two to fifteen strands are involved in the

form ation of a - sheet. Adjacent chains in a -
sheet can run in opposite directions (antiparallel 
sheet), in same direction (parallel  sheet) or both
ways (mix ed  sheets).

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CHAPTER 5
- pleated sheets shown as a folded sheet

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CHAPTER 5
- Pleated Sheets
Anti parallel
Parallel
Hydrogen bonds are form ed between adjacent st rands wit hin a single polypept ide. Note t hat in bet a
sheet s running ant iparallel, hydrogen bonds bet ween NH and CO groups connect each am ino acid t o
a single am ino acid on an adjacent st rand. On t he contrary, in a parallel bet a sheet, hydrogen bonds
connect each am ino acid on one st rand wit h t wo different am ino acids on t he adjacent strand.

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CHAPTER 5
Tertiary Structure of Protein
Diagrammatic representat ion of super secondary motifs (- helix represented as

folded coil, - sheets as wide arrows and loops shown as connecting them).

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CHAPTER 5
Quaternary Structure of Protein

Proteins consisting of more than one polypeptide
chain display quaternary structure.

The protein is known as a multimeric protein or
oligomeric protein. For ex ample, hem oglobin, the
protein that transports ox ygen in the blood is an
oligomeric protein with four polypeptide chains (2 
and 2).

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CHAPTER 5
Denaturation
(a) Native protein (only (b) Denatured protein (note

disulphide bridges are shown that the disulphide linkages
which are intact) are broken)

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CHAPTER 5
How Do Proteins Attain Their Native
Conformation?
Levinthal’s paradox

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CHAPTER 5
Principles of Protein Folding
1. The essence of protein folding is the retention of

partly correct interm ediates.
2. Molten globule
3. Enzym es aiding protein folding:

Disulphide isomerase,
Cis- trans isomerase
4. Chaperone proteins

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CHAPTER 5
Diseases Related to Protein Folding

Prion diseases
Creutzfeldt- Jakob disease
Kuru
Mad cow disease

Alzheimer's disease

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CHAPTER 6
Section II
Molecules of Life
Universities Press
3-6-747/1/A & 3-6-754/1, Himayatnagar
Phone: 040-2766 5446/5447
Rafi M D: Textbook of Biochemistry (4th Edition) Universities
Press
CHAPTER 6
Chapter 6
Nucleotides and Nucleic

Acids

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CHAPTER 6
Nucleotides
Nucleotide, the basic unit of nucleic acids is

com posed of three com ponents.
• Sugars in nucleic acids

• Phosphate group in nucleic acids
• Bases present in nucleic acids
Purines
Pyrim idines

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CHAPTER 6
Structures of major purines (A, G) and
pyrimidines (C, T, U) found in nucleic acids
X = H or ribose or ribose phosphat e

depending upon whet her it is just a
base or a nucleoside or a nucleotide,
respectively.

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CHAPTER 6
Nomenclature of Nucleotides

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CHAPTER 6
Functions of Nucleotides
1. Nucleotides are the building blocks of nucleic

acids (DNA and RNA).
2. ATP is the universal energy currency of living
systems.
3. Cyclic nucleotides such as cAMP and cGMP act

as 'second messengers' .
4. Nucleotides are the structural components of a
num ber of coenzymes.
(cont inued…

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…continues) CHAPTER 6
5. They act as the carriers of certain metabolic

intermediates of carbohydrates, (UDP- glucose),
lipids (CDP- acyl glycerol) and proteins (S- adenosyl
m ethionine- SAM).
6. UDP- Gal UDP- Glu, GDP- Man, CMP- NeuAc, GDPFuc,

UDP- Xyl, UDP- Gal Nac and UDP- Glu Nac - These
eight m olecules are known as 'nucleotide- linked
sugars' and are im portant constituents of
glycoproteins.

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Structures of biologically important
CHAPTER 6
nucleotides

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CHAPTER 6
Synthetic Nucleotides
Therapeutic applications
1. Synthetic nucleosides, cytarabine and vidarabine in
which ribose is replaced by arabinose are used in
chemotherapy to treat cancers.
2. Allopurinol is used in the treatm ent of

hyperuricem ia and gout.
(cont inues…

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CHAPTER 6
…continued)
3. Synthetic analogues such as 6- m ercaptopurine, 5-

fluorouracil, 5- iodouracil, 3- deox yuridine, 5 or 6-
azauridine, 5- or 6- azacytidine, 8- azaguanine, 6-
thioguanine are widely used by oncologists. They are
incorporated into DNA just before cell division, thus
blocking cell proliferation.
3. Drugs such as zidovudine which are used in the

treatment of AIDS are synthetic nucleotide analogues
with alterations in the sugar structure.

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CHAPTER 6
Structure of DNA
The double helical structure of DNA was first proposed by
Jam es Watson and Francis Crick in 1953.

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CHAPTER 6
Watson–Crick Model of DNA
Double Helix

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CHAPTER 6
Watson–Crick Model of
DNA Structure
1. A DNA molecule consists of two chains of
deox yribonucleotides coiled around each other in
the form of a double helix. Two helical DNA chains
wind around each other on the same ax is to form a
right- handed double helix .
2. The two chains are antiparallel – one strand runs

in the 5' to 3' direction, while the opposite is in
the 3' to 5' direction. The term inal residue whose
C5' is not linked to another nucleotide is called the
5' end, and the terminal residue whose C3' is not
linked to another nucleotide is called the 3' end.
(continues…

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CHAPTER 6
…continued )
3. The two strands are held together by hydrogen

bonds between a purine base on one strand and a
pyrimidine base on the opposite strand. The ring
structure of each base occurs in a flat plane
perpendicular to the sugar–phosphate backbone,
resembling the steps on a spiral staircase. The base
pairing maintains a constant distance between the
sugar–phosphate backbones of the two strands as
they twist around each other.
…continues)

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CHAPTER 6
…continued )
4. The hydrogen bonds are form ed between a purine

base and a pyrimidine base only. This specificity is
imposed on the base pairings by the fix ed location
of the hydrogen bonds. If two purines face each
other, they would not fit into the space available
and two pyrimidines would be too far apart to form
hydrogen bonds. Each nucleotide base of one
strand is paired in the sam e plane with a base of
the opposite strand.
(continues…

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CHAPTER 6
…continued )
5. Three hydrogen bonds are form ed between the

purine, guanine and the pyrimidine, cytosine (G–C
or C–G pairing), while only two hydrogen bonds can
be form ed between the purine, adenine and the
pyrimidine, thymine (A–T or T–A pairing). As a
result, G always pairs with C, and A with T. This is
known as complementary base pairing. This
specificity has tremendous significance in DNA self
replication and transcription.
…cont inues)

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CHAPTER 6
…continued )
6. It is more difficult to separate the paired DNA

strands rich in G–C pairing because
GC (three double bonds) pairing is stronger than
A= T (two double bonds) pairing.
7. The deox yribose and phosphate groups (sugar –

phosphate backbone) are hydrophilic in nature and
hence are on the outside of the double helix
orienting towards the surrounding water m olecules,
while the hydrophobic bases are stacked inside.
…continues)

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CHAPTER 6
…continued )
8. The coiling of two strands creates a major groove

and a minor groove on the surface of the duplex .
Proteins interact with DNA at these grooves without
disrupting the double helix .
9 . Geometry of the DNA duplex: The width of a

double helix is 20 Å. Each turn of the helix is 34 Å
with 10 pairs of nucleotides, each pair is placed at a
distance of 3.4 Å.
…continues)

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CHAPTER 6
…continued )
10. Because of their length, DNA chains are described

in terms of base pairs (bp) or thousands of base
pairs (kilobase pairs or kb), e.g. [5 kb (virus),
2,50,000 kb (hum ans)].
11. The two antiparallel strands of DNA are not

identical in either base sequence or com position,
but are complementary to each other. Wherever
adenine occurs in one chain, thym ine is present in
the other and vice versa; similarly, wherever
guanine occurs in one strand, cytosine is found in
the other and vice versa.
…Continues)

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CHAPTER 6
…continued )
12. The DNA duplex is stabilised by two forces; hydrogen

bonding between complementary base pairs and
nonspecific base- stacking interactions.
13. The strand that is transcribed or copied into the RNA

molecule is referred to as the template strand or anti
sense strand. The other strand is the sense or coding
strand of that gene. However it is noteworthy that each
DNA strand can act as a template for the synthesis of its
complem entary strand and hence hereditary information
is encoded in the sequence of bases on either strand .

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CHAPTER 6
Chargaff's Rules
1. The base composition of DNA varies in different species.
2. DNA from different tissues of the same species have

the same base composition.
3. The base com position of DNA in a species does not

undergo any change with the organism's age,
nutritional status or changing environm ent.
4. In all species, the number of adenine is equal to the

number of thymine residues (A= T) and the num ber of
guanine is equal to the number of cytosine residues (G 
C). Thus, the sum of the purine residues equals the sum
of the pyrimidine residues; that is,
A+ G = T + C.

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CHAPTER 6
Different Types (Forms) of DNA
• B- DNA
• A- DNA
• Z- DNA
Unusual structures in DNA
• Hoogsteen pairing
• Triplex DNAs
• Tetraplex
• Palindromes
Denaturation of DNA

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CHAPTER 6
Organisation of DNA
• Supercoiling of DNA
• Topoisomerases
• Nucleoproteins
Histones
Chrom atin
Covalent modifications
Nucleosomes

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CHAPTER 6
Mitochondrial DNA
• Apart from the nucleus, eukaryotic cells also have

other organelles such as m itochondria and
chloroplasts, which contain DNA. Mitochondrial
DNA is capable of encoding certain proteins and
RNA in mitochondria. The synthesis of about 13
proteins of the respiratory chain, are encoded by
m tDNA.
• mtDNA is inherited only from the mother!
• Leber's hereditary optic neuropathy

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CHAPTER 6
Genes
• Can a single gene be ex pressed into m ultiple

gene products?
• Coding and noncoding sequences in genes
Introns
Ex ons
• Transposons

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CHAPTER 6
Centromeres and Telomeres
• The nucleoproteins which link the chromosome to

the mitotic spindle during cell division are anchored
to a specific region on the DNA known as the
centromere.
• This ensures an equal distribution of chromosome

sets to daughter cells.
• The guanine- rich sequences at the ends of

eukaryotic chrom osom es are known as telomeres.
(continues…

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…continued) CHAPTER 6
• Telomeric DNA is synthesised and m aintained by an

enzym e known as telomerase.
The somatic cells of multicellular organism s lack
telomerase activity; however their germ cells have
active telom erase function.
The loss of telomerase activity allows the gradual
shortening of chromosomes with each cycle of DNA
replication and cell division until they reach
senescence (a stage at which there will be no more
division). Perhaps this is the basis of ageing.

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CHAPTER 6
Nucleases
Nucleases are enzym es that are capable of

degrading nucleic acids.
• Endonucleases
• Ex onucleases
• Restriction endonucleases

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CHAPTER 6
Types of RNA
1. Messenger RNA (m- RNA)

Messenger RNA is located in the cytoplasm and
transfers genetic inform ation from the DNA to the
protein- synthesising machinery on ribosomes.
2. Transfer RNA (t- RNA)

It also occurs in cytoplasm , it translates the
information carried by m - RNA into a specific
sequence of amino acids that are incorporated
into protein.
(Cont inues…

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CHAPTER 6
…Continued
3. Ribosomal RNA (r- RNA)

It is a com ponent of ribosomes which are the sites
for protein biosynthesis.
4. Heterogenous nuclear RNA (hnRNA)

It is the prim ary form of RNA in nucleus processed
into m ature m- RNA.

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CHAPTER 6
Messenger RNA (m- RNA)

Messenger RNA is synthesised as the prim ary
transcript from the template strand of DNA and
later processed to m - RNA.

5 capping

Poly ‘A’ tail

Heterogeneous nuclear RNA (hnRNA)

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CHAPTER 6
Transfer RNA (t- RNA)
Transfer RNA is the adapter m olecule that translates

the information carried by m- RNA into specific
sequences of amino acids.
tRNA contains mainly four arms.

• Acceptor arm
• Anticodon arm
• D arm
• T  C arm

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CHAPTER 6
Structure of t- RNA

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CHAPTER 7
Section II
Molecules of Life
Universities Press
3-6-747/1/A & 3-6-754/1, Himayatnagar
Phone: 040-2766 5446/5447
Rafi M D: Textbook of Biochemistry (4 th Edition) Universities
Press
CHAPTER 7
Chapter 7
Enzymes

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CHAPTER 7
Enzymes

Enzymes are highly specialised proteins that act as
catalysts in biological systems (biocatalysts).

Enzym es are highly specific for their substrates

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CHAPTER 7
Nomenclature and Classification
of Enzymes

International Union of Biochemistry (IUB) devised a new
system of nom enclature in 1964.

A four- digit Enzyme Com mission (E.C.) number is
assigned to each enzym e. The first digit represents the
class, the second digit indicates the subclass, the third
digit represents the sub- subclass and the fourth digit
specifies the individual enzym e.

Enzymes are classified into six major classes as per
the IUB system of enzyme classification.

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CHAPTER 7
Classification of Enzymes
(Continues…

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…Continued ) CHAPTER 7
Classification of Enzymes

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CHAPTER 7
Enzyme Structure
• Enzym es are proteins.
• Apoenzym e
• Coenzym e
• Holoenzym e

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CHAPTER 7
Active Site
The small cleft- like portion of an enzym e where the

substrate(s) binds and catalysis occurs is known as the
active site or active centre.
• Substrate binding site

• Catalytic site
A diagrammatic representation of
an enzyme and its active site

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CHAPTER 7
Coenzymes
Coenzymes of B- complex vitamins

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CHAPTER 7
Coenzymes which are not related to vitamins

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CHAPTER 7
Enzyme Specificity
The active site in a specified conform ation on an

intact enzyme m olecule is largely responsible for
the enzym e specificity.
There are three types of specificity:

1. Stereo specificity
2. Substrate specificity
3. Reaction specificity

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CHAPTER 7
How Do Enzymes Work?
• Transition state
• Ground state
• Activation energy
• Enzymes act by reducing the activation energies
• Binding energy

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CHAPTER 7
Effect of enzym e on activation
energy in a reaction
S = substrate
P = product

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CHAPTER 7
To summ arise, the energy required to surm ount the

activation barrier is the activation energy. Enzymes
catalyse the reactions by lowering the activation
barrier. The binding energy resulting from weak
non- covalent interactions between the substrate
and the enzym e reduces the activation energy.
Enzym es do not alter the reaction equilibria, they

only enhance the reaction rates by lowering
activation energies.

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CHAPTER 7
Mechanism of Enzyme Action
1. Lock and key model
2. Induced fit theory
3. Substrate strain theory

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CHAPTER 7
Different models explaining
enzyme–substrate (ES) complex
formation
(A) Lock and key model

(B) Induced fit theory
(C) (C) Substrate strain theory

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CHAPTER 7
Mechanism of Catalysis
• Acid- base catalysis
• Covalent catalysis
• Metal ion catalysis

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CHAPTER 7
Factors Affecting Enzyme Activity
The various factors that influence enzym e activity

are
1. Concentration of enzym e
2. Concentration of substrate
3. Concentration of product
4. Temperature
5. pH

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CHAPTER 7
Effect of Concentration of Substrate
on Enzyme Velocity

The rate of an enzym e catalysed reaction increases
as the substrate concentration increases until it
reaches a m ax imal rate, which remains constant
despite further increases in substrate concentration.

The substrate concentration (ex pressed in m oles/ L)
required to produce half- m ax imum velocity (1/ 2
Vm ax ) is known as Km or Michaelis–Menten constant.

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CHAPTER 7
Effect of substrate concentration on enzyme velocity

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CHAPTER 7
Derivation of Km or Michaelis–Menten Constant
(Continues…

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CHAPTER 7
…Continued
(Continues…

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CHAPTER 7
…Continued

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CHAPTER 7
Effect of Temperature on Enzyme Velocity

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CHAPTER 7
Effect of pH on Enzyme Velocity

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CHAPTER 7
Enzyme Inhibition
Enzym e inhibition can be broadly classified into three

categories:
1. Competitive inhibition
2. Non- competitive inhibition
3. Allosteric inhibition

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CHAPTER 7
Competitive Inhibition

The inhibitor binds with the enzyme through non-
covalent interactions and hence the pro cess is
readily reversible.
 In competitive inhibition, the Km value increases

whereas Vmax is unchanged.

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CHAPTER 7
Velocity versus substrate plot in
com petitive inhibition

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Drugs acting as competitive CHAPTER 7
inhibitors

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CHAPTER 7
Non- competitive Inhibition

The inhibitor binds to the enzyme covalently and
inactivates it making the process essentially
irreversible.
 In non- competitive inhibition, the Km value is

unchanged while V max is lowered.

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CHAPTER 7
Velocity versus substrate plot in non-
com petitive inhibition

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CHAPTER 7
Regulation of Enzyme Activity
1. Allosteric regulation
2. Covalent modulation
3. Proteolytic trimm ing
4. Compartmentation
5. Control of enzyme synthesis and degradation

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CHAPTER 7
Allosteric Regulation

Allosteric enzymes

Allosteric m odulators

Cooperativity of allosteric enzym es

Classification of allosteric enzymes

K class allosteric enzymes

V class allosteric enzymes

Feedback regulation

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CHAPTER 7
Effect of substrate
concentration on allosteric
enzymes

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CHAPTER 7
Examples of allosteric enzymes

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CHAPTER 7
Covalent Modulation

Regulatory enzym es or rate- limiting enzymes

Reversible protein phosphorylation

Protein kinases

Protein phosphatases

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CHAPTER 7
Regulatory enzymes (rate- limiting enzymes) controlled by
covalent modulation

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CHAPTER 7
Isoenzymes
The multiple molecular form s of an enzyme catalysing

the same reaction are called isoenzymes.
They differ in their physical and chemical properties

such as structure, Km , Vmax , pH, electrophoretic mobility
and their susceptibility to inhibitors.

Isoenzyme variants are coded by different genes.

Allelozym es

Hybrid isoenzym es

Isoform s

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CHAPTER 7
Creatine Kinase (CK)


Normal serum levels of CK are 15–100 U/ L in males
and 10–80 U/ L in fem ales.

Lohmann’s reaction

In m yocardial infarction, CK levels (CK–MB
isoenzyme) start to rise within 3–6 hours of
infarction.
Isoenzymes of CK

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CHAPTER 7
Lactate Dehydrogenase (LDH)
Norm al levels of LDH in serum are 100–200 U/ L.
Isoenzymes of LDH

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CHAPTER 7
Alkaline Phosphatase (ALP)
• The norm al serum Isoenzymes of ALP

levels of ALP range
between 40 and 125
U/ L.
• Increased ALP levels

are m ost com m only
associated with
bone disease and
hepatobiliary
disease.

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CHAPTER 7
Clinical Enzymology
• Plasm a specific enzymes

• Plasm a nonspecific enzymes
• Units of enzym e activity

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CHAPTER 7
Diagnostic Markers of Myocardial
Infarction (MI)

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CHAPTER 7
Enzymes Used As Therapeutic Agents

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CHAPTER 7
Enzymes used in Laboratory
Measurements and Gene Transfer

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CHAPTER 8
Section II
Molecules of Life
Universities Press
3-6-747/1/A & 3-6-754/1, Himayatnagar
Phone: 040-2766 5446/5447
Rafi M D: Textbook of Biochemistry (4thEdition) Universities
Press
CHAPTER 8
Chapter 8
Vitamins

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CHAPTER 8
Classification of Vitamins

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CHAPTER 8
Differences between fat- soluble and water- soluble

vitamins

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CHAPTER 8
Vitamin A
• Chemistry
Retinol
Retinal
Retinoic acid
- Carotene

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CHAPTER 8
Summary of kinetics and mechanism of action of vitamin A
(Continues …

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CHAPTER 8
…Cont inued)

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CHAPTER 8
Wald’s Visual Cycle

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CHAPTER 8
Other Biochemical Functions of
Vitamin A
1. Retinol and retinoic acid act like steroid hormones.

They regulate the synthesis of proteins involved in
cell growth and differentiation. Vitam in A is
required for normal reproduction.
2. Vitam in A is required for maintenance of healthy

epithelial tissue. It maintains moist and pliable
epithelium by inhibiting the synthesis of ex cess
keratin (responsible for horny surfaces).
(Continues…

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CHAPTER 8
…Continued)
3. Retinoyl phosphate synthesised from retinoic acid

participates in glycoprotein synthesis.
Glycoproteins are important constituents of
m ucus. In vitamin A deficiency, lack of mucus
secretion results in drying of epithelial tissues.
4. Vitam in A is essential for the formation of

mucopolysaccharides in the ex tracellular matrix .
(Continues…

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CHAPTER 8
…Continued )
5. Vitam in A is essential for the maintenance of the

immune system. Its deficiency causes
keratinisation of the m ucosal lining of the
respiratory, gastrointestinal and genitourinary
tracts m aking them susceptible to frequent
infections.
6. Retinol and retinoic acid are essential for the

synthesis of transferrin, the iron transportaing
protein. Irondeficiency anemia m ay occur in
vitamin A deficiency.

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CHAPTER 8
Daily Requirements and Dietary Sources
1 RE = 1 g retinol
= 6 g - carotene
1 IU = 0.3 g retinol
The RDA for vitamin A are as follows

Men 1000 RE (3500 IU)
Wom en 800 RE (2800 IU)

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CHAPTER 8
Dietary Sources
• Animal foods: Liver, whole milk, eggs, butter,

cheese, fish and m eat. Fish (cod or shark) liver oils
are the richest sources of vitam in A. However, they
are generally used as nutritional supplements rather
than as food sources.
(Continues…

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CHAPTER 8
…Continued)
• Plant foods: Among the best sources are green

leafy vegetables such as spinach and amaranthus,
which are available in all seasons.
The darker the green leaves, the higher is its carotene

content.
Vitam in A also occurs in vegetables and fruits such as

papaya, pumpkin and m ango. Carrots is also a good
source of vitamin A.

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CHAPTER 8
Deficiency Manifestations
The clinical signs of vitamin A deficiency mostly

involve the eye. Vitam in A deficiency is one of the
m ajor causes of preventable blindness.
Night blindness
Night blindness (Nyctalopia) or the inability to see in
dim light is the earliest sym ptom of vitam in A
deficiency. It occurs due to im pairment of dark
adaptation.
(Continues…

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Xerophthalmia
Night blindness, if left untreated, causes dryness of the

conjunctiva and the cornea.
The conjunctiva becom es thick and wrinkled; the

cornea loses its mucosal cells and becom e dull, hazy
due to keratinisation and loses its transparency. These
changes are described as x erophthalmia.

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…Continued CHAPTER 8
Bitot's spots
Bitot's spots are triangular, white patches on the
conjunctiva on either side of the cornea. They are usually
bilateral.
Keratomalacia
If the condition progresses beyond x erophthalmia, there
is ulceration and necrosis of the cornea (keratomalacia)
which results in total blindness. Keratom alacia is one of
the major causes of blindness in developing countries
and is usually associated with protein energy
malnutrition (PEM).
(Continues…

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Manifestation of vitamin A deficiency in the eye

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CHAPTER 8
Vitamin D
Formation and Activation of Vitamin D

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CHAPTER 8
Metabolism and Biochemical
Functions of Vitamin D
(Cont inues…

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CHAPTER 8
…Continued)

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CHAPTER 8
Daily Requirements and Sources

The recom mended dietary allowance (RDA) is about
400 international units (IU) or 10 g. However, in
places with good sunlight (like the Indian
subcontinent), the daily requirem ent is about 200 IU
or 5 g. In growing children, during pregnancy and
lactation, it should be 10 g (400 IU).

Vitamin D can be derived both from sunlight and
food. The human body m anufactures vitam in D with
the help of sunlight. It also occurs m ainly in foods of
anim al origin.
(Continues…

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CHAPTER 8
…Continued)
Oily fish, egg yolk, liver, butter and cheese contain

good quantities. Fish liver oils, although not
considered a food, are the richest source of vitam in
D. Plant sterol, ergosterol provides a dietary source.

Breastmilk contains relatively sm all am ounts of
vitam in D and infants are at risk of developing
vitam in D deficiency, particularly prem ature infants
(since the vitamin is transported across the placenta
mainly in the last trim ester of pregnancy).

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CHAPTER 8
Skeletal deform ities seen in
rickets

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CHAPTER 8
Rickets
• improper mineralisation
• bow- legs, knock- knees, pigeon chest,
kyphoscoliosis.
• ('rickety rosary')
• (Harrison's sulcus)
Osteomalacia
• Renal rickets
• Vitami- D resistant rickets

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CHAPTER 8
Functions of Vitamin E
• Vitamin E plays a vital role in safeguarding the

structural integrity of biological m embranes.
Its antioxidant property prevents the ox idation of

polyunsaturated fatty acids (PUFA) of mem brane
phospholipids.
By inhibiting lipid perox idation in cell m embranes,

it protects the structural integrity of the cell. By
the same mechanism, vitamin E is responsible for
the resistance of RBC to hem olysis (caused by
ox idising agents such as hydrogen perox ide).
(Cont inues…

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It is observed that vitamin E is essential for normal

reproduction and fertility in ex perimental animals.
Hence, it is known as the ‘anti- sterility vitam in’.
It helps in m aintaining intact germinal epithelium

(gonads).
Deficiency of vitamin E adversely affects

spermatogenesis and m otility of sperms. However,
the effects of vitamin E on reproduction in humans
is controversial and evidence of any direct effect is
not fully established.
(Cont inues…

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CHAPTER 8
…Continued)
• It protects low- density lipoproteins (LDL)

from oxidation. (Note that ox idised LDL is
implicated in heart disease.) By doing so,
vitamin E reduces the risk of m yocardial
infarction.

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CHAPTER 8
RDA and dietary sources of vitamin E
Men – 10 mg
Women – 8 mg
Vegetable oils are rich in vitamin E especially wheat

germ oil. Sunflower, safflower, cotton seed oils; corn
and soyabean oils are good sources.

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CHAPTER 8
Vitamin K – Biochemical Functions
Vitamin K is required for blood coagulation.

Clotting factors II (prothrombin), VII, IX and X need
vitamin K for their activation.
Vitamin K acts as a cofactor in the form ation of - carbox yglutamate.

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CHAPTER 8
Biochemical Functions
Vitamin K cycle in hepatocytes

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CHAPTER 8
Daily Requirement and Dietary Sources

Since vitam in K is synthesised by the intestinal
bacteria, its requirem ent in the diet is very low. A
recommended daily allowance (RDA) of 70 –140
mg/ day would suffice for adult hum an needs.

Green leafy vegetables are an ex cellent source of
vitam in K. It is also present in eggs and diary
products.

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CHAPTER 8
●
Vitamin K deficiency leads to prolonged
coagulation and bleeding. Newborns, especially
preterm infants are m ore susceptible to vitamin K
deficiency
(Cont inues…

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Deficiency manifestations
●
Vitam in K deficiency is seen in adults suffering from
obstructive jaundice and other diseases causing
severe fat malabsorption. Patients suffering from
small bowel diseases such as celiac disease, Crohn's
disease suffer from vitam in K deficiency.
Patients on long- term antibiotic therapy (antibiotic

drugs kill vitamin K synthesising bacteria in the
intestine) are also at risk of developing vitam in K
deficiency.

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CHAPTER 8
Vitamin C
Different forms of vitamin C and its derivatives

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CHAPTER 8
Biochemical Role

Collagen formation
Vitamin C plays a role in the maintenance of
normal connective tissue and is essential for
wound healing.
Deficiency of vitamin C results in local hemorrhages

and bones become susceptible to fractures.
Collagen is also a constituent of the ground
substance surrounding capillary walls, hence
vitam in C deficiency causes capillary fragility.
(Cont inues…

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Biochemical Role

Antioxidant: Vitam in C plays a role in elim inating
potentially dangerous free- radicals from the
biological system.

Role in metabolism
Vitamin C plays a role in the metabolism of amino
acids
Tyrosine; Tryptophan
(Cont inues…

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CHAPTER 8
…Continued)
Role in lipid metabolism

Role in iron metabolism
Role in hemoglobin m etabolism

It spares vitamins A, E and some B- com plex
vitam ins from ox idation.

Phagocytosis

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CHAPTER 8

Recomm ended daily allowance is about 60 –75
mg/ day. About 100 m g/ day is required during
pregnancy and lactation. Sm okers, chronic
alcoholics and wom en on oral contraceptives
require up to 125 mg/ day.

The m ain dietary sources of vitamin C are fresh
fruits and green leafy vegetables. Citrus fruits,
Indian gooseberry (amla), guava, cabbage, spinach
are very high in vitamin C content. Germ inating
pulses contain large am ounts of vitam in C.

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CHAPTER 8
Vitamin C deficiency causes scurvy. Symptom s of

scurvy reflect impaired collagen synthesis resulting in
defective connective tissue.

Capillary fragility: Petechiae (sm all, pinpoint
hemorrhages under the skin), ecchymoses or
even hematomas. Internal bleeding into the
gastrointestinal tract, peritoneal cavity,
pericardium and the adrenal glands m ay occur.
(Cont inues…

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• Swollen and painful gums
• Teeth get loosened
• Poor wound healing and anemia
• Osteoporosis
• Barlow's disease

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CHAPTER 8
Thiamine (Vitamin B1 )
Structure of thiamine and thiamine pyrophosphate (TPP)

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CHAPTER 8

Thiam ine ex erts its biochemical functions
through its coenzym e form , thiam ine
pyrophosphate (TPP).

Pyruvate dehydrogenase requires TPP as one of
the coenzym es.

- ketoglutarate dehydrogenase is also dependent
on TPP a a coenzyme.

Ox idative decarbox ylation reaction involving
branched chain am ino acids requires TPP.
(Cont inues…

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
Enzyme transketolase of hex ose m onophosphate
shunt (HMP shunt) utilises TPP as the coenzym e.

TPP plays a role in the transmission of nerve
im pulses.

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CHAPTER 8

The recom mended dietary allowance (RDA) for
adults is 1–1.5 m g/ day.

Whole grain cereals, yeast, legum es, oilseeds and
nuts, especially groundnut, are im portant sources
of thiam ine. Animal foods such as pork, beef and
sheep liver are also good sources.

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CHAPTER 8
• Wet beri- beri

It is characterised by cardiovascular signs and
symptom s such as edema of legs, face, trunk
and serous cavities.
• Dry beri- beri
In this type, the nervous system is affected. It
presents with progressive m uscle wasting,
peripheral neuropathy of the m otor and sensory
systems with diminished reflex es.
• Mixed beri- beri
• Infantile beri- beri
• Wernicke–Korsakoff syndrome

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CHAPTER 8
Riboflavin (Vitamin B2 )
Coenzymes of Riboflavin
Riboflavin ex ists in two active coenzyme forms
1. Flavin mononucleotide (FMN)
2. Flavin adenine dinucleotide (FAD)
Flavin coenzym es mostly occur as prosthetic groups of

ox idoreductase enzymes. These enzym es are known as
flavoproteins.

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CHAPTER 8
Structure of riboflavin and synthesis
of FMN and FAD

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CHAPTER 8
Riboflavin through its coenzym e form s participates in ox idation-

reduction reactions in various m etabolic pathways and in the
respiratory chain
Common reactions involving FMN and FAD as coenzymes in various
metabolisms

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CHAPTER 8
Dietary Sources And Daily
Requirement

Milk and other dairy products, lean meat, fish, eggs
and legum es are good sources.

The recom mended dietary allowance (RDA) for an
adult is about 1.5 mg, with slightly higher amounts
(by 0.2 to 0.5 m g/ day) recomm ended for pregnant
and lactating women.

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CHAPTER 8

Its deficiency usually occurs in association with
deficiencies of other Bcomplex vitamins. It usually
affects the tongue and lips; characterised by
glossitis (magenta tongue), angular stomatitis,
cheilosis (fissure- like lesions at the corners of the
m outh), pharyngitis and genital derm atitis.

Laboratory diagnosis done by measurement of
FAD- dependent glutathione reductase activity in
RBC.

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CHAPTER 8
Niacin
Coenzymes of niacin
1. Nicotinamide adenine dinucleotide (NAD + )
2. Nicotinam ide adenine dinucleotide phosphate

(NADP+ )

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CHAPTER 8

Several enzym es require either NAD + or NADP+ in
ox idation–reduction reactions.

There is a fundamental distinction between NADH
and NADPH
- NADH generated in various catabolic pathways is
ox idised in the respiratory chain to generate ATP.
NADH thus plays a vital role in cellular respiration.
- NADPH serves as a readily available reducing power

in biological system s. It is used as a reductant in
biosynthetic processes.

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CHAPTER 8
Biosynthesis of nicotinamide nucleotides,
NAD + and NADP+
(Continues…

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Biosynthesis of nicotinam ide nucleotides, NAD + and NADP+

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CHAPTER 8
Selected list of enzymes
dependent on NAD + / NADH
(Continues…

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Selected list of enzym es dependent on NAD + / NADH

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CHAPTER 8
Mechanism of ox idation and
reduction of NAD +

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CHAPTER 8
Generators and Consumers of

NADPH
Pentose phosphate pat hway of carbohydrates (HMP shunt) is the major
cont ributor of NADPH in cells

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CHAPTER 8
Selected list of enzym es dependent on NAD +
(Continues…

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CHAPTER 8
Pellagra
Pellagra is a classic disease caused by the deficiency
of niacin.
The disease is characterised by three D's –

dermatitis, diarrhea and dementia.

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CHAPTER 8
Pyridoxine (Vitamin B6 )
Vitam in B6 and its coenzyme [PLP]
(Continues…

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CHAPTER 8
…Cont inued) Vitamin B6 and its coenzyme [PLP]

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CHAPTER 8
Biochemical Functions of B6
• Transamination
Transamination reaction
(Continues…

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CHAPTER 8
…Continued)
• Decarboxylation
Histam ine
Serotonin (hydrox y tryptamine)
(Continued…

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CHAPTER 8
…Continued
Catecholam ines
GABA (gamm a amino butyric acid)
(Continues…

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CHAPTER 8
…Continued)
• Production of niacin coenzymes
• Heme synthesis
• Metabolism of sulphur- containing amino acids
• Phosphorylase
• Deamination

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CHAPTER 8
Dietary Sources and Daily
Requirement

Plants contain B6 as pyridox ine, whereas anim al
tissues contain PLP and pyridox amine phosphate.
Rich sources of B6 include wheatbran, rice bran,
dried yeast, legum es, nuts, meat, fish, milk, eggs
and leafy vegetables.

Requirem ents are related to protein intake as it is
chiefly concerned with am ino acid metabolism . The
RDA for adults is 2 mg/ day. As usual, during
pregnancy and lactation it is slightly higher.

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CHAPTER 8

Neurological symptom s include irritability,
generalised weakness, peripheral neuropathy,
personality changes including confusion and
depression.

Carpal tunnel syndrome
Hematological m anifestations include microcytic,
hypochrom ic anem ia due to diminished hemoglobin
synthesis. Platelet dysfunction m ay also occur.
Dermatological manifestations include seborrheic
derm atitis, stom atitis, glossitis and cheilosis.

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CHAPTER 8
Drug interactions
• Isonicotinic acid hydrazide (INH- isoniazid) inhibits

the enzym e pyridox al kinase, thus affecting the
synthesis of PLP adversely.
• Wom en taking oral contraceptives are prone to

develop B6 deficiency as OCP are known to inhibit
enzymes dependent on PLP.
• Alcoholism m ay result in B6 deficiency, as

acetaldehyde which is form ed from ethanol can
compete with PLP for protein binding.

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CHAPTER 8
Biotin
Biotin is more popularly known as anti- egg white
injury factor.
Structure of
biotin

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CHAPTER 8

Role in gluconeogenesis and Krebs cycle
Conversion of pyruvate to ox aloacetate is catalysed
by biotin- dependent pyruvate carbox ylase.

Role in fatty acid synthesis

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CHAPTER 8

Role in the metabolism of odd- numbered fatty acids
and branched- chain am ino acids

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CHAPTER 8
Reaction mechanism of carboxylation by
biotin coenzyme
The conversion of pyruvate to ox aloacetate by pyruvate carbox ylase is

shown.

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CHAPTER 8

RDA of 200–300 mg/ day.

Liver, soyabean, yeast, egg yolk, peanut, grains are
all rich sources of biotin.
Deficiency manifestations

Biotin deficiency is uncomm on since it is synthesised
by the intestinal microflora and its ubiquitous
occurence in nature.

However, prolonged use of antibiotics that kill the
gut bacteria and ex cess consumption of raw eggs
result in biotin deficiency.

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CHAPTER 8
Pantothenic Acid

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CHAPTER 8
Synthesis of Coenzyme A from Pantothenic Acid
(Continues…

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Synthesis of coenzyme a from pantothenic acid

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CHAPTER 8
Some Examples of Compounds Bound to
Coenzyme A

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CHAPTER 8

More than 70 enzymes have been identified till date that
are dependent on CoA making it the central molecule in
the metabolism of lipids, proteins and carbohydrates. A
few ex amples of enzymes involving coenzyme A are
given below.

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CHAPTER 8
Dietary Sources and Daily Requirements
Pantothenate is widely distributed in plants and

anim als. Ex cellent food sources include egg yolk,
liver, yeast, legum es, whole grains and vegetables.
A daily intake of 10 mg is recomm ended for adults.

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CHAPTER 8
Formation and Fate of Acetyl CoA

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CHAPTER 8
Folic Acid
Folic acid reduced to tetrahydrofolic acid
(Continues…

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Folic Acid
Folic acid reduced to

tetrahydrofolic acid

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CHAPTER 8
• Role in DNA and RNA synthesis: The one- carbon

groups are utilisef for the synthesis of C2 and C8 of
purine ring.
• Role in amino acid metabolism: THF is involved in

the conversion of serine to glycine, catabolism of
histidine and synthesis of methionine.
• Role in protein biosynthesis: THF is required for

the form ation of N- formylmethionine, which
initiates protein biosynthesis.

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CHAPTER 8
Different One- Carbon Units Carried by

THF

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CHAPTER 8
Daily Requirement

The RDA of folic acid in adults is about
200 μ g/ day.

During pregnancy (400 μ g) and lactation
(300 μ g).

An additional supplement is recomm ended during
pregnancy because its deficiency is linked to neural
tube defects and other congenital malform ations.

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CHAPTER 8
Dietary Sources

Folic acid is widely distributed in natural foods.
Green leafy vegetables, cereals, fruits, eggs and dairy

products are rich sources of folic acid.
Bananas, oranges, cauliflower and broccoli contain

high levels of folate.

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CHAPTER 8
• Macrocytic anemia
• Neural tube defects in the fetus
• Hyperhomocysteinem ia
Histidine load test

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CHAPTER 8
Folate Antagonists
Anticonvulsants and oral contraceptives interfere

with folate absorption and prolonged use of these
drugs m ay cause folate deficiency.
• Aminopterin (4- amino folic acid) and amethopterin

(methotrexate) (4- am ino, 10- methyl folic acid) are
structural analogues of folic acid. They competitively
inhibit the enzym e dihydrofolate reductase and block
the formation of THF.
• Trimethoprim inhibits folate reductase and blocks

the formation of THF. Sulphonamides are
compounds that are structurally similar to PABA.

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CHAPTER 8
Vitamin B12
Structure of vitamin B12
[R = substituted by
different groups]

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CHAPTER 8
Absorption, Transport And Storage of
Vitamin B1 2
HC – haptocorrin
IF – intrinsic fact or
TC – t ranscobalam ins

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CHAPTER 8

Synthesis of m ethionine from hom ocysteine
Represent s blockage in B12 deficiency - - Folate trap).

(Continues

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
Conversion of m ethylm alonyl CoA to succinyl CoA
Conversion of m et hylm alonyl CoA

to succinyl CoA involving vit am in
B12 as coenzym e
Blockage in B12 deficiency leads

to t he format ion of et hylmalonic
acid. The latt er is ex cret ed int o
the urine.

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CHAPTER 8
Daily Requirement And Dietary
Sources
Norm al daily requirement is about 1 μg.
Vitamin B12 is also synthesised by bacteria in colon.
It is not found in foods of plant origin. Hence, strict

vegans who do not consume milk products are at risk
of developing B12 deficiency.
Liver, m eat, fish, eggs, milk, curd and cheese are good
sources of vitam in B12 .

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CHAPTER 8
• Megaloblastic anemia
• Subacute combined degeneration
• Demyelination
• Pernicious anemia

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CHAPTER 9
Section III
Physiological Biochemistry
Universities Press
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Phone: 040-2766 5446/5447
Press
CHAPTER 9
Chapter 9
Biochemistry of Blood

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CHAPTER 9
Plasma Proteins
Types of plasma proteins

The norm al total plasma protein concentration is around
6 to 8 g/ dl of blood. The m ajor form s of plasma
proteins are:
1. Albumin constitutes around 60% of all the plasma

proteins (3.5–5 g/ dl).
2. Globulins: Norm al plasma concentration is
2–3 g/ dl. Globulins occur in different forms:
i) a- globulins
ii) b- globulins
iii) g- globulins
3. Fibrinogen (0.2–0.4 g/ dl)

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CHAPTER 9
Separation of Plasma Proteins
1. Salting out technique
2. Electrophoresis
3. Ultracentrifugation technique
4. Cohn's fractionation
5. Gel filtration (m olecular sieving)

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CHAPTER 9
Electrophoretic
patterns: Normal
and abnormal

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CHAPTER 9
Functions of Albumin
1. Maintains colloid osmotic pressure (COP) of plasm a
2. Transport function
3. Buffering action
4. Nutritional function

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CHAPTER 9
Clinical Significance of Albumin

Album in, by binding to certain com pounds in
blood, prevents them from crossing the blood–
brain barrier, e.g., albumin–bilirubin complex ,
albumin–fatty acid com plex .
• Hypoalbum inem ia
• Tissue edem a
• Kwashiorkor
• Cirrhosis of liver
• Nephrotic syndrom e
(Continues…

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Clinical Significance of Albumin
• paucialbum inuria or
• m icroalbuminuria

The normal albumin–globulin ratio (A/ G ratio) is
about 1.2:1 to 2:1. The A/ G ratio is lowered or
reversed either when album in is decreased or
globulins are increased, usually observed in
cirrhosis of the liver, nephrotic syndrome and
m ultiple m yelom a.

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CHAPTER 9
Globulins
Globulins constitute m ultiple proteins that are

separated into four distinct bands (1 , 2 ,  and ) on
electrophoresis.

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CHAPTER 9
Major features and functions of plasma proteins
(Continues

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CHAPTER 9
…Continued) Major features and functions of plasma proteins

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CHAPTER 9
Structure of a Human Ig Molecule
V - Variable region, C - const ant region; each heavy chain is com posed of four unit s - V H ,
CH1 , CH2 , CH3 , while light chain consist s of t wo unit s - V L and CL.
Press
CHAPTER 9
Classes of Immunoglobulins
Depending on the heavy chain make up, the

im munoglobulins are differentiated into five major
classes:
1. Imm unoglobulin G (IgG)
2. IgM
3. IgA
4. IgD
5. IgE

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CHAPTER 9
Major characteristics of human
immunoglobulins

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CHAPTER 9
Multiple myeloma
• Bence- Jones proteins
• Bradshaw’s test
• Amyloidosis

Press
CHAPTER 9
RBC
• Structure
• RBC count
– Men 4.6–6.2 million/ L
– Women 4.2–5.4 million/ L
• Metabolism in RBC

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CHAPTER 9
Biochemical Basis of Diseases Affecting
RBC
Hemolytic anemias
• Hereditary spherocytosis
• hereditary elliptocytosis
• Hemoglobinopathies
• Sickle cell anem ia
• - thalassem ia
• - thalassem ia
• Genetic deficiency of glucose- 6- phosphate

dehydrogenase
Press
CHAPTER 9
WBC
Respiratory burst

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CHAPTER 10
Section III
Universities Press
3-6-747/1/A & 3-6-754/1, Himayatnagar
Phone: 040-2766 5446/5447
Universities
Rafi M D: Textbook of Biochemistry (4thEdition) Press
CHAPTER 10
Chapter 1 0
Hemoglobin - Chemistry of
Respiration
Universities
CHAPTER 10
Structure of Hemoglobin
Diagrammatic representation of tetrameric Hb (2 2 ) (heme is shown as

pink blocks).
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CHAPTER 10
Structure of Heme
Universities
CHAPTER 10
Types of Hemoglobin
Types of normal hemoglobins
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CHAPTER 10
Hemoglobin Derivatives
Apart from the normally found oxyHb and

deoxyHb, hem oglobin also ex ists in a few other
forms:
• Met- hemoglobin
• Carbox y- hem oglobin (CO- Hb)
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CHAPTER 10
Myoglobin
• Myoglobin is a monomeric (with one polypeptide)

heme- containing protein occuring in muscle tissue.
In other words, it is identical to one subunit of Hb
m olecule.
• Myoglobin has higher affinity for oxygen than Hb.

Myoglobin serves as a reservoir for ox ygen and can
be considered an oxygen- storage protein.
• Raised myoglobin levels in blood is considered

to be the earliest marker of myocardial
infarction (MI).
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CHAPTER 10
Blood- Gas Transport
• Respiratory quotient (RQ)
• Partial pressures of gases
• Gas ex change between alveoli and blood
• Gas ex change between tissues and blood
Universities
CHAPTER 10
Transport of Oxygen in Blood
Ox ygen dissociation curve of hemoglobin and myoglobin
Universities
CHAPTER 10
Cooperative Binding of Oxygen to Hb
( = T or taut form; = R or relaxed form).
Universities
CHAPTER 10
Oxygen Transport from Lungs to
the Tissues
Movement of inspired air into the alveoli is by bulk flow; all other
movements of ox ygen across membranes are by diffusion gradient.
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CHAPTER 10
Bohr Effect
Bohr effect on ox ygen dissociation curve with an

increased H+ concentration.
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CHAPTER 10
2 , 3 BPG
• Rapaport- Leubering cycle
• Mechanism of action of 2, 3- BPG
• Clinical significance of 2, 3- BPG
– High altitude
– Obstructive airway disease
– Anem ia
– Fetal hem oglobin (HbF)
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CHAPTER 10
Effect of 2 , 3 - BPG on oxygen dissociation

curve
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CHAPTER 10
Carbon dioxide transport from
tissues to the lungs
Carbon diox ide is

ex haled from lungs
by bulk flow; all
other movement s
of carbon diox ide
across membranes
are by diffusion
gradient.
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CHAPTER 10
Sickle- cell Anemia
RBCs (A) From a normal person; (B) From a patient

with sickle cell anemia.
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CHAPTER 10
• Molecular basis of Hbs

• Homozygous and heterozygous Hbs
Diagram showing single base mutation in DNA (T "A) resulting in amino

acid substitution in the resultant polypeptide (b- globin chain of HbS).
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CHAPTER 10
Mechanism of Sickling in Sickle Cell

Anemia
Diagram showing sticking together of deox y HbS molecules to form linear

multimers that cause sickling of erythrocytes.
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CHAPTER 10
Sickle Cell Trait Protects from Malaria
It is observed that a very high num ber of black

Africans living in tropical areas infested with malaria
are heterozygous for HbS (sickle cell trait). They
possess the abnormal HbS gene in their genomes.
It appears that the sickle cell trait provides resistance

to malaria in these individuals.
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CHAPTER 10
Biochemical basis of the diagnosis and
management of sickle cell disease
1. Sickling test
2. Electrophoresis
3. RDT
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CHAPTER 10
Thalassemias
Types of thalassem ias
(Continues…
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(Continued… CHAPTER 10
Types of thalassemias
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CHAPTER 11
Section III
Universities Press
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Phone: 040-2766 5446/5447
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CHAPTER 11
Chapter 1 1
Heme Synthesis and

Degradation

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CHAPTER 11
Structure of Porphyrin Nucleus
Pyrrole rings are numbered I, II, III

and IV; methyne bridges are
designated , , , and ; side chains
are labelled 1–8 ).

Press
CHAPTER 11
Porphyrins of clinical importance
A = Acetat e (–CH3 COO–)
P = Propionyl (–CH2 CH2 COO–)
M = Met hyl (–CH3 )
V = Vinyl (–CH = CH2 )

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CHAPTER 11
Biosynthesis of Heme
(Continues…

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Biosynthesis of heme

Press
CHAPTER 11
Overview of Heme Synthesis

Press
CHAPTER 11
Summary of Major Features of
Porphyrias

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CHAPTER 11
Degradation of Heme to Bile Pigments
(Continues…

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Degradation of heme to bile pigments

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CHAPTER 11
Summary of Bilirubin Metabolism in Liver
Showing bilirubin transfer from blood to

bile involving three processes - uptake,
conjugation and secretion.
( = represent s blockage at the
respective st eps showing the diseases
that occur due t o the blockage).

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CHAPTER 11
Bilirubin Metabolism- Overview

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CHAPTER 11
Differential diagnosis of jaundice

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CHAPTER 11
Congenital Hyperbilirubinemias

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CHAPTER 12
Section III
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Phone: 040-2766 5446/5447
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CHAPTER 12
Chapter 1 2
Digestion and Absorption

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CHAPTER 12
Functions of the Gastrointestinal Organs

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CHAPTER 12
Digestion and Absorption of Carbohydrates

Digestion in the mouth

Digestion in the small intestine

Non- digestible carbohydrates

Absorption of m onosaccharides

Press
CHAPTER 12
Mechanism of Absorption
Transport of glucose across the intestinalepithelium

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CHAPTER 12
Lactose intolerance
Lactose intolerance is basically due to the deficiency

of the enzym e lactase (- galactosidase). Normally,
lactase hydrolyses lactose into galactose and glucose.
As lactase activity is also rate- limiting for lactose
absorption, deficiency of the enzym e hinders the
digestion and absorption of lactose in the gut.
Signs and sym ptoms of lactose intolerance include

diarrhea, flatulence, abdominal cramps, irritability,
etc.

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CHAPTER 12
How to treat lactose intolerance?

By feeding a lactose free diet to the affected infants and
avoidance of lactose rich foods (milk and dairy
products) in adults. Synthetic preparations of -
galactosidase (lactase) are also available.

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CHAPTER 12
Digestion and Abosrption of Lipids
Digestion in mouth and stomach

• Lingual lipase
• Acid stable lipase
• Gastric lipase
• Enterogastrone
• Satiety value
(Continues…

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CHAPTER 12
…Cont inued)
Digestion in intestine
• Em ulsiffication of lipids
• Role of bile salts
• Role of phospholipids as surfactant
(Continues…

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CHAPTER 12
…Continued)
Digestion of lipids by pancreatic enzymes in the

intestine
The lipolytic enzym es of pancreatic juice include:
1. Pancreatic lipase
2. Cholesteryl esterase
3. Phospholipase A 2
(Continues…

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CHAPTER 12
Digestion of Triacylglycerols (TAGs)
Progressive hydrolysis of TAG

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CHAPTER 12
Digestion of Cholesteryl Esters
Enzymatic degradation of cholesteryl ester

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CHAPTER 12
Digestion of Phospholipids
Enzymatic cleavage of phospholipids
(Continues…

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CHAPTER 12
…Continued)
Enzymatic cleavage of phospholipids

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CHAPTER 12

Absorption of lipids

Bergstrom theory

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CHAPTER 12
Fate of Lipids After Absorption
1. Resynthesis of lipids in the intestinal mucosal

cells
2. Secretion of lipids from the intestinal m ucosal
cells
3. Transport of dietary lipids

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CHAPTER 12
Resynthesis of lipids in intestinal mucosal cells and formation,
secretion and transport of chylomicrons
(Continues …

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(…continued) CHAPTER 12
Resynthesis of lipids in intestinal mucosal cells and formation,

secretion and transport of chylomicrons

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CHAPTER 12
Digestion and Absorption of
Proteins
Proteins are broken down by the hydrolysis of peptide
bonds and hence the enzym es involved are termed as
peptidases, belonging to the class hydrolases.
Peptidases are of two types:
1. Endopeptidases
2. Ex opeptidases
Ex opeptidases are subclassified into
carboxypeptidases (act on C- terminal end) and
aminopeptidases (act on N- term inal end).

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CHAPTER 12
Digestion of Proteins in the Stomach
• Hydrochloric acid
• Pepsin

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CHAPTER 12
Digestion of Proteins – Substrate
Specificity of Proteases

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CHAPTER 12
Meister cycle

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CHAPTER 12
Pancreatic digestion of proteins

• Intestinal digestion of proteins

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CHAPTER 12
Mechanisms of Amino Acid Absorption
• Sodium- dependent active transport

• Di- and tripeptides
• Meister cycle (- glutamyl cycle)

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CHAPTER 13
Section IV
Metabolism
Universities Press
3-6-747/1/A & 3-6-754/1, Himayatnagar
Phone: 040-2766 5446/5447
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CHAPTER 13
Chapter 1 3
A Journey into Metabolism

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CHAPTER 13
Different Phases of Catabolism
(Contiunes…

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CHAPTER 13
…Continued)
Outline of different phases of catabolism [All t he m ajor biom olecules – carbohydrat es, lipids
and prot eins undergo cat abolism t o finally yield acet yl CoA which is furt her ox idised in t he
citric acid (Krebs) cycle releasing carbon diox ide and reducing equivalent s (NADH + H + and
FADH2 ). Reducing equivalent s t hen ent er int o t he respirat ory chain, ult im at ely producing ATP
t hrough t he process of ox idat ive phosphorylation.]

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CHAPTER 13
Experimental Methods to
Study Metabolism

Metabolic inhibitors

Genetic mutations

Genetic manipulations

Radioactive isotopes

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CHAPTER 13
Laws of Thermodynamics

First law of thermodynamics states that the total
amount of energy in the universe remains constant.
However, the form of the energy m ay change, but
within the total system , energy is neither destroyed
nor created during a physical or chemical change.

The second law of thermodynamics states that the
total entropy of a system must increase if a process
is to occur spontaneously.

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CHAPTER 13
Free Energy
The energy available to perform work is known as

free energy. Gibbs proposed the theory of energy
changes during chemical reactions. Gibbs’ change in
the free energy (DG) is used in predicting the
feasibility of chemical reactions.
G = H – TS
where
H = the change in enthalpy (change in the heat

content of the reactants compared to products)
T = absolute temperature in Kelvin

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CHAPTER 13
• Ex ergonic and endergonic reactions

• Chemical coupling

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CHAPTER 13
High- energy Compounds
A list of high- energy and low- energy compounds along with their standard
free energy (G°) on hydrolysis.

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CHAPTER 13
Structure of ATP

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CHAPTER 13
ATP- ADP Cycle
ATP- ADP Cycle: [ATP links the two major components of metabolism –
catabolism (breakdown of biomolecules) and anabolism (synthesis of
biomolecules)].

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Chapter 1 4
Section IV
Metabolism
Universities Press
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Phone: 040-2766 5446/5447
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Chapter 1 4
Chapter 1 4
Central Metabolism:
Biological Oxidation

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Chapter 1 4
Acetyl Coenzyme A: The Central
Molecule of Metabolism
Structure of acetyl CoA

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Chapter 1 4
Sources and Fate of Acetyl CoA

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Chapter 1 4
Pathways linking the breakdown of organic nutrients to
the generation of ATP

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Chapter 1 4
Mitochondrion
(a)Mitochondrion as it appears under an elect ron m icroscope
(b) Diagram m at ic representation of t he mit ochondrion

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Chapter 1 4
Krebs Cycle
Hans Adolf Krebs (1900–1981)

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Chapter 1 4
Location within the Cell and Overview
The enzymes of the TCA cycle are located in the

mitochondrial matrix.

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Chapter 1 4
Reactions of the TCA Cycle
1. Synthesis of citrate
2. Isomerisation of citrate to isocitrate
3. Ox idative decarbox ylation of isocitrate to -
ketoglutarate (- KG)
4. Ox idative decarbox ylation of - KG to succinyl CoA:
5. Formation of succinate
6. Ox idation of succinate to fum arate
7. Formation of malate
8. Conversion of malate to ox aloacetate

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Chapter 1 4
Krebs Cycle
(Continued…

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Chapter 1 4
…Continues)
Krebs cycle

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Chapter 1 4
Summary of the TCA Cycle
The net reaction of the citric acid cycle is as follows:

Acetyl CoA + 3NAD + + FAD + GDP + Pi + 2H2 O
2CO2 + 3 NADH + 3H+ + FADH2 + GTP + CoA

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Chapter 1 4
Inhibitors of the Citric Acid Cycle

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Chapter 1 4
Energetics of the Krebs Cycle

Generation of ATP per one Krebs cycle

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Chapter 1 4
Citric Acid Cycle – An Amphibolic Pathway
Biosynthetic role of the Krebs cycle

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Chapter 1 4
Anaplerotic Reactions of the Citric Acid Cycle
Anaplerotic reactions replenishing interm ediates of TCA cycle

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Chapter 1 4
Glycerol Phosphate Shuttle
Reducing equivalents transported are shown in red colour

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Chapter 1 4
Malate- Aspartate Shuttle
Reducing equivalents are shown in red colour

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Chapter 1 4
Electron Transport Chain
Redox couples

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Chapter 1 4
Redox Potential
Redox potentials (Eo) of some ox idation–reduction systems

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Chapter 1 4
Components of the Respiratory Chain
I. Nicotinamide nucleotides
II. Flavin nucleotides
III. Iron–sulphur proteins
IV. Coenzym e Q
V. Cytochromes

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Chapter 1 4
Organisation of Electron Transport Chain
Route- map of electron flow through ETC

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Chapter 1 4
Route- Map For Electron Flow
Com plex I: NADH to Coenzym e Q

Com plex II: Succinate to Coenzym e Q
Coenzym e Q
Com plex III: CoQ to Cytochrom e C
Cytochrom e C
Com plex IV: Cytochrom e C to Ox ygen

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Chapter 1 4
Inhibitors of the Electron Transport Chain
Site specific inhibitors of ETC

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Chapter 1 4
P:O Ratio
●
The P:O ratio can be defined as the number of
inorganic phosphate (Pi) molecules utilised for ATP
production for every atom of ox ygen consumed.
●
The ox idation of one m olecule of NADH through
ETC and ox idative phosphorylation yields about 2.5
ATP and the ox idation of one m olecule of FADH 2
yields 1.5 ATP.

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Chapter 1 4
Oxidative Phosphorylation
Chemiosmotic hypothesis
●
The electrochem ical gradient or proton gradient thus
developed, acts as the power that drives the
mechanism operating the synthesis of ATP
●
The essence of chem iosmotic theory is, the free energy
generated from the electron flow through ETC is
conserved by pumping protons out of the matrix to
create an electrochemical proton gradient across the
inner mitochondrial mem brane. The electrochemical
potential of this proton gradient is used to synthesise
ATP.

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Chapter 1 4
Chemiosmotic Theory
Diagram ex plaining
the chemiosmotic
theory of ox idative
phosphorylation

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Chapter 1 4
Structutre of ATP Synthase

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Chapter 1 4
Boyer's Binding Change Model
Boyer's binding change model for ATP synthesis by ATP synthase

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Chapter 1 4
Inhibitors of Oxidative Phosphorylation
• Oligom ycin
• Atractyloside
• Ionophores
• Valinom ycin
• Nigercin
• Uncouplers
• 2, 4- dinitrophenol (DNP)

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Chapter 1 4
Disorders Associated with ETC and
Oxidative Phosphorylation
• Alzheim er’s and Parkinson’s diseases

• Leber’s hereditary optic neuropathy
• MELAS (Mitochondrial Encephalopathy, Lactic Acidosis
and Stroke)

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Chapter 1 4
Enzymes Involved in Biological
Oxidation
The crucial enzym es of biological ox idation belong to

the group oxidoreductases. They are classified into
four categories.
1. Ox idases
2. Dehydrogenases
3. Hydroperox idases
4. Ox ygenases

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Chapter 1 4
Oxidases
These enzym es catalyse the rem oval of hydrogen

from a substrate and hand it over to ox ygen to
form water.
• Cytochrome ox idase
• Flavoproteins

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Chapter 1 4
Dehydrogenases
These enzymes transfer hydrogen from one substrate

to another in a coupled ox idation–reduction reaction.
a) Cytochromes:
b) Dehydrogenases dependent on niacin
coenzymes:
NAD+ dependent dehydrogenases
NADP+ dependent dehydrogenases
c) Dehydrogenases dependent on flavin

coenzym es
FMN- dependent dehydrogenases
FAD dependent dehydrogenases
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Chapter 1 4
Hydroperoxidases
Perox idase
Catalase

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Chapter 1 4
Oxygenases
• Diox ygenases
• Monoox ygenases

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CHAPTER
15
Section IV
Metabolism
Universities Press
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Phone: 040-2766 5446/5447
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CHAPTER
15
Chapter 1 5
Metabolism of
Carbohydrates

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CHAPTER
15
Major pathways of glucose
utilisation

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CHAPTER
Facilitative glucose transporters 15
in different tissues
Note that each GLUT occurs in m ore t han one t issue. Only t he predom inant
t issue/ organ is shown in t he t able.

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CHAPTER
Glycolysis 15
Glycolysis is defined as the sequence of reactions

m etabolising one m olecule of glucose to two molecules
of pyruvate or lactate with the concomitant generation
of ATP.

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CHAPTER
15
Reactions of Glycolysis
(Continues…

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…Continued) CHAPTER
15
(Continues…

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15
In t he first half of the pat hway (react ions 1- 5) one

m olecule of glucose is convert ed t o two
glyceraldehyde- 3- phosphat e m olecules in a
series of reactions t hat consum es 2 ATP.
In t he second half of glycolysis t he two

glyceraldehyde- 3- phosphat e m olecules are
convert ed t o t wo
pyruvat e molecules, generating 4 ATP and 2
NADH

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CHAPTER
15
Conversion of Pyruvate to Lactate

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CHAPTER
15
Rapaport- Leubering Cycle in RBC

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CHAPTER
15
PDH Reaction
The reaction m echanism of conversion of pyruvate t o acet yl CoA cat alysed by PDH com plex

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CHAPTER
Amount of ATP generated by the complete 15
oxidation of one molecule of glucose
*This calculation is based on the assumption that NADH produced in glycolysis is transport ed from the cytosol t o mitochondria by t he
glycerol- phosphate shuttle which yields only 1.5 ATP per NADH. If the NADH is t ransported by the malate- aspartat e shut tle, t he yield is
2.5 ATP per mole of NADH and t he total ATP per mole of glucose will be 32. However, the shutt le operative is tissue- specific (see
Chapt er 14, Mitochondrial Shuttles).
** There is a gain of an additional ATP during anaerobic glycolysis if t he start ing point of glycolysis is glycogen inst ead of free glucose,
since step I catalysed by hex okinase that utilises an ATP is bypassed.

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CHAPTER
15
Gluconeogenesis
(Continues…

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15
The pathway of gluconeogenesis
[The substrates for gluconeogenesis are shown in . The key enzym es of gluconeogenesis are shown in .
The enzym es catalysing the irreversible steps in glycolysis are shown in . The num bers represent the entry of
glucogenic am ino acids. 1 Alanine, glycine, serine, cysteine and tryptophan; 2 Aspartate and asparagine; 3
Glutam ate, glutam ine, arginine, histidine and proline; 4 Valine, isoleucine, threonine and m ethionine; 5
phenylalanine and tyrosine.
Note that a few reactions of gluconeogenesis occur in m itochondria which are shown in a separate com partm ent —
area shaded in blue. The rem aining of the pathway occurs in cytosol.]

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CHAPTER
15
Reactions of Gluconeogenesis
1. Conversion of pyruvate to phosphoenolpyruvate

2. Conversion of fructose- 1, 6- bisphosphate to
fructose- 6- phosphate
3. Conversion of glucose- 6- phosphate to free glucose

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CHAPTER
15
Cori cycle and glucose–alanine cycle
The Cori cycle (black)

Glucose–alanine cycle (peacock blue)
Other reactions are common for both cycles.

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CHAPTER
15
Glycogenesis
1. Synthesis of UDP- glucose

2. Initiation of glycogen synthesis
3. Elongation of the glycogen chain
4. Form ation of ‘glycogen tree’

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CHAPTER
15
Glycogenesis
St eps in glycogen synt hesis

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CHAPTER
15
Glycogenesis
Glycogen synt hesis from
glucose )
(Continues…

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CHAPTER
…Continued) 15
Glycogen synthesis from glucose

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CHAPTER
15
Glycogenolysis
1. Action of glycogen phosphorylase

2. Action of debranching enzym e
3. Formation of glucose- 6- phosphate
4. Fate of glucose- 6- phosphate

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CHAPTER
15
Glycogenolysis
Steps in glycogenolysis

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CHAPTER
Glycogenolysis 15
Degradation of
glycogen
(Continues…

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CHAPTER
15
…Continued) Degradation of glycogen (glycogenolysis)

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CHAPTER
15
Multiple Fates of Glucose- 6 - Phosphate

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CHAPTER
Regulation of Glycogenesis by 15
Covalent Modification

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CHAPTER
Regulation of Glycogenolysis by Covalent 15
Modification
Calm odulin- dependent prot ein

kinase is not shown in figure for
t he sake of clarit y.

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CHAPTER
15
Glycogen Storage Diseases
(Continues…

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15
Glycogen Storage Diseases

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Overview of glycogen m etabolism in CHAPTER
15
glycogen storage disease

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CHAPTER
15
Blood Glucose Homeostasis
Factors contributing to blood glucose

1. Dietary carbohydrates absorbed as glucose in the
intestine
2. Glucose released by glycogen breakdown from the
liver
3. Gluconeogenesis
Factors that remove glucose from the blood

1. Utilisation by the tissues for energy
2. Storage as glycogen
3. Conversion of surplus glucose into fat

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CHAPTER
15
Glycemic Status

In norm al healthy individuals, fasting plasma
glucose levels are 7 0 –1 1 0 mg/ dl.

When the concentration ex ceeds the norm al range,
it is called hyperglycemia. When the values are
below the normal limits it is known as
hypoglycemia .
• Diabetes mellitus
• Hypoglycemia

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CHAPTER
15
Blood glucose homeostasis

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CHAPTER
Role of Hormones in Blood 15
Glucose Regulation
Hypoglycemic effect
Insulin
• Prom otes tissue uptake of glucose
• Enhances glycolysis
• Favours glycogen synthesis
• Depresses glycogen breakdown
• Inhibits gluconeogenesis
• Inhibits glucagon release from the pancreas and
depresses glucagon gene ex pression
• Increases HMP shunt

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CHAPTER
Role of Hormones in Blood 15
Glucose Regulation
Hyperglycemic effect
Glucagon
• Promotes glycogen breakdown in liver
• Enhances gluconeogenesis
• Depresses glycogen synthesis
• Inhibits glycolysis
(Continues…

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CHAPTER
15
…Continued)
Epinephrine

Stim ulates glycogenolysis

Decreases glucose use

Increases gluconeogenesis

Stim ulates glucagon secretion and inhibits
insulin secretion by the pancreas
(Continues…

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CHAPTER
15
…Continued)
Cortisol
• Stim ulates gluconeogenesis
Growth hormone
• Stim ulates gluconeogenesis
• Decreases glycolysis
• Antagonises insulin prom oted glucose uptake

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CHAPTER
15
Hexose Monophosphate Shunt
(Continues…

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CHAPTER
15
…Continued) The reactions of hex ose monophosphate shunt
(Continues…

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15
The reactions of hex ose m onophosphate shunt

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CHAPTER
15
An overview of HMP pathway
(Continues…

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CHAPTER
15
…Continued)

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CHAPTER
15
Significance of the HMP Shunt
• HMP shunt is associated with the generation of two

im portant products, NADPH and pentose sugars .
• In the pentose phosphate pathway, five- carbon

sugars are formed, the most im portant being
ribose- 5 - phosphate. Ribose- 5- phosphate and its
derivatives are used as components in the synthesis
of DNA, RNA, ATP and nucleotide coenzymes such
as NAD + , FAD and coenzyme A.
(Continues…

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CHAPTER
15
…Continued)
• NADPH is used as the reducing power in various

anabolic processes. It plays the role of a reductant
in biosynthetic processes such as fatty acid
biosynthesis, cholesterol and steroid biosynthesis,
nucleotide biosynthesis and neurotransm itter
biosynthesis.
• Antioxidant role

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CHAPTER
15
Uronic Acid Pathway

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CHAPTER
15
Metabolism of Fructose
(Continues…
Rafi M D: Textbook of Biochemistry (4th Edition) (Continues… Universities

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CHAPTER
15
…Continued)

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CHAPTER
15
Metabolism of Galactose
- Enzyme defect causes galactosemia.

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CHAPTER
15
Types of mucopolysaccharidoses
MR = m ent al ret ardat ion; CS = chondroit in sulfate; KS = kerat in sulfat e; HS = heparin

sulfat e; DS = derm at an sulfat e

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CHAPTER
16
Section IV
Metabolism
Universities Press
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Phone: 040-2766 5446/5447
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CHAPTER
16
Chapter 1 6
Metabolism of Lipids

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CHAPTER
16

Lipids, apart from their prim e function as a
storage form of energy, also act as structural
elem ents. Being hydrophobic, lipids are
unique am ong biomolecules.

The lipids in the body ex ist in three forms.
1. Structural lipids
2. Storage lipids
3. Plasm a lipids and transport lipids

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CHAPTER
16
Plasma Lipids
Lipids in human blood plasma

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CHAPTER
16
Summary of Lipid Metabolism

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CHAPTER
16
Synthesis of Triacylglycerols
(Continues…

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CHAPTER
16
…Continued)

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CHAPTER
16
Lypolysis
Complet e hydrolysis of TAG (lipolysis)

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CHAPTER
16
Lipolysis in Adipose Tissue Effected by Hormones
(Continues…

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CHAPTER
16
…Continued)

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CHAPTER
16
Synthesis of Glycerophospholipids
(Continues…

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16

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CHAPTER
16
Summary of Biosynthesis of Ether Lipids

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CHAPTER
16
An outline of the synthesis of sphingom yelin

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CHAPTER
Site- specific Hydrolytic Activity of 16
Phospholipases on a Phospholipid
Substrate

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CHAPTER
16
Degradation of Sphingomyelin

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CHAPTER
Biosynthesis of cerebrosides
16
Biosynthesis of cerebrosides

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CHAPTER
Degradation of sphingomyelins and cerebrosides with associated 16
inborn errors
Depict s enzym e deficiency

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CHAPTER
16
Sphingolipidoses or lipid storage diseases

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CHAPTER
Eicosanoids 16
The structures of common eicosanoids and their parent molecule arachidonate
A subscript numeral indicat es t he num ber of double bonds (e.g., PGE2 with t wo double
bonds),which also represent s t he group which t hey belong to. All t he above compounds are
Group 2 eicosanoids.

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CHAPTER
16
Synthesis of Eicosanoids

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CHAPTER
16
Biosynthesis of Fatty Acids
Raw material for fatty acid biosynthesis

Acetyl CoA is the chief precursor and the source of
carbon atom s, NADPH (a reduced electron carrier) is
used as a reductant and ATP provides energy for fatty
acid biosynthesis.

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CHAPTER
Shuttling of acetyl CoA from mitochondria to 16
cytosol for fatty acid biosynthesis
PDH = Pyruvate dehydrogenase; MDH = Malate dehydrogenase

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CHAPTER
Conversion of Acetyl CoA to 16
Malonyl CoA

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CHAPTER
16
Fatty Acid Synthase Multienzyme Complex
The complex is a dim er with two ident ical polypept ides 1 and 2 running ant iparallel t o each
other, each consist ing of seven enzym e act ivit ies and an acyl carrier prot ein– ACP).

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CHAPTER
16
Biosynthesis of Fatty Acids
(Continues…

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Biosynthesis of Fatty Acids CHAPTER
16
…Continued)
(Continues…

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16
…Continued)
(Continues…

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16
…Continued)
KS = Ket oacyl synthase

ACP = Acyl carrier prot ein

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CHAPTER
16
Fatty Acid Oxidation
The - ox idation of fatty acids occurs in three stages.
1. Activation of fatty acids in the cytosol

2. Transport of fatty acids from cytosol to
mitochondria
3. Reactions of - ox idation in the mitochondrial
matrix .

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Activation of fatty acid to
CHAPTER
16
acyl CoA

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CHAPTER
16
Transport of acyl CoA into mitochondria through the carnitine shuttle

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CHAPTER
16
- oxidation of Fatty Acids
(Continues…

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- Oxidation of Fatty Acids 16
Further processing of acetyl CoA through ETC and ox idative phosphorylation is briefly shown.

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CHAPTER
An overview of the com plete ox idation of 16
palmitic acid

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CHAPTER
16
Energetics of Palmitic Acid Oxidation

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CHAPTER
16
Oxidation of Odd- chain Fatty Acids
Conversion of propionyl CoA to succinyl CoA

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CHAPTER
16
Synthesis of ketone bodies

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CHAPTER
16
Degradation of Ketone Bodies
(Continues…

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CHAPTER
16
…Continued)
Degradation of ketone bodies to acetyl CoA

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CHAPTER
16
Ketone Bodies
Formation, utilisation and ex cretion of ketone bodies

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CHAPTER
16
• Biochemical basis of ketosis in starvation and

diabetes mellitus
• Diabetic ketoacidosis

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CHAPTER
16
Biosynthesis of Cholesterol
The synthesis of cholesterol can be studied

conveniently in five stages.
1. Form ation of HMG CoA

2. Synthesis of mevalonate
3. Generation of isoprenoid units
4. Synthesis of squalene
5. Form ation of cholesterol

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CHAPTER
16
(Continues…

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CHAPTER
16
…Continued)
(Continues…

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CHAPTER
16
…Continued
(Continues…

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CHAPTER
16
…Continued)

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CHAPTER
16
Sum m ary of cholesterol synthesis

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CHAPTER
Hormonal Regulation of Cholesterol 16
Synthesis

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CHAPTER
Esterification of cholesterol in HDL 16
by LCAT

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CHAPTER
16
Degradation of Cholesterol
• Synthesis of bile acids

• Synthesis of steroid hormones
• Synthesis of vitam in D

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CHAPTER
Outline of Synthesis and Fate 16
of Bile Acids

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CHAPTER
16
Cholesterol – Clinical Significance
• Plasma cholesterol
In healthy persons, the levels of total plasma
cholesterol vary between 1 5 0 –2 0 0 mg/ dl.

Principle of estimation
• HDL- C and LDL- C ratio
• Hypercholesterolemia
• Cholesterol and heart disease

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CHAPTER
16
Management of Hypercholesterolemia
• Mevastatin (compactin)
• Lovastatin (mevinolin)
• Provastatin
• Simvastatin
• Starvostatin
• Cholestyram ine
• Cholestipol

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CHAPTER
16
Lipoprotein
Diagram showing a lipoprotein particle

[Note the hydrophilic ex terior and hydrophobic interior.]

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CHAPTER
16
Classification of Lipoproteins
Plasm a lipoproteins are separated by electrophoresis

into different fractions
1. Chylomicrons
2. Very low density lipoproteins (VLDL)
3. Low density lipoproteins (LDL)
4. High density lipoproteins (LDL)
5. FFA–Albumin com plex

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CHAPTER
16
Lipoproteins
Representation of comparative sizes of different lipoproteins

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CHAPTER
Characteristics and com positions of hum an 16
plasm a lipoproteins

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CHAPTER
16
Apoproteins
The protein part of lipoproteins is known as

apolipoprotein, or sim ply, apoprotein.
Functions of major apoproteins

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CHAPTER
16
Lipoproteins
Summary of lipid transport by lipoproteins

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CHAPTER
Transport of dietary lipids by 16
chylomicrons
Apoprot eins - A- I, B- 48, C- II and E; TAG – Triacylglycerol; C – Cholest erol; CE –

Cholest eryl est er; PL - Phospholipid; FFA - Free Fat ty acids; LPL - Lipoprot ein lipase;
CETP - Cholest eryl ester t ransfer prot ein; HL - Hepat ic lipase]

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CHAPTER
Very Low Density Lipoproteins (VLDL) 16
Low Density Lipoproteins (LDL)
Transport of lipids by VLDL and LDL.

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Cellular uptake of LDL- cholesterol by
CHAPTER
16
different m echanisms
The sm all red- balls represent protein clat hrin, sm all yellow- balls represent free cholest erol.

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CHAPTER
16
High Density Lipoproteins (HDL)
Reverse cholesterol transport

The major function of HDL is to collect the ex cess
cholesterol from tissues and to transport it back to the
liver where it is ex creted in bile. This process is known
as reverse cholesterol transport.

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Metabolism of HDL enabling reverse
CHAPTER
16
cholesterol transport
Metabolism of HDL enabling reverse cholesterol transport.

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CHAPTER
16
Disorders of Lipoproteins
Classification and characteristics of hyper and hypolipoproteinemias
(Continues…

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CHAPTER
16
…Continued)

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CHAPTER
16
Sum mary of Hyperlipoproteinem ias

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CHAPTER
16
Fatty Liver
Though lipids are synthesised by the liver they are not

stored by it.
The term fatty liver represents the ex cess deposition of
fats in the liver beyond its capacity to utilise or
transport them.
Causes
• Increased synthesis of TAG: High fat intake
• Reduced utilisation and rem oval of fats
• In protein- energy malnutrition (PEM)

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CHAPTER
16
To summ arise:
Increased synthesis, mobilisation and decreased

oxidation of fatty acids coupled with impaired
lipoprotein secretion by the liver leads to fatty
liver.

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CHAPTER
16
Causes of fatty liver

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CHAPTER
16
Lipotropic Factors
The substances that play active role in the removal of

fats from the liver by different mechanisms, thus
preventing the risk of developing fatty liver are known
as lipotropic factors. They include
• Choline
• Inositol
• Methionine
• Betaine
• Vitamin E and selenium
• Om ega- 3 fatty acids

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CHAPTER
Lipid Metabolism and Cardiovascular 16
Disease
• Plasma lipid profile

• Risk factors for cardiovascular disease
• Atherosclerosis

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CHAPTER
16
Prevention of Cardiovascular Disease
• Avoid smoking
• Ex ercise regularly and maintain norm al body
weight
• Dietary m easures

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CHAPTER
16
Alcohol Metabolism

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CHAPTER
17
Section IV
Metabolism
Universities Press
3-6-747/1/A & 3-6-754/1, Himayatnagar
Phone: 040-2766 5446/5447
Press
CHAPTER
17
Chapter 1 7
Metabolism of Amino Acids

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CHAPTER
17
Protein Turnover

Body protein content is not static but ex ists in
a dynamic steady state.

It is estimated that about 300–400 g of
protein is degraded and synthesised a day,
which represents the body protein turnover.

How does a cell recognise proteins that are
to be degraded?
• Ubiquitin
• Proteosome

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CHAPTER
17
Amino Acid Pool

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CHAPTER
17
Amino Acid Metabolism – An Overview

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CHAPTER
Metabolism of Amino Acids – Fate of 17
Amino Group
Transmination
The transfer of an - amino group from an -
am ino acid to an - keto acid is known as
transam ination. The reaction involves the
interconversion of a pair of am ino acids and
a pair of keto acids. The overall process is
catalysed by a group of enzym es known as
transaminases or aminotransferases. The
derivative of vitamin B6 - pyridoxal
phosphate (PLP) acts as the coenzym e.

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CHAPTER
17
• Mechanism of transamination
• Significance of transm ination
Transamination reaction

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CHAPTER
17
Transamination
(Continues…

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CHAPTER
Mechanism of transam ination 17
…Continued)
A and B : Reactions catalysed by ALT and AST, respectively. C : Formation of Schiff base

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CHAPTER
17
Deamination
• Ox idative deam ination

Ox idative deam ination by glutam ate
dehydrogenase
Ox idative deamination of glutamate by GDH.

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CHAPTER
17
Oxidative deamination by amino acid oxidase

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CHAPTER
17
Non- oxidative deamination

• Hydrox y amino acids
• Sulphur am ino acids

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CHAPTER
17
• Deamination of histidine

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CHAPTER
17
Balance in Ammonia Metabolism

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CHAPTER
17
Transport of Ammonia
• Glutamine as a ‘store- house’ of amm onia

• Transport of amm onia through alanine
Synthesis of glutamine and its conversion to glutamate.

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CHAPTER
17
Ammonia Toxicity

Ammonia toxicity

Removal of ammonia
Amm oniotelics
Uricotelic
Ureotelic

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CHAPTER
17
Urea Cycle
Outline of the urea cycle

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CHAPTER
17
Reactions of the Urea Cycle
• Formation of carbamoyl phosphate

• Formation of citrulline
• Synthesis of argininosuccinate
• Cleavage of argininousuccinate
• Urea form ation

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CHAPTER
17
Urea Cycle
(Continues…

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CHAPTER
17
…Continued)
Reactions of the urea cycle
Note that in the

synthesis of urea,
one amino group is
obtained from the
ammonium ion,
while the other is
contributed by
aspartate; carbon
atom comes from
CO2 (as bicarbonate).
This is represented
in different colours.
The first two
reactions of the cycle
occurs in the
mitochondria.
The rest of the cycle

occurs in cytosol.
(NAG — N- acetyl
glutamate).

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CHAPTER
Metabolic Link Between Urea Cycle and 17
TCA Cycle

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CHAPTER
17
Metabolic Defects in the Urea Cycle

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CHAPTER
17
Blood Urea – Clinical Application

Urea levels in the blood are estim ated to assess
kidney function. In healthy individuals, the normal
blood urea concentration ranges between 15 –40
m g/ dL.

The term uremia is used to refer to elevated blood
urea due to renal disease. The conditions in which
blood urea levels are elevated are classified into
three categories.

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CHAPTER
17
1 . Pre- renal
Severe dehydration, fevers, diabetic coma,
thyrotox icosis, severe burns and so on are
pre- renal causes of elevated urea levels.
2 . Renal
Blood urea is increased in kidney diseases such as,
acute glomerulonephritis, pyelonephritis, polycystic
kidney, nephrotic syndrom e and so on.

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CHAPTER
17
3 . Post- renal
When there is an obstruction to the flow of urine
(e.g. stones, tumours, stricture urethra,
enlargm ent of prostate and so on) blood urea is
elevated, due to the increased reabsorption in the
renal tubules.
Blood urea nitrogen (BUN)

BUN = ½ NPN (approx .)

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CHAPTER
17
Summary of Glycine
Metabolism

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CHAPTER
17
Metabolism of Glycine

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CHAPTER
Biologically important compounds 17
synthesised from glycine
1 . Synthesis of purine ring: Glycine contributes to the

C4 , C5 and N7 of the purine ring.

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CHAPTER
17
2 . Synthesis of heme
3 . Formation of glutathione
4 . Glycine as conjugating agent
5 . Glycine as a neurotransmitter

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CHAPTER
17
Formation of Creatine
(Continues…

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CHAPTER
17
...Continued…
Synthesis and degradation of creatine

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CHAPTER
Differences between Creatine and 17
Creatinine

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CHAPTER
17
Metabolic Errors of Glycine
• Primary hyperox aluria

• Glycinem ia and glycinuria

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CHAPTER
17
Summary of Branched Chain Amino Acids
(Continues….

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CHAPTER
17
…Continued)

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CHAPTER
Diseases of the metabolism of 17
branched- chain amino acids
1 . Maple syrup urine disease: This metabolic disorder

occurs due to a defect in the enzym e branched-
chain - keto acid dehydrogenase
2 . Isovaleric acidemia: Deficiency of the enzyme,

isovaleryl CoA dehydrogenase
3 . Hypervalinemia

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CHAPTER
17
Serine
Metabolic role of serine

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CHAPTER
17
Threonine
Outline of threonine metabolism

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CHAPTER
17
Sulphur Amino Acids
Outline of metabolism of sulphur amino acids

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CHAPTER
Outline of metabolism of sulphur 17
amino acids

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CHAPTER
S- Adenosylmethionine (SAM) 17
Form ation, utilisation and

regeneration

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CHAPTER
17
Formation of Cystine

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CHAPTER
17
Synthesis of Cysteine
Synthesis of cysteine from methionine

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CHAPTER
17
Metabolism of Cysteine
(Continues…

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CHAPTER
17
…Continued)

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CHAPTER
17
Formation of PAPS

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CHAPTER
17
Significance of cysteine metabolism

Taurine

PAPS (3’- Phosphoadenosine- 5-
phosphosulphate)

Glutathione

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CHAPTER
17
Inborn Errors of Sulphur Amino
Acids

Cystinuria

Cystinosis

Hom ocystinurias
Homocystinuria type I: The defect in the enzym e

cystathionine synthase causes abnormal
accumulation of homocysteine and methionine.

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CHAPTER
17
Cystathionuria: It occurs due the deficiency of

cystathionase.
It results in mental retardation, endocrine

disorders, anemia and so on.
Deficiency of vitamin B6 may sometimes be

associated with this disorder.

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CHAPTER
Outline of aspartate and 17
asparagine metabolism

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CHAPTER
Outline of glutamate and glutamine 17
metabolism

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CHAPTER
Synthesis of biologically important 17
substances from glutamate
1. N- acetylglutam ate (NAG)

2. Glutathione
3. - Carbox yglutamate
4. Glutam ate as a neurotransm itter
5. - Am inobutyric acid (GABA)

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CHAPTER
17
Lysine
Summ ary of lysine degradation

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CHAPTER
Metabolism of glutamate, glutamine, 17
histidine, arginine and proline

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CHAPTER
17
…Continued)
All t he five amino

acidsglutam at e, glut am ine,
hist idine, arginine and
proline are degraded t o -
ket oglut arate.

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CHAPTER
17
Metabolism of Arginine
Metabolism of arginine - overview

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CHAPTER
17
Nitric Oxide (NO)
Functions of nitric oxide

1. Nitric ox ide is a potent vasodilator.
2. NO acts as a neurotransmitter
3. It stimulates the release of hormones (GHRH, CRH)
from the hypothalam us.
4. It is involved with the potential bactericidal actions
of macrophages

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CHAPTER
17
Therapeutic effects of NO
1. NO as nitroglycerine is used in the treatm ent of

angina pectoris.
2. Inhalation of NO gas is em ployed in the treatment of

pulmonary hypertension.

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CHAPTER
17
Phenylalanine and Tyrosine
Summary of phenylalanine and tyrosine metabolism

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CHAPTER
Summ ary of phenylalanine and 17
tyrosine metabolism

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CHAPTER
17
Conversion of Phenylalanine to Tyrosine
Form ation of tyrosine from phenylalanine. ( – Block in phenylketonuria).

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CHAPTER
Degradation of Phenylalanine and Tyrosine 17
The circled numbers indicate sites of metabolic defects.

1 Phenylketonuria ; 2 Tyrosinemia type II ; 3 Neonatal tyrosinemia ; 4 Alkaptonuria
(Continues…

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CHAPTER
17
…Continued)
The circled numbers indicate

sites of metabolic defects.
5 and 6 Tyrosinemia (type I)

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CHAPTER
17
Synthesis of Catecholamines from

Tyrosine

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CHAPTER
Synthesis of Thyroid Hormones 17
from Tyrosine
Not e that t yrosine residues occur as a part of t hyroglobulin, a huge glycoprot ein t hat
occurs in t he lum en of t hyroid follicles.
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CHAPTER
17
Synthesis of Melanin from Tyrosine

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CHAPTER
Metabolic Defects of Phenylalanine and 17
Tyrosine
Phenylketonuria
Enzyme defect in PKU: Phenylketonuria results from a
deficiency of the enzym e, phenylalanine hydroxylase.

Altered metabolism in PKU

Clinical features of PKU

Diagnosis of PKU
– Phenylalanine levels in blood
– Ferric chloride test
– Guthrie test
– DNA probes

Management of PKU

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CHAPTER
17
Alkaptonuria

Enzym e defect

Biochemical changes in alkaptonuria

Diagnosis and treatm ent
Albinism
Tyrosinem ias
Tyrosinem ia type I (hepatorenal tyrosinemia)
Tyrosinem ia type II (Occulocutaneous tyrosinem ia)

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CHAPTER
17
Sum mary of phenylalanine m etabolism

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CHAPTER
17
Outline of tryptophan metabolism

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CHAPTER
17
Tryptophan
The metabolism of tryptophan can be studied in two

different pathways.
I.Kynurenin–Nicotinate pathway
II. Serotonin–Melatonin pathway

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CHAPTER
17
Kynurenin–Nicotinate Pathway
(Continues…

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CHAPTER
17
…Continued)
(Continues…

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CHAPTER
17
…Continued)
PLP – pyridox al phosphate; PRPP – phosphoribosylpyrophasphate; QPRT – quinolinate phosphoribosyl transferase

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CHAPTER
17
Summary of kynurenin–nicotinate pathway

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CHAPTER
17
Serotonin–Melatonin Pathway
(Continues…

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CHAPTER
17
…Continued)

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CHAPTER
17
Serotonin

Synthesis of serotonin

Degradation of serotonin

Biological role of serotonin
1. Serotonin is a potent vasoconstrictor and
causes sm ooth muscle contraction in
arterioles and bronchioles.
2. It serves as an ex citatory neurotransmitter in
the brain
(Continues…

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CHAPTER
17
…Continued)
3. Serotonin causes peristalsis and causes the release

of peptide horm ones from GI tract.
4. It is capable of influencing the behavioural patterns
in humans.

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CHAPTER
17
Degradation of Tryptophan
Degradation of tryptophan
to indoleacetate
Indo le ace tate

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CHAPTER
17
One- Carbon Metabolism
(Continues…

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CHAPTER
17
…Continued)
Summary of one- carbon metabolism

(Some ex perts consider even CO2 as a one- carbon carrier.
Carbon diox ide is involved in several carbox ylation reactions dependent
on biotin. For instance, conversion of pyruvate ox aloacetate in
gluconeogenesis)

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CHAPTER
17
Polyamines
Biosynthesis of polyamines

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CHAPTER
17
Biologically important compounds generated from amino acids

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CHAPTER
Summary of Inborn Errors of Amino Acid 17
Metabolism

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CHAPTER
Summary of the Metabolic Fate 17
of Amino Acids
Note that some amino acids appear more than once, reflecting different fates for different parts of their
carbon skeletons. Glucogenic amino acids depicted in black, ketogenic in red and both glucogenic and
ketogenic in green.

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CHAPTER
Classification of amino acids based on the 17
metabolic fate of their carbon skeletons

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CHAPTER
17
The Nitrogen Cycle

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CHAPTER
Outline of Biosynthesis of 17
20 Amino acids
(Continues…

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CHAPTER
17
…Continued)
Out line of biosynt hesis of 20 am inoacids in plants and microorganism s. Not e that t he
precursors from glycolysis (red), HMP pat hway (blue) and t he citric acid cycle (green) are
shaded, and am ino acids derived from t hem are shown in box es of corresponding colours).

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CHAPTER
18
Section IV
Metabolism
Universities Press
3-6-747/1/A & 3-6-754/1, Himayatnagar
Phone: 040-2766 5446/5447
Press
CHAPTER
18
Chapter 1 8
Metabolism of Nucleotides

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CHAPTER
18
Biosynthesis of Purine Nucleotides
The sources of nitrogen and carbon atoms of the purine ring

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CHAPTER
18
De Novo Synthesis of Purine Nucleotides
(Continued…
(Continues…

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18
(Continued…
(Continues…

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…Continued CHAPTER
18
(Continued…
(Continues…

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…Continued CHAPTER
18
(Continued…

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CHAPTER
18
Synthesis of AMP and GMP from IMP
(Continues…

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CHAPTER
18
…Continued) Synthesis of AMP and GMP from IMP

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CHAPTER
18
Salvage Pathways
Free purine bases, derived from the norm al turnover of

nucleic acids are recycled in tissues incapable of de novo
synthesis such as the brain, RBC and neutrophils.
These free purine bases are reconverted to their

corresponding nucleotides and this process is known as
the salvage pathway.
Salvage reactions require far less energy compared to de

novo synthesis.

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CHAPTER
18
Role of Phosphoribosyl Pyrophosphate
(PRPP)
Phosphoribosyl pyrophosphate (PRPP) plays a crucial
role in salvage pathways by acting as the donor of
ribose 5- phosphate.
PRPP - Phosphoribosyl pyrophosphat e; PPi - Inorganic pyrophosphat e; AMP -

Adenosine monophosphat e; GMP - Guanosine monophosphat e; IMP - Inosine
monophosphat e

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CHAPTER
18
Salvage pathways of purine nucleotide
synthesis
* Deficiency of HGPRT causes Lesch–Nyhan syndrom e.

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CHAPTER
18
Synthesis of Deoxyribonucleotides
Formation of deox yribonucleotides from ribonucleotides

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CHAPTER
18
Degradation of Purine Nucleotides
(Continues…

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18
Degradation of Purine Nucleotides
Not e that purine ribonucleot ides and deox yribonucleotides are degraded by t he same
pat hway and enzymes).

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CHAPTER
18
Sources and Excretion of Uric Acid

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CHAPTER
18
Disorders of Purine Metabolism
Most of the disorders of purine metabolism are

associated with hyperuricemia, a term that refers to
elevated levels of uric acid in the blood – above 7.0
mg/ dl in men and above 6.0 m g/ dl in women.

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CHAPTER
Gout 18
• Primary gout
• Hyperactivity of PRPP synthetase
• Superactivity of PRPP glutamyl am idotransferase
• HGPRT deficiency
• Glucose- 6- phosphatase deficincy
• Secondary gout
• Treatment of gout

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CHAPTER
18
Lesch–Nyhan Syndrome

This genetic disorder is characterised by spasticity,
m ental retardation, self- injurious behaviour and
gout. It was first described in 1964 by a m edical
student, Michael Lesch, and his professor, William
Nyhan.

Inherited deficiency of the purine salvage enzyme,
hypoxanthine- guanine
phosphoribosyltransferase (HGPRT), is
responsible for the Lesch–Nyhan syndrom e.

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CHAPTER
Immunodeficiency diseases associated with 18
purine metabolism

Severe combined imm unodeficiency (SCID)

Gene therapy

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CHAPTER
18
Sources of Pyrimidine Ring
Sources of carbon and nitrogen atoms in

the pyrimidine ring

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CHAPTER
De novo synthesis of pyrimidine 18
nucleotides
(Continues…

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CHAPTER
18
…Continued De novo synthesis of pyrimidine nucleotides

Press
CHAPTER
19
Section IV
Metabolism
Universities Press
3-6-747/1/A & 3-6-754/1, Himayatnagar
Phone: 040-2766 5446/5447
Rafi M D: Textbook of Biochemistry (4th Edn) Universities
Press
CHAPTER
19
Chapter 1 9
Integration of Metabolism

Press
CHAPTER
19

The reactions occurring in various m etabolic
pathways do not occur in isolation; they occur in an
integrated, well- coordinated manner based on the
overall requirements of the organism.

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CHAPTER
19

The pathways of all three classes of biomolecules
are interlinked through som e common
intermediates.
All these biom olecules are catabolised by the Krebs
cycle, electron transport chain and generate energy
(ATP) through ox idative phosphorylation.
Each class of these biomolecules provides the raw
materials for the synthesis of the components of
other classes.

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CHAPTER
19
The inter- relationships between the metabolic pathways of
carbohydrates, lipids and proteins

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CHAPTER
Summary of pathways of 19
various metabolisms
(Continues…

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CHAPTER
19
…Cont inued)
Summary of pathways of various metabolisms
(Cont inues…

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CHAPTER
19
…Cont inued)
Summary of pathways of various metabolisms

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CHAPTER
19
Fast- Feed Cycle
What happens in the body in a day?

Studying the metabolic changes after an evening
m eal, fasting at night and the re- fed state after
breakfast is known as the fast–feed cycle or starve–
feed cycle.

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CHAPTER
19
The Fed State

The biochemical changes following the
consum ption of food constitute the well- fed or
post- prandial state.

The carbohydrate content of the diet after
digestion is mostly absorbed as glucose and enters
the blood. The rise in blood glucose levels induce
the beta cells of the islets of Langerhans in the
pancreas to secrete insulin.
(Cont inues…

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CHAPTER
19
…Continued)

Within the cells, the glucose is converted to pyruvate
or lactate by glycolysis or it is used in the HMP
shunt for the formation of NADPH and pentose
sugars.

Availability of a plentiful supply of glucose in the
well- fed state coupled with the high insulin levels
result in the storage of glucose as glycogen
(glycogenesis) in the liver and m uscle. The liver, by
thus storing glucose as glycogen during the fed
state, releases glucose into blood from its stores
(glycogenolysis) in between meals.
(Cont inues…

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CHAPTER
19
…Continued)

Dietary proteins are digested and absorbed as
amino acids in the intestine. Very small amounts
are used by the intestinal cells and the rest is
transported through portal blood to the liver and all
other tissues where they are prim arily used for
protein synthesis.
(Cont inues…

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CHAPTER
19
…Continued)

The dietary lipids are packaged into chylomicrons
in the intestinal epithelial cells and are transported
to the blood through the lym phatic system.
Most of the triacylglycerols (TAG) present in

chylom icrons are hydrolysed by lipoprotein lipase
(LPL) in various tissues, especially in adipose tissue.
(Cont inues…

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CHAPTER
19
…Continued)

The TAG thus produced from dietary fat in the liver
along with sm all am ounts of TAG produced from
glucose and amino acids by de novo synthesis
(endogenous fat) are packaged into very low density
lipoproteins (VLDL) and secreted by the liver into the
blood.

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CHAPTER
19
Metabolism in the Fed State

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CHAPTER
19
The Fasting State

The fasting state constitutes the period after an
evening m eal, ex tending into the fasting state
during the night until breakfast the nex t m orning.

The glucose levels in the blood start dropping after
a few hours during the post absorptive period. The
fall in blood glucose decreases insulin secretion and
stim ulates glucagon secretion. Glucagon stimulates
glycogen breakdown and inhibits glycogen
synthesis.
(Cont inues…

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CHAPTER
…Continued) 19

As a result, glycogenolysis in the liver contributes
to blood glucose in the early fasting state.

In addition, glucagon also inhibits glycolysis
Glucagon also inhibits fatty acid synthesis

The liver glycogen levels are depleted by late night
and the early hours of the morning (8–10 hours
after supper), and the body switches over to hepatic
gluconeogenesis to maintain the blood glucose
levels.
(Cont inues…

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CHAPTER
…Continued) 19

Since the fatty acids cannot be used for the
synthesis of glucose, the net glucose synthesis by
gluconeogenesis derives its carbon source from
glycerol and m uscle proteins.

A substantial amount of acetyl CoA produced by
fatty acid ox idation is used for the formation of
ketone bodies

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CHAPTER
19
Metabolism in the Fasting State

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CHAPTER
19
The Re- fed State

After breakfast, fat is metabolised as described in
the normal fed state. But glucose is metabolised
differently. The liver does not use the glucose which
is available aplenty in the blood, but leaves it for
the peripheral tissues. Interestingly, the liver
continues to remain in the gluconeogenic mode for
3–4 hrs after feeding. The glucose synthesised by
gluconeogenesis is used to replenish the liver ’s
glycogen stores.
(Cont inues…

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CHAPTER
…Continued) 19

The gluconeogenic mode of the liver com es to an
end and the liver starts synthesising glycogen
directly from fresh blood glucose.

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CHAPTER
19
Metabolism in Starvation

When the fasting state is prolonged, it results in
starvation. During starvation, the entire body
m etabolism is reorganised for the survival of the
organism.

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CHAPTER
19
Metabolism in Starvation

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CHAPTER
20
Section IV
Metabolism
Universities Press
3-6-747/1/A & 3-6-754/1, Himayatnagar
Phone: 040-2766 5446/5447
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CHAPTER
20
Chapter 2 0
Metabolism of Xenobiotics:
Detoxification

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CHAPTER
20

Human beings are ex posed to a number of foreign
chemicals in their day- to- day life. This includes food
additives, pesticides, cosmetics, environmental
pollutants and most important, drugs. All these
foreign chem icals are collectively called
'x enobiotics’.

When drugs and other x enobiotics gain access to the
hum an biological system, they undergo chemical
alteration, a process known as biotransformation.

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CHAPTER
How Does the Body Handle Foreign 20
Compounds?

Even though all tissues m etabolise x enobiotics, the
liver is the major organ involved.

The m etabolism of x enobiotics is considered in two
phases.
• Phase I reactions
Hydrox ylation, Reduction and Hydrolysis
• Phase II reactions
• Conjugation reactions

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CHAPTER
Phase I Reactions 20
1 . Oxidation or Hydroxylation
Cytochrome P45 0 hydroxylase cycle

Fe2 S2 (iron- sulphur prot ein), e– (elect ron), CO (carbon m onox ide)inhibit s t he indicat ed st ep,
* represent s an addit ional elect ron

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CHAPTER
20
Reduction
The nitro compounds are reduced to their am ines and
aldehydes, ketones are reduced to alcohols, e.g.,

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CHAPTER
20
Hydrolysis
Many of the tox ic m olecules in the body are broken
down into smaller molecules by hydrolysis. The
hydrox yl group and the hydrogen ion of H 2 O are
transferred to different fragm ents of the substrate.
Bonds such as glycoside, ester, amides and so on are

bio- transformed by hydrolysis. Aspirin, the popular
analgesic, is metabolised by hydrolysis

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CHAPTER
20
Phase II Reactions
A x enobiotic m etabolised into a polar com pound in

phase I reactions m ay not be sufficiently polar to be
eliminated easily from the body. Usually, these
interm ediates need to undergo more biotransform ation
reactions. Phase II reactions, m ostly conjugating
reactions, metabolise these hydrox ylated byproducts
into even m ore hydrophilic m olecules. As a result, they
are easily elim inated from the body.

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CHAPTER
20
Conjugation
Conjugation involves the addition of body metabolites

to x enobiotics rendering them more polar, non- tox ic
and easily ex cretable com pounds.
Glutathione

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CHAPTER
20
Glycine
As a part of norm al body m etabolism, glycine
conjugates bile acids. It also detox ifies m any other
substances, e.g. benzoic acid (a food preservative).

Press
CHAPTER
20
Glucuronic acid
Sulphation

Press
CHAPTER
20
Acetylation
Methylation

Press
CHAPTER
21
Section V
Nutrition
Universities Press
3-6-747/1/A & 3-6-754/1, Himayatnagar
Phone: 040-2766 5446/5447
Rafi MD: Textbook of Biochemistry (4th Edition) Universities
Press
CHAPTER
21
Chapter 2 1
Minerals

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Nutrients are of two categories


Macronutrients

Micronutrients
About 17 inorganic elements serve as essential

dietary constituents for humans. These minerals are
broadly classified as
(1) major elements that include electrolytes and

(2) trace elem ents.

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List of mineral elements

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Calcium
Dietary sources
Milk and m ilk products are rich sources for calcium .
Leafy vegetables, beans, egg and fish (eaten with
bones) also contain good amounts of calcium.
Cereals (wheat, rice) contain only small amounts of
calcium.
But cereals are the staple diet in India. Therefore,
cereals form the m ajor source of calcium in our diet.

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Calcium
Daily requirement
An adult needs 800 mg/ day and a child about 1.2
g/ day.
Requirem ent m ay increase to 1.5 g/ day during
pregnancy and lactation.
After the age of 50, there is a general tendency to
develop osteoporosis, especially in post- menopausal
wom en, which may be prevented by increased
calcium (1500 mg/ day) plus vitam in D (20 m g/ day).

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Factors promoting calcium
absorption
i) Vitamin D
ii) Parathyroid hormone
iii) Acidity
iv) Am ino acids
v) Lactose

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Factors inhibiting calcium absorption
1. Phytic acid
2. Ox alates
3. Malabsorption syndrome
4. Phosphate
5. Alkalinity

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Calcium: Biochemical Functions
1. Development of bones and teeth

2. Muscle contraction
3. Blood coagulation
4. Nerve im pulse transm ission
5. Activation of enzymes
6. Calcium as second messenger

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Calcium in Blood
i) Normal blood level

Norm al calcium level in plasm a 9–11 mg/ dl
ii) Ionised calcium
About 5 mg/ dl of calcium is in the ionised form and
is m etabolically active.

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Different proportions of various forms 21
of circulating calcium

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Regulation of Blood Calcium Levels
There are effective controls to maintain the narrow

range (9–11 m g/ dl) of blood- calcium . The horm ones–
calcitriol, parathyroid hormone (PTH) and calcitonin
play a major role in blood calcium hom eostasis.

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Vitamin D

Vitamin D and absorption of calcium

Vitamin D and bone

Vitamin D and renal tubules

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Parathyroid Hormone (PTH)

PTH on bones

PTH on kidney

PTH on intestine
Calcitonin

Calcitonin, calcitriol and PTH act together to
achieve calcium homeostasis

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Disease States
Hypercalcemia
Increased serum Ca level is associated with
hyperparathyroidism
Hypocalcemia
Hypocalcemia is m ostly due to hypoparathyroidism

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Phosphorus
Requirement and sources

The requirement of phosphorous is the same as of
calcium (1:1, that is 800 m g/ day). Milk is a good
source
Blood levels of phosphorus

Adults 3–4 m g/ dl
Children 5–6 m g/ dl

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Biochemical Functions of Phosphorous
1. Phosphorous helps in the form ation of bone and

teeth.
2. It serves as a com ponent of high- energy

phosphate com pounds such as ATP, GTP and
creatine phosphate.
3. In the synthesis of nucleotide coenzymes such as

NAD+ , NADP+ and PLP.

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Magnesium
Dietary sources and requirements

Cereals, vegetables, nuts, fruits, m ilk and meat are good
sources.
An adult requires about 300 mg per day.

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Biochemical functions of
magnesium
1. Mg 2+ serves as an essential cofactor for many
enzym es particularly those utilising ATP, e.g.,
hex okinase, phosphofructokinase, alkaline
phosphatase, adenyl cyclase and cAMP dependent
kinases.
2. Along with calcium and phosphate, magnesium is

utilised for bone and teeth form ation.

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Sulphur
Sulphur mostly occurs in proteins as sulphur-

containing am ino acids (methionine and cysteine).
Biochemical functions
1. Sulphur as a component of iron–sulphur (Fe–S)
proteins plays a crucial role in mitochondrial
cellular respiration.
(Continues…

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…Continued)
2. The sulphur- containing amino acids are essential

components of many proteins. The disulphide
bonds involving sulphydryl groups stabilise the
protein structure.
3. The vitamins thiamine, biotin, lipoic acid and
coenzyme A of pantothenic acid contain sulphur.
The sulphydryl group (–SH) of coenzyme A plays a
crucial role in various metabolic reactions.
4. Keratin, the fibrous protein present in hair and
nails is rich in sulphur.

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Iron
The total amount of iron in an adult body is 3 to 5 g.

About 75% of this occurs in the RBC as a constituent of
hemoglobin.
Dietary requirements
Adult male – 10 mg/ day
Menstruating wom an – 20 mg/ day
Pregnant and lactating wom en – 40 mg/ day
Dietary sources
Jaggery, organ meats (e.g. liver, heart), leafy
vegetables, pulses, cereals, apples, dry fruits and fish.

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Absorption, Transport and Storage of Iron
Iron absorption, transport and storage

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Factors Affecting Iron Absorption
1. Acidity, ascorbic acid, alcohol, fructose and

cysteine promote iron absorption.
2. In iron deficiency anemia, Fe absorption is
increased and is almost ten times that of normal
absorption.
3. Amino acids and small peptides favour iron uptake
by mucosal cells.
4. Phytates (in cereals), ox alates (in leafy vegetables)
and tannins (in tea) inhibit Fe absorption by
forming insoluble iron salts.

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Biochemical Functions of Iron
1. The major role of iron in humans is to carry O2 to

tissues and CO2 to lungs as part of heme protein
that in turn is part of hemoglobin. Ox ygen is also
bound by another iron- containing hem e protein in
m uscle, m yoglobin.
2. Iron plays a vital role in m itochondrial electron
transport (cellular respiration) as a component of
cytochrom es and iron–sulphur proteins.
3. Iron serves as a component of the enzyme
catalase in RBC which ex erts a antiox idant
role.

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Iron: Disease States

Iron deficiency anemia

Hemosiderosis

Hemochromatosis

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Copper
Copper mostly distributed in the liver, brain, kidneys,

heart, bone, m uscle and hair.

Organ meats, cereals, fruits, green leafy vegetables,
shellfish, poultry are sources of dietary copper.
Adults 2–3 mg/ day
Infants and children 0.5 –2 m g/ day

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Copper: Biochemical Functions
1. Copper serves as the metal cofactor for many enzymes.
2. As a com ponent of cytochrom e ox idase (complex IV of

ETC), it plays a role in cellular respiration.
3. As a constituent of superox ide dism utase and catalase,

it is involved in antiox idant function.
4. As a cofactor of ALA synthase (heme synthesis), it is
required for the formation of hem oglobin, m yoglobin
and cytochromes.
5. The hydrox ylation of lysine and proline (required for

collagen cross- linking) requires copper as an essential
cofactor.

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Some important enzymes that 21
contain copper

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Copper: Disease States
Menke's kinky hair syndrome

It is an X- linked condition (affecting only male
infants). It is said to be due to m utations in the gene
for a copper - binding ATPase. As a result, copper
cannot be transported in the blood.
(Continues…

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…Continued)
Wilson’s disease
It is a genetic disease (autosom al recessive) which
occurs due to mutations similar to that of Menke's
disease but in a different gene (a gene encoding a
copper- binding ATPase ex pressed only in liver cells).
Hence, the condition is also known as hepatolenticular
degeneration.
Ceruloplasmin levels in blood are decreased .
(Continues…

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…Continued)
Kayser–Fleischer ring
Patients of Wilson's disease are treated with lifelong

adm inistration of penicillamine and British antilewisite
(BAL) which chelate copper and are subsequently
ex creted in urine.

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Iodine
Occurs in the thyroid gland.

Dietary sources
Seafoods and plant foods grown near the sea are the
best sources. Iodine content in the soil and water is
very low in high altitudes.
There are m any areas around the world, endemic for
iodine deficiency.
In all other areas, drinking water, vegetables and
fruits contain m oderate amounts of iodine.
Nowadays, iodised salt is used for cooking in most
households.

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Dietary requirements of iodine

Adults 100–150 mg/ day
Pregnant and lactating women 200 mg/ day
●
Protein- bound iodine (PBI)

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Biochemical functions of iodine

Iodine is used for the synthesis of thyroid hormones
namely, thyrox ine (T4 ) and triiodothyronine (T3 ).
Disease states of iodine

Goitre (enlargement of thyroid gland). Hypothyroidism
and myx edem a occur in iodine deficiency. In children,
cretinism m ay occur.

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Zinc
Most of it occurs in the prostate and salivary glands.

Meat, fish, nuts, legumes, eggs and milk are good
sources of bioavailable zinc.
Adults 10–15 m g/ day
Pregnancy 20 mg/ day
(Continued…

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…Continued) Zinc
1. Zinc is an integral component of m any enzym es
(over 300 metalloenzymes) in the body, e.g.
carbonic anhydrase, alkaline phosphatase, alcohol
dehydrogenase.
2. It plays a crucial role in the synthesis and
stabilisation of DNA, RNA and proteins. It is an
integral component of DNA and RNA polym erases.
It forms zinc fingers.
(Continues…

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…Continued) Zinc
3. As a component of carbonic anhydrase, zinc plays

an important role in blood- gas transport and
renal regulation of pH.
4. Zinc is present in gusten, a salivary protein
im portant for the taste sensation.
5. It is required for norm al wound healing.
6. Zinc is required for normal sperm atogenesis.
7. It is required for embryonic development and fetal
growth.

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Manganese
1. Mn serves as a cofactor for a number of enzymes,
e.g. pyruvate carbox ylase, hex okinase, superox ide
dism utase, glutamine synthetase, arginase.
2. Mn as a component of glycosyl transferases plays a

role in the synthesis of glycoproteins and
glycosam inoglycans.
(Continues…

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…Continued)
Manganese
Dietary sources and requirement

Nuts, cereals, leafy vegetables and fruits are good
sources. Tea leaves are high in Mn content. The
requirement is about 5 mg/ day.

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Selenium
1. Selenium ex erts synergistic antiox idant function
along with vitamin E.
2. Selenium is a component of glutathione
perox idase.
3. As a constituent of iodothyronine deiodinase,
selenium is involved in the rem oval of iodine from
thyroid hormones.
4. Selenium is also a constituent of thioredox in
reductase

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…Continued) Selenium

Seafood, muscle meat and all kinds of cereals are rich
sources of selenium .
Dietary requirem ent is about 50–100 µg/ day.

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Fluorine
Fluoride is popular for its role in the prevention of

dental caries. It is im portant for the m aintenance of
the structure of teeth and bone. But when present in
ex cess, it is potentially dangerous.
1. Fluoride increases the resistance of enamel to acid
attacks.
2. Fluoride is essential for the normal development of
bony structure.
(Continues…

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…Continued) Fluorine
Dietary sources
Drinking water is the m ain source of fluoride.
Disease states

Dental caries

Fluorosis

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Chromium
1. Chrom ium potentiates the action of insulin.
2. Com ponent of a functional complex termed glucose
tolerance factor (GTF).
3. Cr lowers cholesterol levels in blood.
Dietary sources, requirements and metabolism

Brewer's yeast, meat and wholegrains are good sources.
Safe intakes of Cr are 50–200 µg/ day for adults.

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COBALT
The only role of cobalt in the hum an body is as a
component of vitamin B12 .
MOLYBDENUM
Molybdenum is an essential cofactor of a number of
enzymes, in particular, x anthine ox idase which converts
the end products of purine metabolism to uric acid.
It participates in nitrogen fix ation reactions as a
component of nitrite reductase.

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Section V
Nutrition
Universities Press
3-6-747/1/A & 3-6-754/1, Himayatnagar
Phone: 040-2766 5446/5447
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Chapter 2 2
Principles of Nutrition

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Humans derive their energy from food. Foods provide

both building m aterial and energy in the form of
nutrients to the body. Nutrients are substances that
are not synthesised in the body in sufficient amounts
and hence need be obtained through the diet.

Macronutrients

Micronutrients

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Calorific Value of Foods
Calorific value is the amount of heat energy generated

by burning 1 g of the food com pletely in the presence
of ox ygen.
Calorific values of major nutrients

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Respiratory Quotient (RQ)
Respiratory quotient is the ratio of the volum e of carbon

diox ide produced to the volume of ox ygen consumed
during the ox idation of food.
Carbohydrates: The volum e of carbon diox ide produced
is equal to the volume of ox ygen utilised when a
carbohydrate diet is consumed (RQ is 1).

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Lipids: The RQ of fats is relatively lower and is about

0.7
Proteins: The RQ of proteins cannot be m easured

directly. By indirect m ethods it is calculated to be
about 0.8.
Mixed diet: The RQ of a m ix ed diet is dependent upon

the relative proportions of carbohydrates, lipids and
proteins in it and in a well balanced diet, it is
approx imately around 0.8.

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Energy Expenditure
For weight to remain stable, energy intake must match

energy output. Energy ex penditure in man is attributed
to the following processes that occur in the body.
1. Basal m etabolic rate

2. Specific dynam ic action
3.Physical activity
Apart from these three energy determ inants, certain

physiological conditions such as age, pregnancy,
lactation, convalescence and so on demand ex tra
supply of energy.
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Basal Metabolic Rate
BMR is defined as the minim um am ount of energy

required by the body to m aintain life at complete
physical and mental rest.
While determining the BMR, the person should be

awake and in a post- absorptive state.

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Normal values of BMR

●
A healthy adult male has a BMR of about 35–38
Cal/ m 2 / h and adult female about 32–35 Cal/ m 2 / h.
●
More comm only, BMR is ex pressed as Cal/ day. For an
adult male, BMR is around 24 Cal/ kg body wt/ day
and for an adult fem ale, it is 22 Cal/ kg body wt/ day.

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Factors Affecting BMR

Age

Gender

Body surface area

Built

Clim ate

Race

Physical activity and ex ercise

Starvation
(Continues…

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…Continued) Factors affecting BMR

Fever

Hormones

Stress
Significance of BMR

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Specific Dynamic Action
The fact that the consumption of food is associated

with an ex penditure of energy is well established.
Utilisation of food is associated with the ex penditure of
energy resulting in increased heat production following
the ingestion of food.
This is known as specific dynamic action (SDA) or the
thermic effect of food.
Mechanism of SDA
(Continues…

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SDA …Continued)
SDA for different foods

The SDA for carbohydrates is 5%, for lipids 15% and
for proteins, it is about 30%.
SDA for a m ix ed diet is taken at 10% of overall
calories.
Significance of SDA

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Physical Activity

Provided the BMR and SDA are more or less constant,
physical activity is highly variable in different
individuals.

Energy requirements in average adults are roughly as
follows (energy required for BMR, SDA and physical
activity).
Light work 2000–2500 Cal/ day
Moderate work 2500–3000 Cal/ day
Heavy work 3000–4000 Cal/ day

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Total Daily Energy Expenditure (TDEE)
The body functions by producing energy and utilising it.

Even at rest, our body uses energy.
The total daily energy expenditure (TDEE) is calculated to

find out how the body ex pends energy or burns calories.
To calculate the TDEE or the energy requirem ent in humans,

three factors are taken into consideration:
(Continues…

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TDEE …Continued)
1 . Basal metabolic rate (BMR)

Also called resting metabolic rate (RMR), it is the
amount of energy the body uses during rest and is
approx im ately 60–70% of TDEE.
Three organs use m ost of the calories at rest. They
are the liver, brain and skeletal muscle, which
burn 2 7 , 2 0 and 1 8 per cent of the BMR
respectively .
2 . Specific dynamic action (SDA)

Also known as the thermic effect of food (TEF), it is the
energy the body uses to convert food into energy or to
move it to a location where it can be stored (as fat) for use
at later times. This accounts for about 10% of TDEE.
(Continues…

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TDEE …Continued)
3 . Thermic effect of physical activity (TEPA)

It varies greatly in individuals, based on the duration,
intensity and the type of physical activity they indulge
in.
TEPA makes up about 15–30% (could go up to 50% during

heavy work) of TDEE.
Post- exercise oxygen consumption (EPOC) is the

amount of energy the body burns after ex ercise to
return to its normal state. EPOC is considered as a part
of TEPA.

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TEPA Types
TEPA is of two types:
i.Planned ex ercise: It is the physical ex ercise undertaken in

a gymnasium or other sports- like activities. These activities
burn calories heavily.
ii. Non- ex ercise physical activities (NEPA): These are the

physical ex ertions that occur routinely in activities such as
standing up from a seated position and domestic work.

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Calculation of calorie 22
expenditure in humans

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Non- exercise Activity Thermogenesis (NEAT)
Ex pendit ure of energy in NEPA

Non- ex ercise activity therm ogenesis Energy ex pendi-
(NEAT), also called as non- ex ercise ture (Cal/ h)
physical activity (NEPA), refers to the

energy ex pended in all routine
physical activities other than planned
gym ex ercises and sports activities.
The current practice of calculating the

daily energy requirem ents considers
NEAT as a significant contributor to
TDEE.
Daily energy requirement =

BMR + SDA + NEAT
(Continues…

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NEAT . . . Continued)…
Usually, about 60-70% of calories are spent on BMR, 10-15%

towards SDA, and about 10-15% towards NEAT. In active
domestic workers, NEAT may go up to 50%.
Thus, NEAT plays a vital role in keeping the body fit and healthy.
Impairment in NEAT significantly contributes to obesity. This can

be observed from the fact that obesity is less common in active
homemakers and domestic helpers.

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Nutritional Importance of Carbohydrates
Dietary carbohydrates are major sources of energy.

Carbohydrates

Provide 50%–60% of the total calories.
Energy content of carbohydrates

The energy content of dietary carbohydrate is
approx im ately 4 Cal/ g.

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Dietary sources of
carbohydrates
1. Digestible carbohydrates
They are the chief energy sources of the
body. E.g. starch, glycogen, sucrose,
lactose, glucose, fructose.
2. Non- digestible carbohydrates
Also known as dietary fibre. Though they
are non- calorigenic they ex ert certain
beneficial effects in the body, e.g. cellulose,
hemicellulose, pectins, gum s.
(Continues…

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…Continued)
Common sources of digestible carbohydrates

include
Cereals (rice, wheat) (60%–80%)

Pulses (50%–60%)
Roots and tubers (20%–40%)
Bread (50%–60%)
Meat (40%–50%)
Vegetables and fruits (30%–50%)
Table sugar (100%), honey (80%)
(Continues…

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…Continued)
Sources of carbohydrates

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Dietary Fibre
Certain non- starch polysaccharides are resistant to

digestion by enzymes of the human small intestine
and are popularly known as dietary fibre.
Role of fibre in the body

1. Function on GIT
2. Beneficial in preventing cancers
3. In diabetes m ellitus
4. Hypolipidemic effect
5. Effect on obesity
(Continues…

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…Continued
Sources of dietary fibre

Leafy vegetables, fruits, other vegetables, legumes and
rice bran are rich sources of fibre.

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Glycemic Index (GI)
It is a nutritional measure of different carbohydrate

foods on blood glucose levels. Glycemic index of a food
item refers to the extent of the rise of blood glucose
following its intake.
Glycemic index is measured by performing the glucose

tolerance test (GTT) after ingesting the food item to
be assessed and comparing it with a reference meal.
The reference meal is usually taken as 50 g of
glucose.
(Continues…

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…Continued) Glycemic Index (GI)
Glycemic index is defined as the incremental area

under the GTT curve after eating 50 g of the test
meal (food item to be assessed) compared to the
incremental area of the GTT curve with an equal
quantity of the reference meal (50 g glucose).
(Continues…

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…Continued) Glycemic Index (GI)
Glycemc index curve

(Continues…

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Glycemic Index (GI)
…Continued)
Glycemic index of some common foods
(Continues…

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…Continued)
Glycemic index
Significance of Glycemic Index

1. Foods that digest quickly after ingestion and release
glucose rapidly into the blood have a high GI. These
foods with high GI speedily contribute to an
increase in blood sugar levels.
Foods with high glycemic index (GI > 70) include white
bread, table sugar (sucrose), all tubers (e.g.,
potatoes), polished rice, free glucose and maltose
powders, etc.
Note that most of the foods with high GI contain refined

sugars. (Cont inues…

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…Continued)
Glycemic index
2. Foods that digest slowly after ingestion and hence

release glucose slowly into the blood have a low GI
and contribute to blood glucose levels in tandem
with the insulin release.
This is because the digestion and absorption of
complex (raw) carbohydrates is slow because of the
large amounts of fibre in them and contribute to an
increase in blood sugar only slowly.
For ex ample, the GI of a complex carbohydrate (starch)
is lower than that of a refined carbohydrate
(glucose). Foods with low GIs include vegetables
and legumes, fruits, milk, whole grain cereals
(e.g., brown rice), ice cream, etc.
(Continues…

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…Continued) Glycemic index
3. When carbohydrates occur in the combined

form with fibre, fats and protein, the glycemic
index is low because of the delay in digestion
and absorption.
Ice cream has a low GI despite its high sugar
content because it also contains fat, which delays
its absorption.

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Glycemic Index - Clinical Significance
Glycemic index is a useful tool for form ulating

therapeutic diets for diabetic patients.
Foods with low glycem ic index prevent a steep
rise in blood sugar concentration after a meal
and are hence preferred.
Such foods also tend to potentiate the insulin
sensitivity of tissues.

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Nutritional Importance of Fats and Lipids
Sources of dietary lipids

Food item s such as butter, ghee, various vegetable oils
and fish oils, m ilk and m ilk products, egg, meat,
pulses and cereals are the dietary sources of fats and
lipids.

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Composition of oils and fats

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Essential Fatty Acids
Linoleic acid and linolenic acid are considered to be

two essential fatty acids (EFA) because neither can be
synthesised in the human body.

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Cholesterol and Heart Disease
●
Cereals, pulses and vegetables do not contain
cholesterol, which is obtained only from foods of
animal origin. It is well known that high
cholesterol levels (> 240 m g/ dl) are associated
with increased risk of coronary artery disease.
●
In norm al individuals, the cholesterol level in the
blood ranges between 150–200 mg/ dl (desirable
< 180 m g/ dl).
(Continued…

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…Continues) Cholesterol and heart disease
Cholesterol content of various foods of animal

origin

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Nutritional Importance of Proteins
Essential am ino acids

Amino acids and their nutritional requirements

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Nitrogen Balance

Negatie nitrogen balance

Positive nitrogen balance
Nitrogen balance in the body

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Nutritional Value of Proteins
Dietary sources of proteins

Meat, eggs, fish, m ilk are good sources of protein.
Legum es, beans, peanuts, peas, soyabean are the best
plant sources.
How much protein do we need?
The recom mended dietary allowance (RDA) is 0.75 g/ kg
body weight/ day is sufficient for a norm al adult.

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Common dietary sources and their protein content

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Biological Value (BV)
Biological value is the ratio of the am ount of nitrogen

retained in the body and nitrogen absorbed.
Though BV is a widely used index to ex press the

nutritional value of proteins, it has certain inherent
defects. It does not take into account the nitrogen which
is lost during digestion and hence it may not provide an
accurate nutritional assessm ent of proteins.

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Net Protein Utilisation (NPU)
This is a better nutritional index com pared to BV as it

takes into account the factor of digestion. It is the ratio
between the nitrogen retained in the body to total
nitrogen ingested .

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Protein Efficiency Ratio (PER)
The quality of a protein can be determined by its ability

to produce growth. It is the assessm ent of weight gain
per gram of protein taken. PER is the m easurement of
the ability of a protein to prom ote growth in an animal.

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Balanced Diet
A balanced diet is com posed of a variety of foods

which contain all the essential nutrients such as
carbohydrates, fats, proteins and micronutrients in the
right proportions
A mix ed diet for an

average adult
(weighing 60 kg)

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Principles of prescribing a diet

Calorie requirem ent has to be assessed based on
age, gender, build, health status and physical
activity.

Protein requirem ent has to be assessed based on
age, gender, build, health status and physical
activity.
(Continues…

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…Continued) Principles of prescribing a diet

Protein requirement can be calculated as
0.8 g/ kg body weight/ day. During growing age,
pregnancy and lactation it can be m ore than 2 g/ kg
body wt. Protein can be obtained as anim al protein
or m ix ed vegetable protein. The calorie yield from a
protein diet should be 10%–15% of total calorie
requirem ents.
(Continues…

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CHAPTER
22
●
Fats can provide 20%–25% of total calorie requirem ents
of the individual. Visible (fats and oils) should be
restricted to not m ore than 20 g/ day for a healthy
adult.
However, in various physiological states it can be

enhanced, such as growing children (25 g), pregnant
women (30 g) lactating mothers (45 g). It is advisable
that obese individuals, old people and persons at risk
of cardiovascular disease restrict fat content to less
than 20 g a day. Different vegetable oils can be used to
balance the composition of SFA, MUFA and PUFA to
1:1:1.
(Continues…

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CHAPTER
22
●
Choose a diet with plenty of whole grains,
vegetables and fruits and green leafy foods which
contain both digestible carbohydrates and dietary
fibre. Ideally 55%–65% of calories can be obtained
from carbohydrate diet.

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CHAPTER
22
Food Guide Pyramid
Food pyramid : A
guide to
balanced diet

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CHAPTER
22
Dietary Guidelines during Pregnancy
What a pregnant woman eats or drinks during

pregnancy forms her baby’s main source of nutrition.
Hence, it is essential that her diet contain a variety of
foods that provide the required nourishm ent for the
baby’s optim um growth and developm ent.
It should be noted that all the regular nutrients of

an adult have to be supplied in increased
quantities, as pregnancy makes additional
demands on the same for the baby’s growth—
especially more protein, calcium, iron and folic acid.
(Continues…

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CHAPTER
22
…Continued) Dietary guidelines during pregnancy
Protein: Since protein is considered a ‘builder

nutrient’, adequate amounts of protein are needed
during pregnancy. Food sources rich in proteins
include meat, poultry, fish, eggs, nuts, dried beans
and peas.
Calcium: This m ineral is crucial to build a baby’s

bones and teeth. If a pregnant woman does not
consume enough calcium, the mineral will be drawn
from her bones to meet the extra demands of
pregnancy.
(Continues…

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CHAPTER
22
…Continued) Dietary guidelines during pregnancy
Nowadays, many dairy products are fortified with

vitamin D, another essential nutrient that works with
calcium to develop the baby’s bone and teeth.
Pregnant wom en need to consum e about 1 ,2 0 0 - 1 ,5 0 0

mg of calcium daily.
Food sources include milk, yogurt, cheese, fish (eaten

with bones), leafy vegetables, eggs and other calcium-
fortified foods.
(Continues…

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CHAPTER
22
…Continued)
Dietary guidelines during pregnancy
Iron: Pregnant women need up to 4 0 mg of iron per day,

which is almost double the amount needed normally. This
is essential to m aintain an optim um supply of ox ygenated
blood to the baby.
Insufficient intake of iron during pregnancy can lead to

anem ia, which increases the risk of infections during
pregnancy.
Foods rich in vitamin C, e.g., orange juice, when consumed

with iron- rich foods, increases the absorption of iron.
Food sources for dietary iron include jaggery, meat, poultry,

fish, leafy vegetables, peas, dry fruits and iron- fortified
cereals. (Continues…

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CHAPTER
22
…Continued)
Dietary guidelines during pregnancy
Folic acid: The B- com plex vitamin folate is known to

prevent birth defects in the baby’s brain and spinal
cord (neural tube defects).
It has been observed that the diet alone is not always

able to provide the required folate. Hence, a daily
folate supplement of 400 m icrogram s is
recom mended during pregnancy.
Food sources include green leafy vegetables, beans,

fruits, fortified cereals, eggs and dairy products.

Press
CHAPTER
Dietary Guidelines in Coronary Artery 22
Disease (CAD)
• Limit the cholesterol in the diet to less than 200 mg per
day.
• Take adequate amounts of PUFA. They are required for
the esterification of cholesterol and its subsequent
rem oval. ω - 3 fatty acids reduce LDL- C levels.
• Restrict the saturated fats and trans- unsaturated fatty
acids to less than 10% of the total calorie intake. Ex cess
consumption of these tends to interfere with the
lipoprotein metabolism. They increase the levels of
LDL- C and decrease HDL- C. They also reduce
mem brane fluidity.
(Continues…

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CHAPTER
22
…Continued) Dietary guidelines in coronary artery disease (CAD)
• Restrict the overall intake of fat to no more than 20%

of the total energy intake.
• Add good am ounts of fibre in the diet. They increase

the reabsorption of bile salts and decrease the
absorption of dietary cholesterol.
• Restrict the consumption of refined carbohydrates and

sucrose. Sucrose steeply raises the TAG levels. Refined
carbohydrates, when consumed in ex cess, are easily
converted to fats and stored.
(Continues…

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CHAPTER
22
…Continued) Dietary guidelines in coronary artery disease (CAD)
• Consum e sufficient am ounts of vitamin C, E and beta

carotene. These are natural antiox idants that prevent
the formation of ox idised LDL. Low dietary intake of
folate, B12 and B6 elevates homocysteine
concentrations, which is dangerous.
• A m oderate intake of alcohol (2–4 units a day) offers

some protection from coronary artery disease. However,
restricting the intake of alcohol to moderate amounts is
easier said than done. Heavy drinking can lead to
diseases of the heart m uscle, called cardiomyopathy,
besides m any other health problems.

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CHAPTER
22
Diabetic Diet
Doctors are frequently called upon to give dietary advice

to diabetic patients. The aim of the diet is to keep the
blood glucose level as close to normal values as
possible. To achieve this, certain dietary guidelines
have to be followed by the patient.
• Refined carbohydrates, sweets, sugar are to be

avoided.
• Foods with a low glycemic index are to be chosen so

that elevation in blood glucose level is not rapid.
(Continues…

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CHAPTER
22
…Continued) Diabetic Diet
• Frequent, sm all meals are recommended with a proper

distribution of calories.
• Roots and tubers should be restricted. Leafy vegetables

can be taken liberally as they contain large am ount of
dietary fibre.
• The intake of cholesterol- rich foods should be reduced.

PUFA should constitute about one- third of the total fat
intake.

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CHAPTER
22
Obesity
Obesity is the term used to describe individuals with

ex cess body fat. Ex cess calorie intake coupled with the
lack of sufficient physical activity results in obesity.
Body mass index (BMI): It is used to assess the degree of

by taking the weight of a person (in kg) and
dividing it by the person’s height squared (in m 2 ) .
(Continues…

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CHAPTER
22
…Continued) Obesity
Obesity is the result of the ingestion of food in excess of the

body’s needs.
It could occur during childhood (juvenile-onset) or in adults.
In juvenile-onset obesity, there is an increase in both the

number and size of the adipocytes, whereas in adult
obesity, fat is accumulated only in already existing
adipocytes.
A certain degree of genetic predisposition exists in the

development of obesity (if one of the parents is obese, there
is a 50% chance of their wards becoming obese)

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CHAPTER
22
Protein- Energy Malnutrition (PEM)
PEM is m ostly seen in young children due to

malnutrition. The clinical picture of PEM is manifested
in two forms.
1. Kwashiorkor; 2. Marasmus
Kwashiorkor
Kwashiorkor represents protein malnutrition in children
characterised by edema.
Marasmus
Marasmus is the result of severe m alnutrition in infants
characterised by severe m uscle wasting.
(Continues…

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CHAPTER
22
…Continued) PEM
Comparison between kwashiorkor and marasmus

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CHAPTER
23
Section VI
Clinical Biochemistry
Universities Press
3-6-747/1/A & 3-6-754/1, Himayatnagar
Phone: 040-2766 5446/5447
Limited
Rafi MD: Textbook of Biochemistry (4thEdition) Universities Press
CHAPTER
23
Chapter 2 3
Water and Electrolytes
Limited
CHAPTER
23
Water

Role of water

Distribution of water in the body

Water balance

Water input

Water output
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CHAPTER
23
Distribution of Water in the Body
Limited
CHAPTER
23
Water Balance in Humans
Limited
CHAPTER
23
Electrolytes – Distribution and Balance

Electrolytes are well distributed in body fluids to
maintain osmotic equilibrium and water balance.
Na+ is the principal cation of ECF, while K+ is the
chief cation of ICF.

Osmolarity: Osmotic pressure ex erted by the
num ber of m oles per litre of solution.

Osmolality: Osmotic pressure ex erted by the
num ber of m oles per kg of solvent. Osm olality is
more comm only used than osmolarity in clinical
practice.
(Continues…
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CHAPTER
23
…Continued Electrolytes – distribution and
balance
Composition of electrolytes in body fluid compartments (mEq/ L)
(Continues…
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CHAPTER
23
…Continued) Electrolytes – distribution and
balance
Osmolality of plasm a: The osmolality of plasm a is in

the range of 280–300 milliosm oles/ kg.
(Continued…
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CHAPTER
23
…Continued)
Constituents contributing to plasma osmolality
(Continues…
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CHAPTER
23
…Continued)

Regulation of water and electrolyte balance

Aldosterone

Anti- diuretic hormone (ADH)

Renin- Angiotension system
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CHAPTER
23
Dehydration
Causes of dehydration
Dehydration m ay occur as a sequel to diarrhea,
vom iting, ex cessive sweating, fluid loss in burns,
adrenocortical dysfunction, kidney diseases (e.g. renal
insufficiency), deficiency of ADH (diabetes insipidus).
Features of dehydration
The clinical m anifestations of severe dehydration include
increased pulse rate, low blood pressure, sunken
eyeballs, decreased skin turgor, lethargy, confusion and
eventually coma.
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CHAPTER
23
Treatment of Dehydration
●
Intake of plenty of water is a simple and effective
method of treating dehydration. In persons who
cannot ingest it orally, water can be given via a
nasogastric tube.
●
If this is not possible, water should be given
intravenously (IV) in an isotonic solution, usually as
5% glucose (5% dex trose). If there is an associated
electrolyte depletion, dex trose- saline (4% dex trose,
0.18% NaCl) should be given intravenously.
(Continues…
Limited
CHAPTER
23
Treatment of dehydration
…Continued)
●
When sodium depletion alone is the problem , giving
intravenous isotonic fluid – normal saline (0.9% NaCl)
or plasma ex panders or album in is the first line of
managem ent of salt deficit.
●
After restoring the intravascular volume, the
underlying cause m ust be treated.
Limited
CHAPTER
23
…Continued) Treatment of dehydration
Oral Rehydration Therapy (ORT)

Oral rehydration therapy is used to treat acute
diarrheas due to all etiologies and in all age groups.
The aim of oral fluid therapy is to prevent dehydration
and reduce m ortality.
Glucose and salts (electrolytes) in water are the

ingredients of oral rehydration solutions (ORS)
commonly used.
(Continues…
Limited
CHAPTER
23
…Continued)
Oral Rehydration Therapy (ORT)
It is a sim ple mix ture made of table salt (one level

teaspoon) and sugar (6 level teaspoons) dissolved in
one litre of drinking water. The solution should be
made freshly daily and used within 24 hours. It should
not be boiled or otherwise sterilized. These simple
home- made ORS are life- saving in cases of acute
dehydration.
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CHAPTER
23
Electrolytes - Sodium
Sodium is the principal cation of the ex tracellular

fluid.
Dietary sources
Comm on salt (NaCl) used in cooking medium is the
major source of sodium. Whole grains, nuts, eggs,
leafy vegetables, milk and bread are good sources of
Na+ .
(Continues…
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CHAPTER
23
…Continued) Electrolytes - Sodium
Dietary requirements
The recom mended dietary allowance (RDA) of sodium
is about 5–10 g/ day.
Sodium in ECF: The normal concentration of sodium in

plasm a is 135–145 m Eq/ L.
(Continues…
Limited
CHAPTER
23
1 . Sodium regulates the body's acid–base balance
along with chloride and bicarbonate. It is involved in
forming a bicarbonate buffer system (NaHCO3 –
H2 CO3 ) and a phosphate buffer system (NaH 2 PO4 –
Na2 HPO4 ). These buffer systems play an im portant
role in the acid–base balance.
2. Sodium plays a role in maintaining osmotic pressure

and fluid balance.
(Continues…
Limited
CHAPTER
23
3. Sodium is important in muscle ex citability and is

necessary for initiating and maintaining the
heartbeat.
4. Sodium plays a role in cell permeability.
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CHAPTER
23
Diseases States
Hyponatremia
When the plasma sodium level falls below normal, the
condition is referred to as hyponatrem ia. The causes
include
1. Vomitting
2. Diarrhea
3. Burns
4. Addison's disease (adrenocortical insufficiency)
5. Renal tubular acidosis (tubular reabsorption of
sodium is defective)
6. Severe sweating
(Continues…
Limited
CHAPTER
23
…Continued)
Hypernatremia
This condition is marked by an elevation in the plasm a
sodium level.
Causes of hypernatremia include:
1. Cushing's syndrom e (adrenocortical hyperactivity)

2. Prolonged cortisone therapy
3. Pregnancy (steroid hormones cause sodium
retention in the body to som e ex tent)
4. Dehydration (when water is lost, the blood volume
is decreased with apparent increase in the
concentration of sodium)
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CHAPTER
23
Potassium
Potassium is the m ajor intracellular cation.
Dietary sources: Banana, orange, pineapple, potato,

beans, chicken and organ meat (liver) are good sources
and coconut water has very high content of potassium.
RDA: About 3–4 g/ day.
Potassium in the ECF and ICF: The plasma

concentration of potassium is 3.5–5.0 mEq/ L.
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CHAPTER
23
Biochemical Functions of Potassium
1. Potassium maintains intracellular osmotic pressure.

Movem ent of water across the biological m embranes
is dependent on the osmotic pressure differences
between ICF and ECF. In a healthy state, the osmotic
pressure of ECF (mainly due to Na+ ions) is equal to
the osmotic pressure of ICF (contributed by K+ ions).
As such, there is no net passage of water molecules
in or out of the cells maintaining osm otic
equilibrium.
(Continues…
Limited
CHAPTER
23
…Continued) Biochemical functions of potassium
2. It plays a role in the regulation of acid –base and

water balances in the cells.
3. The enzyme, pyruvate kinase (glycolysis) is

dependent on K+ for its optimal activity.
4. Potassium is required for the transmission of nerve

impulses.
Limited
CHAPTER
23
Disease States
Hypokalemia
Hypokalem ia is manifested as muscular weakness,
impaired m yocardial contractility leading to cardiac
arrhythmias and even cardiac arrest.
Hypokalem ia is observed in hyperactivity of the adrenal

cortex (Cushing's syndrom e).
(Continues…
Limited
CHAPTER
23
…Continued)
Hyperkalemia
Hyperkalem ia, leads to ventricular arrhythmia,
ventricular fibrillation, bradycardia and m ay lead to
cardiac arrest .
Decreased potassium ex cretion can occur in

mineralocorticoid deficiency (Addison's disease) and by
potassium- sparing diuretics.
Limited
CHAPTER
24
Section VI
Universities Press
3-6-747/1/A & 3-6-754/1, Himayatnagar
Phone: 040-2766 5446/5447
Limited
Rafi MD: Textbook of Biochemistry (4 th Edition) Universities Press
CHAPTER
24
Chapter 2 4
Acid- Base Balance
Limited
CHAPTER
Source of Hydrogen Ion 24
Gains and Losses
Limited
CHAPTER
24
Hydrogen Ion Homeostasis
There are three lines of defence operative in

maintaining blood pH.
I. Buffer system s
II. Respiratory m echanism
III. Renal regulation
Limited
CHAPTER
24
Buffer Systems

Buffers are the solutions which resist change in pH
on the addition of sm all amounts of acids or bases.
Any substance that can reversibly bind hydrogen
ions can be labelled as a buffer.

Buffers can neither remove H + ions from the body
nor add them to it. They can only keep H + ions in a
temporarily suspended form. The H + ions have to be
ultimately eliminated by the kidneys.
Limited
CHAPTER
24
Major Buffer Systems
There are three m ajor buffer systems operative in

the human body.
1. Bicarbonate buffer
2. Phosphate buffer
3. Protein buffer
Limited
CHAPTER
24
Bicarbonate Buffer
 Bicarbonate and carbonic acid pair (NaHCO3 – H2 CO3 )

is the predominant buffer system in the ex tracellular
fluid.

The concentration of the bicarbonate ion is not
directly m easured; it is calculated from the
Henderson–Hasselbalch equation.
Limited
CHAPTER
24
Handerson–Hasselbalch Equation
(Continues…
Limited
CHAPTER
…Continued) 24
This equation is known as

the Henderson–
Hasselbalch equation
and is applicable for any
buffer pair.
(Continues…
Limited
CHAPTER
…Continued) 24
The concentration of H2 CO3 = 40 × 0.03 = 1.2 mmol/ L.
The Henderson–Hasselbalch equation for the bicarbonate

buffer pair is
Substituting the values (plasma pH = 7.4; pKa for H 2 CO3 = 6.1;

HCO3 – = 24 mmol/ L; H2 CO3 = 1.2 mmol/ L), we can see that
(Continues…
Limited
CHAPTER
…Continued) 24
From this, it is seen that the concentration of bicarbonate is

almost 20 times more than that of carbonic acid in the
blood.
This is referred to as the alkali reserve, and it is essential

for meeting the challenge of buffering the H + ions
generated from the huge body acid–load.
Limited
CHAPTER
24
Respiratory Regulation of pH
The respiratory system and the kidneys together

maintain the body pH. The respiratory response to
the altered plasma pH is very swift (within minutes)
compared to the slow but steady response of the
kidneys (hours to days).
Limited
CHAPTER
24
Chloride Shift
Generation of bicarbonate by the erythrocytes
Limited
CHAPTER
24
Renal Regulation of pH
The basic mechanisms of renal regulation of pH

include:
1. Reabsorption of bicarbonate
2. Addition of new bicarbonate to the plasma
3. Ex cretion of amm onium ions
4. Ex cretion of free H+ ions.
Limited
CHAPTER
24
Reabsorption of Bicarbonate
Limited
CHAPTER
Addition of New Bicarbonate to 24
the Plasma
Limited
CHAPTER
24
Excretion of Ammonium Ions
Limited
CHAPTER
24
Excretion of Free H + Ions
Limited
CHAPTER
Clinical Causes and Biochemical 24
Mechanisms of Acid- Base Disorders
Limited
CHAPTER
24
Anion Gap
The difference between the sums of the concentrations

of the measured cations (Na+ and K+ ) and that of
measured anions (C1 – and HCO3 –) is known as the anion
gap.
Thus, the anion gap represents unmeasured anions in

the plasma. It can be calculated as:
(Continues…
Limited
CHAPTER
…Continued) 24
The anion gap in normal health is about 12 –18 mEq/ L.
The alterations in the anion gap are useful in the

clinical assessm ent of acid–base disorders.
Limited
CHAPTER
24
Acid- Base Disorders
Metabolic acidosis
The primary abnormalities in m etabolic acidosis are:

Increased production or decreased ex cretion of H +
ions or both at once.
 Loss of HCO3 – from the body
Causes of metabolic acidosis


Severe uncontrolled diabetes mellitus

Diabetic ketoacidosis (DKA).

Starvation ketosis, lactic acidosis and renal failure
(Continues…
Limited
CHAPTER
24
…Continued)
Management
Metabolic acidosis can be effectively m anaged by
identifying and treating the underlying cause. This
includes treatm ent of diabetes, control of diarrhea,
correction of shock and so on.
Limited
CHAPTER
24
Metabolic Alkalosis
The primary abnorm ality in metabolic alkalosis is an

increase in plasma HCO3 – concentration with a
consequent reduction in H + ion.
Causes

Sustained vom iting

Bicarbonate administration for prolonged periods as
a m eans of treating indigestion and overuse of
antacids for treatment of dyspepsia result in
metabolic alkalosis.

Overdose of diuretic drugs
(Continues…
Limited
CHAPTER
24
…Continued)

Mineralocorticoid ex cess (Cushing's syndrome,
Conn's syndrome), alkalosis occurs due to increased
H+ ion ex cretion in the urine

Principles of management include correcting the
underlying cause, adequate intravenous fluid
administration and replacem ent of K+ correcting
hypokalem ia.
Limited
CHAPTER
24
Respiratory Acidosis
The primary abnorm ality in respiratory acidosis is

retention of CO2 ( H2 CO3 ) due to im paired alveolar
ventilation resulting in a rise in pCO2 .
Causes
• Airway obstruction and pulm onary disease – chronic
obstructive airway disease (emphysema, bronchitis
and so on)
– severe asthma
– bronchopneumonia
– respiratory distress syndrom e (RDS)
– flail chest
(Continues…
Limited
CHAPTER
…Continued 24
- Depression of the respiratory centre by the

administration of overdose of anesthetics,
sedatives and narcotics.
- Neurom uscular diseases like poliomyelitis,

Guillian–Barre syndrome, motor neuron disease
and so on.
Management involves correction of causative
factors. The general principle of treating
respiratory acidosis is to improve alveolar
ventilation and lower pCO2 .
(Continued…
Limited
CHAPTER
24
Respiratory Alkalosis
The prim ary abnormality in respiratory alkalosis is a

decrease in H2CO3 concentration. A fall in pCO2,
reduces the ratio of carbonic acid to bicarbonate
resulting in acidosis.
Causes
• Hyperventilation resulting from hysteria and anx iety
states cause respiratory alkalosis.

Raised intracranial pressure and brain stem lesions
cause hyperventilation by stim ulating the
respiratory centre. Ingestion of certain drugs
(salicylates) also stimulates the brain stem
respiratory centre.
(Continues…
Limited
CHAPTER
24
…Continued)

Hypox ia occurring in high altitudes, severe anaem ic
conditions and pulmonary disease
Principles of management include

correcting the underlying causes, alleviating
anx iety. In acute cases, the patient is
subjected to re- breathing into a closed bag
to allow CO2 levels to rise.
Limited
CHAPTER
Acid–base disorders with primary 24
changes and compensatory

mechanisms
Limited
CHAPTER
25
Section VI
Universities Press
3-6-747/1/A & 3-6-754/1, Himayatnagar
Phone: 040-2766 5446/5447
Limited
CHAPTER
25
Chapter 2 5
Organ Function Tests
Limited
CHAPTER
25
Liver Function Tests
Major Functions of the Liver
1 .Metabolic function
Carbohydrate metabolism: Glycolysis, glycogen synthesis,

breakdown, gluconeogenesis and HMP shunt.
Lipid metabolism: Fatty acid synthesis, ketogenesis,

cholesterol synthesis and ex cretion, bile acid synthesis, 25-
hydrox ylation of vitam in D and lipoprotein synthesis.
Protein metabolism: Urea synthesis from am m onia, synthesis

of plasm a proteins (barring im m unoglobulins) and synthesis of
som e coagulation factors.
Hormone metabolism: Metabolism and ex cretion of steroid

horm ones and m etabolism of peptide horm ones. (Continues…
Limited
CHAPTER
…Continued) 25
2 . Synthesis function
Synthesis of plasm a proteins, clotting factors, cholesterol,
TAG and lipoproteins.
3 . Excretory function
Ex cretion of bile pigm ents and bile salts into the bile.
4 . Detoxification
Am m onia is detox ified to urea; drugs and other x enobiotics
are detox ified and ex creted.
5 . Regulation of blood glucose levels

By hepatic glycogenolysis and gluconeogenesis.
6 . Storage function
Glycogen, vitam ins A, D, K, B12 and iron.
Limited
CHAPTER
25
Classification of Liver Function Tests
A. Biochemical classification
I. Tests based on liver excretory function
i. Serum- bilirubin: Total, conjugated and

unconjugated
ii. Urine: Bile pigm ents, bile salts and urobilinogen
(Continues…
Limited
CHAPTER
25
…Continued
II. Liver enzymes

i. Alanine am inotransferase (ALT)
ii. Aspartate am inotransferase (AST)
iii. Alkaline phosphatase (ALP)
iv. Gamm a glutamyl transpeptidase (GGT)
III. Tests for synthesis function of liver

i. Total proteins
ii. Serum albumin, globulins, A/ G ratio
iii. Prothrom bin tim e
(Continued…
Limited
CHAPTER
25
…Continued
IV. Special tests

i. Ceruloplasmin
ii. Ferritin
iii. 1 - Antitrypsin
iv. - Fetoprotein
(Continues
Limited
CHAPTER
25
…Continued
B. Clinical classification
I. Markers of liver dysfunction
i. Serum bilirubin: Total, conjugated
ii. Urine: Bile pigments, bile salts and
urobilinogen
iii. Total protein, serum albumin and
A/ G ratio
iv. Prothrom bin tim e
v. Blood ammonia
(Continued…
Limited
CHAPTER
25
…Continued
II. Markers of hepatocellular injury

i. Alanine aminotransferase (ALT)
ii. Aspartate aminotransferase (AST)
III. Markers of cholestasis (obstruction to the flow

of bile)
i. Alkaline phosphatase (ALP)
ii. Gamma glutam yl transpeptidase (GGT)
Limited
CHAPTER
25
Bilirubin
Serum bilirubin
Norm al serum bilirubin level varies from 0.2 to 1.0
mg/ dl. Of this, the unconjugated bilirubin (bilirubin –
albumin complex ) varies from 0.2 to 0.4 mg/ dl and
conjugated bilirubin varies from 0.2 to 0.6 m g/ dl.
Van den Bergh reaction

Jaundice is the m ost com mon characteristic clinical
manifestation occurring due to fluctuations in serum
bilirubin.
(Continues…
Limited
CHAPTER
25
…Continued
There are three types of jaundice
1. Hemolytic jaundice (pre- hepatic jaundice)
2. Obstructive jaundice (posthepatic jaundice)
3. Hepatic jaundice (intra- hepatic jaundice)
Limited
CHAPTER
25
Causes of Different Types of Jaundice
Limited
Tests for the differential
CHAPTER
25
diagnosis of jaundice
Limited
CHAPTER
25
Metabolic Function

Galactose tolerance test

Plasma amino acid test

Anti- pyrine breath tests
Limited
CHAPTER
25
Synthesis Function

Serum albumin

Ceruloplasmin

A1- Antitrypsin

Haptoglobin

A2- Macroglobin

Transferrin

A- Fetoprotein

Prothrom bin tim e (PT)

Electrophoretic separation of plasma proteins
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CHAPTER
25
Tests Based on Enzyme Estimations
Aminotransferases or transaminases

alanine aminotransferase (ALT)

aspartate aminotransferase (AST)
The levels of aminotransferases in serum are elevated in

all liver diseases. The normal concentrations of ALT and
AST in plasma are 10–35 IU/ L and 8–20 IU/ L respectively
(Continues…
Limited
CHAPTER
25
…Continued
Alkaline phosphatase (ALP)

Bile duct obstruction induces the synthesis of ALP by
biliary tract epithelial cells. Hence, high levels of ALP
(normal 3–13 KA units/ dl) indicate
obstructive/ posthepatic jaundice.
Gamma glutamyl transpeptidase (GGT)
Elevated levels of GGT are associated with chronic
alcoholism . Its levels are also increased (norm al 5–40
IU/ L) in biliary obstruction, pancreatic disease and
chronic obstructive pulm onary disease (COPD) Its
activity is usually parallels to that of ALP.
(Continues…
Limited
CHAPTER
25
…Continued)
Nucleotidase
The activity of 5'- nucleotidase (norm al 2 –15 IU/ L) is
elevated in obstructive liver disease
Limited
CHAPTER
25
Tests Based on Detoxification Function
Blood Ammonia Test

Blood amm onia estim ation serves as an index of urea
synthesis by the liver. Raised am monia in the plasma is
suggestive of cirrhosis. Arterial blood should be used for
blood amm onia estim ation. Estim ation of am monia is
used to ex clude or diagnose hepatic failure in patients
with unex plained stupor or coma. In neonates with
suspected urea cycle disorders, amm onia estim ation is
indicated.
Limited
CHAPTER
25
Renal Function Tests
Diagrammatic representation of a brief summ ary of nephron function

Limited
CHAPTER
25
Classification of Renal Function Tests
Kidney function tests m ay be divided into four groups.

I. Tests to assess glomerular function
All the clearance tests (creatinine, urea and so on) are
useful in measuring the glomerular filtration rate
(GFR). Measuring the proteinuria reflects glom erular
perm eability.
II. Tests to assess tubular function
Urine concentration and dilution tests, urine
acidification test and secretion tests are used to tex t
tubular function.
(Continues…
Limited
CHAPTER
25
…Continued
III. Analysis of blood/ serum

Estimation of blood urea, serum creatinine, protein
and electrolytes are often used to assess renal
function.
IV. Complete urine analysis
Ex am ination of urine for volume, pH, specific gravity,
osmolality and presence of any abnormal constituents
is also useful to assess the kidney function.
(Continues…
Limited
CHAPTER
25
Creatinine Clearance Test
Creatinine clearance m ay be defined as the volum e of

plasm a that would be com pletely cleared of creatinine
per m inute.
U = Urine concentration of creatinine

V = Urine output in ml/ min
(24- hour urine volume divided by 24 × 60)
P = Plasm a concentration of creatinine
(Continues…
Limited
CHAPTER
25
…Continued
Reference volumes: The norm al range of creatinine

clearance is about 120–145 m l/ min.
Diagnostic application: A decrease in creatinine

clearance (< 75% normal) indicates a decreased GFR
reflecting renal dam age.
Limited
CHAPTER
25
Estimated GFR (eGFR)
An indirect and much sim pler method of estim ating

creatinine clearance (and thereby GFR) is by using
serum creatinine level. It is called as eGFR, and a
formula known as the Cockcroft- Gault equation is
em ployed to estim ate it.
(Continues…
Limited
CHAPTER
25
…Continued
The factor 0.85 is used to estimate the creatinine

clearance in fem ales based on the estim ation that they
have 15% less muscle m ass compared to m ales. eGFR
is particularly used in patients of chronic kidney
disease.
Creatinine coefficient: It is the amount of creatinine

and creatine put together ex creted in mg/ kg body
weight.
Normal range Males: 20- 28 m g/ kg
Females: 15- 22 mg/ kg
Its value is elevated in muscular dystrophy.
Limited
CHAPTER
25
Urea Clearance Test
Urea clearance is defined as the volum e of plasma that

would be completely cleared of urea per m inute. It is
calculated by:
Cm = Max imum urea clearance

U = Urea concentration in urine (mg/ dL)
V = Urine ex creted per minute in mL
P = Urea concentration in plasm a (m g/ mL)
(Continues…
Limited
CHAPTER
25
…Continued)
The above calculation is applicable when the output of

urine is m ore than 2 m L per minute. This is referred to
as m ax imum urea clearance (Cm ) , and the norm al
value is about 75 mL/ min.
Diagnostic interpretation: A urea clearance value below
75% normal indicates renal damage.
Limited
CHAPTER
25
Inulin Clearance Test
Inulin clearance (Cin ) is calculated by:
Uin = Urine concentration of inulin

V = Urine output in m L/ min
Pin = Plasm a concentration of inulin
Diagnostic importance: The norm al inulin clearance

rate is 125 ml/ min. A comparison of the clearance of
a given substance with the clearance of inulin
provides information about the renal transport
processes.
Limited
CHAPTER
25
Tests for Tubular Function
• Specific gravity and osmolality of urin

• Urine concentration test
• Urine dilution test
Limited
CHAPTER
25
Analysis of Blood/ Serum
Estimation of blood urea (15–40 mg/ dl) and serum

creatinine (0.7–1.5 m g/ dl) are prescribed to assess
the kidney function. Serum creatinine is more
sensitive than blood urea
Complete urine analysis

Physical ex am ination, chemical analysis and analysis
for abnorm al constituents in urine provide clues
about the status of renal function.
Limited
CHAPTER
25
Gastric Function Tests

Fractional test meal (FTM)

Pentagastrin stimulation test

Augm ented histam ine test

Alcohol test meal

Tubeless gastric analysis
Limited
CHAPTER
25
Pancreatic Function Tests

Measurement of pancreatic enzymes

Secretin–cholecystokinin test
Limited
CHAPTER
26
Section VI
Universities Press
3-6-747/1/A & 3-6-754/1, Himayatnagar
Phone: 040-2766 5446/5447
Limited
Rafi M D: Textbook of Biochemistry (4 th Edn) Universities Press
CHAPTER
26
Chapter 2 6
Biochemistry of Cancer
Limited
CHAPTER
26
Etiology of Cancer
I. Predisposing factors

Age

Heredity

Environmental factors
– Lifestyle
– Diet
– Occupation
– Iatrogenic

Acquired pre- cancerous disorders
Limited
CHAPTER
26
Carcinogenic Agents
Carcinogens that cause cancer can be divided into

three broad groups.

Physical: Radiant energy

Chemical: A large variety of chemical com pounds

Biological: Oncogenic viruses
Limited
CHAPTER
26
Radiant Energy
Radiations can cause cancer mainly in two ways.
i) Direct effect:
Radiation dam ages DNA by events such as single or
double strand breaks, elim ination of
purine/ pyrimidine bases, cross linking of strands
and formation of pyrim idine dim ers.
By inflicting injuries by the above m echanisms,
radiations like X- rays, - rays, UV- rays harm to the
DNA and are m utagenic.
(Continues
…
Limited
CHAPTER
26
…Continued
ii) Indirect effect: Ex cessive radiation by - rays and X-

rays produce free radicals (reactive ox ygen species)
which interact subsequently with DNA and other
m acromolecules in the cells causing molecular
dam age
Limited
CHAPTER
26
Chemical Carcinogens
A selected list of chemical carcinogens
(Continues…
Limited
CHAPTER
26
…Cont inued)
A selected list of chemical carcinogens
Limited
CHAPTER
26
Oncogenic Viruses
A selected list of viruses causing human cancers
Limited
CHAPTER
26
Moecular Basis of Cancer
●
Certain genetic alterations in cells cause their
uncontrolled m ultiplication. Two types of regulatory
genes – oncogenes and antioncogenes have been
identified as crucial players in the development of
cancer.
Limited
CHAPTER
26
Oncogenes
●
The genes that can cause cancer are known as
oncogenes. They were first identified in tum our-
causing viruses (Rous sarcoma virus). These viral
oncogenes were later found to be intim ately similar
to certain genes present in normal host cells which
are called protooncogenes. These are the genes that
norm ally encode for growth- regulating proteins in
the human body. The conversion of protooncogenes
to oncogenes is the crucial factor in the causation of
cancer.
Limited
CHAPTER
26
A selected list of oncoproteins, protooncogenes and associated human

cancers
Limited
CHAPTER
Activation of Protooncogenes to 26
Oncogenes
The conversion of protooncogenes to oncogenes

occurs by several mechanism s.
Promoter insertion
Limited
CHAPTER
26
Enhancer insertion
Limited
CHAPTER
26
Chromosomal translocation
Diagrammatic representation of reciprocal translocation in Burkitt's

lym phoma.
Limited
CHAPTER
26
Growth Factors
●
Growth factors norm ally stimulate the proliferation
of cells. They regulate cell division by signal
transduction across the cell mem brane to the
interior portions of the cell including the nucleus. It
is widely accepted that growth factors play a key
role in carcinogenesis.
Limited
CHAPTER
26
A selected list of polypeptide growth factors and their
functions in human tissues
Limited
CHAPTER
26
Mechanism of Action
Mechanism of action of ras P21 prot ein

GRF = guanine nucleotide releasing factor; GAP = GTPase activat ing protein
Limited
CHAPTER
26
Anti- oncogenes
●
Genes other than oncogenes (opposing their effects)
have been found to play a major role at least in som e
types of cancers. They are also called tum our
suppressor genes since they are m ostly involved in
the regulation of cell proliferation.
●
An im portant m odel for understanding their role is
retinoblastoma which occurs in children.
(Continues…
Limited
CHAPTER
26
…Continued)
●
Loss of antioncogenes producing cancer has been
incrim inated also in various other types of cancers
which include Wilm 's tum our of kidney, small cell
carcinoma of lung, and a specific type of breast
cancer.
Limited
CHAPTER
Diagrammatic representation of the role of m ultiple factors in 26
the development of cancer.
Limited
CHAPTER
26
Tumour Markers
Tum our cells produce certain substances whose

detection in the blood indicates the presence of
tum our in the body.
These are called tum our m arkers.
Limited
CHAPTER
26
Tumour markers and the cancers they are associated

with
Limited
CHAPTER
26
Cancer Therapy
Chemotherapy is the use of certain drugs to treat

cancers.
Anticancer drugs regularly used in chemotheraphy
Limited
CHAPTER
27
Section VII
Molecular Biology
Universities Press
3-6-747/1/A & 3-6-754/1, Himayatnagar
Phone: 040-2766 5446/5447
Limited
CHAPTER
27
Chapter 2 7
DNA Metabolism
Limited
CHAPTER
27
Replication of DNA
Principles of Replication
1. Copying is accurate
2. Replication is semiconservative
3. Supercoils and DNA topoisom erases
4. Replication fork
5. Replication is sim ultaneous and bidirectional
6. DNA synthesis is catalysed by DNA polymerases
Limited
CHAPTER
27
Replication fork
Limited
CHAPTER
27
Replication in Prokaryotes

Most of the inform ation about the replication in
prokaryotes is obtained from the studies made on
the intestinal bacterium , E.coli.

The process of replication can be studied
conveniently in three stages.
1. Initiation
2. Elongation
3. Termination
Limited
CHAPTER
27
Initiation

Helicase enzym es

Replication fork

Topoisomerases (also called as DNA gyrases)

Single- stranded DNA binding (SSB) proteins

Primer

Primase

DNA polymerase III
Limited
CHAPTER
27
Diagrammatic representation of the process of replication
Limited
CHAPTER
27
Elongation

Okazaki fragments

DNA polymerase I

Proof- reading ability
Limited
CHAPTER
27
Discovery of Okazaki fragm ents
Limited
CHAPTER
27
Termination

Terminus sequence

DNA polymerase II
Limited
CHAPTER
27
Replication in Eukaryotes
There are five distinct DNA polym erases identified in

eukaryotes. They are numbered by Greek alphabets.
1. DNA polymerase  synthesises primers for both

leading and lagging strands of DNA.
2. DNA polymerase  has sim ilar function as that of

DNA polymerase II of prokaryotes –DNA repair.
3. DNA polymerase  ex clusively replicates

mitochondrial DNA.
(Continues…
Limited
CHAPTER
27
…Continued)
4. DNA polymerase  is analogous to that of DNA

polymerase III of prokaryotes. It takes part in the
synthesis of both leading and lagging strands with an
intrinsic proof- reading activity.
5. DNA polymerase  is com parable in its action to the

prokaryotic DNA polymerase I. It is involved in the
rem oval of RNA prim ers of Okazaki fragm ents on the
lagging strand. Occasionally, it replaces the DNA
polymerase d and performs its function.
(Continues…
Limited
CHAPTER
27
…Continued)
Like bacterial replication, replication in eukaryotic cells

can also be studied in three stages.
1. Initiation
2. Elongation
3. Termination
Limited
CHAPTER
27
Initiation

Helicases

Topoisomerases

Replication protein A (RPA)

Replication fork

Multiple sites of origin of replication

Primers
Limited
CHAPTER
27
Overview of DNA replication on the lagging strand in eukaryotes
(leading strand is not shown)
Limited
CHAPTER
27
Elongation

Replication factor C (REC)

Proliferating cell nuclear antigen (PCNA)

Sliding clamp

DNA polymerase 

Okazaki fragments

RNase H

FENI (flap endonuclease I)

DNA polymerase 
Limited
CHAPTER
27
Termination

Telomeres

Telomerases

RNA dependent DAN synthesis

Reverse transcriptase
Limited
CHAPTER
27
Inhibitors of Replication
●
The differences between the DNA replication in
bacteria and hum an cells give scope for developing
antibiotics that target bacterial replication but do not
affect hum an cells. Several drugs (e.g., ciproflox acin,
nalidix ic acid ) act by inhibiting the enzym es of
bacterial replication.
(Continues…
Limited
CHAPTER
27
…Continued)
Drugs targeting human replication enzym es such as

topoisom erases and telomerases arrest new DNA
synthesis and thus the cell division. For ex ample,
dox orubicin, adriam ycin, etoposide.
Certain nucleotide analogues such as 6-

m ercaptopurine and 5- fluorouracil inhibit human
DNA polym erases. They are also used as anticancer
drugs.
Limited
CHAPTER
27
Cell Cycle
• S (Synthetic) phase
• M (m itotic) phase
• Gap1 (G1 ) and gap2 (G2 )
• Dormant phase (G0)
• Cyclins
• Cyclin- dependent kinases (CDKs)
Limited
CHAPTER
27
Cancer and the cell cycle
Cell cycle in mammalian cells
S (synt hesis phase)

M (m it otic phase)
Gap1(G1 phase)
Gap2 (G2 phase)
Dorm ant phase (G0)
Limited
CHAPTER
27
Apoptosis
Apoptosis
A programm ed cell suicide is referred to as apoptosis
Limited
CHAPTER
27
Recombination
The ex change of genetic inform ation between

various DNA segm ents is known as
recombination. Apart from rearranging genetic
inform ation, it also repairs dam aged DNA.
There are two types of recombinations.
1. Hom ologous recom bination: The ex change of

genetic inform ation takes place between
identical DNA sequences (parts of
chromosomes). The most com m on ex am ple is
recom bination between the m aternal and
paternal chrom osom es of a pair. (Continues…
Limited
CHAPTER
27
…Continued)
2. Non- hom ologous recom bination: In this type,

the ex change of genetic inform ation occurs
randomly (need not be between identical
sequences). Genetic rearrangem ent usually
takes place by this process (transposition). It
also involves the ex perim ental installation of
foreign DNA into a host's chrom osom e.
Limited
CHAPTER
27
Homologous Recombination
Diagrammatic representation of homologous recombination
Limited
CHAPTER
27
Three m odels have been proposed to ex plain

homologous recom bination.
i) Holliday m odel
ii) Meselson–Radding m odel
iii) Double- strand break model
Limited
CHAPTER
Holliday model of homologous recombination (the areas shown in 27
dotted box es are heterologous regions)
Limited
CHAPTER
27
Non- homologous Recombination
Three types of non- hom ologous

recombination have been identified.
i) Transposition
ii) Site- specific recom bination
iii) Non- hom ologous end joining.
Limited
CHAPTER
27
A diagrammatic representation of retrotransposition
Limited
CHAPTER
27
DNA Damage
The types of damage inflicted on the DNA

molecule by various physical and chem ical
agents (mutagens) are broadly classified into
four categories
(Continued…
Limited
CHAPTER
27
Types of DNA Damage
Limited
CHAPTER
27
Repair of DNA
Four m ajor repair system s are involved in

rectifying the DNA damage.
1. Mism atch repair
2. Base ex cision repair
3. Nucleotide ex cision repair
4. Double–strand break repair.
Limited
CHAPTER
27
Mechanisms of DNA Repair
Limited
CHAPTER
27
Mismatch Repair
●
Even though replication occurs with high
fidelity, defects do occur during copying.
Mism atch repair corrects errors involving
single base pair or a sm all region of
unpaired DNA
Limited
CHAPTER
27
A diagrammatic representation of mismatch repair
Limited
CHAPTER
27
Base Excision Repair
Diagram matic representation
Limited
CHAPTER
27
Nucleotide Excision Repair
●
Xeroderma pigmentosum
Limited
CHAPTER
27
Double- strand Break Repair

●
Double- strand breaks are com mon during
im munoglobulin gene rearrangem ent which
are repaired forthwith. Otherwise, double-
strand breaks occur due to the free radicals,
radiation and chem otherapeutic agents.
Limited
CHAPTER 28
Section VII
Molecular Biology
Universities Press
3-6-747/1/A & 3-6-754/1, Himayatnagar
Phone: 040-2766 5446/5447
Limited
CHAPTER 28
Chapter 2 8
RNA Metabolism
Limited
CHAPTER 28
In 1958, Francis Crick formulated the m uch publicised

central dogm a of life where he proposed the flow of
biological inform ation
Limited
CHAPTER 28
●
Cells contain three major types of RNA:
●
ribosom al RNA (rRNA) is the chief constituent of
ribosom es;
●
messenger RNA (m RNA) carries the information
required for protein biosynthesis in its
nucleotide sequences;
●
transfer RNA (tRNA) delivers amino acids to the
ribosom es for protein biosynthesis of rRNA.
Limited
CHAPTER 28
Principles of Transcription
●
Transcription is the synthesis of the RNA molecule
from DNA. Unlike replication where the entire
sequence of DNA is duplicated, during
transcription only certain segments of DNA are
ex pressed. These transcribable portions of the DNA
are referred to as genes.
(Continues…
Limited
CHAPTER 28
…Continued)
●
Only one strand of the duplex DNA takes part at a
tim e in the synthesis of RNA.
●
The sequence of ribonucleotides in the newly
synthesised RNA m olecule is com plementary to the
sequence of deox yribonucleotides in this strand of
DNA m olecule and is called the tem plate strand.
●
The other DNA strand which does not take part in
transcription is referred to as the coding strand.
(Continues…
Limited
CHAPTER 28
…Continued

Transcription initiation site (TIS)

Downstream

Upstream
The figure shows that the genes occur on both strands of the
DNA and are always transcribed in the 3' to 5' direction (note
the direction of the arrows)
(Continues…
Limited
CHAPTER 28
…Continued)
Diagrammatic representation of the process of transcription

(Note the unwinding of certain portions of DNA enabling the
transcription to occur freely).
(Continues…
Limited
CHAPTER 28
…Continued)

RNA polym erase (RNAP)

How does RNAP know where to start the process of
transcription in a stretch of DNA?

Promoters
(Continues…
Limited
CHAPTER 28
…Continued)
Complementary base pairing in transcription
Limited
CHAPTER 28
Transcription in Prokaryotes
The transcription in prokaryotes is relatively sim ple

compared to that of eukaryotes. The enzyme RNA
polymerase (RNAP) catalyses the synthesis of all types
of RNAs in prokaryotes.
Transcription occurs in three stages:
1. Initiation
2. Elongation
3. Termination
(Continued…
Limited
CHAPTER 28
…Continued)
Initiation
The process of RNA synthesis in bacteria begins with
the binding of RNA polymerase (RNAP) m olecule on the
DNA.
RNAP recognises the transcription start site on the
tem plate strand of DNA with the help of certain
specific regions on the DNA known as prom oter
elem ents (PE).

Pribnow box (TATA box )

The ‘–35’ sequence
(Continues…
Limited
CHAPTER 28
…Continued)
Prom oter regions in prokaryotes. Note that by convention the PE

regions are shown to occur on coding strand. However these regions
are functional only in an intact double- stranded DNA.
(Continues…
Limited
CHAPTER 28
…Continued
Elongation
●
The elongation of the RNA molecule occurs from the
5' to its 3' end, antiparallel to the template strand.
RNAP assembles ribonucleotides in a
complem entary sequence to the tem plate strand by
Watson- Crick base pairing rules
Termination
• Rho () factor
• Palindrom es
(Continues…
Limited
CHAPTER 28
Synthesis of RNA from DNA - the process of

transcription
TIS = Transcription
initiation site
RNAP = RNA polymerase
Sigma factor
Termination factor
Limited
CHAPTER 28
Transcription in Eukaryotes

Eukaryotic transcription is m ore complex com pared
to that of prokaryotes. There ex ist three distinct RNA
polymerases transcribing different types of RNAs
compared to the single RNAP in prokaryotes.

RNAP I catalyses the formation of large ribosom al
RNAs. RNAP II synthesises the prim ary transcript
form of m RNAs. RNAP III takes part in the synthesis
of tRNAs and small ribosomal RNAs.
(Continues…
Limited
CHAPTER 28
…Continued)
The transcription occurs in three stages.

1. Initiation
2. Elongation
3. Termination
Initiation
Transcription begins with the RNAP recognising the
promoter site. Two types of sequence elements are
identified as eukaryotic promoters which occur
prox imal to the TIS.
(Continues…
Limited
CHAPTER 28
…Continued)
1. TATA box
This sequence is the Goldberg- Hogness box .
Determines where ex actly the transcription has to
begin along the template strand of DNA.
2 . CAAT box
This region determines how frequently the
transcription has to occur and at what speed .
(Continues…
Limited
CHAPTER 28
…Cont inued)
Promoter regions in eukaryotes
Limited
CHAPTER 28
Pre- Initiation Complex (PIC)
The assembly of preinitiation complex (PIC)

or basal transcription complex (BTC)
[The scale (–30 t o + 30 ) shows the ex t ent of
BTC].
Limited
CHAPTER 28
Elongation
The RNAP II moves along the DNA template

incorporating ribonucleotides one by one as per the
Watson–Crick base pairing rules. The synthesis of
mRNA proceeds 5' to 3' end, in other words, the
tem plate strand of DNA is from the 3' to the 5' end.
• Enhancers and silencers

• Termination
Limited
CHAPTER 28
Post- Transcriptional Modifications
●
The mRNA in prokaryotes is fully functional as soon
as it is synthesised.
●
On the contrary, all types of eukaryotic RNA
produced by transcription as prim ary transcripts are
non- functional as such. They undergo ex tensive
structural alterations to produce the mature
functional molecules. The phenom ena of RNA
processing in eukaryotes are known as post-
transcriptional m odifications.
(Continues…
Limited
CHAPTER 28
…Continued)
Messenger RNA
• Heterogeneous nuclear RNA (hnRNA)
• Mature mRNA
• The cap
• The tail
• Splicing
• m RNA editing
(Continues…
Limited
CHAPTER 28
…Continued )
Splicing of mRNA from hnRNA
Limited
CHAPTER 28
Inhibitors of Transcription
●
There are a number of drugs and tox ins capable of
inhibiting the process of transcription at various
stages, in bacteria.
Rifampicin, a drug used in the treatment of tuberculosis

and leprosy binds to the RNA polym erase and blocks
the bacterial transcription.
Actinomycin D, an antibiotic, binds to the tem plate

strand of DNA and obstructs the movement of RNAP,
thus blocking bacterial transcription.
(Continues…
Limited
CHAPTER 28
…Continued)
●
- Amanitin, a mushroom poison binds to the RNAP II
and inhibits the transcription in eukaryotes. Heparin,
a glycosaminoglycan binds to RNAP and inhibits the
transcription.
Limited
CHAPTER 28
RNA acting as enzyme

Reverse transcriptase
RNA degradation
Limited
CHAPTER 29
Section VII
Molecular Biology
Universities Press
3-6-747/1/A & 3-6-754/1, Himayatnagar
Phone: 040-2766 5446/5447
Limited
CHAPTER 29
Chapter 2 9
Protein Biosynthesis
Limited
CHAPTER 29
Genetic Code
The three nucleotide (triplet) base sequences in mRNA that

code for amino acids in protein are called codons or genetic
code.
(Continues…
Limited
CHAPTER 29
…Continued)
Genetic code with corresponding amino acids
Limited
CHAPTER 29
Features of Genetic Code
•
Universality
•
Specificity
•
Non-overlapping
•
Degeneracy
Limited
CHAPTER 29
Genetic Code
Limited
CHAPTER 29
Codon–Anticodon Pairing
Limited
CHAPTER 29
Wobble Hypothesis
●
A single tRNA can recognise more than one codon. This is
possible because the third base (3' base) in the codon
sometimes fails to recognise its complementary base in
the anticodon (5' base).
Limited
CHAPTER 29
Mutations
Classification of mutations
Mutations can be classified into two major categories.
1. Point mutations: It is defined as change in a single

nucleotide in DNA. These are subclassified as
i) Transitions: In this type, a purine is replaced by
another purine or a pyrimidine is substituted by another
pyrimidine.
ii) Transversions: In this case, a purine is replaced by a
pyrimidine or vice versa.
(Continues…
Limited
CHAPTER 29
…Continued)
2. Frameshift mutations: Mutations of this type result

when one or more base pairs are either inserted in or
deleted from the DNA sequences. Hence, they are also
called insertion and deletion mutations.
(Continued…
Limited
CHAPTER 29
…Continued
Different types of mutations
(Continues…
Limited
CHAPTER 29
…Continued
Effects of point mutations

•
Silent mutation
•
Missense mutation
The mistaken (or missense) amino acid incorporated
into a protein may be acceptable, partially acceptable or
unacceptable
•
Nonsense mutation
Effects of frameshift mutations
Limited
Link between amino acids and CHAPTER 29
nucleic acids
The link between amino acids and nucleic acids is first made by
enzymes called aminoacyl tRNA synthetases.
Limited
CHAPTER 29
Translation in Prokaryotes
Translation occurs in three stages

1. Initiation
2. Elongation
3. Termination
Limited
CHAPTER 29
Initiation
Initiation of translation in bacteria (see textbook for abbreviations)
Figure
continued
Limited
CHAPTER 29
Elongation
•
Binding of an incoming aminoacyl-tRNA
•
Peptide bond formation
•
Translocation
Limited
CHAPTER 29
Elongation and termination of bacterial translation (see

textbook for abbreviations)
Figure
continues
(Figure continues…
Limited
CHAPTER 29
…Continued)
Limited
CHAPTER 29
…Continued)
Limited
CHAPTER 29
Termination
•
Stop codons UAA, UAG, UGA
•
Release factors (RF-1, RF-2, and RF-3)
Limited
CHAPTER 29
Translation in Eukaryotes
Eukaryotic translation is more complex. As in prokaryotes, it

also occurs in three stages.
1. Initiation
2. Elongation
3. Termination
Limited
CHAPTER 29
Initiation
It occurs in three steps.

i) Dissociation of the ribosome
ii) Formation of the preinitiation complex
iii) Formation of the initiation complex
Limited
CHAPTER 29
Initiation of Translation in Eukaryotes
(see textbook for abbreviations)
Limited
CHAPTER 29
…Continued)
Limited
CHAPTER 29
Elongation
It occurs in three steps.
1. Binding of aminoacyl-tRNA to the A site on the

ribosome
2. Peptide bond formation
3. Translocation
Limited
CHAPTER 29
Elongation and Termination in
Eukaryotic Translation
See textbook for
abbreviations.
A site, P site and

E site are shown
separately on the
left-hand side of
the diagram for
more clarity.
Note that there is

no E in
eukaryotes; it
exists only in
prokaryotes.
However, an E
site is shown in
this figure for
better
understanding.
Limited
CHAPTER 29
...Figure continued
Limited
CHAPTER 29
...Figure continued
Limited
CHAPTER 29
Termination
•
Stop codons (UAA, UAG, UGA)
•
Release factor (eRF)
Limited
CHAPTER 29
Protein Synthesis and Antibiotics
Inhibitors of protein synthesis

•
Streptomycin, neomycin
•
Gentamycin
•
Tetracyclins
•
Chloramphenicol
•
Erythromycin
•
Purmycin
•
Diphtheria toxin
Limited
CHAPTER 29
Post-translational Modifications
•
Trimming
•
Structural reorganisation
•
Modifications at the N-terminal and
C-terminal ends
•
Covalent modifications
Phosphorylation
Hydroxylation
Glycosylation
(Continued…
Limited
CHAPTER 29
Covalent Modifications of the Regulatory Enzymes of Major

Metabolic Pathways
Limited
CHAPTER 29
Protein Targeting
•
Signal sequence
•
Signal recognition particle (SRP)
•
Endoplasmic reticulum (ER)
•
Transport vesicles
•
SNARE proteins
•
Receptor-mediated endocytosis
Limited
CHAPTER 29
Protein destination by different pathways
Limited
CHAPTER 29
Some conformational diseases due to
mutations affecting protein targeting
Limited
CHAPTER
30
Section VII
Molecular Biology
Universities Press
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Phone: 040-2766 5446/5447
Limited
CHAPTER
30
Chapter 3 0
Regulation of Gene
Expression
Limited
CHAPTER
30
Principles of Gene Regulation
• Induction and repression – enhancers and silencers
• Housekeeping and invisible genes
• One cistron–one subunit concept
• Genome organisation–size and gene num ber
• DNA organisation and gene ex pression

• Covalent modifications of histones control gene
ex pression
Limited
CHAPTER
30
DNA- Protein Interactions
• Helix - turn- helix m otif

• Zinc finger motif
• Leucine zipper motif
Limited
CHAPTER
30
Diagrammatic representation of motifs mediating DNA–protein
interactions
Limited
CHAPTER
Regulation of gene expression in 30
prokaryotes
• LAC operon
• Structure of lac operon
• How does the lac operon operate?
Limited
CHAPTER
30
Lac operon
Diagrammatic representation of the modus operandi of the lac operon in E.coli
(Continues…
Limited
CHAPTER
30
…Continued)
(CAP-cAMP = catabolite gene activator protein bound to cAMP; RNAP=RNA

polymerase)
Limited
CHAPTER
30
Tryptophan Operon
Tryptophan operon in E.coli (for abbreviations, refer to the tex tbook)
Limited
CHAPTER
Regulation of Gene Expression in 30
Eukaryotes
1. Chrom atin structure

2. Gene amplification
3. Gene rearrangem ent
4. Regulatory elem ents
5. Transcription
6. RNA processing
7. Alternate m RNA splicing
8. Transport of mRNA from nucleus to cytoplasm
9. Degradation of m RNA
10. Gene regulation at the level of translation
Limited
CHAPTER
30
Gene Amplification
A diagram matic representation of the am plification of

genes.
Limited
CHAPTER
30
Gene Rearrangement
Limited
CHAPTER
31
Section VIII
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Immunology
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CHAPTER
31
Chapter 3 1
Recombinant DNA
Technology
Limited
CHAPTER
Recombinant DNA Technology 31
Protocol
1. Isolation of the desired gene

2. Isolation of vector
3. Formation of chim eric DNA
4. Uptake of chimeric DNA (Transfection)
5. Selection of cells containing chim eric DNA
6. Ex pression of the gene to produce the desired
product
Limited
CHAPTER
31
Basic mechanism of recombinant DNA technology
Limited
CHAPTER
31
Diagrammatic representation of selection of recombinant clones
Limited
CHAPTER
Tools of Recombinant DNA 31
Technology
• Enymes
• Vectors
• Hosts
• DNA to be cloned
Limited
CHAPTER
31
Enzymes
• Nucleases
• Ex onucleases
• Endonucleases
• Restriction endonucleases
Limited
CHAPTER
31
Restriction Endonucleases
• Nomeclature of RE
• Recognition sequences
• Restriction m ap
• Cleavage patterns
Limited
CHAPTER
31
Action of RE SmaI/ HpaI produces blunt ends
Limited
CHAPTER
31
Action of RE BmHI/ EcoRI produces sticky ends
Limited
CHAPTER
31
Some restriction enzymes with their sources, recognition
sequences and the products formed
Limited
CHAPTER
31
Enzymes commonly used in rDNA technology/ genetic engineering
Limited
CHAPTER
31
Vectors – The Cloning Vehicles
• Once the desired fragments of DNA are obtained,

they are inserted into a suitable vector to produce
indefinite num ber of copies of genes. This is
known as gene cloning. They are self- replicating in
an appropriate host cell.
• An ideal cloning vector should have the following

properties:
(Continues . . . )
Limited
CHAPTER
31
(. . . Continued)
• It must have low molecular weight.

• It must have a unique cleavage site for the activity of
restriction enzym es at a single point.
• It must be able to replicate truly inside a host cell
after its introduction.
• It must contain genes which provide resistance to
antibiotics. This helps in the selection of
transformed cells from untransformed cells.
Limited
CHAPTER
31
Different cloning vectors with the corresponding hosts and
max imum sizes of foreign DNA inserts
Limited
CHAPTER
Vectors are of the following types

31
1) Plasmids
2) Bacteriophages
3) Cosmids
4) Artificial chromosome vectors
5) Ex pression vectors
Limited
CHAPTER
31
Cloning capacities of common cloning vectors
Limited
CHAPTER
31
Larger DNA fragments in cosmids and other vectors
Limited
CHAPTER
31
Hosts
• Prokaryotic hosts
• Eukaryotic hosts
Limited
CHAPTER
31
Some host cells used in genetic engineering
Limited
CHAPTER
31
Gene Cloning Strategies
• Cloning from genomic DNA or mRNA

• Methods of gene transfer
– Transform ation
– Conjugation
– Electroporation
– Liposom e- mediated gene transfer
– Direct transfer of DAN
• Gene libraries
• Screening m ethods
Limited
CHAPTER
31
Gene Banks
●
Gene banks are the outcome of novel concepts
where certain centres have been developed for the
storage of an individual's DNA for future use (e.g.,
in the diagnosis of diseases)
Limited
CHAPTER
31
Applications of rDNA Technology
Insulin
Technique for production
of recombinant insulin
Limited
CHAPTER
31
• Hepatitis- B vaccine
• Growth hormone
Limited
CHAPTER
31
Ex pression of human growth hormone in bacteria
(Continued…
Limited
CHAPTER
31
…Continues)
Limited
CHAPTER
31
DNA Vaccine
Mechanism of action of DNA vaccine in developing immunity
Limited
CHAPTER
31
DNA Finger Printing/ DNA Typing
Process of DNA typing

The process of DNA typing
Limited
CHAPTER
31
• Interferons
• Antibiotics
• Recom binant factor VIII
• Tissue plasm inogen activator (tPA)
• Monoclonal antiboides
• Diagnosis of HIV infection
• Gene therapy
• Transgenic animals
• In agriculture
Limited
CHAPTER
31
Som e transgenic animals and their applications
Limited
CHAPTER
31
Bioreactor/ pharma anim als and their uses
Limited
CHAPTER
31
Therapeutic human proteins produced by recombinant DNA technology
(Continues…)
Limited
CHAPTER
31
(. . .Continued)
(Continues…)
Limited
CHAPTER
31
(. . .Continued)
Limited
CHAPTER
Selected list of diseases for which recombinant vaccines have 31
been or are being developed
(Continues . . .)
Limited
CHAPTER
31
(. . . Continued)
Limited
CHAPTER
32
Section VIII
Biotechnology and
Immunology
Universities Press
3-6-747/1/A & 3-6-754/1, Himayatnagar
Phone: 040-2766 5446/5447
CHAPTER
32
Chapter 3 2
Molecular Biology
Techniques

CHAPTER
32
Hydridisation
DNA–DNA
hybridisation

CHAPTER
32
Probes
A probe is a single- stranded fragment of DNA, usually

labelled with a radioisotope, the nucleotide sequence
of which is complementary to the target DNA.
Application
DNA libraries contain thousands or even m illions of
irrelevant DNA fragm ents. It is difficult to pick out a
specific gene (a DNA sequence of interest) from these
fragments. Probes are used to search and locate a
specific gene from DNA libraries.

CHAPTER
Identification of DNA sequences 32
using labelled probes

CHAPTER
32
Blotting Techniques
Blotting techniques are com m only used analytical tools for the
identification of desired DNA or RNA fragm ents.
Blotting is the process of im m obilisation of sam ple nucleic acids

on solid support.

CHAPTER
32
Types of Blotting
The most comm only used blotting techniques are

listed below.
1. Southern blotting for DNA
2. Northern blotting for RNA
3. Western blotting for proteins
4. Dot blotting (DNA/ RNA)

CHAPTER
Southern Blotting for Identification 32
of DNA Fragments
It is a technique to transfer DNA species separated by

agarose gel electrophoresis to a filter for analysis.
Applications of southern blotting

1. Mutant genes such as Hbs, cystic fibrosis,
Duchenne muscular dystrophy, phenylketonuria,
presence of viral DNA (hepatic virus B and C) can be
identified by this m ethod.
(Continues…

CHAPTER
32
…Continued)
2. It is used to detect minute quantities of DNA which

is mostly useful for forensic purposes (e.g.
determining paternity, identifying thieves and
rapists).
3. Useful for the determ ination of restriction fragment
length polym orphism (RFLP) associated with a
pathological condition.
4. It is used for the confirmation of DNA cloning
results.
(Continues…

CHAPTER
32
…Continued) Southern blotting technique

CHAPTER
32
Northern Blotting for RNA
Northern blotting is used for the specific identification

of RNA m olecules.
There will be RNA–DNA hybridisation. This is used to

detect the gene ex pression in a tissue.
(Continues…

CHAPTER
32
…Cont inued)
Applications

It is an ideal technique for determ ining the number
of genes (through mRNA) present on a given DNA.
But this is not practically applicable since each gene
may give rise to two or more RNA transcripts.

It is used to size and quantitate specific RNA
molecules.
(Continues…

CHAPTER
32
…Continued) Northern blotting technique

CHAPTER
32
Western Blotting for Proteins
Proteins are identified by this technique and are isolated

from the tissue and subjected to electrophoretic
separation.
Application
It is currently being used as one of the tests for
detecting the AIDS virus. In this case, the presence of
viral proteins in the blood is detected by antibodies. In
Western blot analysis, antibodies against different
components of the virus are analysed so it is considered
to be the confirm atory test for diagnosis.

CHAPTER
32
Dot Blotting for DNA/ RNA
●
It is a modification of the Southern and Northern
blotting techniques. In this technique, the nucleic
acids (DNA/ RNA) are directly spotted on to the filters
(not subjected to electrophoresis). The hybridisation
procedure is the sam e as that of the blotting
techniques.
●
It is useful in accum ulating data for the evaluation of
gene ex pression.

CHAPTER
32
Comparison of blot transfer techniques

CHAPTER
32
Polymerase Chain Reaction (PCR)
PCR is basically a DNA amplification technique. It is an

in vitro procedure which generates large quantities of
DNA segm ents. Millions of identical copies of any DNA
sequence of interest can be produced by PCR.
Principle and technique of PCR

Step 1 – Denaturation
Step 2 – Renaturation (annealing)
Step 3 – Polymerisation (synthesis)
(Continues…

CHAPTER
32
Polymerase chain reaction (PCR) – diagram matic
representation of the process
…Continued)
(Continues…

CHAPTER
32
…Continued)

CHAPTER
32
Applications of PCR

Diagnosis

Cancer detection

Prenatal diagnosis

Medico- legal cases

Genetic disorders

PCR in DNA sequencing

Fossil studies

Com parative studies of genom es

CHAPTER
32
Hybridoma Technology
●
Production of monoclonal antibodies in vitro is called
hybridom a technology.
●
The hybridoma cells formed retain the properties of
both the parent cells, that is, antibody secretion
property of B- lymphocytes and uncontrolled division
property of cancer cell.

CHAPTER
32
A diagram depicting production of monoclonal antibodies

CHAPTER
32
Applications of Monoclonal Antibodies
1. It is used in the quantitative preparation of the

specific type of cells used in the ELISA tests.
2. Is used in the quantitative preparation of pure

antigens.
3. It is helpful in the early detection of pregnancy and

in the treatm ent of cancers.
4. It is also used in the diagnosis of leprosy.
5. It is used in the enumeration of lymphocyte

subpopulations.
(Cont inues…

CHAPTER
32
…Continued) Applications of Monoclonal Antibodies
6. It is used to suppress rejection reactions in organ

transplantation.
7. It can be used to institute of platelet clum ping in

patients who have undergone angioplasty and in the
treatm ent of autoim mune diseases.
8. It can be used to detect chlam ydia (a streptococcal

bacterium ), HIV causing AIDS and tox in and drug
levels in the body.

CHAPTER
32
DNA Sequencing
Techniques employed for DNA sequencing

1. Max am –Gilbert technique
2. Interrupted enzym atic cleavage method or the

dideox ynucleotide m ethod by Sanger
3. Automated DNA sequencing
4. DNA chips – micro arrays

CHAPTER
32
Maxam and Gilbert method for DNA sequencing

CHAPTER
32
DNA Markers in Disease Diagnosis
The DNA markers are used for genetic m apping

of the genome and for diagnostic purposes.
There are four types of DNA sequences which

can be used as m arkers.
(Continues…

CHAPTER
32
…Continued)
1. Restriction fragment length polym orphisms

(RFLPs)
2. Minisatellites (or) variable number tandem

repeats (VNTRs)
3. Microsatellites (or) simple tandem repeats

(STRs)
4. Single nucleotide polym orphisms (SNPs).

CHAPTER
Restriction Fragment Length 32
Polymorphisms (RFLPS)
Methods of RFLP scoring

Two methods are comm only used for the detection of
RFLPs. They are
i) Southern hybridisation
ii) PCR

CHAPTER
32
RFLPs by Southern hybridisation

CHAPTER
32
Applications of RFLPS
i) RFLPs in the diagnosis of diseases

ii) RFLPs can be used to detect hum an genetic
defects in prospective parents (or) fetal
tissue

CHAPTER
32
RFLPs by polymerase chain reaction

CHAPTER
Variable Number Tandem 32
Repeats (VNTRS)
The pattern of VNTRs and RFLPs is used in DNA finger

printing, as the individual genome has many different
VNTRs and RFLPs which are unique to the individual.
Diagrammatic representation of VNTRs

CHAPTER
32
Applications of VNTRs
• VNTRs are useful in detecting the genetic diseases

which are linked to changes in the degree of
repetition of microsatellites, e.g. Huntington's
chorea is a disease observed when the VNTRs
ex ceed 40 repeat units.
• When DNA molecules are subjected to restriction

endonuclease action, the VNTR sequences are
released, and they can be detected due to variability
in repeat sequence copies
• Microsatellites (simple tandem repeats)

• Single nucleotide polymorphisma (SNPs)

CHAPTER
32
Mutation Detection Techniques
1. Single strand confirm ation polymorphism (SSCP)

2. Heteroduplex analysis
SSCP analysis

CHAPTER
Conformation Sensitive Gel 32
Electrophoresis (CSGE)

CHAPTER
32
Protein Truncation Test (PTT)
Protein truncation test (or) IVSPA

CHAPTER
32
• Denaturation high performance liquid

chromatagraphy (DHPLC)
• Ame’s test to detect mutagenicity

CHAPTER
32
Nanotechnology
Nanotechnology is a rapidly evolving new science

that deals with matter on a very small scale, just
bigger than atoms and m olecules.
It is used to understand the structure and dynam ics

of matter at dimensions between 1 nanometre (10 –9
m) and 100 nm .

CHAPTER
32
Applications of
Nanotechnology

Drug delivery

Therapy

Cellular repair

Anti- microbial actions

Diagnostic medicine

CHAPTER
32
ELISA
●
Enzyme linked imm une sorbent assay (ELISA) is
based on the imm unochem ical principles of the
antigen–antibody reaction.
●
Types of ELISA
●
Antibody detection by ELISA (Indirect ELISA)
(Continues…

CHAPTER
32
Indirect Elisa to detect antibody
…Continued)
(Continued…

CHAPTER
Antigen Detection by ELISA 32
(Sandwich ELISA)

CHAPTER
32
ELISA

CHAPTER
Radioimmunoassay
32
A known concentration of labelled

antigen with sat urat ed am ount of
specific antibodies is added to a
num ber of tubes cont aining serial
dilutions of the pat ient’s serum (only
one dilution is shown).
The unlabelled antigen (if present)

from t he serum displaces t he labelled
antigen at tached t o ant ibodies.
The com plex es are precipit at ed by

protein A or anti- IgG antibodies and
t he radioactivit y of t he supernat ant is
counted.
A standard graph is generat ed using

known concentrat ion of ant igens vs.
radioact ivit y.
Concentrat ion of ant igen in t he serum

of the pat ient is quantified wit h t he
help of the standard graph.
CHAPTER
32
Radioimmunoassay (RIA)
As the nam e im plies, radioim munoassay is a

combination of the principles of radioactivity of isotopes
and the im munological reactions of antigen and
antibody.
Applications
Radioim munoassay is widely applied in the diagnosis of
cancers, hormonal disorders and also for therapeutic
m onitoring of drugs. It is an ex tensively used tool in
biomedical research.
(Continues…

CHAPTER
32
…Continued)
Haptens are the substances that make other substances

antigenic, which in turn, elicit specific antibody
responses. By this m ethod, the concentrations of several
compounds can be estimated by RIA, which include
steroid horm ones, peptides, nucleic acids, vitamins,
drugs including several antibiotics.

CHAPTER
32
Applications of Radioactive Isotopes
1. The sequence of reactions and chemical changes in

the metabolic pathways is studied by using isotope
tracers. By labelling the substrates with these tracers
(e.g., 14 C and 15 N), it is possible to know the sequence
of reactions and m olecular changes from substrate to
end- product in a metabolic pathway, e.g., Krebs
cycle, glycolysis, urea cycle, fatty acid synthesis and
ox idation.
2. The rate of synthesis and degradation of a substance
in the body (turnover rate) is studied by using
radioisotope tracers.
(Continues…

CHAPTER
32
…Continued) Applications of Radioactive Isotopes
By this the biological half- lives of various substances

are studied (e.g., half- life of album in is 21 days).
3. Radioisotopes are used to measure the total quantity
of a particular substance in the body (e.g., amino acid
pool) and its m etabolic turnover.
4. Radioisotopes are used to study the nucleic acid
synthesis (32 P), endocrine studies (125 I) and carbon
dating to study the age of fossils (14 C).
5. Radioisotopes are widely used in drug research and
xenobiotic metabolism.

CHAPTER
33
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Biotechnology and
Immunology
Universities Press
3-6-747/1/A & 3-6-754/1, Himayatnagar
Phone: 040-2766 5446/5447
CHAPTER
33
Chapter 3 3
Biochemical Techniques

CHAPTER
33
Electrophoresis
●
Electrophoresis is the migration of charged
particles in an electric field.
●
Electrophoresis is widely used for the separation of
proteins in body fluids (mostly the plasma proteins
including immunoglobulins, lipoproteins,
hemoglobins, isoenzymes and nucleic acids).

CHAPTER
33
Electrophoresis apparatus – basic components

CHAPTER
33
Types of Electrophoresis
• Zone electrophoresis
• Imm unoelectrophoresis
• Isoelectric focusing
• High voltage electrophoresis
• Capillary electrophoresis

CHAPTER
33
Diagram m atic representation of im m unoelectrophoresis

CHAPTER
33
Chromatography
Chromatography is a popular analytical technique that

is used to separate a m ix ture of closely related
compounds.
Principle and technique
●
Chrom atography consists of two imm iscible phases,
a m obile phase and a stationary phase. In the
mobile phase, the sample mix ture of substances to
be separated are dissolved in a liquid or gas.
(Continues…

CHAPTER
33
…Continued)
●
In the stationary phase, there is a porous solid
medium through which the sample mix ture in the
moving solvent (m obile phase) percolates.
The physicochem ical interaction that occurs between

the m obile and stationary phases results in the
separation of the com pounds from the mix ture.

CHAPTER
33
Types of Chromatography
HPLC – High
performance liquid
chromatography;
TLC – Thin layer

chromatography)

CHAPTER
33
Paper (Partition) Chromatography

CHAPTER
33
Centrifugation
Ultracentrifugation is a process through which

particles of very low molecular weight are separated.
It is made possible by applying very high speeds.
Applications of ultracentrifugation include separation

of serum proteins, lipoproteins, living cells and
organelles, determination of m olecular weights,
checking the purity of colloids and so on.

CHAPTER
33
Principles of Colorimetry
• Coloured solutions have the property of absorbing

certain wavelengths of light (monochromatic light)
and reflecting (transm itting) certain others. For
ex am ple, a solution of hem oglobin absorbs blue-
green light and reflects the red colour and thus the
solution appears red in colour.
• In colorimetry, filters are selected accordingly, so that
the absorption is max imum at the chosen
wavelength.
(Continues…

CHAPTER
33
…Continued)
• In colorimetry, only coloured com pounds, or those

compounds capable of forming coloured derivatives
by reacting with test reagents can be analysed. In
these estim ations, the colour is directly proportional
to the concentration of the substance in the sam ple.
(Continues…

CHAPTER
33
…Continued) Principles of Colorimetry
Electromagnetic spectrum
Components of a colorimeter
(Continued…

CHAPTER
33
Beer–Lambert Law
Beer’s law
The amount of transm itted light diminishes
ex ponentially with an increase in the concentration of
absorbing m aterial.
Lambert’s law
The amount of transm itted light decreases
ex ponentially with the increase in the thickness of the
medium.

CHAPTER
33
• Spectrophotom eter
• Flam e photometer
• Fluorimeter
• Auto Analyser
• Atomic absorption spectrophotom etry

CHAPTER
33
Schematic diagram of atom ic absorption spectrophotom eter

CHAPTER
34
Section VIII
Biotechnology and
Immunology
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Rafi Press (India) of
M D: Textbook Private Limited
Biochemistry (4th Edition) Universities Press
CHAPTER
34
Chapter 3 4
Immunochemistry
©
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Universities
CHAPTER
34
Immunology is the study of the defence mechanism s

ex erted by the body to recognise non- self or foreign
matter.
Types of immunity
Immune defences or imm unity of the body operates in
two different ways, which are interdependent to some
ex tent.
• Innate (or natural) imm unity
• Adaptive (or acquired) im munity
©
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CHAPTER
34
Adaptive immunity is of two types.

1. Humoral im munity
2. Cell- mediated imm unity
©
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CHAPTER
34
Organisation of the Immune System
Cells involved in the immune system

B-lymphocytes and T-lymphocytes
B-lymphocytes
B-lymphocytes specifically recognise each antigen and
produce antibodies (immunoglobulins) against each of them.
B-cells are involved in humoral immunity.
(Cont inues
©
Limited
Universities
CHAPTER
34
…Continued)
T-lymphocytes
T-lymphocytes identify viruses and microorganisms from the
antigens displayed on their surfaces. T-cells are involved in
cell-mediated immunity.
Cytokines
They regulate the host cell division (mitosis) and also
mediate communication between different cell types of the
immune system.
©
Limited
Universities
CHAPTER
34
Innate Immunity
Innate immunity defends against foreign cells in general

without recognising their specific identities (non-specific
immunity). Mechanisms of innate immunity include defence at
the body surfaces, inflammation and through interferons.
• Defences at body surfaces

• Inflammation
• Major histocompatibility complex (MHC)
• Complement system
• Interferons
©
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CHAPTER
34
Adaptive Immunity
Adaptive imm unity is m ost specific in its action and
ex erts a m ore effective immune response.
It responds to m acrom olecules (mainly proteins), but not
to small foreign molecules unless these are attached to
large ones. These foreign macrom olecules are called
antigens, and antibodies are produced in response to
them . Lymphocytes mediate the adaptive imm une
response.
Adaptive imm une system is of two types.
1. Hum oral immunity

2. 2. Cell- mediated immunity
©
Limited
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CHAPTER
Acquired Immunodeficiency 34
Syndrome (AIDS)
Mode of transmission
1. Majority of cases (about 80%) are through sex ual
contact. Men indulging in homosex ual relationships
are particularly at high risk. Heterosex ual
intercourse presents a relatively lower risk.
2. Intravenous drug abuse (by the sharing of needles).
3. Unprotected blood transfusions.
4. Vertical transm ission (mother to infant during
pregnancy)
©
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CHAPTER
34
Structure of HIV
©
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CHAPTER
34
Immunology of AIDS
• Macrophages and monocytes carry the virus to

various organs, including the CNS.
• T- helper (CD4) count falls drastically (< 400/ cu.mm

of blood). This not only reduces their cytotox ic
activity, but also leads to reduced availability of all
the other imm unological effectors.
• Antibody response against a foreign antigen is

reduced. Lymphokines such as interferon,
interleukin- 2 are also lowered.
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CHAPTER
34
Auto- immune diseases with corresponding autoantibodies
• Auto- immune disorders

• Mechanism of autoim munity
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CHAPTER
34
Clonal selection theory
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CHAPTER
34
Cells of the imm une system
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CHAPTER
34
Hematopoiesis
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CHAPTER
34
Important cytokines and their functions
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CHAPTER
34
Phases of adaptive immune
response
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CHAPTER
34
In vivo immune response
(…Cont inues
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CHAPTER
34
In vivo im m une response
. . . Continued)
DCs encounter the invading microbes at the site of infection.

The microbes or particulate/ soluble antigens of the microbes
are picked up by DCs. The DCs and free antigens are taken to
the T cell- rich area in the regional lymph nodes through the
afferent lymphatic vessel. The antigen- processed DCS and
resident macrophages are presented to T H cells in the contex t
of class II MHC molecules. T H cells proliferate, travel to the B
cell area and produce cytokines which induce proliferation and
differentiation of B cells (with the help of T H2 cells) and CD 8 -
positive T C cells (with the help of T H1 cells) to antibody-
producing plasma and CTLs respectively. The effector cells and
antibodies leave the lymph nodes through the efferent
lymphatic vessel and reach the site of infection.
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Auto- imm une diseases with corresponding
CHAPTER
34
autoantibodies
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CHAPTER
35
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Biochemical Perspective of
Endocrinology
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CHAPTER
35
Chapter 3 5
Hormone Action

CHAPTER
35
Classification of Hormones
Based on mechanism of action

This is the most widely used classification. Hormones
are broadly classified into two m ajor groups based on
the location of receptors and nature of the signal used
to m ediate the action of hormone.

CHAPTER
35
Group I Hormones
These horm ones are m ostly lipophilic.
The receptors for these hormones are located inside

the cell (intracellular receptors). The hormone–receptor
complex enters the nucleus, binds to the DNA and
causes selective gene ex pression, e.g., steroids,
thyroids and so on.

CHAPTER
35
Group II Hormones
These horm ones are m ostly hydrophilic.

Receptors for this group of hormones are located on
the cell surface (plasma membrane receptors).
Since the hormone cannot enter the cell, the hormone–
receptor complex stimulates the release of certain
m olecules known as second m essengers in the cell,
which ex ert the horm one's biochem ical function.
Here, the hormone itself is considered to be the first
m essenger.

CHAPTER
35
Classification of hormones by their mechanism of
action

CHAPTER
35
Diagrammatic representation of mechanism of action of
Group I hormones

CHAPTER
Promoter element (PE) and 35
hormone response element (HRE)
Hum an genes have two distinct regulatory regions.
1. Prom oter element (PE)

2. Horm one response element (HRE)

CHAPTER
35
Diagrammatic representation of gene, promoter element (PE) and
hormone response element (HRE) in human genome

CHAPTER
Diagrammatic representation of coregulators 35
linking HRU to PIC
The interaction between HRU and PIC is highly complex .
HRU = Hormone response unit; PIC = Pre- initiation complex ; RNAP = RNA polymerase; TFIID = Transcription factor
IID; HRE = Hormone response element, HR complex = Hormone- receptor complex , AF= Accessory factors, DE =
DNA element, ?= Unknown DNA regulatory elements

CHAPTER
35
Different signal tranduction cascades function through coregulators (CBP/ P300)
Coregulators establish communication

between HRU and PIC, resulting in
integration of complex metabolic
responses.

CHAPTER
35
Mechanism of action of group II hormones
The hormone response occurs in four stages.

i) Binding of horm one to receptor
ii) Activation of G- proteins
iii) Generation of second messengers
iv) Protein phosphorylation

CHAPTER
35
●
G- Proteins
●
Generation of second messengers – cyclic amp
●
Cyclic GMP

CHAPTER
Mechanism of action of group II hormones using cAMP 35
as second messenger
Horm one signal t ransducing t hrough HR com plex → G- prot eins → Adenylyl cyclase → cAMP
→ Prot ein kinase A → Prot ein phosphorylat ion, result ing in the ult im at e biochem ical response
(Continues…

CHAPTER
35
…Continued)

CHAPTER
35
Formation of cAMP from ATP and its inactivation to 5 AMP

CHAPTER
Calcium and phosphatidyl inositols 35
as second messengers
• Inositol 1, 4, 5 trisphosphate (IP3)

• Diacylglycerot (DAG)
• Calcium and calmodulin

CHAPTER
Formation of DAG and IP3 by the action of 35
phospholipase C

CHAPTER
35
The mechanism of action of group II hormones using DAG, IP3
and Ca 2 + as second messengers.
(Continues…

CHAPTER
35
…Continued)

CHAPTER
Kinase- phosphatase signal 35
transduction cascades
●
Several receptors have enzyme activity (intrinsic protein
kinase activity) that is activated by the binding of the
hormone to the receptor.
These kinases autophosphorylate the tyrosine residues,
triggering a cascade of phosphorylation–dephosphorylation
reactions involving several other protein kinases and
phosphatases inside the cell.
(Continues…

CHAPTER
35
…Continued)
●
Different signal transducing m olecules such as Jak,
STATs, Ras, MAP kinase SH2 domains are involved in
a com plex array of m olecular interactions in the
cytosol and inside the nucleus.
●
Horm ones such as insulin, growth hormone,
prolactin, erythropoietin, leptin and so on initiate
their action by activating intrinsic tyrosine kinase
activity.

CHAPTER
36
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CHAPTER
36
Chapter 3 6
Hypothalamic and Pituitary

Hormones

CHAPTER
36
Hypothalamic Hormones
Hypothalamic hormones directly control the production

and release of the anterior pituitary horm ones. Hence,
they are more com monly known as hypothalamic
releasing factors.
• Corticotropin- releasing horm one (CRH)

• Thyrotropin- releasing horm one (TRH)
• Gonadotropin- releasing hormone (GnRH)
• Growth hormone- releasing hormone (GHRH)
• Growth hormone release- inhibiting hormone (GHRIH)
• Prolactin release- inhibiting hormone (PRIH)
CHAPTER
36
Regulatory feedback loops between hypothalamus -
pituitary - peripheral endocrine glands
For abbreviations see t he t ex t book .

CHAPTER
36
Anterior Pituitary Hormones
The hormones produced by adenohypophysis can be

broadly classified into three groups, based on their
mode of synthesis and mechanism of action.
I. The growth hormone – prolactin group

II. The glycoprotein hormones
III. The pro- opiom elanocortin peptide fam ily

CHAPTER
36
The growth hormone- prolactin group
• Growth hormone (GH)

• Mechanism of action and molecular dynamics

CHAPTER
Signal transduction pathways mediat ing the biochemical actions of 36
growth hormone at sub- cellular level
GHR = Growth hormone receptor

JAK = Janus activator kinase
IRS = Insulin receptor substrate
PI- K = Phosphatidyl inositol kinase
STATs = Signal transducers and
activators of transcription
MAPK = Mitogenic activator protein
kinase
PLC = Phospholipase C
DAG = Diacyl glycerol

PKC = Protein kinase C
GLUT = Glucose
transporter

CHAPTER
Biochemical and physiological 36
functions of GH
• Effects on growth
• Effects on protein metabolism
• Effects on carbohydrate m etabolism
• Effects on lipid m etabolism
• Effects on mineral m etabolism
• Effects on lactation

CHAPTER
36
Disorders of GH production
Deficiency of GH
• Dwarfism GH-deficient dwarfs
• Laron type dwarfs and pygmies
Excess of GH
• Gigantism in children and acromegaly in adults

CHAPTER
36
Prolactin
Prolactin or lactogenic hormone is primarily involved in the

initiation and maintenance of lactation
Diagnostic chemistry and pathophysiology:

Prolactin measurements are indicated to investigate
amenorrhea (cessation of menses), infertility and
galactorrhea (spontaneous, inappropriate lactation).
Tumours of prolactin secreting cells cause the problems

mentioned above. Overproduction of prolactin causes
gynecomastia (breast enlargement in men).

CHAPTER
36
The glycoprotein hormones
Horm ones secreted by the anterior pituitary that are

glycoprotein in nature are classified under this
category.
These horm ones, four in number, are structurally
sim ilar but have diverse functions.
1. Thyroid stimulating horm one (TSH)

2. Follicle stimulating horm one (FSH)
3. Luteinising hormone (LH)
4. Hum an chorionic gonadotropin (HCG)

CHAPTER
The pro- opiomelanocortin (POMC) peptide 36
family
1. ACTH
2. - lipotropin (- LPH)
3. - MSH

CHAPTER
36
Adrenocorticotropic hormone (ACTH)
• Biochemical role of ACTH

• Ex cess production of ACTH

CHAPTER
36
Derivation of hormones from POMC cleavage
See the tex tbook for abbreviations.

CHAPTER
36
●
–lipotropin (–LPH)
Endorphins and enkephalins
●
Melanocyte- stimulating horm one (MSH)

CHAPTER
36
Posterior pituitary hormones
●
Two hormones, nam ely ox ytocin and antidiuretic
hormone (ADH), are released from the posterior
pituitary gland (neurohypophysis).
These two horm ones are originally synthesised in the

hypothalamus and are carried with specific carrier
proteins to the posterior pituitary from where they
are released into circulation.

CHAPTER
Structures of posterior pituitary horm ones

36
The amino acids that are different in ox ytocin and ADH are shown in red.

CHAPTER
36
Oxytocin
Biochemical and physiological functions
• Oxytocin stimulates uterine contractions during parturition
• Oxytocin acts on the myoepithelial cells of the breast and

produces a squeezing effect on the ducts, enhancing milk
flow through the ducts to the nipples (milk ejection or milk
let-down response).

CHAPTER
36
Antidiuretic hormone (ADH)
As is obvious from the name, ADH stim ulates the

kidneys to retain water.
It is also called vasopressin because it causes a
generalised vasoconstriction.
ADH is released in response either to a rise in plasma
osmololity or to a fall in ex tracellular fluid volume.

CHAPTER
36
Biochemical and physiological functions of ADH
• ADH causes the kidney tubules to reabsorb water
• ADH induces a generalised vasoconstriction (hence the

name, vasopressin) that leads to a rise in blood pressure.

CHAPTER
36
Abnormalities of ADH production and action
●
Abnormalities of ADH production or action cause diabetes
insipidus, a condition characterised by the excretion of
high volumes of dilute urine (polyuria).
●
Nephrogenic diabetes insipidus

CHAPTER
37
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CHAPTER
37
Chapter 3 7
Thyroid Hormones

CHAPTER
37
The thyroid gland produces two m ajor horm ones,

nam ely, triiodothyronine (T3 ) and tetraiodothyronine or
thyrox ine (T4 ).
It also produces calcitonin, a hormone involved in the

regulation of calcium levels in the blood.

CHAPTER
37
The structural formulae of thyroid

hormones

CHAPTER
37
Biosynthesis of thyroid hormones
A protein nam ed thyroglobulin and the trace elem ent

iodine are essential for the synthesis of thyroid
hormones.
• Uptake and activation of iodide
• Thyroglobulin
• Organification
• Coupling
• Phagocytosis (Continues…

CHAPTER
37
…Continued
• Hydrolysis of thyroglobulin
• Deiodination and recycling of iodine
• Release of T3 and T4

CHAPTER
37
Biosynthesis and
release of T 3 and
T4 in the thyroid
gland
See the tex tbook

for the details of
steps 1 - 8
(Continued…

CHAPTER
Biochemical and physiological 37
functions
• Stim ulation of basal metabolic rate (BMR)

• Effect on protein synthesis
Protein synthesis and cause positive
nitrogen balance
• Effect on carbohydrate m etabolism
Intestinal absorption of glucose
Glycogenolysis and gluconeogenesis
• Effect on lipid m etabolism
Lipolysis and lipid turnover

CHAPTER
37
Disorders of thyroid function
Goitre
Hyperthyroidism
• The m ost com mon cause of hyperthyroidism is
Grave's disease
Hypothyroidism
• Cretinism
• Myx oedema

CHAPTER
Laboratory diagnosis of 37
thyroid function
Reference values of thyroid hormones

CHAPTER
38
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CHAPTER
38
Chapter 3 8
Hormones of the
Adrenal Gland

CHAPTER
38
The adrenal glands are two small organs (each weighing

about 10 g) located above the kidneys (hence also called
suprarenal glands).
They have two functionally distinct parts, of different

embryological origin – the outer cortex (with three zones)
and an inner medulla

CHAPTER
38
Adrenal gland with cortex (three zones) and medulla

CHAPTER
38
Hormones of the adrenal cortex
The adrenal cortex secretes three classes of steroid

hormones.
• Glucocorticoids
• Mineralocorticoids
• Sex steroid hormones

CHAPTER
38
Synthesis of adrenocorticosteroids
Adrenal steroidogenesis (biosynthetic pathway of major steroids in
adrenal cortex )
(Continues…

CHAPTER
38
…Continued)

CHAPTER
38
Salient features of adrenal steroidogenesis
• Mineralocorticoid synthesis
• Glucocorticoid synthesis
• Androgen synthesis

CHAPTER
Biochemical and physiological functions of 38
glucocorticoids
Cortisol, cortisone and corticosterone are the

important glucocorticoids.
1. Effects on carbohydrate metabolism :

Glucocorticoids increase the supply of amino acids for
gluconeogenesis. Cortisol also increases hepatic
glycogenesis
2. Effects on lipid metabolism: Glucocorticoids

promote the breakdown of triacylglycerols (Lipolysis)
(Continues…

CHAPTER
38
…Cont inued
3 . Effects on protein and nucleic acid metabolism:

Glucocorticoids promote RNA synthesis and protein
synthesis
4 . Effects on body defence: They suppress the host

imm une response
5 . Other physiological effects: Maintain water–

electrolyte balance. Maintain normal blood pressure
and cardiac output.

Biochemical and physiological
CHAPTER
38
functions of mineralocorticoids
Aldosterone is the most potent among mineralocorticoids.
Aldosterone acts on the distal renal tubule to promote Na +

reabsorption in exchange for urinary loss of H+, K+ and NH4+
ions.
Its effect on sodium makes it an important determinant of

ECF volume.

CHAPTER
38
Diseases of adrenal cortical function
Hypofunction of the adrenal cortex
Disorders resulting from adrenal insufficiency is known

as Addison's disease.
Addison's disease is characterised by hypoglycemia,

intolerance to stress, weight loss, anorex ia, low blood
pressure, severe weakness, low Na+ and high K+ levels
in plasm a, increased skin pigm entation and so on.

CHAPTER
Hyperfunction of the adrenal cortex

38
Hyperfunction of the adrenal cortex resulting in

glucocorticoid ex cess is known as Cushing's
syndrome.
The condition is characterised by hyperglycemia,

muscle wasting causing negative nitrogen balance,
fatigue, ex cess deposition of fat in the trunk region
giving a typical 'buffalo hum p' appearance,
osteoporosis, hypokalemia and hypernatremia.

Laboratory diagnosis of adrenocortical
CHAPTER
38
function
The androgens are ex creted in urine as

17- ketosteroids (5–17 mg/ day)
Synacthen test

CHAPTER
Horm ones of the adrenal m edulla

38
Adrenal m edulla is an ex tension of the sympathetic

nervous system.
It produces two important hormones:
- epinephrine (formerly known as adrenaline) and
- norepinephrine (form erly noradrenline).

CHAPTER
38
Synthesis of catecholamines
Synthesis of catecholamines from tyrosine
(Continues…

CHAPTER
38
…Continued)

CHAPTER
38
Mechanism of action
The catecholamines act through two classes of
receptors.
1.- adrenergic receptors
2. - adrenergic receptors

CHAPTER
Biochemical and physiological functions of 38
catecholamines
• Response to stress
Fight-or-flight response
• Effects on carbohydrate metabolisms
Hyperglycemic effect
• Effects on lipid metabolism
Lipolysis
• Other physiological functions

Abnormalities of catecholamine
CHAPTER
38
Production
Pheochromocytomas
●
The disease is manifested by signs and sym ptoms
characteristic of catecholam ine ex cess (e.g.,
hypertension, palpitations, severe headache,
ex cessive sweating).
●
The tum our cells produce more norepinephrine than
epinephrine.
●
The levels of vanillylmandelic acid (VMA) in urine
(normal < 8 mg/ day) are elevated in patients of
pheochromacytom a. This is m easured in clinical
chemistry laboratories.

CHAPTER
39
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CHAPTER
39
Chapter 3 9
Hormones of the
Gonads

CHAPTER
39
The primary reproductive organs, the testes (in males)

and the ovaries (in females), collectively known as
gonads, perform two types of functions.
1. Gametogenesis (the production of gametes –

spermatozoa in males and ova in females)
2. Synthesis of sex hormones (gonadal steroids)

CHAPTER
39
Sex hormones are categorised into three groups.
• Androgens
• Estrogens
• Progestins

CHAPTER
39
Androgens
In the m ale, testosterone is the principal and the most

potent androgen.
Biosynthesis of androgens

CHAPTER
39
Biosynthesis of Androgens and Estrogens from
Cholesterol
For the enzymes of the initial steps of the pathway see Fig. 37.2
(Continues…

CHAPTER
39
…Continued)

CHAPTER
Biochemical and physiological functions 39
of androgens
1. Development and m aintenance of the male

reproductive system .
2. Metabolic effects
3. Androgens along with FSH induce Sertoli cells to
produce a hormone called inhibin.
Inhibin plays a role in promoting sperm atogenesis and
testosterone production by stim ulating the secretion
of FSH through a negative feedback loop.
(Continues…

CHAPTER
39
…Continued)
4. Testosterone is required for the initiation and

maintenance of spermatogenesis through Sertoli
cells.
5. Testosterone induces differentiation of m ale

accessory reproductive organs and maintains their
function.

CHAPTER
Abnormalities associated with 39
androgen defects
• Hypogonadism
• Testicular feminisation syndrome
• Castration

CHAPTER
Biosynthesis of estrogens and 39
progesterones
 Ovaries produce estrone (E1 ), estradiol (E2 ) while

placenta synthesises E1 , E2 and estriol (E3 ).

Corpus luteum

Placenta

CHAPTER
Biochemical and physiological role 39
of estrogens
1. They stimulate the growth of the ovary and follicles.
2. Estrogens prepare the endom etrium for

progesterone actions by stimulating progesterone
receptors; increase myometrial contractions in the
uterus; stimulate secretion of cervical mucus.
3. Estrogens prom ote prolactin secretion but inhibit

prolactin's milk producing effect on the mamm ary
glands.
(Continues…

CHAPTER
39
…Continued )
4. Estrogens increase lipogenesis in adipose tissue and

cause a 'female pattern of fat distribution' (fat
deposition on the hips and breasts).

CHAPTER
Biochemical and physiological role of 39
progesterone
1. Progesterone converts the estrogen- prim ed

endometrium to an actively secreting tissue that will
allow the implantation of the blastocyst; it
stimulates the secretion of a thick, sticky cervical
m ucus. It has an overall effect of m aintaining the
pregnancy.
2. It decreases proliferation of the vaginal epithelium;

decreases m yometrial contractions and movem ents
of the fallopian tubes.
(Continues…

CHAPTER
39
…Continued )
3. Progesterone stim ulates breast growth, particularly

of glandular tissue; inhibits the milk- inducing
actions of prolactin.
4. It increases the body tem perature.

CHAPTER
39
●
Role of estrogens and progesterone in
maintaining the menstrual cycle

Role of estrogens and progesterone during
pregnancy
• Contraception
• Menopause

CHAPTER
Hormonal disorders of the female 39
reproductive system
• Prim ary hypogonadism
• Secondary hypogonadism
• Turner’s syndrome
• Polycystic ovary disease (PCOD) (Stein- Levinthal
syndrom e)
• Congenital adenal hyperplasia
• Precocious puberty
• Amenorrhea
• Dysm enorrhea (painful m enstruation)

CHAPTER
40
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CHAPTER
40
Chapter 4 0
Hormones of the
Pancreas and Diabetes
Mellitus

CHAPTER
40

The pancreas is unique in that it contains two
different organs within its structure; an ex ocrine
gland and an endocrine gland.

The ex ocrine part secretes enzymes that are used for
digestion.

The endocrine portion contains about 1–2 m illion of
the islets of pancreas containing different cell types
(, ,  and so on). They secrete different horm ones.

CHAPTER
40
1. - cells secrete glucagon
2. - cells secrete insulin
3. - cells secrete somatostatin
4. F cell type secrete sm all am ounts of pancreatic

polypeptide

CHAPTER
40
Insulin
Biosynthesis of insulin
Proteolytic
processing of
proinsulin to
insulin

CHAPTER
Mechanism and Molecular Dynamics of Insulin 40
Action at the Cellular Level
Diagrammatic
representation
of signalling
pathways
downstream
from the insulin
receptor .
For
abbreviations,
see the
tex tbook .
(Continues…

CHAPTER
40
…Continued)

CHAPTER
40
Metabolic Effects of Insulin
Insulin acts as an anabolic hormone in the body
1. Effect on carbohydrate metabolism

• Promotes glucose uptake
• Favours glucose utilisation
• Discourages glucose production
2. Effects on lipid metabolism
• Favours lipogenesis
• Decreases lipolysis
• Discourages ketone body formation
(Continues…

CHAPTER
40
…Continued)
3. Effects on protein metabolism

• Insulin stimulates translation process

CHAPTER
Glucagon 40
Metabolic effects of glucagon

The metabolic effects of glucagon are in general, opposite
to that of insulin.
1. Effects on carbohydrate metabolism

2. Effects on lipid metabolism
3. Effects on protein metabolism

CHAPTER
40
Diabetes Mellitus

It is a metabolic disorder resulting either from deficiency of
insulin or resistance to its action causing increased blood
glucose levels (hyperglycemia) which lead to several
systemic complications.

Diabetes mellitus is broadly classified into two categories
based on etiology rather than treatment.
1. Type I diabetes mellitus

2. Type II diabetes mellitus

CHAPTER
40
Type 1 Diabetes Mellitus
Based on treatment, it was formerly known as insulin-

dependent diabetes mellitus (IDDM).
It is also called Juvenile-onset diabetes.
Type 1 DM is considered an autoimmune disease.

CHAPTER
40
Type 2 Diabetes Mellitus
It is also called adult-onset diabetes.
It was formerly known as non-insulin dependent diabetes

mellitus-NIDDM.
●
Metabolic syndrome

CHAPTER
40
Metabolic Abnormalities in Diabetes
Diabetic ketoacidosis
• Diabetic ketoacidosis is characterised by hyperglycemia,
hyperketonemia and metabolic acidosis.
• Kussmaul’s respiration
• Sweet smell
• Hyperglycemia
• Ketosis
(Continues…

CHAPTER
40
…Continued)
• Metabolic acidosis
• Severe dehydration
• Electrolyte loss
Hypersomolar nonketotic coma
Lactic acidosis

CHAPTER
40
Long- term Complications in Diabetes
These include
• Atherosclerosis
• Retinopathy
• Nephropathy
• Neuropathy

CHAPTER
Laboratory Diagnosis and Monitoring of 40
Diabetes Mellitus
• Blood glucose estimation
Random blood sugar (RBS)
Fasting Blood Sugar (FBS)
Post Lunch or post- prandial

CHAPTER
Urine Testing for Glucose, Ketones 40
and Proteins
• Testing urine for glucose is com monly done in

medical wards by using glucose- specific dipsticks
based on Benedict's principle.
• Detection of ketone bodies in urine is done by
Rothera's test.
• Testing for urinary albumin is performed to assess
renal dam age.
• Microalbuminuria
• Blood lipids and other parameters

CHAPTER
40
Glucose Tolerance Test (GTT)
• Normal response
• Diabetes mellitus
• Impaired glucose tolerance (IGT)
• Renal glycosuria
• Alim entary glycosuria
• Gestational diabetes mellitus (GDM)

CHAPTER
40
●
Glycated hemoglobin
The concentratio of HbA1 c reflects the mean
blood glucose concentration of the preceding 8 –
1 0 weeks.
●
Fructosamine

CHAPTER
40
Reference values of
HbA1 c

CHAPTER
40
Hypoglycemia
• Whipple’s triad
• Post- prandial hypoglycem ia
• Reactive hypoglycemia
• Fasting hypoglycem ia

CHAPTER
41
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CHAPTER
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Chapter 4 1
Extracellular Matrix
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CHAPTER
41
Composition of extracellular matrix
The ECM is made up of three major classes of biom olecules:
1 .Structural proteins such as collagen, elastin and fibrillin

which constitutes the major chunk of the ground substance.
2. Specialized proteins such as fibronectin and laminin,

which form a mesh of fibres resulting in a porous structure.
3. Proteoglycans, consisting of long chains of repeating

structures of disaccharides (glycosaminoglycan– or GAG,
known earlier as mucopolysaccharides), attached to specific
proteins.
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CHAPTER
41
Functions of ECM
• It holds the cells together in the tissues and plays a role in

normal growth and development of the latter.
• The ground substance (ECM), a porous structure, provides a

pathway for the sim ple diffusion of blood gasses between
cells and blood capillaries.
• It serves as a medium for the supply of nutrients to

individual cells.
• It plays a role in the development of inflammation.
• It plays an important role in the ageing process reflected in

the age- related changes in ECM.
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CHAPTER
41
Biomedical significance of ECM
• Ex tracellular matrix is believed to be associated with the

spread of cancer. Recent research suggests that
alterations in the structures of glycoproteins and
proteoglycans on the surfaces of cancer cells result in
metastasis.
Metastasis is the phenomenon whereby cancer cells leave

their site of origin (e.g., the breast) and m igrate through
the blood stream to distant sites in the body (e.g., the
brain) and grow there in an uncontrolled m anner, causing
deleterious effects in advanced- stage cancer patients.
(Continues…
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CHAPTER
41
. . . Continued )
• Involvement of certain components of ECM is established

in two of the major connective tissue diseases –
rheumatoid arthritis (RA) and osteoarthritis (OA).
Arthritis is a term com monly used to describe inflam mation

of joints. There are different kinds arthritis other than the
above two types. Although RA and OA both affect joints,
they are very different from each other.
RA is an autoimmune disorder in which the body’s own

imm une system attacks the body’s joints, while OA is a
degenerative condition caused by age- related
mechanical wear and tear of joints.
(Continues…
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CHAPTER
41
. . . Continued)
• Several diseases (such as osteogenesis imperfector and

various types of Ehlers–Danles syndrome) occur due to
genetic derailment of the synthesis of collagen.
• Genetic deficiencies of the lysosom al hydrolases involved

in the degradation of glycosaminoglycans (GAGs) cause
certain diseases called mucopolysaccharidoses, so called
on the basis of the older nam e for GAGs.
These disorders occur due to im paired degradation of GAGs

and hence their abnorm al accumulation in various tissues.
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Functions of Collagen
1. Collagens that are present in all tissues and organs

play a m ajor role in determining size and shape of
the organs. It provides structural strength and
support to tissues.
2. As a major com ponent of ECM, it enables proper

alignment of cells and thus remodels organs and
tissues.
3. It also plays a role in the proliferation and

differentiation of cells into different tissues and
organs.
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Schematic of the structure of collagen and fibril
(X and Y represent am ino acids ot her t han glycine.)
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Types of collagen and their distribution
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Diseases due to impaired collagen

synthesis
About 30 genes encode the collagen and eight enzymes

take part in its biosynthesis. Thus, it is not surprising
that several diseases occur due to mutations in
collagen genes or in the genes that encode the
enzymes involved in collagen synthesis (mostly
during post- translational modifications).
Some of the clinically significant genetic diseases are

described nex t.
(Continues . . .
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. . . Continued) Diseases due to impaired collagen synthesis
1 . Osteogenesis imperfecta (Brittle bone disease)
The condition is caused by m utations in the genes encoding type I

and II collagens.
The disease is characterised by increased fragility of bones that lead
to multiple fractures in infancy and childhood. The sclera
becomes extremely thin and appear blue owing to a deficiency
of collagen. Eight types (I- VIII) of this condition have been
recognised.
Am ong these, type II is very severe and infants with this condition do
not survive. In other types, there is replacement of glycine with
som e other am ino acid which affects the form ation of the triple
helix resulting in polypeptides assem bling into abnormal fibrils.
This leads to degradation of the m olecule and is known as
procollagen suicide. (Continues . . .
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. . . Continued) 41
Diseases due to impaired collagen synthesis
2 . Ehlers–Danlos syndrome
This group of disorders are characterised by hyperextensiblility

of skin and hypermobility of the joints. (The contortionist
known as the rubber man of Gem ini Circus could twist his body
into am azing shapes owing to his being a victim of Ehlers–Danols
syndrom e.)
Other sym ptom s of this condition include vascular fragidity,

kyphoscoliosis, arthrochalasis and derm atosparax is. Type I, III and
V collagens are affected.
(Continues . . .
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3 . Alport syndrome (Hereditary nephritis)
It is due to m utations affecting the synthesis of type IV collagen

found in the basem ent m em branes of the renal glom eruli, inner ear
and eye. The early sign is hem aturia (ex cretion of blood in urine)
followed by ocular lesions and hearing loss. The patient finally
develops end- stage renal disease.
4 . Epidermolysis bullosa
This condition which shows blisters and cracks in the skin following
sm all injuries is caused by m utations in the gene encoding type VII
collagen.
(Continues . . .
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5 . Scurvy
Scurvy, caused by vitam in C deficiency, is not a genetic disorder.

Vitam in C, however, is essential for the hydrox ylation of proline
and lysine to form hydrox yproline and hydrox ylysin respectively,
which are integral to the structure of collagen. In vitam in C
deficiency (scurvy), collagen synthesis is adverse affected,
resulting in defective connective tissue.
This results in capillary fragility, which, in turn, leads to bleeding

under the skin. This is why patients of scurvy suffer from swollen
and bleeding gum s, subcutaneous haem orrhages and poor wound
healing capability.
(Continues . . .
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6 . Lathyrism
Like scurvy, lathyrism is also not a genetic disorder but a condition
characterised by lack of strength in or inability to m ove lower lim bs
observed in people who consum e pulse lathyrus sativa or sweet pea
(kesari dal) regularly as a part of their diet. This pulse contains a
tox in called β - ox alyl am inoalanine (BOAA) which inhibits the
enzym e lysyl ox idase, which adversely affects the cross- linking of
lysine in the collagen structure.
The result is weakening and brittleness of bones.
7 . Menkes disease
Deficiency of copper results in defective cross- linking of collagen
and elastin by the copper- dependent enzym e lysyl ox idase.
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Genetic Abnormalities of Elastin
Williams–Beuren syndrome: This is a m ultisystem disorder

characterised by heart defects and unusual facial features. It occurs
due to deletion of elastin gene resulting in defective connective
tissue which also affects the central nervous system . Supravalvular
aortic stenosis occurs due to defective elastin synthesis,
ham pering blood flow.
Scleroderma: This is a skin disorder associated with accum ulation

of elastin in the skin.
Pulmonary emphysema: Decreased elastin synthesis results in

pulm onary em physem a.
Aging of skin: Fragm entation or decreased elastin form ation results

in cutis laxa and prem ature aging of skin.
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Marfan Syndrome
Mutations in the gene for fibrillin cause Marfan syndrome.
It is inherited as an autosomal dominant trait characterised by

hyperextensibility of joints.
Patients of this syndrome show disproportional skeletal growth.
They are tall and have large body frames; even the digits of their
hand are extra-long.
They also exhibit cardiovascular signs such as weakness of the aorta

leading to dilation of the ascending aorta (aortic aneurism), and
dislocation of eye lens (ectopia lentis).
(It is conjectured that American President Abraham Lincoln could have

been a victim of Marfan syndrome.)
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Structure of Aggrecan
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Functions of glycosaminoglycans
1. GAGs, as part of proteoglycans, serve as the structural

components of extracellular matrix (ECM). They interact
with other components of ECM such as collagen, elastin,
fibrillin, fibronectin and laminin.
2. They play a role in cell- to- cell adhesion and facilitate

cell migration. Some of them serve as receptors in cell
membranes.
3. They contribute to the characteristic turgor of different

tissues.
4. They are responsible for the porous structure of ground

substance and serve as molecular sieves in the ECM.
(Cont inues . . .
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. . . Cont inued) 41
Functions of
glycosaminoglycans
6. They are the components of synaptic vesicles.
7. As a component of glom erular mem brane, they play a role

in glomerular filtration.
8. They play a role in maintaining corneal transparency.

They are responsible for maintaining the structural
integrity of sclera of the eyes.
9. They serve as anticoagulant and prevent intravascular

clotting.
10. They facilitate fertilization by clearing the gel around

ovum and also play a role in morphogenesis in embryos.
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Heteropolysaccharides
Glycosaminoglycans (GAGs)
GAGs, also known as mucopolysaccharides, are made

up of repeating units of disaccharides containing amino
sugars (glucosamine or galactosam ine) and uronic
acids (glucuronic or iduronic acids).
Amino sugars are further acetylated and sulphated.
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The major GAGs include:
1. Hyaluronic acid
2. Chondroitin sulphates
3. Keratan sulphates
4. Heparin and heparan sulphate
5. Dermatan sulphate
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Structures of Glycosaminoglycans
Hyaluronic acid
Chondroitin 4- sulphate
(Continues…
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…Continued) Structures of glycosaminoglycans
Keratan sulphate
Heparin
(Continues…
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. . .Continued) Structures of glycosaminoglycans
Dermatan sulphate
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Important Mucopolysaccharidoses (MPS)
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Types of Glycoproteins
Glycoproteins are classified into three major classes.
O- linked glycoproteins
They are form ed by O-glycosidic linkage with the –OH of
serine or threonine.
Usually, the glycosidic linkage is between N- acetyl

galactosam ine and serine or threonine. Mucins belong to
this group.
They are distributed on the surfaces of epithelial cells of the

respiratory, gastrointestinal and genitourinary tracts.
(Continues…
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. ...Continued) 41
Types of glycoproteins
N- linked glycoproteins
The linkage is between the N of the amide group of

asparagine and N- acetyl glucosam ine.
The glycosylation occurs in the endoplasmic reticulum

and the Golgi complex .
Plasma proteins belong to this group.
(Continues…
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. ...Continued) Types of glycoproteins
GPI- linked glycoproteins
This group of glycoproteins are anchored to the

plasm a mem brane by GPI (glycosyl phasphatidyl
inositol).
They are called sticky foot structures.
Ex amples include enzyme acetyl cholinesterase

anchored on the RBC membrane and alkaline
phosphatase on the intestine and placental structures.
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Disorders associated with glycoproteins
Congenital glycosylation disorders are a group of

autosomal recessive disorders resulting from mutation
in the genes encoding the enzymes required for the
synthesis of N- linked glycoprotein or the processing of
N- glycan chain.
Som e of these disorders are given nex t.
(Continues . . .
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. . . Continued)
Paroxysmal nocturnal hemoglobinuria
Parox ysmal nocturnal hemoglobinuria (PNH) is

characterised by anaem ia and the presence of
hemoglobin in urine due to RBC hem olysis.
This is due to a defect in the genes of the enzymes

required for attaching alucosamine to
phosphatidylinositol in GPI- linked glycoproteins,
leading to defective attachment of the protein to the
RBC membrane.
RBCs are hence easily hemolysed.

(Continues . . .
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. . . Continued)
I- cell disease or inclusion cell disease

I- cell disease is a m ultisystem ic disease involving CNS, leading to
psychological and m otor abnorm alities with m ental retardation.
Patients do not usually live beyond their first decade of life.
The lack of lysosm al enzym es in cells causes the accum ulation of
m any different types of undergraded m olecules in the lysosom es,
which results in the form ation of inclusion bodies.
A defect in the targeting of the glycoprotein enzym e hydrolase is
also present, which results in the enzym e protein not reaching its
destination, i.e., the lysosom es, but getting secreted instead in the
plasm a.
(Continues . . .
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. . . Continued)
Mannosidosis and related disorders
Degradation of glycoproteins requires many enzymes

such as neuram inidases, galactosidase, mannosidases,
and fucosides.
Mannosidosis is caused by the specific deficiency of α-

m annosidase.
Most diseases have CNS involvement leading to mental

retardation and enlargement of liver, spleen, etc. These
are autosom al recessive disorders which vary from m ild to
severe form.
At present, there is no definite treatm ent for them.

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Chapter 4 2
Reactive Oxygen Species

and Antioxidants
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The products of partial reduction of molecular ox ygen

which are unstable and highly reactive can dam age
the tissues. These potentially dangerous molecules
are called reactive ox ygen species (ROS).
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Types of reactive oxygen species (ROS)
1. Most of the ROS are free radicals in nature.
A free radical is a m olecule or molecular species

capable of independent ex istence that contains one
or more unpaired electrons (shown as a ‘dot’) in its
outer orbital, e.g., the hydrox yl radical . (Note
that it is different from the hydrox yl ion OH –),
hydroperox yl radical , lipid perox ide radical
, , nitric ox ide (NO) and so on.
2. Certain ions e.g. hypochlorite ion (CIO–).

(Continues…
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…Cont inued)
3. A combined form of free radical and ion , e.g.,

superox ide anion
4. Certain highly reactive m olecules (which do not

contain an unpaired electron), e.g., hydrogen
perox ide (H2 O2 ), singlet ox ygen (1 O2 ).
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Sources and generation of ROS
1 . Respiratory chain
About 2% of inhaled ox ygen undergoes partial
reduction liberating superox ide anion and perox ide
radical which are potentially destructive.
(Continues…
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…Cont inued)
2 . Respiratory burst
Macrophages and neutrophils engulf bacteria, utilising
large amounts of ox ygen in the process. This
phenomenon is called respiratory burst. As a result of
this phagocytic activity, about 10% of the consumed O 2
is diverted to the generation of ROS such as superox .ide
. and hypochlorite ion
(O2 –), H2 O2 , hydrox yl radical (OH)
(CIO–).
Interestingly, the generated ROS ex erts a potent
bactericidal action (killing the bacteria).
(Continues…
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…Cont inued)
3 . Lipid peroxidation
Initiation phase
Propagation phase
Termination phase
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Other sources of ROS
• Superox ide anion and H2 O2

• During - ox idation in perox isomes
• In WBC and platelets, the enzyme lipo- ox ygenase
• Haber- Weiss reaction
• During drug metabolism
• Ionising radiation (e.g. UV radiation, - rays)
• Tobacco sm oking
• Reperfusion- induced injury
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Mechanism of action of ROS
• Effect of ROS on lipids

• Effect on proteins and carbohydrates
• Effect on DNA
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ROS and disease
• Ageing
• Cardiovascular disease
• Cancer
• Inflam matory diseases
• Respiratory diseases
• Diabetes
• Infertility
• Beneficial effects of ROS
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Protection against ROS – 42
the role of antioxidants
• Preventive antiox idants
• Chain breaking antiox idants
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Antioxidant Enzymes
• Superox ide dism utase (SOD)
• Catalase
• Glutathione perox idase and reductase
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Antioxidant Vitamins
• Vitamin E
• Vitamin C
• - Carotene
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Other Antioxidants
• Selenium
• Glutathione
• Uric acid
• Bilirubin
• Ceruloplasmin, transferrin and haptoglobin
• Lipoic acid
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Antioxidant Foods
●
Unprocessed vegetable oils (like soyabean oil, rice
bran oil, cotton seed oil, sunflower oil), whole grains,
green leafy vegetables, legum es, fruits (like citrus
fruits, guava, am la), green vegetables (like spinach,
cabbage, cauliflower), carrots, papaya, waterm elon,
tom atoes have a high content of various antiox idants.
(Continues…
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…Cont inued)
●
Grapeseed, turm eric, walnuts, onions, beverages
such as green tea, red wine and organ meats contain
different polyphenols, catechins, curcuminoids,
proanthocyanidins, phenolic acids have antiox idant
properties. Certain food additives such as
propylgallate, butylated hydrox yl anisole, butylated
hydrox yl toulene are used to prevent lipid
perox idation in packed foods.
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Chapter 4 3
Biochemical Genetics
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Development of genetics – Mendel’s principles

Heredity – make- up in humans
●
Alleles
●
Diploid
●
Haploid
●
Aneuploid
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Genetic diseases and modes of 43
inheritance
Genetic diseases can be categorised into three

groups.
1. Chromosomal
2. Gene disorders
3. Multifactorial disorders
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Monogenic traits occur in different patterns, depending

on whether the m utant gene is dom inant or recessive
and whether it is on an autosom e or a sex chromosome.
1. Autosomal dom inant

2. Autosomal recessive
3. Sex or X–linked dom inant
4. Sex or X–linked recessive
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• Inborn errors of m etabolism

• Genetics in cancer
• Prenatal diagnosis
• Eugenics
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Differences between monozygotic and dizygotic twins
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Chapter 4 4
Environmental Pollutants,
Toxins and Biomedical
Waste Management
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Types of Pollution
Pollution can be broadly categorised into the

following types.
1. Air pollution
2. Water pollution
3. Soil pollution
4. Noise pollution
5. Thermal pollution
6. Nuclear pollution
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Properties and Classification of Pollutants
Classification based on type of damage
1. Physical pollutants
2. Chem ical pollutants
3. Biological pollutants
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Classification based degradability
1. Biodegradable pollutants
2. Slowly degradable pollutants
3. Non- biodegradable pollutants
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Air Pollution
• Sulphur diox ide

• Nitrogen ox ides
• Ozone
• Am monia
• Carbon monox ide
• Carbon diox ide
• Volatile organic com pounds
• Persistent organic pollutants
• Airborne particles
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Water Pollution
• Effects of water pollution
• Measures to prevent water pollution
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• Soil pollution
• Noise pollution
• Therm al pollution
• Nuclear pollution
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Toxins and Poisons
• Lead
• Mercury
• Aluminium
• Cadm ium and arsenic
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Toxins in Foods
• Enzyme inhibitors
• Neurotox ins and m ycotox ins
• Goitrogens
• Biogenic am ines
• Anti- vitam ins
Pesticides
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• Biochemical Waste Management
Colour- coded bags for biomedical waste segregation as per 2016 rules
Cont inues…
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… Continued
Colour- coded bags for biomedical waste segregation as per 2016 rules
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45
Chapter 4 5
Human Genome Project and

Bioinformatics

CHAPTER
45
What is the Human Genome Project?
The human genom e project (HGP) is an international

scientific research project with the goal of
determ ining the complete sequence of nucleotides
of the human DNA, and of indentifying and mapping
all the genes of the hum an genome.

CHAPTER
45
The specific goals of HGP
• Estim ating the total number of genes; identifying and

locating the positions of all these genes in the hum an
genom e.
• Understanding the dynam ics of gene ex pression

which will improve our ability to provide better
diagnosis, treatm ent and prevention of diseases.
• Determ ining the sequences of the three billion

nucleotides in the DNA, storing this inform ation in
databases and developing tools for data analysis.

CHAPTER
Mapping and sequencing of the 45
human genome
• Mapping protocols
– Probes
– Hierarchical shotgun approach
– RFLP
– VNTR
– Cytogenetic maps
– FISH
(Continues…

CHAPTER
45
…Continued)
• Sequencing protocols
– Cloning
– rDNA technology
– PCR
– Gel electrophoresis
– Max am - Gilbert m ultiplex technique
– Sanger sequencing
– Chromosome walking
– Chromosome jumping

CHAPTER
45
Results of HGP
– Number of genes
– Orphan genes
– Non- coding RNAs
– Junk DNA
– Sizes of genes
– Genes associated with diseases
– Single nucleotide polym orphisms
– Satellite DNAs
(Continues…

CHAPTER
45
…Continued)
– Short tandem repeats (STRs)
LINEs
SINEs (ALU family)
LTRs

CHAPTER
45
Various Elements in the Human Genome

CHAPTER
Applications and proposed benefits of 45
HGP
Applications in medicine
The hum an genome project, through its sequencing of
the DNA, helps in understanding the root causes of
diseases, enabling us to develop effective treatment
strategies.
• Applications in biotechnology
• Agriculture
• Environm ental cleanup
• ELSI of HGP

CHAPTER
45
Bioinformatics
Bioinform atics is the combination of biology, com puter

science and inform ation technology.
Scope of bioinformatics
• DNA sequencing data
• Gene ex pressions profiles
• RNA and protein sequences
• Protein 3- D structure analysis
• Microarrays (DNA chips)
• Data from patients’ clinical trials
• Drug designing

CHAPTER
45
Biological Databases
• Primary databases
• Secondary databases

CHAPTER
45
• Nucleotide sequence databases

• Protein sequence databases
• Macrom olecular structure databases
• Other databases

Some Popular Database Website
CHAPTER
45
Addresses

CHAPTER
45
Applications of Bioinformatics
• Bioinform atics can be applied to the identification of
nucleotide sequences of protein- encoding genes. Since
amino acid sequencing is a tedious task, the base
sequence of the gene for a given protein can be
translated into an am ino acid sequence using the genetic
code.
However, this is easier said than done because eukaryotic

genes are interrupted by introns, making it difficult to
deduce the ex act location of the gene sequence that codes
for a particular protein.
The ends of introns, however, have specific base sequences
that help in locating the ex act positions of genes.
(Continues…

CHAPTER
45
…Continued)
• Specific locations in the DNA can be identified and cut by

restriction enzym es.
• By making a comparative study of the genomes of various

organism s, the evolutionary relationships
• between genes can be elucidated.
• The 3- D structure of proteins can be predicted and the

highly conserved sequences (which represent the
• functional domain portions) can be traced.
• Using the inform ation in databases, it is possible to

develop drugs to treat a disease or a diagnostic test to
detect diseases at an early stage.
(Continues…

CHAPTER
45
…Continued)
• Bioinformatics can be used in clinical trials and for

studying the complex m etabolic pathways in cells.
• In agriculture, it can be used to implant disease- and

insect- resistant crops to increase crop yields and to
monitor the nutritional content of plants that serve as
food. The cum ulative genetic health of a breeding pool
and livestock can be improved.
• Molecular modelling of biomolecules can be attem pted

using the available database.
• The complete DNA sequences or the genomes of

endangered species can be preserved (recall the
• recreation of dinosaurs in the movie, Jurassic Park).
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46
Chapter 4 6
Gene Therapy

CHAPTER
46
Gene therapy involves correcting defective genes. Gene

therapy involves isolating normal genes and inserting
them into diseased cells, so that the norm al genes are
ex pressed, allowing the diseased cells to return to a
normal state.
Gene delivery protocols
• Ex - vivo gene transfer
• In- vivo gene transfer

CHAPTER
46
Gene Transfer Techniques
Viral vectors
• Retroviruses
• Adenoviruses
• Herpes viruses
Transfer through liposomes
Receptor- mediated endocytosis
Artificial chromosome vectors
Antisense therapy
Target tissues for gene transfer

CHAPTER
46
Some of the diseases treated by gene therapy

CHAPTER
46
Use of retrovirus for gene therapy
The retrovirus carries an RNA copy of the therapeutic gene into the cell. The endosome containing virus
dissolves and the viral RNA and reverse transcriptase are released into the cell. The enzyme copies the RNA into
a double- stranded DNA that eventually integrates into the host DNA. Transcription and translation of this DNA
(the therapeutic gene) produces the therapeutic protein. Note that the virus does not multiply since its genes
were removed and replaced by the RNA copy of the therapeutic gene.

CHAPTER
46
Diseases treated with gene therapy

A defect in the adenosine deaminase (ADA) gene
located on chrom osom e 20 causes severe com bined
im munodeficiency syndrom e (SCID).

Familial hypercholesterolem ia

Cystic fibrosis

CHAPTER
46
• Gene therapy in cancer
• Antisense therapy

CHAPTER
46
The process of RNA
interference

CHAPTER
46
RNA interference, a natural mechanism evolved

against the invasion of RNA viruses,
is also used to block the ex pression of oncogenes.

CHAPTER
46
Future scope of gene therapy
The advent of gene therapy in the past few decades

offers the promise of many revolutionary changes in
clinical medicine.
• Curing genetic disorders perm anently

• Correcting cancer genes and inducing cancerous cells
to m ake tox ins so that they kill themselves
• Blocking viral genes (e.g., HIV)
• Creating stem cells from som atic cells

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CHAPTER 1

Rafi MD: Textbook of Biochemistry (4th Edition) Universities Press

Anatomy is the study of structure, Physiology is the

Rafi MD: Textbook of Biochemistry (4th Edition) Universities Press

Energy in Living Organisms

Every organism needs energy for its survival.

Energy flow in the environment

Essential Elements in the Body

Rafi MD: Textbook of Biochemistry (4th Edition) Universities Press

Polar and Non- polar Molecules

• Hydrophilic, hydrophobic and amphipathic

Rafi MD: Textbook of Biochemistry (4th Edition) Universities Press

Major Biomolecules in the Body

Rafi MD: Textbook of Biochemistry (4th Edition) Universities Press

The Cell and Biological

Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press

Structure of a Human Cell

Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press

Mem branes are made of a double layer of lipid

Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press

Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press

The ex tracellular surface of the plasm a m embrane

Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press

Fluid Mosaic Structure

Mem branes are organised into a structure known as

Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press

Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press

Transport Across Biological Membranes

Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press

Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press

1 . Primary Active Transport

2 . Secondary Active Transport

Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press

3 . Carrier Type Transport

Carrier type processes

Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press

4 . Vesicular Transport or Transcytosis

Sequence of events in endocytosis

Sequence of events in ex ocytosis

Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press

Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press

Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press

Classification and Nomenclature

Carbohydrates are classified into three major classes based

Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press

They are categorised into two groups based on the

Aldoses: Monosaccharides with an aldehyde group

are known as aldoses. For ex am ple, ribose (5C), glucose

are called ketoses. For ex am ple, fructose (6C).

Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press

Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press

The spatial arrangement of –H and –OH groups on the

If the –OH group is on the right side, the sugar

Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press

Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press

Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press

Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press

Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press

Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press