VK Malhotra - Biochemistry For Students, 12th Edition

BIOCHEMISTRY
for
Students
BIOCHEMISTRY
for
Students
12th Edition
VK Malhotra PhD (Gold Medalist)
Department of Biochemistry
Maulana Azad Medical College (MAMC)
New Delhi, India
Foreword
Nancy Kaul
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Biochemistry for Students
2012, Jaypee Brothers Medical Publishers
All rights reserved. No part of this publication should be reproduced, stored in a
retrieval system, or transmitted in any form or by any means: electronic, mechanical,
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This book has been published in good faith that the material provided by the author
is original. Every effort is made to ensure accuracy of material, but the publisher,
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of any dispute, all legal matters are to be settled under Delhi jurisdiction only.
Previous Editions: 1978, 1980, 1982, 1984, 1985, 1987, 1989,
1991 (Reprint 1993), 1996, 1998, 2003 (Reprint 2006, 2008)
Twelfth Edition: 2012
ISBN 978-93-5025-504-9
Typeset at JPBMP typesetting unit
Printed at
Foreword
Biochemistry has been playing a very important role in day-
to-day life of medical students. The book Biochemistry for
Students written by Dr VK Malhotra, Gold Medalist, serves
as a quick reading material being purposefully written in clear,
lucid and precise manner. This book will certainly serve the
needs of medical students.
Dr (Mrs) Nancy Kaul
Ex-Head, Department of Biochemistry
Lady Hardinge Medical College
New Delhi, India
Preface to the Twelfth Edition
This book is revised keeping in view all categories of students
and it addresses their needs in a simple and practical manner
as biochemistry tries to explain the mystery of life in the
language of chemistry.
I hope the book will be received warmly by the students
as well as teachers for both desire maximum benefits out of
it. All the chapters are revised to gain understanding and
clarity. Suggestions to improve the future editions are most
welcome and will be highly appreciated.
I would like to thank Shri Jitendar P Vij (Chairman and
Managing Director) and Mr Tarun Duneja (Director Publishing)
of M/s Jaypee Brothers Medical Publishers (P) Ltd, New Delhi
for the publication of this book. Mr Subrata Adhikary (Author
Coordinator) deserves special praise for this venture.
VK Malhotra
Preface to the First Edition
Biochemistry currently occupies an eminent position parti-
cularly among medical subjects. However, there are few texts
in the market at present which enable the students to acquire
a working knowledge of the subject.
Having been connected with the teaching profession for
the past few years, I am well acquainted with the difficulties
encountered by the students while trying to master the subject.
The present book is the result of my humble attempt to
overcome these handicaps and present the subject in a simple
and easily comprehensible form.
Attempts have been made to illustrate the subject matter
with diagrams and chemical formulae wherever necessary.
Special thanks to my publisher Shri Jitendar P Vij, without
whose help, this book could not have seen the light of the
day.
VK Malhotra
Contents
1. Biophysics ......................................................................... 1
Hydrogen Ion Concentration, pH ................................................. 1
Osmosis and Osmotic Pressure .................................................... 12
Colloids ............................................................................................ 16
Surface Tension ............................................................................... 17
Absorption....................................................................................... 18
Viscosity ........................................................................................... 18
2. Chemistry of Carbohydrates ..................................... 19
Carbohydrates ................................................................................ 19
Functions of Carbohydrates ......................................................... 19
Classification of Carbohydrates ................................................... 19
Oligosaccharides ............................................................................. 40
Polysaccharides ............................................................................... 45
Heteropolysaccharides .................................................................. 49
3. Chemistry of Lipids .................................................... 53
Simple Lipids ................................................................................... 54
Compound Lipids ........................................................................... 62
Derived Lipid................................................................................... 68
4. Chemistry of Amino Acids and Proteins ............ 74
Chemistry of Amino Acids ........................................................... 74
Proteins ............................................................................................ 85
5. Hemoglobin.................................................................. 102
Porphins ......................................................................................... 102
Porphyrins ..................................................................................... 103
xii BIOCHEMISTRY FOR STUDENTS
Hemoglobin .................................................................................. 103
Porphyria ....................................................................................... 114
6. Enzymes ........................................................................ 120
Enzymes ......................................................................................... 120
Factors Influencing the Rate of Enzymatic Reactions ............. 124
Enzyme Activity ........................................................................... 129
Enzyme Inhibitions ...................................................................... 130
Catalytic Site or the Active Sites of the Enzymes .................... 134
Enzyme Induction ........................................................................ 135
Diagnostic Value of Plasma Enzymes ....................................... 137
7. Biological Oxidation ................................................. 140
Biological Oxidation...................................................................... 140
Mixed Function Oxidases ............................................................. 142
High Energy Compounds ........................................................... 143
Respiratory Chain ........................................................................ 144
8. Metabolism of Carbohydrates............................... 151
Glycolysis ....................................................................................... 151
Citric Acid Cycle ........................................................................... 155
Energetics ....................................................................................... 158
Glycogenesis .................................................................................. 163
Gluconeogenesis ........................................................................... 168
Galactose Metabolism .................................................................. 169
Fructose Metabolism.................................................................... 172
Lactose Synthesis .......................................................................... 173
Uronic Acid Pathway ................................................................... 174
Regulation of Blood Glucose ....................................................... 175
9. Metabolism of Lipids ............................................... 184
Plasma Lipoproteins ..................................................................... 184
CONTENTS xiii
10. Metabolism of Proteins ........................................... 210
Digestion and Absorption ........................................................... 210
Urea Cycle (Krebs-Henseleit Cycle) .......................................... 214
Glycine ............................................................................................ 221
Methionine ..................................................................................... 226
Cysteine and Cystine ................................................................... 227
Phenylalanine and Tyrosine ........................................................ 229
Tryptophan.................................................................................... 237
Leucine, Isoleucine and Valine .................................................... 240
11. Nucleic AcidChemistry and Metabolism...... 241
Nucleic Acids ................................................................................. 244
12. Vitamins ....................................................................... 259
Fat Soluble Vitamins .................................................................... 261
Vitamin A....................................................................................... 261
Vitamin D....................................................................................... 264
Vitamin E ....................................................................................... 265
Vitamin K....................................................................................... 266
Water Soluble Vitamins ............................................................... 268
Vitamin C....................................................................................... 268
Thiamine ........................................................................................ 270
Riboflavin....................................................................................... 272
Niacin.............................................................................................. 273
Pantothenic Acid ........................................................................... 275
Pyridoxine ...................................................................................... 275
Biotin............................................................................................... 277
Folic Acid........................................................................................ 279
Cyanocobalamin........................................................................... 281
Antivitamins .................................................................................. 283
13. Acid-base Balance ..................................................... 284
Acid-base Balance ......................................................................... 284
xiv BIOCHEMISTRY FOR STUDENTS
14. Water and Mineral Metabolism........................... 292
Biological Importance of Water .................................................. 292
Minerals .......................................................................................... 295
15. Xenobiotics .................................................................... 306
16. Nutrition ....................................................................... 310
Food Values ................................................................................... 319
1500 Calories Diabetic Diet Chart .............................................. 322
17. Organ Function Tests ............................................... 326
Liver Function Tests ..................................................................... 326
Renal Function Tests .................................................................... 330
Pancreatic Function Test .............................................................. 335
Gastrointestinal (Git) Function Test ........................................... 338
18. Immunology ................................................................. 339
Introduction................................................................................... 339
Functions of T Cells ...................................................................... 343
19. Cancer ............................................................................ 356
20. Hormones...................................................................... 360
Insulin ............................................................................................. 364
Glucagon ........................................................................................ 367
Triiodothyronine (T
3
) and Thyroxine (T
4
) ................................. 367
Calcitonin ....................................................................................... 368
Parathormone ............................................................................... 369
Thyroid Gland ............................................................................... 370
CONTENTS xv
21. Protein Biosynthesis ................................................. 371
Activation Step .............................................................................. 372
Initiation of Polypeptide Chain (In Ribosomes) ...................... 374
Elongation...................................................................................... 376
Termination ................................................................................... 378
Codon............................................................................................. 380
Regulation of Gene Expression .................................................. 381
22. Instrumentation .......................................................... 385
Colorimetry ................................................................................... 385
Electrophoresis .............................................................................. 386
Isotopes and their Application.................................................... 387
Electrometric Determination of pH ........................................... 388
Estimation of Nitrogen Content by Micro-Kjeldahl Method..... 390
Chromatography ......................................................................... 393
Index ........................................................................................ 397
HYDROGEN ION CONCENTRATION, pH
Acids are substances which furnish hydrogen ions (H
+
) in the
solution, whereas bases are substances that furnish hydroxide
ions (OH
) in the solution. Substances that dissociate in water

into a cation (positively charged ion) and an anion (negatively
charged ion) are classified as electrolytes. Whereas sugar or
alcohols which dissolve in water but do not carry a charge
or dissociate into species with a positive and negative charge
are classified as nonelectrolytes.
Strong electrolytes are completely ionized in aqueous solut-
ions whereas weak electrolytes are partially ionized in aqueous
solutions.
pH of a solution is defined as the negative logarithm of
its hydrogen ion concentration.
pH = log
10
[H
+
]
=
10
1
log [H ]
+
Pure water has equal concentration of H
+
and OH
ions,
the concentrations of each is very small and each being equal
to 10
7
moles/liter at room temperature.
Water dissociates into:
H
2
O H
+
+ OH
From the Law of Mass action, the dissociation of water

can be represented as:
K
w
=
+
2
[H ] [OH ]
[H O]
Biophysics
CHAPTER
1
2 BIOCHEMISTRY FOR STUDENTS
The bracket indicates the concentration of each component
in moles per liter.
The concentration of undissociated water is so large as
compared to the concentration of H
+
and OH
ions, so that
for all the practical purposes it is fairly constant. This simplifies
the above equation to:
[H
+
] [OH
] = K [H
2
O]
[H
+
] [OH
] = K
w
Where K
w
is ionic product of water or the dissociation
constant of water. Electrical conductivity measurements have
shown that dissociation constant of water is constant at a given
temperature and changes with the change in temperature.
Ionic product of water is usually taken as 10
14
at the room
temperature (25C).
Then
[H
+
] [OH
] = 10
14
Taking logarithm of both sides
log [H
+
] + log [OH
] = 14
By rearrangement
log [H
+
] log [OH
] = 14
According to the definition of pH, the above equation
simplifies to:
pH + pOH = 14
At neutrality, both hydrogen and hydroxide ions have
equal concentration, i.e.
pH = 7
pOH = 7
There exists an inverse relationship between [H
+
] and
[OH
] ions in solution. As hydrogen ion concentration incr-

eases, the hydroxide ion concentration decreases and vice
versa.
The acidity or alkalinity of a solution is determined by
the amount of [H
+
] and [OH
] ions present.
BIOPHYSICS 3
A solution having hydrogen ions concentration of one nor-
mality (1 N) will have a pH 0, and other having hydroxide
concentration of one normality (1 N) will have pH 14.
It should also be kept in mind that a change of one pH
unit brings a ten-fold change in acidity or alkalinity, i.e. a
solution of pH 5 has ten times more the hydrogen ion
concentration than that of a solution of pH 6 and a hundred
times more than that of a solution of pH 7. If hydrogen ion
concentration is doubled, the pH falls by 0.3 units.
The average pH values of some of body fluids are:
Gastric juice 1.4
Saliva 6.8
Urine 6.0
Milk 7.1
Tears 7.2
Blood 7.4
Pancreatic juice 8.0
Q. Calculate the pH of a solution of which hydrogen ion
concentration is 4.6 10
9
M.
Ans. pH = log
10
[C
H
+]
= log
10
[4.6 10
9
]
= log
10
4.6 + 9 log
10
10
= 0.66 + 9
= 8.34.
Q. Calculate the hydrogen ion concentration of a solution,
the pH of which is 4.50.
Ans. pH = log
10
[C
H
+]
log
10
[C
H
+
] = pH = 4.50 = 5.50
[C
H
+] = Antilog 5.50
[C
H
+
] = Antilog 0.5 antilog 5.00
= 3.16 10
5
M.
Buffers
Buffers are the solutions, which resist changes in pH, when
small amount of acid or alkali is added to them. The best
buffer is the one which gives the smallest change in pH. Buffers
act like shock absorber against the sudden changes of pH.
Acetic acid: sodium acetate (CH
3
COOH; CH
3
COONa) and
carbonic acid: sodium carbonate (H
2
CO
3
; NaHCO
3
) are exam-
ples of buffer systems. Physiologic buffers include bicarbonate,
orthophosphate and proteins.
A buffer is a pair of weak acid and its salt with a strong
base or a pair of weak base and its salt with a strong acid.
If either free H
+
or free OH
are added to a solution of such

a pair they will be partially converted to the unionized form.
Thus
B
+ H
+
BH
or HB + OH
H
2
O + B
Where HB denotes a weak acid and B
its conjugate base.

The combination of a weak acid and the base that is formed
on dissociation is referred to as a conjugate pair. Ammonium
ion NH
4
+
is

an acid because it dissociates to yield a H
+
and
NH
3
which is conjugate base. Phosphoric acid (H
3
PO
4
) is an
acid and PO
4
3
is a base.
NH
4
+
H
+
+ NH
3
(acid) (conjugate base)
H
3
PO
4
3H
+
+ PO
4
3
The ability to buffer hydrogen ions is more important to
the body than the buffering of hydroxyl ions.
The most commonly used buffers in the laboratory are:
Acetate buffer (Sodium acetate/acetic acid).
Phosphate buffer (Na
2
HPO
4
/KH
2
PO
4
).
Citrate buffer (Sodium citrate/citric acid).
BIOPHYSICS 5
Barbitone buffer (Sodium diethyl barbiturate/diethyl
barbituric acid).
The pH of a buffer solution is calculated by the Henderson-
Hasselbalch equation.
Suppose the solution is composed of a weak acid [HA] and
its salt with a strong base [BA].
The dissociation of weak acid [HA] and salt [BA] can be
represented as follows:
HA H
+
+ A
Weak acid Proton + Conjugate base

Conjugate base (A
) is the ionized form of a weak acid

[HA] [H
+
] + [A
+
] ...(1)
[BA] [B
+
] + [A
] ...(2)
[HA] dissociates less because it is a weak acid, whereas
[BA] dissociates completely because it is a salt of a strong
base.
Larger the ka, the stronger the acid, because most of the
HA will be converted into H
+
and A
. Conservely, smaller
the ka, less acid will be dissociated and hence weaker the
acid.
The dissociation constant of equation (1) is represented
as:
K
a
=
+ -
[H ] [A ]
[HA]
By rearrangement
[H
+
] [A
] = K
a
[HA]
[H
+
] =
a
K [HA]
[A ]
+
As the acid [HA] is weak acid, it will be very slightly
ionized, and most of it will be present as [HA], whereas the
salt [BA] will be highly ionized, the concentration of [A
] can
be taken as the total concentration of [BA].
[H
+
] =
a
K [HA]
[BA]
Taking logarithm of both sides
log [H
+
] = log K
a
+
[HA]
log
[BA]
log [H
+
] = log K
a
+
[BA]
log
[HA]
pH = pK +
[BA]
log
[HA]
pH = pK +
[Salt]
log
[Acid]
This equation is called Henderson-Hasselbalch equation.
If the value of K (the dissociation constant) is known, the
pH of a buffer solution of a given composition can be readily
calculated.
The above equation indicates that the pH of the buffer
solution depends on the ratio of the concentrations of the salt
and the acid.
The buffering power of a mixture of a weak acid and its
salt is greatest when the two substances are present in equi-
valent proportions. Then the buffer has its maximum capacity
to absorb either H
+
or OH
ions. So that pH is approximately

equal to the pK of the acid, i.e. when the acid is half neutralized.
[salt] = [acid]

[salt]
acid
= 1

[salt]
log
acid
= log 1 = 0
Therefore pH = pK
For example
BIOPHYSICS 7
The effective range of a buffer is 1 pH unit higher or lower
than the pKa. The pKa value of most of the acids produced
in the body is well below the physiological pH, hence, they
ionizes, immediately and add H
+
to the medium.
The effect of dilution on the pH of a buffer mixture and
on the apparent pK of the acid is slight. The pH depends upon
the ratio of salt: acid and this ratio is not much affected by
dilution.
The pH of the buffer solution is determined by the pK
and the ratio of salt to acid concentration. Lower the pK value,
lower is the pH of the solution; whereas the ratio of salt to
acid concentration may vary with no change in pH as long
as the ratio remains the same. When the ratio between the
salt and the acid is 10:1, the pH will one unit higher than the
pKa whereas when the ratio between salt and acid is 1:10
the pH will be one unit lower than the pKa. Maximum buffering
capacity occurs 1 pH unit on either side of pKa.
Buffers are of main importance in regulating the pH of
the body fluids and tissues within limits consistent with life
and normal function. Many biochemical reactions, including
those catalysed by enzymes, require pH control which is
provided by buffers.
Dissociation constant and pK of acids of importance in bio-
chemistry.
Compound Dissociation constant pK
Acetic acid 1.74 10
5
4.76
Citric acid 8.12 10
4
3.09
Lactic acid 1.38 10
4
3.86
Pyruvic acid 3.16 10
3
2.50
Water 1 10
14
14
Succinic acid 6.46 10
5
4.19
Q. A mixture of equal volumes of 0.1 M NaHCO
3
and 0.1 M
H
2
CO
3
shows a pH of 6.1. Calculate the pKa of H
2
CO
3
.
Ans. Concentration of H
2
CO
3
, i.e. acid = 0.1 M.
Concentration of NaHCO
3
, i.e. salt = 0.1 M.
Applying Henderson-Hasselbalch equation
pH = pK acid +
3
2 3
[NaHCO ]
log
[H CO ]
6.1 = pK acid +
0.1
log
0.1
6.1 = pK acid + log 1
[log 1 = 0]
pK acid = 6.1
Q. Phosphate buffers are prepared by mixing together 0.1 M
Na
2
HPO
4
and 0.1 M KH
2
PO
4
in different ratios. Calculate the
expected pH of the buffer solution prepared by mixing the
salt and the acid in the above system in the ratio 2:1 (Given
log 2 = 0.30 and pK
2
of phosphoric acid 6.7)
Ans. Concentration of Na
2
HPO
4
(i.e. salt) = 2 0.1 M.
Concentration of KH
2
PO
4
(i.e. acid) = 1 0.1 M.
Applying Henderson-Hasselbalch equation
pH = pK phosphoric acid +
2 4
2 4
[Na HPO ]
log
[KH PO ]
= 6.7 +
2 0.1
log
1 0.1
= 6.7 + log 2
= 6.7 + 0.3
= 7
So the epected pH of the buffer solution is 7.
Q. You are provided with ample supply of carbonic acid and
sodium bicarbonate. How would you prepare a buffer solution
of pH 6.1. Give the theoretical basis of the procedure to be
followed (pKa of carbonic acid = 6.1).
Ans. Applying Henderson-Hasselbalch equation:
pH = pK +
[Salt]
log
[Acid]
pKa of carbonic acid = 6.1
The buffer solution to be prepared should have a pH of 6.1.
This can be achieved if the concentration of sodium
carbonate and carbonic acid is the same.
BIOPHYSICS 9
So buffer solution of pH 6.1 can be made by mixing equal
volume of sodium carbonate and carbonic acid of same
concentration.
Q. What would be the pH of 100 cm
3
of a 0.2 M acetic acid
solution to which has been added 10 cm
3
of 1.5 M sodium
hydroxide. (Given the pK for acetic acid 4.74.).
Ans. Before the addition of NaOH,
The number of moles of acetic acid present is:
100
0.2
1000
= 0.02 M
Also the number of moles of sodium hydroxide present
in 10 cm
3
of 1.5 M NaOH solution are:
100
1.5
1000
= 0.015 M
Before the start of reaction, the concentration of acetic acid
is 0.02 M and that of sodium hydroxide is 0.015 M.
When the reaction takes place, i.e. 0.015 M NaOH neutralizes
0.015 M of CH
3
COOH to form 0.015 M of sodium acetate.
After the reaction is over, the concentration of CH
3
COOH
left behind 0.02 M 0.015 M = 0.005 M.
Reaction CH
3
COOH + NaOH CH
3
COONa + H
2
O
Now, applying Henderson-Hasselbalch equation
pH = pK +
10
[Acetate]
log
[Acetic acid]
= 4.74 +
10
0.015
log
0.005
= 4.74 + log
10
3
= 4.74 + 0.48
= 5.22
Blood Buffers
The buffer systems of blood are:
1. Bicarbonate-carbonic acid (BHCO
3
: H
2
CO
3
)
2. Hemoglobinate-hemoglobin (BHb : HHb)
3. Oxyhemoglobinate-oxyhemoglobin (BHbO
2
: HHbO
2
)
4. Phosphate buffer (B
2
HPO
4
: BH
2
PO
4
)
5. Protein buffer (B Protein : H Protein).
The most important buffer of plasma is bicarbonate-carbonic
acid system. It is present in high concentration. It is of great
importance in the acid-base balance of the extracellular fluid
and in the maintenance of the blood pH within normal limits.
The bicarbonate system is of prime physiological importance,
and acts cooperatively with other buffers.
The hemoglobinate-hemoglobin and oxyhemoglobinate-
oxyhemoglobin buffer, i.e. hemoglobin buffers are of prime
importance in the erythrocytes. Hemoglobin actually absorbs
60 percent of the hydrogen ions produced by H
2
CO
3
.
Hemoglobin is a better buffer than most proteins at pH
7.4 because of relatively high concentration of imidazole group
(pKa approximately 7) of the constituent histidine molecules.
Deoxyhemoglobin is a better buffer than oxyhemoglobin.
The converse is also true, i.e. the hydrogen ions decrease the
affinity of hemoglobin for oxygen.
Protein and phosphate buffers are of little importance in
the blood, i.e. they are the minor buffering systems in the
blood. Proteins are present in much higher concentrations in
cells than in plasma. They are probably important in buffering
H
+
ions before their release from cells. But phosphate buffer
is of importance in raising the plasma pH through excretion
of H
2
PO
4
by kidney. It is an important urinary buffer and
works cooperatively with the bicarbonate system.
Approximate contribution of individual buffers to total
buffering in whole blood is given below.
Individual buffers Percent buffering in whole blood
Hemoglobin and oxyhemoglobin 35
Organic phosphates 3
Inorganic phosphates 2
Plasma proteins 7
Plasma bicarbonate 35
Erythrocyte bicarbonate 18
BIOPHYSICS 11
Indicators
Indicators are substances which change in color with change
in the pH of the solution in which they are present. Indicators
are dyes which are weak acids or weak bases and have the
property of dissociating in solution. Their ionized form have
one color and their unionized form have another color. The
color of an indicator solution depends on the relative amounts
of its acid and base form present in the solution.
An indicator which is a weak acid, is undissociated in acid
solution and gives the acid color. In the presence of alkali,
it forms a salt which dissociates and displays alkali color.
Indicators are used in:
1. Determining the end point in acid-base titrations.
2. Determining pH of solutions.
Universal Indicator
It is a mixture of a number of indicators which gives a variety
of color changes over a wide-range of pH.
Some common indicators useful for biological pH range
are:
Color
Indicators pK pH range In acid In alkaline
solution solution
1. Thymol blue 1.65 1.22.8 Red Yellow
(acid range)
2. Methyl yellow 2.94.0 Red Yellow
(Topfers reagent)
3. Methyl orange 3.46 3.14.4 Red Orange
Yellow
4. Methyl red 5.00 4.36.1 Red Yellow
5. Phenol red 7.81 6.78.3 Yellow Red
6. Thymol blue 8.90 8.09.6 Yellow Blue
(alkaline range)
7. Phenolphthalein 9.70 8.210 Colorless Pink
OSMOSIS AND OSMOTIC PRESSURE
Osmotic flow occurs whenever a semipermeable membrane
separates a solution and its pure solvent or between two
solutions differing in concentrations. Water molecules pass
through the membrane until the concentration on both sides
becomes same. Such a movement of solvent molecules from
a pure solvent or dilute solution through a semipermeable
membrane is called osmosis.
Osmotic Pressure
Osmotic pressure is the pressure that must be applied on a
solution to keep it in equilibrium with the pure solvent when
the two are separated by semipermeable membrane or osmotic
pressure is the force required to oppose the osmotic flow.
Hypertonic solutions: If the osmotic pressure of the surround-
ing solution is high, water passes from the cell to the stronger
solution outside, this causes the cell to shrink away.
Isotonic solutions: If external solution has the same osmotic
pressure, no flow of water takes place and hence no effect
upon the cell protoplasm is observed.
Hypotonic solutions: If the osmotic pressure of the surrounding
solution is low, water passes into the cell from the surrounding,
the cells become turgid and rupture.
Vant Hoffs law of osmotic pressure:
1. The osmotic pressure of a solution is directly proportio-
nal to the concentration of the solute in the solution.
2. The osmotic pressure of a solution is directly proportio-
nal to the absolute temperature.
Thus indirectly they follow Boyles and Charles Law.
Osmotic pressure is given by the formula.
= CRT
where =Osmotic pressure
C =Concentration in moles per liter
R =Gas constant
T =Absolute temperature
BIOPHYSICS 13
Osmotic pressure is dependent upon the number of
dissolved particles (i.e. on concentration) and is independent
of the size or weight of the particle.
According to the law of osmotic pressure, 1 molar solution
exerts an osmotic pressure of 22.4 liters at 0C.
The osmotic pressure of substances which ionizes is given
by the formula.
= i CRT
where i the isotonic coefficient is given by:
i = 1 + (n1)
where = degree of ionization
n = number of ions obtained on ionization
The value of i, depends upon the degree of dissociation
of the electrolyte, which varies from one electrolyte to another.
It increases as the dilution increases and depends upon the
number of ions formed.
Since osmotic pressure is proportional to the total number
of solute particles in solution, the substances which ionize,
will have the higher osmotic pressure as compared to those
substances which do not ionize.
The osmotic pressure exerted by colloidal solutions is
always less as compared to that of crystalloids of similar
concentrations in gram per liter because the magnitude of
osmotic pressure depends upon number of particles present
in unit volume of the solution. Solutions that exert the same
osmotic pressure are called isomotic.
The osmotic pressure of 1 M NaCl will be double, as
compared to the osmotic pressure of 1 M sucrose or glucose
solution because each molecule of NaCl on ionization gives
two ions, i.e. Na
+
and Cl
ions and each ion will exert the

respective osmotic pressure.
The unit of osmotic pressure is osmol or milliosmol. An
osmolar solution is defined as one exerting the osmotic
pressure of a molar solution of a nondissociated solute in one
liter of solution. Thus the number of osmoles of a undissociated
substance in a liter of solution would be the weight in grams
divided by its molecular weight. The milliosmolar concen-
tration of glucose in a sample of plasma containing 90 mg per
100 ml therefore would be:
90 mg per 100 ml 10
180 (Mol. wt of glucose)
= 5 milliosmol per liter

For nonelectrolytes such as glucose or sucrose, 1 millimol
is equal to 1 milliosmol. For electrolytes such as NaCl, one
millimol of NaCl is equivalent to 2 milliosmol (Na
+
and Cl).
Q. 1 Molar solution of glucose has an osmotic pressure of 22.4
atmosphere at 0C. Calculate the osmotic pressure of 0.1 M
sucrose and 0.1 M NaCl at the same temperature. Assume
100% dissociation of NaCl.
Ans. 1 molar solution exerts an osmotic pressure of 22.4
atmosphere.
So 0.1 Molar solution will exert an osmotic pressure of 2.24
atmosphere.
So 0.1 M sucrose will have an osmotic pressure of 2.24
atmosphere.
In case of sodium chloride, each molecule of NaCl on disso-
ciation gives Na
+
ions and Cl
ions. Each ion, i.e. Na

+
and
Cl
will exert an independent osmotic pressure. Also the

dissociation of sodium chloride is 100%.
So 0.1 M solution of NaCl will exert an osmotic pressure
of 2 2.24, i.e. 4.48 atmospheres.
Q. Calculate the osmolarities of:
i. 0.1 M NaCl solution
ii. 0.1 M sucrose solution
Ans. The term milliosmol is used in connection with osmotic
pressure.
0.1 M solution of NaCl will have an osmotic pressure of
0.1 2 = 0.2 milliosmol (because each molecule of sodium
chloride on ionization gives two ions).
Whereas 0.1 M sucrose will have an osmotic pressure of
0.1 milliosmol.
Milliequivalent
One milliequivalent is one thousandth of an equivalent and
is the same as millimol as long as the valency is one.
BIOPHYSICS 15
For valence 1; 1 millimol = 1 milliequivalent
How to calculate millimols?
millimol =
milligrams per liter
Formula weight
Example:
78 mg of K
+
ions per liter = 78/39, i.e. 2 millimols
= 2 milliequivalent
= 2 milliosmols
Whereas
100 mg of Ca
++
per liter = 100/40, i.e. 2.5 millimols
= 2.5 milliosmols
= 5 milliequivalent
222 mg of CaCl
2
per liter = 222/111, i.e. 2 millimols
of CaCl
2
= 6 milliosmols.
Ca = 40, 2Cl
= 2 35.5
= 71
Gibbs Donnan Equilibrium
Gibbs Donnan equilibrium is concerned with the distribution
of electrolytes in systems separated by membranes which are
impermeable to certain components. This resultant unequal
distribution of diffusible ions due to the presence of nondi-
ffusible ions on one side of the membrane is called Gibbs
Donnan Effect.
Example: Consider a semipermeable membrane separating
a solution of NaCl and Protein (NaR). The membrane is
permeable to Na
+
and Cl
but not to R
.
Na
+
Na
+
Na
+
Na
+
R
Cl
Cl
Cl
In the beginning (A) At equilibrium (B)

When the equilibrium is attained, the product of concentra-
tions of diffusible ions (Na
+
and Cl
) on one side of membrane

is equal to the product of concentrations of same ions on the
other side, i.e.
(Na
+
)(Cl
) > (Na
+
)(Cl
)
The concentration of diffusible positive ion is greater on
the side of membrane containing nondiffusible ion, i.e.
[Na
+
]
1
> [Na
+
]
2
Donnan effect is of physiological significance in biological
systems involving ion exchanges across permeable membranes
when the fluid on one side of the membrane contains a non-
diffusible component. This results in difference of concen-
tration of diffusible ions which leads to junction potential
across the membrane, which is a driving force for most of
the body reaction. Donnan effect is also involved in absorption,
secretion and maintenance of different electrolyte concen-
trations between various compartments of the body.
COLLOIDS
Graham classified substances into:
1. Crystalloids: Substances which pass through parchment or
animal membrane.
2. Colloids: Substances which do not pass through parchment
or animal membrane.
But nowadays, the size of the molecule or particle deter-
mines whether they will form crystalloidal or colloidal sol-
utions.
According to modern concept.
True solution Colloidal solution Suspension solution
where the size where the size is where the size is
(diameter) of the between 1-20 m more than 200 m
particle is less
than 1 m
Properties of Colloidal Solutions
1. Dialysis: The process of separation of crystalloids from
colloids by diffusion through a membrane by osmotic force
BIOPHYSICS 17
is called dialysis. Dialysis has an important application in
medicine in the artificial kidney. This device is inserted
into the patients circulation and diffusible material parti-
cularly urea passes out from the blood substituting for the
action of the faulty kidneys.
2. As the size of the colloidal particle is large, few particles
are present in small concentration, the osmotic pressure
of the colloidal solution will be very small. This is of prime
importance in driving the passage of water and other
substances through cell membranes.
3. Precipitation: Colloids possess net charge at the surface
which arises from ionisable groups on the particle surface
and also from absorption of ions and can be precipitated
by neutralizing the charge.
4. Brownian motion.
5. Tyndall effect.
SURFACE TENSION
The force with which the surface molecules are held in a
solution is called surface tension. Some substances such as bile
salts have the property of lowering the surface tension of the
medium in which they are present. This effect is used in the
absorption of fats from the intestine.
Other properties of surface tension are formation of drops
of liquids falling through air; rise of liquid in a capillary tube
and formation of meniscus at the surface of liquids. Surface
tension decreases with increase in temperature.
Role of Surface Tension
Substance which lower the surface tension becomes concen-
trated in the surface layer whereas substances which
increase surface tension are distributed in the interior of
the liquid.
Soaps, oils, proteins and bile acids reduce the surface
tension of water, while sodium chloride and inorganic salts
increase the surface tension.
Surface tension leads to better adsorption.
ABSORPTION
Certain substances have the power of making water insoluble
substances soluble in water without any apparent chemical
alteration of the dissolved substance.
The substances having such quality are called hydrotropic
substances.
Among the insoluble substances which are brought into
the solution are fats, phospholipids, sterols, calcium carbonate,
magnesium phosphate, etc.
Substance which bring about the solubility are cholic acids,
benzoic acid, hippuric acid, soaps of higher fatty acids, etc.
The biological importance of the solution of an insoluble
substance in hydrotropic substances lie in the fact that the
substances so dissolved are diffusible through membranes.
VISCOSITY
Viscosity of a liquid is the resistance to flow. Viscosity of blood
is 4.5 times more than water. Viscosity of blood is lowered
in anemia, nephritis, leukemia, malaria, diabetes mellitus,
jaundice, whereas excessive sweating and shock leads to
increase of blood viscocity.
CARBOHYDRATES
Carbohydrates are defined as the aldehydic or ketonic deriva-
tives of polyhydroxy alcohols and their polymers having
hemiacetal glycosidic linkages.
The general formula for carbohydrates is C
n
(H
2
O)
n
.
Carbohydrates are the main source of energy in the body.
Brain cells and RBCs are exclusively depend on carbo-
hydrates (glucose) as the energy source.
The sugar is a carbohydrate and is sweet to taste, soluble
in water and chars on heating. Glucose (Grape sugar), fructose
(fruit sugar), sucrose (cane sugar), lactose (milk sugar), and
maltose (malt sugar) are few examples of sugar. All sugars
are carbohydrates but all carbohydrates are not sugars. Gly-
cogen and inulin are carbohydrates but not sugars.
FUNCTIONS OF CARBOHYDRATES
1. Provides energy, i.e. as major source of energy to the body.
Their calorific value is 4 kcal per gm.
2. As structural components of membranes.
3. As structural basis for DNA and RNA (Ribose/Deoxyribose).
4. As structural basis for nucleosides and nucleotides.
5. As source of carbon skeltons for some amino acids.
6. As basis of some intracellular messenger systems.
CLASSIFICATION OF CARBOHYDRATES
Monosaccharides
Monosaccharides consists of single polyhydroxy aldehyde or
ketone unit which cannot be broken down to simpler sub-
stances on acid hydrolysis. They are also called simple sugars.
Monosaccharides are further divided into:
i. Aldoses, i.e. Aldo sugars
ii. Ketoses, i.e. Keto sugars.
Chemistry of
Carbohydrates
CHAPTER
2
Aldoses
Monosaccharides containing aldehydic group as the functional
group are called aldoses.
They are classified according to the number of carbon atoms
present. Monosaccharides containing three to seven carbon
atoms are called trioses, tetroses, pentoses, hexoses and hepto-
ses respectively.
Trioses : D-glyceraldehyde (aldotriose)
Dihydroxy acetone (ketotriose)
Tetroses : D-Erythrose (aldotetrose)
Pentoses : D-Xylulose (ketopentose)
: D-Ribose (aldopentose)
: D-Deoxyribose (aldopentose)
: D-Xylose (aldopentose)
: D-xylulose (aldopentose)
Hexoses : D-Glucose, D-Galactose,
D-Mannose (aldohexose)
: D-Fructose (ketohexose)
Structures of Erythrose, Ribose, Glucose, Galactose,
Mannose are:
CHEMISTRY OF CARBOHYDRATES 21
Ketoses
Monosaccharides containing ketonic group as the functional
group are called ketoses.
Examples: Xylulose, Ribulose, Fructose, etc.
Stereochemistry
The presence of asymmetric carbon atoms (an asymmetric
carbon atom is one to which four different atoms or groups
are attached) in the compound results in the formation of
isomers of that compound. The number of isomers of a
compound depends on the number of asymmetric carbon atoms
and is given by 2
n
, where n indicates the number of asymmetric
carbon atoms in that compound.
If the hydroxyl group on the highest asymmetric carbon
atom or on the penultimate carbon atom is on the right hand
side, than the compound will belong to D-Series. If the hydroxyl
group is on the left side, than the compound will belong to L-
Series.
The D-and L-forms of glucose are given below:
Two compounds that resemble each other but are different
because their carbons are asymmetric. The relationship exhibi-
ted by each compound is called stereoisomerism and the two
compounds are called stereoisomers or enantiomorphs.
Stereoisomers are those compounds which have the same
composition but differ in spatial arrangements.
Carbohydrates exhibit the property of optical activity and
exist as optical isomers.
Glucose with four asymmetric carbon atom will have 2
4
,
i.e., 16 isomers. 8 of these isomers will belong to D-series and
other 8 to L-series.
(Where X denotes that particular carbon atom is asym-
metric).
In the open chain structure of D-glucose, C
2
, C
3
, C
4
, and
C
5
are the asymmetric carbon atoms. But in nature, D-glucose
exists in 32 stereoisomers, i.e. 32 isomers of D-glucose has
been isolated. The 32 isomers can be best explained if there
is one more asymmetric center in the D-glucose. This is
possible if glucose exists in ring or cyclic structure. The cyclic
structure involves the formation of hemiacetal linkage
between aldehyde group (i.e. C
1
) and hydroxyl group at C
4
.
In the process, a new asymmetric centre C
1
is created at
glucose.
In the ring form of D-glucose, C
1
, C
2
, C
3
, C
4
, and C
5
are
asymmetric and will have 2
5
, i.e., 32 stereoisomers.
During the process of cyclization a six membered ring
consisting of five carbon atoms and an oxygen atom is formed
in case of glucose. This ring structure is also called pyranose
structure.
Similarly a five membered ring consisting of four carbon
atoms and an oxygen atom is formed in case of fructose. This
ring structure is also called furanose structure.
The planar formula of sugars is also called Fischer formula
and the ring formula is called Haworth formula.
Epimers: Carbohydrates that differ in their configuration about
a specific carbon atom other than the carbonyl carbon atom
are called epimers.
Glucose and galactose are epimers as they differ in their
configuration at C-4 carbon atom. Similarly, glucose and
mannose are epimers as they differ at C-2 carbon atom.
The process of interconversion of glucose and galactose
is known as epimerization.
In glucose, the hydroxyl group at C-4 is on the right hand
side whereas in galactose, the hydroxyl group at C-4 is on
the left hand side.
Anomers: Carbohydrates that differ only in their configu-
ration around the carbonyl carbon atom are called anomers.
The carbonyl carbon atom is called the anomeric carbon
atom.
-D-glucose and -D-glucose are the anomeric forms of
D-glucose.
In -D-glucose, the hydroxyl group at C-1 (i.e. carbonyl
carbon atom) is on the right hand side whereas in -D-glucose,
the hydroxyl group at C-1 is on the left hand side.
Anomeric form arises as a result of cyclization or ring for-
mation. During the process of cyclization, the C-1 carbon atom
which is symmetrical in the open chain formula of glucose
is converted into asymmetric carbon atom.
The presence of asymmetrical carbon atom give rise to optical
activity. When a beam of plane polarized light is passed through
a solution of carbohydrates, it will rotate the light either to
left or to the right. Depending upon rotation, molecules are
called dextrorotatory (+) or (d), levorotatory () or (l).
A compound that rotates the plane of polarized light in
a clockwise direction is said to be dextrorotatory (+), whereas
that which rotates the plane of light in a anticlockwise direction
is said to be levorotatory ().
Amino Sugars
The amino sugars occurring most frequently are glucosamine
and galactosamine. They occur as N-acetyl compounds.
Glucosamine is present in chitin, shells of insects and mam-
malian polysaccharides whereas galactosamine is present in
polysaccharides of cartilage and chondroitin.
Reactions of Monosaccharides
1. Action of acids
2. Mutarotation
3. Reducing property
4. Osazone formation
5. Action of dilute alkali
6. Oxidation
7. Reduction
8. Glycoside formation.
Action of Acids
This is a general test for carbohydrates. Monosaccharides on
treatment with concentrated sulphuric acid undergoes dehy-
dration to give furfural or furfural derivatives which on
condensation with -naphthol yield a violet or purple colored
complex. Pentoses yield furfural whereas hexoses yield
5-hydroxy furfural.
Mutarotation
Mutarotation is defined as the change in specific rotation of
optically active solution without any change in other properties.
When glucose is dissolved in water, the optical rotation
of the solution gradually changes and attains an equilibrium
value. This change in optical rotation is called mutarotation.
Mutarotation occurs due to the cyclization of open chain
form of glucose into or form with equal probability. This
and cyclic form of glucose have different optical rotations.
This is because, the and form are not mirror images of
each other. They differ in configuration about the anomeric
carbon (C
1
) but have the same configuration at C
2
, C
3
, C
4
,
and C
5
asymmetric carbons. These cyclic forms are in
equilibrium with open chain structure in aqueous solution.
Such a change from a single form to an equilibrium mixture
that includes its other form is called mutarotation.
+112
o
+52-5
o
+19
o
-D-glucose Equilibrium mixture -D-glucose
contains , and
open chain forms
-form 36%, -form 63% and open chain form 1%. The
predominance of the -form in aqueous solution is due to its
more stable conformation relative to the -form.
Biologically this change is catalyzed by the enzyme, muta-
rotase.
In aqueous solution, many monosaccharides behave as if
they have one more asymmetric center than is given by open
chain structure.
Ring formation involves the formation of internal hemi-
acetal linkage between the aldehyde group, i.e. C-1 and the
hydroxyl group at C-5 and a new asymmetric carbon at C-
1 is created in glucose. In this cyclic form, there are now five
asymmetric carbon atoms (i.e. C-1, C-2, C-3, C-4, C-5) which
best explains about the existence of 2
5
, i.e. 32 isomers of glucose.
Reducing Property
Monosaccharides by virtue of free aldehydic or ketonic group
in their structure, i.e. presence of free anomeric carbon atom,
reduces certain heavy metallic cation, e.g. Cu
++
ions in alkaline
solution at high temperature.
So all the reducing sugars will give Benedicts qualitative
test and Fehling test positive.
The reaction is as follows:
The color of the solution or precipitate gives an approximate
(rough) amount of reducing sugars present in the solution.
Green color......up to 0.5% (+)
Green precipitate.....0.5-1% (++)
Green to yellow precipitate.....1.0-1.5% (+++)
Yellow to orange precipitate.....1.5-2.0% (++++)
Brick red precipitate....more than 2%
Benedicts qualitative reagent contains cupric sulfate, sodium
carbonate and sodium citrate whereas Fehling solution contains
cupric sulfate, sodium hydroxide and sodium potassium tartrate
(Rochelle salt).
Sodium citrate in Benedicts reagent and sodium potassium
tartrate (Rochelles Salt) in Fehling solution prevent the preci-
pitation of cupric hydroxide or cupric carbonate, by forming
a deep blue soluble slightly dissociated complexes with the
cupric ions. These complexes dissociate sufficiently to provide
a continuous supply of readily available cupric ions available
for oxidation.
Benedicts qualitative reagent is preferred above Fehling
solution because it is more stable. Also traces of sugar which
is destroyed by the strong alkali of Fehling solution is not
destroyed by Benedicts reagent.
Osazone Formation
Reducing sugars can be distinguished from one another by
phenylhydrazine test when characteristic osazones are formed.
These osazones have characteristic crystal structures, melting
point, precipitation time and show different crystalline forms
under a microscope and hence, are valuable in the identification
of reducing sugar.
Glucose, fructose and mannose give the same osazones and
hence, they cannot be differentiated from each other by this
test.
In the osazone formation only first two carbon atoms, i.e.
C-1 and C-2, take part in the reaction. So reducing sugars
which differ in their configuration at C-1 and C-2 and have
rest of the structure same, i.e. C-3, C-4, C-5 and C-6 have
the same configuration, give the same osazones because during
osazone formation, the structural dissimilarity at C-1 and
C-2 disappears and the rest of the molecule structure is the
same.
Three molecules of phenylhydrazine are required to
produce one molecule of osazone.
The formation of osazones of glucose is explained below.
Fructose reacts with phenylhydrazine in a similar manner.
Glucose osazone, fructose osazone and mannose osazone
are identical with respect to its crystal structure and chemical
structure. Glucose, fructose and mannose give the needle shape
osazones whereas maltose gives sunflower and lactose gives
cotton ball shape osazones.
Appearance of yellow crystals takes place. Observe the
shape of crystals under microscope.
Lactose (Cotton Ball) Maltose (Sunflower)
Osazone of maltose and lactose
Action of Dilute Alkali
Monosaccharides on treatment with dilute alkali undergo a
variety of molecular transformation through enediol for-
mation. The enediols of sugars are good reducing agents and
form the basis of reducing action of sugars in alkaline medium.
When glucose is treated with dilute alkali for several hours,
the resulting mixture obtained contains both fructose and
mannose in addition to glucose. A similar mixture of same
sugars is obtained with any of the other two sugars. This
interconversion of related sugars by the action of dilute alkali
is termed as Lobry de Bruyn-van Ekenstein rearrangement
(see page 36 for reaction).
Whereas sugars on boiling with strong alkalis are carame-
lized to give yellow to brown resinous product. That is the
reason why Benedicts reagent containing sodium carbonate
is preferred to Fehling solution containing sodium hydroxide.
Oxidation
Aldoses are oxidized under variety of conditions to the
following:
i. Aldonic acid: Whereby the first carbon atom (C-1) is
oxidized to carboxyl group only. The rest of the molecule
structure remains unaffected.
ii. Uronic acid: Whereby the terminal carbon atom is oxidized
to carboxyl group only. The first carbon atom, i.e. alde-
hydic group and the rest of the molecular structure
remains unaffected. Uronic acid derivatives are
particularly important in detoxification process, i.e.,
bilirubin is excreted as bilirubin diglucuronide. Besides
this, D-glucuronic acid, D-galactouronic acid, D-mannou-
ronic acid, L-induronic acid are important components
of polysaccharides.
iii. Aldaric or saccharic acid: Whereby both the first carbon atom,
i.e. aldehydic group and the terminal carbon atom, i.e.
primary alcoholic group are oxidized to carboxyl group.
Galactose undergoes oxidation to form a dicarboxylic acid,
mucic acid. This reaction is often important in the identification
of galactose.
Example: The oxidation products of glucose under different
conditions are given on Page 37.
Glucose Oxidase: The substrate for glucose oxidase is -
D-glucopyranose. Blood glucose which is an equilibrium
mixture of - and -anomers of D-glucose is qualitatively
determined by the formation of hydrogen peroxide by the
reaction (P-39).
Two very important uronic acids occuring in carbohydrates
are D-glucuronates and L-iduronate (from hexose idose).
The only difference between these two molecule is that
the carboxyl group is above the ring for D-glucuronate and
below the ring for L-iduronate.
This requires that the -D-glucopyranose be rapidly
isomerized by mutarotation into the -D-isomer. This reaction
is fast without catalyst.
Q. A reducing carbohydrate gives a positive reaction with
Barfords test and mucic acid crystals on oxidation. Give the
structure of that carbohydrate. Would it exhibit property of
mutarotation. If so, what products are formed at equilibrium.
Ans. Since Barfords test is positive. It indicates that reducing
carbohydrate is monosaccharide.
Also mucic acid crystals are obtained on oxidation sugges-
ting that the given reducing carbohydrate is galactose as it
is the galactose which on oxidation gives mucic acid crystals.
D-galactose will show mutarotation due to the cyclization
of open chain form of D-galactose into - and - form with
equal probability. The products at the equilibrium are:
Reduction
Glucose on reduction gives sorbitol. Whereas fructose on
reduction gives a mixture of sorbitol and mannitol.
Mannose gives mannitol, galactose is reduced to dulcitol
and ribose to ribotol.
Fermentation: Fermentation is the process of breakdown of
complex organic substances into smaller substances with the
help of enzymes. Glucose is fermented to ethyl alcohol and
carbon dioxide by yeast. Hence this process is called alcoholic
fermentation as alcohol is produced.
Glycosides Formation
Glycosides are sugar derivatives in which hydrogen of the
hydroxyl group of hemiacetal or hemiketal form of the sugar
is replaced by an organic moiety. A molecule of water is
eliminated when this reaction takes place. Glycosides are not
reducing sugars and do not show mutarotation.
If the organic moiety is derived from another monosac-
charide, the product formed is disaccharide. If the organic
moiety is a noncarbohydrate, then it is called aglycone.
Aglycone: The noncarbohydrate portion of the glycoside is
called the aglycone or aglucone.
Glycosides do not reduce alkaline copper sulphate because
sugar group is combined, i.e. aldehyde group is converted
to an acetal group.
Glycosides = Carbohydrate + Carbohydrate part
or
noncarbohydrate part (aglycone)
Examples
Cardiac glycosides = Carbohydrate + Digoxin or digitoxin
(aglycone)
Indican = Carbohydrate + Indoxyl (aglycone)
Amygdalin = Carbohydrate + Benzaldehyde (aglycone)
OLIGOSACCHARIDES
Oligosaccharides are arbitrarily defined as carbohydrates that
contains two to ten monosaccharide units per molecule joined
by glycosidic linkages. On hydrolysis they yield monosac-
charides.
Depending upon the number of constituent monosaccharide
units, the oligosaccharides are called disaccharides, trisaccha-
rides, etc.
Oligosaccharides are reducing sugars if one of the carbonyl
group is free (not involved in glycosidic linkage). The reducing
power of carbohydrate decreases as the number of their sugar
components increases.
Disaccharides
Disaccharides consist of two monosaccharides joined by a
glycosidic linkage. The most common and important disaccha-
rides are maltose, Lactose and Sucrose. Maltose and lactose
are reducing disaccharides whereas sucrose is nonreducing
disaccharide.
In general, the properties of disaccharides are similar to
those of monosaccharides. Reducing disaccharide sugars are
not as reducing agents as monosaccharide because of the lower
ratio of reducing groups to carbon atoms.
Maltose
Maltose consists of two molecules of D-glucose joined by
(1,4)-glycosidic linkage. The anomeric carbon of one glucose
molecule is joined to the C-4 carbon of the second glucose
molecule. The anomeric carbon of the second glucose molecule
is free. So maltose is a reducing disaccharide.
Maltose or malt sugar does not occur in free state but is
formed as an important transitory intermediate product of
the digestion of starch and glycogen.
Maltose reduces heavy metallic ions in alkaline solution
(e.g. Benedicts reagent), undergoes mutarotation and
forms sunflower crystals of maltosazone with phenyl-
hydrazine.
Lactose
Lactose consists of galactose and glucose joined by (1,4)-
glycosidic linkage. The anomeric carbon of D-galactose is
joined to 4-carbon of D-glucose. The anomeric carbon of D-
glucose is free, so lactose is a reducing disaccharide.
Lactose is glucose galactoside.
Lactose or milk sugar is an animal disaccharide and is
present to the extent of 5% in milk only. It is synthesized
in mammary gland and during lactation may appear in the
urine.
Lactose on treatment with concentrated nitric acid gives
mucic acid crystals.
Lactose reduces Benedicts reagent, undergoes mutarotation
and forms cotton ball lactosazone crystals with phenyl-
hydrazine.
Sucrose
Sucrose is a non-reducing disaccharide. Sucrose consists of
glucose and fructose joined by (1) (2) glycosidic linkage.
The anomeric carbon (C-1) of glucose molecule in
configuration is linked to anomeric carbon (C-2) of fructose
in configuration. So sucrose is a nonreducing disaccharide
as both the reducing groups of glucose and fructose are linked
together and hence not available for reduction.
Sucrose or sugar cane is a plant disaccharide and is present
in high concentration in sugar cane and sugar beet. Sucrose
is used for sweetening purpose.
Sucrose does not reduce Benedicts reagent, does not show
mutarotation and does not form osazone with phenyl-
hydrazine.
Invert Sugar
Sucrose on hydrolysis yields equimolecular amounts of glucose
and fructose. Since this mixture is levorotatory whereas the
original sucrose is dextrorotatory, the process is known as
inversion because of the inversion of the sign of rotation, and
the mixture of glucose and fructose obtained is called as invert
sugar.
H
+
Sucrose Glucose + Fructose
(+65.5) (+52.7) (92)
Honey contains large amount of invert sugar.
Isomaltose
Isomaltose, a disaccharide is derived from the branch point
of starch. Isomaltose has (1 6)-D-glucosidic linkage to a
second D-glucose residue.
POLYSACCHARIDES
Polysaccharides are the polymers of monosaccharide units
which are joined in linear or branched chain fashion by
glycosidic linkages.
Polysaccharides contain a large number of sugar components
per free carbonyl group. In a branched polysaccharides, there
is only one reducing end and multiple nonreducing ends. Thus
these free carbonyl groups are not sufficiently potential to
reduce the Benedicts Reagent, etc.
By convention polysaccharides are given names ending in
an attached to the particular monosaccharide that make up
the polymer. Thus a name for a polysaccharide in general is
glycans from glucose. Examples are mannans, xylans and
arabans which are polymers of mannose, galactose, xylose and
arabinose.
Polysaccharides have two important biological functions.
1. As storage form of fuel (i.e., glycogen of animal origin and
starch of plant origin). Glycogen and starch are both storage
form of glucose; glycogen is used by animals to store
glucose and starch is used by plants.
2. As structural components, e.g. Cellulose.
The structural polysaccharides have -linkage and the
storage polysaccharides have an -linkage. The -linkage
keeps the molecular linear whereas -linkage tends to fold
the molecule, forming a gloublar structure then linear one.
Polysaccharides can be divided into two groups:
a. Homopolysaccharides
b. Heteropolysaccharides.
Homopolysaccharides
They contain only one type of monosaccharides as the rep-
eating unit and on hydrolysis gives only one type of sugar.
Example: Starch, cellulose, glycogen, dextrins, etc.
Starch
Native starch is a mixture of two polysaccharides.
a. Amylose
b. Amylopectins.
Amylose
Amylose is a linear unbranched molecule in which D-glucose
units are linked by (14) glycosidic linkages. It is water
soluble and gives blue color with iodine.
Amylopectin
Amylopectin is a branched chain molecule in which D-glucose
units in addition to -(1,4) linkages are branched by -(1,6)
glycosidic linkages. This branching occurs on an average of
24 to 30 D-glucose units. It is water insoluble and gives violet
color with iodine.
Starch is a nonreducing polysaccharide, tasteless substance
and gives blue color with iodine. Starch on hydrolysis with
dilute mineral acids, i.e. with hydrochloric acid gives glucose
only.
Action of amylases on starch: Amylases are hydrolytic enzymes
which hydrolyze polymers of glucose containing -(1 4)
glycosidic linkages,
Amylases are of two types:
1. -Amylases.
2. -Amylases.
-amylases are present in saliva and pancreatic juice. They
act on starch, hydrolyzing -(1,4) glycosidic linkages in a
random manner to yield glucose, free maltose and smaller
units of starch called starch dextrins. These starch dextrins
contain the original -(1,6) glycosidic linkages. -amylase
cannot hydrolyze the -(1,6) linkages at the branched point
of amylopectins. The -amylases are activated by chloride
ions.
-amylases present in barley malt, cleave successive maltose
units beginning from nonreducing ends of starch to give mal-
tose. -amylase yield only maltose with amylose and smaller
branched polysaccharides, known as limit dextrins, as well
as maltose with amylopectin. -amylases also cannot hydrolyze
-(1,6) linkages at the branched point of amylopectin.
Cellulose
Cellulose is a linear polymer of D-glucose units joined
together by (1,4) glycosidic linkages. On partial hydrolysis,
cellulose yields -1,4 disaccharide cellobiose instead of
maltose. Cell-ulose is water insoluble, nonreducing and gives
no color with iodine.
Unlike starch and glycogen which are readily digested,
cellulose cannot be utilized for energy purposes by human
beings, because the enzyme which cleavage -(1,4) linkage is
missing in the gastrointenstinal tract and hence, merely
provide bulk to the diet. Cellulose is present in plant leaves,
stems, and outer coverings of fruits and vegetables. Cellulose
is a component of fiber (nondigestible carbohydrate) in the
diet. Cellulose is present in plant leaves stems and outer cover-
ings of fruits and vegetables. Cellulose aids intestinal mobility
and acts as an stool softener and reduces bowel cancer. The
nutrition value of cellulose is nil. Celluloses are the most
abundant organic compound on earth. Celluloses are the major
components of plants comprising 20 to 45% of this cell wall mass.
Glycogen
Glycogen is the carbohydrate reserve of the body. Glycogen
is also called animal starch, because it serves as nutritional
reservoir in animal tissues.
Glycogen is a highly branched chain molecule in which
glucose unit in addition to linear -(1,4) linkages are also
linked by -(1,6) at the branched point. This branching repeats
after every 8-10 glucose units.
Glycogen is water soluble and has no reducing property.
It gives red color with iodine.
Glycogen is stored in liver and muscle. About three-fourth
of all the glycogen in the body is stored in muscle.
Difference between starch and glycogen.
1. Starch is of plant origin whereas glycogen is of animal
origin.
2. Glycogen is much more branched than the starch. In starch,
the branching is after every 24 to 30 glucose units, whereas
in glycogen, the branching is after every 8 to 10 glucose units.
3. Starch gives blue color with iodine solution whereas glyco-
gen gives red color.
Dextrins
They are the partial hydrolytic products of starch by -amylase,
-amylase and acids. Dextrins formed from amylases have
unbranched chains while those formed from amylopectins are
branched. All dextrins have free sugar group and accordingly
reduce alkaline copper sulphate solution.
HETEROPOLYSACCHARIDES
Heteropolysaccharides are made up of mixed disaccharides
repeating units and on hydrolysis gives a mixture of more
than one product of monosaccharides and their derivatives
of amino sugars and sugar acids.
They are the essential components of the tissues where
they are present in combination with proteins as mucoproteins.
They are also called mucopolysaccharides or glycosamino
glycans (CAG).
The other suitable name for such heteropolysaccharides
is Glycosaminoglycan or CAG. Glycosaminoglycans are un-
branched polysaccharides consisting of repeating dissaccharide
units comprising a sugar linked to either N-acetylglucosamine
or N-acetylgalactosamine.
They can be divided into:
1. Neutral mucopolysaccharides
2. Acidic mucopolysaccharides.
Acid mucopolysaccharides are present in connective tissues.
They contain hexosamine as the repeating disaccharide unit.
The repeating structure of each disaccharide contains alternate
1,4 and 1,3 linkages.
The most common CAGs are:
Hyaluronic Acid
Hyaluronic acid is present in the connective tissues, synovial
fluid and vitreous fluid in combination with proteins.
It is an unbranched polymer. The repeating disaccharide
is made up of D-glucuronic acid and N-acetyl D-glucosamine.
The monosaccharide subunits are linked by -(1,4) and -(1,3)
glycosidic linkages. Glc UA-(1 3) Glu NAc connected
by (1 4) linkages.
On acid hydrolysis it gives an equimolar quantities of glucu-
ronic acid, glucosamine and acetic acid.
Hyaluronates form viscous lubricants of joints and gel like
substance inside the eyes-vitreous humor.
Heparin
Heparin is glucosaminoglycans. Heparin is an acidic mucopoly-
saccharide in which both the amino and the hydroxyl groups
are combined with sulphuric acid, which causes it to be slightly
acidic substance.
Heparin is present in liver, lungs, thymus, spleen and blood.
Heparin is blood anticoagulant. Heparin contains D-gluco-
samine, D-glucuronic acid or L-iduronic acid as the repeating
disaccharide units. The glucosidic linkage is (1,4) involving
the glucuronic acid anomeric carbon hydroxyl with hydroxyl
group at C-4 of glucosamine.
Chondroitin Sulfates
They are present in connective tissues and serve as a structural
material such as cartilage, tendons and bones.
Chondroitin sulfates are sulfated polysaccharides. Chond-
roitin sulfate is galacto aminoglycans. The acid hydrolysis of
chondroitin sulfate yield D-galactose, D-glucuronic acid, acetic
acid and sulfuric acid.
Sialic Acids
Sialic acids are N-acetyl derivatives of neuraminic acid and
are widely distributed in tissues such as mucins are present
in blood group substances.
Neuraminic acid is a condensation product of pyruvic acid
and mannosamine.
Examples Repeating units
Hyaluronic acid Glucuronic acid; N-Acetyl glucosamine
Chondroitin Glucuronic acid; N-Acetyl galactosamine
Chondroitin-4- Glucuronic acid;
sulfate N-Acetyl galactose-4-sulphate
(Chondroitin
sulfate A)
Heparin Glucosamine-6-SO
4
; glucuronic
acid-SO
4
; iduronic acid
Other CAGs
1. Chondroistin sulfate and dermatan sulfate are galactosa-
mine glycam.
2. Heparin sulfate, heparin and keratan sulfate are glucosa-
mine glycam.
Mucoproteins and Glycoproteins
If the carbohydrate associated with protein is greater than
4%, then the complex protein is called mucoprotein. If the
carbohydrate content is less than 4%, then is called glyco-
protein.
Plasma
1
and
2
globulins are glycoproteins.
Blood Group Substances
They are water soluble, high molecular weight substances,
made up of polysaccharides and proteins. They are present
in saliva, gastric mucin, erythrocyte membranes, etc.
The immunological specificity resides in oligosaccharide
part. The residues present in the oligosaccharides are L-fucose,
D-galactose, N-acetyl-D-galactosamine and N-acetyl gluco-
samine.
According to Bloor, lipids are defined as a group of naturally
occurring substances consisting of the higher fatty acids, their
naturally occurring compounds and substances found naturally
in association with them. It includes a wide variety of subs-
tances with different structures. They are insoluble in water
but are soluble in so-called fat solvents such as ether, acetone,
chloroform, benzene, etc. Associated with them are various
fat soluble, non-lipid substances which includes carotenoid
pigments and certain vitamins, i.e. vitamins A, D, E and K.
Lipids are widely distributed throughout both plant and
animal kingdom and are essential constituents of cell mem-
brane.
Fats are said to be protein sparing because their availability
in the diet reduces the need to burn proteins for energy.
Lipids have several important biological functions.
1. They serve as the reservoir of energy because of their:
a. High energy content. The calorific value is 9 kcal/gm
as compared to carbohydrates which have calorific value
of 4 kcal/gm.
b. Storage in concentrated form in water free state (anhy-
drous) in the tissues as compared to carbohydrates
which are highly hydrated and cannot be stored in such
concentrated form.
2. As structural components of cell membranes.
3. As transport forms of various metabolic fuel.
4. As protective coating on the surface of many organs such
as kidney, against injury.
5. To facilitate the absorption of the fat soluble vitamins A,
D, E and K.
Chemistry of Lipids
CHAPTER
3
Dietary fat can be divided into two types:
a. Visible fat or fat consumed as such, e.g. butter, oils, ghee.
b. Invisible fat or fat present as part of other foods items,
e.g. egg, fish, meat, cereal, nuts, etc.
Classification and Functions of Lipids
Classification Functions
1. Fatty lipids Metabolic fuel, building block for
other lipids
2. Triglycerides Fatty acid storage, transport
3. Phospholipids Membrane structure, storage of
arachidonic acid
4. Sphingolipids Membrane structure
5. Ketone bodies Fuel
SIMPLE LIPIDS
They are esters of fatty acids with various alcohols.
If the alcohol is glycerol, then they are called fats or neutral
fats and are also called triglycerides as all the three hydroxyl
groups of the glycerol are esterified.
If the fat is liquid at ordinary temperature it is called an oil.
Triglycerides are given by the formula
R = Same or different
All of the three fatty acids can be same or different.
If all the three fatty acids are same, then they are called
simple triglycerides. If the fatty acids are different, then they
are called mixed triglycerides. In nature, mixed triglycerides
are more abundant than the simple triglycerides.
CHEMISTRY OF LIPIDS 55
If the alcohol is high molecular weight instead of glycerol
then they are called waxes.
Comparison of simple and compound lipids is terms of their
composition.
Lipid Components
Simple lipids 1. Triglycerides Glycerol + Fatty acids
2. Waxes Alcohol + Fatty acids
(Both long chain)
Compound 1. Phospholipids Glycerol + Fatty acids
lipids + Phosphate
2. Sphingomyelins Sphingosine + Fatty acid
+ Phosphate + Choline
3. Cerebrosedes (glycolipids) Sphingosine + Fatty acid
+ Simple sugar(s)
4. Gangliosides (glycolipids) Sphingosine + Fatty acid
+ 2-6 simple sugars one of
which is sialic acid
Fatty Acids
Fatty acids in nature as such are not very abundant but are
present as ester.
Fatty acids are represented by general formula RCOOH.
A fatty acid is a long chain aliphatic carboxylic acid.
General points about them.
1. They are monocarboxylic acids.
2. Number of carbon atoms are even, though odd number
fatty acids exist but are very rare.
3. They may be saturated or may be unsaturated.
If unsaturated they can be monounsaturated acid or
poly-unsaturated acid.
Mammals and plants contain both monosaturated and poly-
unsaturated fatty acids whereas all the fatty acids containing
double bonds that are present in bacteria are monounsat-
urated. Plant and fish fats contain more polyunsaturated fatty
acids than animal fats. The double bonds in a polyunsaturated
fatty acid are neither adjacent nor conjugated since this would
make the structure to easily oxidisable when exposed to
environment oxygen. Rather the double bonds are three carbon
apart; this provide somewhat greather protection against
oxidations.
Fats obtained from animals are generally saturated and
those from plants are commonly polyunsaturated. However,
these are some exceptions: coconut, palm oils are highly
saturated.
The most common among the saturated fatty acids are
palmitic acid (C
16
), stearic acid (C
18
) and among the unsaturated
fatty acid, oleic acid (C
18
). Unsaturated fatty acids have lower
melting point than saturated fatty acids of same chain length.
Fatty acids with odd number of carbon atoms occur in trace
amounts in terrestrial and marine animals.
Fatty acids with one to eight carbons are liquids at room
temperature while those with more carbon atoms are solids.
The most common fatty acids in neutral fats are:
No. of atoms Formula
Butyric acid 4 CH
3
(CH
2
)
2
COOH
Caproic acid 6 CH
3
(CH
2
)
4
COOH
Lauric acid 12 CH
3
(CH
2
)
10
COOH
Palmitic acid 16 CH
3
(CH
2
)
14
COOH
Stearic acid 18 CH
3
(CH
2
)
16
COOH
Oleic acid 18 CH
2
(CH
2
)
7
CH=CH
(CH
2
)
7
COOH
Fats as an Energy Source
Fats/oils are tremendous source of energy and 40% of total
calories are provided by fatty acids that come from trigly-
cerides and phospholipids.
Naturally occurring straight chain saturated fatty acid
No. of Common name Type Systematic name
C atoms
2 Acetic acid Short n-Ethanoic acid
3 Propionic acid chain n-Propanoic acid
4 Butyric acid n-Butanoic acid
Contd...
8 Caprylic acid Medium n-Octanoic acid
10 Capric acid chain n-Decanoic acid
12 Lauric acid n-Dodecanoic acid
14 Myristic acid Long n-Tetradecanoic acid
16 Palmitic acid chain n-Hexadecanoic acid
18 Stearic acid n-Octadecanoic acid
20 Arachidic acid n-Eicosanoic acid
The presence of double bond in the molecule gives rise
to geometric isomerism. All naturally occurring unsaturated
long chain fatty acids are found in cis isomer.
Most plant fats are liquid since they contain a large
proportions of unsaturated fatty acids with melting points.
Animal fats, on the other hand, contain a high proportion of
palmitic and stearic acids, and are solid or semi-solid at room
temperature. Milk fat is unusual in containing a high proportion
of shorter chain (C
4
-C
14
) fatty acids.
Essential Fatty Acids
They are also called polyunsaturated fatty acids. They are not
synthesized in the body and hence, have to be provided in the
diet. Although linolenic acid and arachidonic acid are syn-
thesized by the body from linoeic acid, but they are synthesized
in insufficient quantity for our needs.
The deficiency of essential fatty acids in humans gives rise
to dry, scaly skin, hair loss, poor wound healing, failure of
growth and increase in metabolic rate. These essential fatty
acids requirement is about 1% of the caloric intake be in the
form of essential fatty acids. Essential fatty acids are needed
for proper cell membrane formation and for synthesis of
prostaglandins prostacyclins, thromboxanes and leukotrienes.
Essential fatty acids are:
No. of No. of Position of Dietary
carbon double double bonds source
atoms bonds from carboxyl end
1. Linoleic acid 18 2 9, 12 Vegetable oils
2. Linolenic acid 18 3 9, 12, 15 Vegetable oils
3. Arachidonic acid 20 4 5, 8, 11, 14 Vegetable oils
4. Timnodonic acid 20 5 5, 8, 11, 14, 17 Fish oils
Contd...
Vegetable oils are oils and have many double bonds hence
polyunsaturated appears on the label of must vegetable oils.
Butter, on the other hand, is a fat and hence would be expected
to have saturated fatty acids, i.e. no double bonds.
Two of the essential fatty acids, linoleic and linolenic acids
are not synthesized by the mammal but are synthesized by
plants. As long as adequate amounts of linoleic acids are
available mammals can synthesize other essential acids.
Structures
Linoleic acid
CH
3
(CH
2
)
4
CH=CHCH
2
CH=CH=(CH
2
)
7
COOH
Linolenic acid CH
3
CH
2
CH=CHCH
2
CH=CHCH
2
CH
=CH(CH
2
)
7
COOH
Arachidonic acid CH
3
(CH
2
)
4
(CH=CHCH
2
)
4
(CH
2
)
2
COOH
Essential fatty acids are necessary in the biosynthesis of
prostaglandins and for proper cell membrane formation.
Prostaglandins are hormone-like compounds which in small
amounts have profound effect.
Important fatty acids in mammalian tissues:
Common name No. of carbon Double Position of
atoms bonds double bonds
Acetic acid 2 0
Lauric acid 12 0
Myristic acid 14 0
Palmitic acid 16 1 9
Stearic acid 18 0
Oleic acid 18 1 9
Linoleic acid 18 2 9, 12
Linolenic acid 18 3 9, 12, 15
Arachidonic acid 20 4 5, 8, 11, 14
Prostaglandins
Prostaglandins are the derivatives of prostanoic acid which
are the cyclic derivatives of unsaturated fatty acids having
twenty carbon atoms.
Prostaglandins are synthesized from essential fatty acids
such as linoleic acid, linolenic acid and arachidonic acid. Five
type of rings are found in the naturally occurring prostag-
landins giving rise to prostaglandins of A, B, E, F and G or
H series. The prostaglandins which are widely distributed
in the body are PGE
1
, PGE
2
, PGE
3
, PGF
1
, PGF
2
and PGF
3
.
Linolenic acid is the precursor to PGE
3
and PGF
1
, Arachi-
donic acid is the precursor to PGF
2
and PGF
2
.
Prostaglandins are synthesized and released by all mamma-
lian cells and tissues except RBC. Also prostaglandins are not
stored in cells but are synthesized and released immediately.
Biological function of prostaglandins:
1. They lower blood pressure (PGE, PGA, PGI
2
).
2. They are used in the induction of labor, termination of
pregnancy and prevention of conception (PGE
2
).
3. They are used in treatment of gastric ulcer (PGE).
4. They are used to prevent inflammation.
5. They are used in asthma.
6. They are used in congenital heart disease.
7. They inhibit platelet aggregation (PGI
2
) whereas PGE
2
pro-
mote clotting process.
Eicosanoids: Fatty Acid Derivatives
Eicosanoids are all derived from C-20 carbon arachidonic acid.
Prostaglandins, Thromboxanes and Leukotrienes are collecti-
vely referred as eicosanoids.
They have a variety of extreme potent hormone like action
on various tissues. These compounds are involved in the regu-
lation of blood pressure, diuresis, blood platelet aggregation,
effects on immune and nervous systems, gastric acid secretion
and muscle contraction.
Properties of Fats
1. Acrolein formation: When glycerol is heated in the presence
of a dehydrating agent such as potassium bisulphate, acrolein
is produced.
Acrolein has a characteristic unpleasant odor and is easily
identified on the basis of this smell. This reaction occurs
whether glycerol is in free or esterified form as occurs in the
triglycerides.
2. Hydrogenation: Unsaturated fats can be hydrogenated by
the addition of hydrogen across the double bonds of the fatty
acids in the presence of nickel as catalyst to give fully saturated
fats. The above process is called Hardening of oils whereby
vegetable oils are hydrogenated to produce commercial cooking
fats.
3. Saponification: Hydrolysis of a fat by alkali is called
Saponification. The products of hydrolysis are glycerol and
alkali salts of fatty acids, which are called soaps. Since the
common fats contain palmitic acid, stearic acid and oleic acid
predominantly, the soaps used for washing consist largely of
sodium salts of these acids. While these fatty acids are insoluble
in water their sodium and potassium salts are water soluble.
4. Rancidity: Rancidity is a chemical change resulting in unpl-
easant odor and taste on storage when fats are exposed to light,
heat, air and moisture. Rancidity is more rapid at high temp-
erature. Rancidity may be due to hydrolytic or oxidative change
taking place at the double bonds of the unsaturated fatty acids
resulting in short chain aldehydes or ketones which have
unpleasant odor.
The addition of certain substances, called antioxidants such
as ascorbic acid and vitamin E prevents rancidity whereas
addition of proxidants like copper, lead and nickel quickens
rancidity.
The oxidation of unsaturated bonds in fatty acids when
the are exposed to oxygen in the environment is referred to
as either auto oxidation or peroxidation. Rancid fats are those
that contain an appreciable amount of peroxidized fatty acid.
Antioxidants are generally added to many food fats to
improve their storage quantities.
Characterization of Fats
Saponification number: Saponification number is defined as the
milligrams of KOH required to saponify 1 gm of fat. Since
fats are mixtures of triglycerides largely of mixed type so the
saponification number of a fat indicates the average molecular
weight (average chain length) of the fatty acids constituting
or comprising the fat.
Saponification number is inversely proportional to the
average chain length of the fatty acids. Higher the saponification
number, the shorter will be the chain lengths of the fatty acids
and vice versa.
The saponification number of some of the fats is given below:
Fat Saponification number
Butter fat 210-230
Human fat 195-200
Olive oil 185-195
Cottonseed oil 194-196
Linseed oil 188-195
Coconut oil 250-260
Castor oil 175-185
Iodine number: Iodine number of a fat is defined as the number
of gm of iodine absorbed by 100 gm of the fat. Halogens,
e.g. iodine or bromine are taken up by the fats because of
the presence of double bonds present in the fatty acid part
of the fat.
Iodine number is a measure of the degree of unsaturation
of fat.
Iodine number of some of the fats is given below:
Fat Iodine number
Butter fat 26 - 28
Human fat 65 - 70
Olive oil 80 - 90
Peanut oil 85 - 100
Corn oil 105 - 115
Soyabean oil 135 - 145
Linseed oil 170 - 200
Acid number: Acid number is defined as the milligrams of KOH
required to neutralize the free fatty acids present in 1 gm.
of fat. This is used in determining the rancidity due to free
fatty acids.
Acetyl number: The acetyl number is defined as the milligrams
of KOH required to neutralize acetic acid liberated by the
saponification of 1 gm of fat after it has been acetylated.
Since acetylation takes place at the hydroxy groups of the
hydroxy fatty acid residues in the fat, so acetyl number is
a measure of the hydroxy fatty acids in the fat content.
Polenske number: The ml of N/10 KOH required to neutralize
the insoluble fatty acids from 5 gm. of fat which are not steam
volatile.
Reichert Meissel number: This represents the ml of N/10 KOH
required to neutralize the volatile acid obtained from 5 gm
of fat which has been saponified then acidified to liberate the
fatty acids and then steam distilled.
Butter fat, which contains shorter chain fatty acids has a
Reichert Meissel number of 26 to 30.
COMPOUND LIPIDS
They are the esters of fatty acids containing nitrogen base
in addition to an alcohol and fatty acids.
A molecule which has changed and an unchanged portion
is called an amphipathic molecule.
Phospholipids
They are also known as phosphatides. Phospholipids act as
a detergent and increase the solubility of other lipids. They
are present in all cells as well as in the plasma.
Phospholipids include the following groups:
Phosphatidic Acid
The general structure of phosphatidic acid.
They are important intermediates in triglyceride synthesis.
Phosphatidic acid on hydrolysis yield glycerol, fatty acid and
phosphoric acid.
Lecithins
The structure of lecithins are:
Lecithin contains saturated fatty acid residue at the -posi-
tion and unsaturated fatty acid residue at the -position of
the glycerol.
Lecithins on hydrolysis give glycerol, fatty acid, phosphoric
acid and choline.
Cephalins
The structure of cephalins are:
Cephalins differ from lecithins with respect to base attached
to phosphoric acid.
If the base is ethanol amine then it is called phosphatidyl
ethanolamine or ethanolamine cephalin.
If the base is amino acid serine then it is called phosphatidyl
serine which is also called serine cephalin.
Cephalins on hydrolysis yield glycerol, fatty acids, phos-
phoric acid, ethanol amine or serine.
Phosphatidyl Inositol
The structure of phosphatidyl inositol is:
It contains inositol in place of base.
Cardiolipin
An important phospholipid of mitochondrial membrane is
cardiolipin. It is a diphosphatidyl glycerol in which two phos-
phatidic acids are joined by a molecule of glycerol.
These phospholipids are particularly rich in the polyunsatu-
rated fatty acids especially linoleic acid.
Plasmalogens
These compounds possess fatty aldehyde in place of fatty acid
at the -position, with the result the normal ester linkage is
replaced by the ether linkage on the C
1
carbon. In some cases,
bases like choline, serine or ethanol amine are also found.
They are found in brain and heart.
Sphingomyelins
Phospholipids containing sphingosine are called sphingo-
myelins. They contain, a complex base sphingosine in addition
to phosphoryl choline. A fatty acid is attached to the amino
group of the sphingosine. No glycerol is present.
Sphingomyelins are present in all tissues especially in brain
and other nervous tissues.
Sphingomyelins on hydrolysis yield sphingosine, fatty acid,
phosphoric acid and choline.
Increased concentration of sphingomyelins occur in liver,
spleen, etc. in a condition known as Niemann-Picks disease.
Cerebrosides or Glycolipids
Glycolipids are carbohydrate-glyceride derivatives containing
sugar, sphingosine and a fatty acid. These compounds do not
contain phosphoric acid. If the sugar component is galactose,
the lipid is termed galactolipid. The term cerebroside is used
because it is found in large quantities in brain tissues
particularly in white matter.
Structure of Sphingomyelins
On hydrolysis cerebrosides give sphingosine, a fatty acid
and galactose. Cerebrosides are differentiated on the basis
of fatty acid present.
Examples:
Kerasin: It contains Lignoceric acid
Cerebron: It contains Hydroxy Lignoceric acid
Nervon: It contains Nervonic acid
Oxynervon: It contains Hydroxy Nervonic acid
Cerebrosides occur in large amounts in the white matter
of brain and in the myelin sheaths of nerves.
In Gauchers disease, large amount of cerebroside accumu-
lates in the liver and spleen.
Gangliosides
They are found in nerve tissues. They contain carbohydrates,
N-acetyl galactosamine and N-acetyl neuraminic acid.
Cerebrosides
Sulfatides (Sulpholipids)
They are cerebrosides having a sulfate group attached to the
galactosyl residue.
DERIVED LIPID
Those substances which are derived from the above two
groups by hydrolysis. These include fatty acids of various
series, steroids, bile acids and substances associated with lipids
in nature such as carotenes, vitamin A, D, E and K.
Lecithins are hydrolyzed by certain enzymes, phospholi-
pases or lecithinases. The nature of hydrolysis depends upon
the type of phospholipase used.
Phospholipase A: Present in snake venom (cobra) hydrolyzes
fatty acid in or 1-position of glycerol in the lecithin to form
lysolecithins. In the similar manner it acts on cephalin.
Phospholipase B: Hydrolyzes the remaining fatty acid of lyso-
lecithin present at or 2-position to form glyceryl phosphoryl-
choline.
Phospholipase C: Hydrolyzes phosphorylcholine from lecithins
to form diglycerides. Phospholipase C catalyses the hydrolysis
at the glycerol side of the phosphate group.
Phospholipase D catalyses the hydrolysis on the phosphate
side of the phosphate group.
Phospholipase D: Hydrolyzes choline from phosphatidyl ethano-
lamine (cephalin) form phosphatidyl serines.
There are two classes of nonsaponificable lipids.
Terpenes
They are linear or cyclic compounds formed by condensation
of two or more isoprene units.
Other important terpenoid compounds are:
a. Tocopherol (vitamin E)
b. Coenzyme Q (also called ubiquinone)
c. Vitamin K (a naphthaquinone)
They include vitamins A, E, K and carotenes, etc.
Cyclopentano-perhydro-phenanthrene ring
(Steroid nucleus)
Steroids
The term steroids includes many compounds which have
however one feature in common, the steroid skeleton. Steroids
are the derivatives of cyclopentano-perhydro-phenanthrene
ring (consists of four fused rings). This is a saturated (per-
hydro) pheranthrene ring with a cyclopentane ring attached.
Steroids are steroidal alcohol. The most important member
of this group is cholesterol. The four rings that make up
perhydro-cyclopentano-phenanthrene are named alphabetic-
ally from left to right.
Despite popular belief, cholesterol is not a poison but a very
necessary part of our cell membranes and the basis of sexual
hormones (androgens, estrogens, etc). Cholesterol is only a
problem if it is in excess and in this respect we do not need
cholesterol in our diets because body can synthesis it.
Steroids belong to the class of important biological com-
pounds with diverse physiological activities.
Some of the biologically important steroids are:
a. Ergosterol: UV radiation causes rupture of
ring B to produce vitamin D.
b. Bile acids: In lipid metabolism.
c. Adrenal cortex Corticosterone and cortisol.
steroids:
d. Female hormones: Progesterone and estrogen.
e. Male sex hormones: Testosterone and androsterone.
Cholesterol is an animal fat and it does not occur in plants.
Cholesterol contains hydrogen group at C-3, methyl groups
at C-10 and C-13, a double bond at C-5 and an 8C branched
alkyl group attached to C-17. This marks a total of 27C.
This ring structures are lipid soluble and hydroxyl group of
C-3 is hydrophilic.
Plants have stigmasterol and -sitosterol which differ only
in the alkyl group side chain attached at C-17.
The Antioxidant System
In healthy individuals, the antioxidant system defends tissues
against free radical attack. Antioxidants are known to prevent
cellular damage and enhance repair. Three classes of antioxi-
dants have been identified.
a. Primary antioxidants: They prevent the formation of new
free radical species, e.g. superoxide dimutase, glutathione
peroxidase, ceruloplasmin, transferrin, ferritin.
b. Secondary antioxidants: They remove newly formed free
radicals before they can initiate chain reactions. These chain
reactions can lead to cell damage and further free radical
formations, e.g. vitamin E, vitamin C, -carotene, uric acid,
bilirubin, albumin.
c. Tertiary antioxidants: They repair cell structures damaged
by free radicals attack, e.g. DNA repair enzymes,
methionine sulphoxide reductase.
Deficiency in the antioxidant system can develop for a
number of reasons:
a. Low intake of dietary antioxidants
b. Total parenteral nutrition
c. Decreases that reduce the absorption of antioxidant
nutrients from food, e.g. Crohns disease
d. Renal dialysis
In these situations the antioxidant system struggles to
protect the body from free radical attack and as a result the
risk of free radical-mediated disease increases.
Increased antioxidant status by supplementation may
indeed reduce the risk of certain diseases.
i. High intake of vitamin E has been associated with reduced
risk of mortality from ischemic heart disease.
ii. High incidence of vitamin C and -carotene have been
associated with a reduced incidence of some cancers.
iii. Dietary supplementation of vitamin E, -carotene and
selenium significantly reduces mortality from esophageal
cancer.
iv. Within one week on antioxidant rich, low fat diet reduces
lipid peroxide levels and increased aborrhic acid level in
patient in the acute myocardial infarction.
Free Radicals
A free radicals is defined as any atom or molecule that possesses
an unpaired elactron. It can be anionic, cationic, or neutral.
Free radicals are highly reactive molecules generated by the
biochemical redox reactions that occur as part of normal cell
metabolism and by exposure to environmental factors such
as UV light, cigarette smoking, environmental pollutions and
gamma radiations.
Human body is constantly under attack from free radicals.
Some toxic compounds can result in the production of free
radicals which include anticancer drugs, anaesthetics, anal-
gesics, etc.
The free radicals species which occur in the human body
are:
a. Superoxide radical (
O
2
)
b. Hydroxyl radical (OH
)
c. Nitric oxide radical (NO
)
d. Peroxyl radical (ROO
).
Once formed, free radicals attack cell structures within the
body. As a result, free radicals have been implicated in
numerous diseases such as atherosclerosis, cancer, AIDS, liver
damage, rheumatoid arthritis, Parkinsons disease, etc.
Process of Lipid Peroxidation
This process is responsible for randicity of food. This process
involves:
i. Initiation
ii. Propagation
iii. Termination.
Initiation
ROOH + Metal
n+
ROO
+ Metal
(n-1)
+
+ H
+
X
+ RH R
+ HX
Propagation
R + O
2
ROO
ROO
+ RH ROOH + R
Termination
2ROO
ROOR + O
2
ROO
+ R
ROOR
R
+ R
RR
Eicosanoids
Eicosanoids are formed from C
20
polyunsaturated fatty acid.
Arachidonate and some other C
20
fatty acids give rise to eico-
sanoids which includes prostaglandins, thromboxanes, leuko-
trienes, lipoxins. There are two pathways of their formation:
1. Cyclooxygenase pathway
2. Lipooxygenase pathway.
CHEMISTRY OF AMINO ACIDS
Naturally occurring amino acids are amino acids containing
amino group and carboxyl group on the same alpha carbon
atom and are represented by the general formula:
Chemistry of Amino
Acids and Proteins
CHAPTER
4
All amino acids found in living systems, plant and animal
proteins are L--amino acids. Glycine is the only amino acid,
which is optically inactive and cannot be resolved into D-or
L-form because of symmetry on the -carbon atom. All other
amino acids are optically active.
The configuration of L--amino acid is:
A variety of classification of amino acids are possible. Either
they can be classified according to the presence of acidic, basic
or neutral groups or upon their chemical structures, i.e., pre-
sence of polar groups, nonpolar groups, sulphur containing
groups, aromatic groups, heterocyclic ring, branched chain and
so on.
CHEMISTRY OF AMINO ACIDS AND PROTEINS 75
Classification of Amino Acids
1. Aliphatic amino acids
2. Aromatic amino acids
3. Heterocyclic amino acids.
Tryptophan
Besides these there are number of amino acids which are
obtained in free or combined form but do not occur in protein
molecules, e.g. thyroxine, triiodothyronine, ornithine, citruline,
-aminobutyric acid, -alanine, etc. Their structures are given
here.
A dipeptide has two amino acids joined by a single peptide
bond; a tripeptide is composed of three amino acids joined
by two peptide bonds: a polypeptide is one in which any
number (n) of amino acids or (AA)
n
are linked together by
(n-1) peptide bonds.
Examples of relatively smaller peptides that possess biologi-
cal activity are glutathione, oxytocin, vasopressin, hypertensin,
etc.
Glutathione is a tripeptide consisting of glutamic acid,
cystine and glycine and is found in red blood cells.
Oxytocin and vasopressin are produced by the posterior
of the pituitary gland. Each is made up of nine amino acids.
Oxytocin causes contraction of smooth muscle and it is used
in obstetrics to initiate labor whereas vasopressin raises blood
pressure and reduces the secretion of urine.
Angiotensin I has 10 amino acids and angiotensin II has
8 amino acids. They cause hypertension.
Functions of Amino Acids
Amino acids serve as:
1. Building block of proteins
2. Precursors of:
a. Hormones. (peptide and thyroid)
b. Purines
c. Pyrimidines
d. Porphyrins
e. Vitamins
3. Neurotransmitter such as tryptophan (sertonin).
4. Transport of nitrogen: Alanine, glutamine.
5.Substrates for protein synthesis: Those for which there is
a codon.
Essential Amino Acids
Those amino acids which are not synthesized in the body and
hence have to be provided in the diet. They are also called
indispensible amino acids. There are eight essential amino
acids.
They are leucine, isoleucine, threonine, tryptophan, pheny-
lalanine, valine, methionine and lysine.
Adequate amounts of essential amino acids are required
to maintain the proper nitrogen balance.
Deficiency of one or more essential amino acids in the diet
gives rise to decrease in protein synthesis resulting in failure
in growth of the child, negative nitrogen balance in adults
and fall in plasma proteins and hemoglobin levels.
Semiessential Amino Acids
Those amino acids which are synthesized partially by the body
but not at a rate to meet the requirement of the body are
called semiessential amino acids.
Semiessential amino acids are arginine and histidine.
Nonessential Amino Acids
Those amino acids which are synthesized by the body. These
amino acids are derived from carbon skeletons of lipids and
carbohydrates during their metabolism or from the transfor-
mation of essential amino acids.
Nonessential amino acids are alanine, arginine, asparagine,
aspartic acid, cysteine, glutamic acid, glutamine, glycine, pro-
line, serine and tyrosine.
Nitrogen Balance
The ratio of:
Intake N
Output N
=
> 1, i.e. positive nitrogen balance, e.g. during
pregnancy, convulsions and growth.
< 1, i.e. negative nitrogen balance, e.g. in mal-
nutrituion and in certain wasting diseases
where, there is tissue breakdown.
1, i.e. nitrogen equilibrium. Normal adults
are in nitrogen equilibrium.
Ninhydrin Reaction
All amino acids (except proline and hydroxyproline), proteins
or protein derivatives containing free amino group and a free
carboxyl group react with ninhydrin to give a blue-violet
colored compound called Rheumanns purple, whereas amino
acids, proline and hydroxyproline, give a yellow color with
ninhydrin.
Reaction with Nitrous Acid
-amino acids are deaminated to the corresponding -hyd-
roxy acids with nitrous acid. Each amino group yields one
molecule of nitrogen which can be measured accurately.
Hence, this reaction is used for the estimation of free amino
groups in amino acids, peptides and proteins.
Formal Titration
Sorensens formal titration method is used for the estimation
of free carboxyl group in amino acid and mixtures of amino
acids. By this method one can determine the rate of digestion
of proteins by determining the increase in carboxyl groups
which accompanies during enzymatic hydrolysis. Amino acids
by virtue of Zwitter ion formation are neutral in solution. If
formaldehyde is added to a solution of amino acid, an adduct
is formed at the amino group, leaving the carboxyl group free
and the molecule acidic in reaction. In other words the presence
of formaldehyde decreases the basicity of the amino group,
permitting free carboxyl group to exert its maximum acidity.
Free carboxyl group thus can be titrated.
Isoelectric Point of Amino Acids (pl)
pl is defined as that pH at which the amino acid does not
migrate in an electric field. At this pH, the amino acid molecule
exists in the Zwitter ion form, in which the sum of the positive
charges are equal to the sum of the negative charges and the
net charge on the molecule is zero.
pl is calculated as:
where pK
1
is the pH at which the carboxyl group is half-
titrated and pK
2
is the pH at which the N
+
H
3
group is half-
titrated.
Amino acids are amphoteric electrolytes, i.e. they exhibit
properties of both an acid and a base. The acidic groups of
amino acids are carboxylic group (COOH COO+ H
+
)
and protonated -amino group (N
+
H
3
NH
2
+ H
+
).
Basic groups of amino acids are dissociated carboxyl group
(COO + H
+
COOH) and -amino group (NH
2
+ H
+
N
+
H
3
).
Amino acids in aqueous solutions have been shown to occur
as a dipolar species or zwitter ion (Molecules which have both
a negative and a positive change).
As every amino acid has at least two ionizable groups, it
can exist in different ionic forms depending on the pH of the
medium.
In aqueous solution a neutral amino acid is in the zwitter
ion form which is dipolar. It is therefore an amphoteric
electrolyte. Ampholytes are those molecules that act as both
an acid and a base.
In strongly acid pH, it is in cationic form while in strongly
alkaline pH, it is in anionic form.
At isoelectric pH, the solubility and buffering capacity is
minimum.
Similarly, protons exist as cations in the acid media and
anion in the alkaline media of the isoelectric pH. Hence, protein
acts as buffers on both sides of isoelectric pH. So a proton
is an anion at pH values above the pl and is a cation at pH
values below the pl.
At the isoelectric pH, glycine exists as Zwitter ion. Addition
of acid converts it into cation and addition of alkali converts
it into anion. Therefore amino acids depending on the medium
pH carry net zero, positive or negative charges.
Q. Show the formula of isoelectric glycine. Indicate by
formulae what happens on the addition of (a) acid and
(b) base to the isoelectric molecule.
Ans. At isoelectric point the glycine exists as:
PROTEINS
Proteins are defined as compounds of high molecular weight
made up of -amino acids linked to one another by peptide
linkages. Proteins contain 20 odd individual amino acids
present in characteristic proportions and linked in a specific
sequence in each protein.
Proteins are linear polymers consisting of L--amino acids.
The amino acids are joined together by peptide bonds. The
peptide bond is formed by the union of carboxyl group of
one amino acids with amino group of other amino acid with
an elimination of water molecule.
Classification of Proteins
Proteins are classified on the basis of their composition.
Simple Proteins
Simple proteins are made up of amino acids only and on
hydrolysis yield constituent amino acids mixture only.
Example:
1. Fibrous proteins:
These are animal proteins which are highly resistant to
digestion by proteolytic enzymes. They are water insoluble.
a. Collagens It contains high proportion of hydroxy
proline and hydroxylysine. It is a major
protein of connective tissues. On boi-
ling with water it forms gelatin.
b. Elastins It is present in tendons and arteries.
c. Keratins It contains large amount of sulphur as
cystine. It is present in hair, wool, nails,
etc.
2. Globular proteins:
a. Albumins Serum albumin and ovalbumin of egg
white. It is water soluble. It is precipi-
tated from solution by full saturation
of ammonium sulfate. It is coagulated
by heat.
b. Globulins Serum globulins, fibrinogens and mus-
cle myosin. It is soluble in dilute salt
solutions. It is precipitated from solu-
tion by half saturation of ammonium
sulphate. It is coagulated by heat.
c. Glutelins Cereal proteins such as glutelins of
wheat, oxyzenin from rice and zein of
maize. It is soluble in weak acids or
bases but insoluble in neutral aqueous
solutions.
d. Gliadins Gliadin from wheat and zein from
(Prolamines) corn. It is water insoluble but soluble
in ethanol.
e. Protamines Salmine from salmon sperm cells con-
tains high proportion of arginine.
f. Histones Globulin in hemoglobin. It contains
high proportion of basic amino acid. It
is water soluble.
Conjugated Proteins
They are proteins which contain nonprotein group (also called
prosthetic group) attached to the protein part. On hydrolysis
they give nonprotein component and amino acid mixture.
Conjugated Protein = Protein part + Prosthetic group.
Conjugated proteins are classified according to the nature
of the nonprotein group attached to the protein part.
Derived Proteins
They are formed from simple and conjugated proteins by
physical and chemical means.
The products of partial hydrolysis of proteins are often
classified as derived proteins.
1. Primary derived These are formed as a result of slight
protein change in structure with little or no
hydrolytic cleavage of peptide bonds.
a. Protein Fibrin from fibrinogen.
b. Metaprotein They are soluble in dilute acids and
bases but insoluble in neutral solvents.
c. Conjugated Formed by the action of heat, alcohol,
proteins UV light, X-rays. Example, cooked egg
white and egg albumin.
2. Secondary derived These are formed by the progressive
protein hydrolytic cleavage of the peptide
bonds of protein molecules. They are
water soluble and are not coagulated
by heat.
a. Proteoses
b. Peptones
c. Peptides.
These proteins are formed as a result of various deep seated
changes in the structure or composition of the proteins.
Separations of proteins by ion exchange resins in a chro-
matography is also an important technique for the separation
and characterization of proteins by change. Ion exchange resins
Proteins Prosthetic group Example
1. Nucleoproteins = Nucleic acid Virus proteins
2. Phosphoproteins = Phosphoric acid Casein of milk
(Serine residues
are phosphory-
lated), ovovitellin
of egg yolk.
3. Glycoproteins = Carbohydrate or Mucin of saliva
a derivative of
carbohydrate
4. Lipoproteins = Lipids (Lecithin, Serum
Cephalin, lipoproteins
cholesterol, etc).
5. Flavoproteins = Riboflavin Biological oxida-
tion reduction
reactions
6. Metalloproteins = Metals (Zinc, Carbonic anhy-
iron and copper) drases, catalase,
cyctochrome
oxidase
are prepared of insoluble materials such as agarose, polyacry-
lamide, cellulose, etc. that contains negatively changed ligands
(such as CH
2
COO, C
3
H
6
SO
3
) or positively charged legands
such as diethyl amino. The degree of retardation of a protein
or amino acid by a resin will depend on the magnitude of
the charge on the protein at a particular pH of the experiment.
Molecule of the same charge as the resin are eluded first in
a single band, followed by proteins with an opposite charge
to that of the resin.
Electrophoresis
If a solution of a mixture of proteins is placed between two
electrodes, the charged particle will migrate to one electrode
or the other at a rate that depends on the net change and,
depending on the supporting medium used, on the molecular
weight.
Structure of Proteins
Proteins exhibit four levels of organization:
Primary structure Refers to amino acid sequence.
Secondary structure Refers to folding of polypeptide chain
into specific coiled structure which
is repititive in one direction.
Tertiary structure Refers to arrangement and interrela-
tionship of twisted chain into a three
dimensional structure.
Quaternary structure Refers to the association of different
monomeric subunit into a composite
polymeric protein.
Primary Structure
It determines the sequence of amino acids in the protein
molecule. It indicates the number of amino acids, type of amino
acids and in which fashion they are linked up.
The sequence of amino acids in proteins can be found out
by Sangers and Edmans degradation method.
Sangers Method
This method is used to determine the N-terminal amino acid
of proteins. The reagent used is 2, 4-dinitrofluorobenzene (DNFB).
DNFB reacts with free amino group of the terminal amino
acid of proteins to give a yellow colored 2, 4-dinitrofluoro-
benzene derivative which on hydrolysis, give the terminal
amino acid as the yellow 2,4-dinitro derivative and all the
other amino acids of protein are obtained as free amino acids.
The yellow derivative is separated and identified by paper
chromatography, by comparison with known 2,4-DNP amino
acid.
Edmans Method
N-terminal amino acid residue of proteins can also be identified
by Edmans method. The reagent used is Phenylisothiocyanate
(PITC). It reacts with free alpha amino group of the N-terminal
amino acid of proteins to give the phenylisothiocarbamate
derivative of the protein which cyclizes in acid medium giving
N-terminal amino acid as phenylthiocarbamyl amino acid
(PTCA), leaving the rest of the protein chain intact, but shorter
by one amino acid. PTCA then cyclizes to give the correspon-
ding phenylthiohydration derivative, which is separated and
identified by chromatography.
The reaction with phenylisothiocyanate is then repeated
on the shortened peptide. The amino acid sequence is thus
determined from the N-terminal end of the peptide one by
one.
Phenylthiohydantoin Derivative
Edmans method is superior over Sangers method. Edmans
degradation involves the removal of one amino acid at a time
from the amino end of a peptide or protein chain, leaving
the remaining peptide chain intact. The process can be repeated
and the sequence of amino acid from N-terminal end is
obtained.
Whereas in Sangers method, only the N-terminal amino
acid is identified because after the removal of N-terminal acid
with DNFB, the remaining peptide chain breaks into amino
acid mixture.
Another reagent often used is Dansyl chloride (Dimethyl
aminonaphthalene-5-sulphonyl chloride).
The procedure with this reagent is the same as that used
with DNFB. A covalent bond is formed with the free N-ter-
minal amino group. The dansylated protein is hydrolyzed with
acid and dansylated amino acid is separated and identified
by chromatography.
C-terminal residues are usually identified with enzyme
carboxypeptidase. This enzyme attack only the peptide bond
joining the last residue with a free -carbonyl group of the
peptide chain. Amino acids released are identified by chrom-
atography.
Also the polypeptide is treated with the anhydrous hyd-
razine, which breaks peptide bonds forming hydrazides with
the carbonyl carbons. The C-terminal residue does not form
a hydrazide because its carboxyl group is free. After the rem-
oval of the hydrazides, this amino acid is then identified chrom-
atographically.
The repetition of Edman reactions under favorable condi-
tions can be carried out for 30 to 40 amino acids into the poly-
peptide chain from the NH
2
-terminal end. Since most
polypeptide chains in proteins contain more than 30 to 40 amino
acids, they have to be hydrolyzed into smaller fragments and
sequenced in sections.
Both enzymatic and chemical methods are used to break
polypeptide chains into smaller polypeptide fragments.
Trypsin and chymotrypsin are proteolytic enzymes that
are used for partial hydrolysis of polypeptide chains in
sequencing. Enzyme trypsin catalyse the hydrolysis of peptide
bond on the -COOH side of the basic amino acid residues
of lysine and arginine with the polypeptide chains. Chymo-
trypsin hydrolyzes peptide bonds on the -COOH side of
amino acid residues with larger apolar side chains.
The chemical reagent cyanogen bromide cleaves peptide
bonds on the carboxyl side of methionine residue with poly-
peptide chains.
R
1
Reagent
Phenylalanine
Tyrosine Chymotrypsin
Tryptophan
Anginine, Lysine Trypsin
Methionine Cyanogen bromide
Tryptophan O-Iodosobenzoic acid
Secondary Structure
The polypeptide back-bone does not assume a random three-
dimensional structure, but instead generally forms regular
arrangements of amino acids that are located near to each
other in linear sequence. These arrangements are called as
secondary structure of proteins. The durameter of helix is 10.
This is of following types:
1. -helix: This is most common type of secondary structure,
it is spiral structure. -helix is stabilized by extensive
hydrogen bonding and it consists of 3-6 amino acid per
turn. Proline disrupts the -helical structure because it
is imino acid and geometricallly not compatible with
helical structure.
2. -sheet: In this surface appears pleated. So also known as
-pleated sheet. The two or more chains may be parallel
or antiparallel.
Amyloid protein deposited in brains of individuals with
Alzheimers disease is composed of -pleated sheet.
3. -bends: -bends reverse the direction of a polypeptide
chain, helping it to form a compact, globular shape. These
are usually found on the surface of protein molecules.
4.Nonrepetitive secondary structure: About half of an average
globular protein is organized into repetitive structures.
These are not random but have a less regular structure.
5.Supersecondary structures: These mainly form core, i.e. interior
to molecule. These are also known as motifs. The common
ones are -- unit, greek key and meander.
Tertiary Structure
Tertiary structure refers to the coiling of several helical portion
of single helix into a three-dimensional structure.
The tertiary structure of proteins is stabilized by:
1. Hydrogen bonding: It is formed by sharing of hydrogen
atom between electronegative oxygen atoms, nitrogen
atoms or combination of two.
2. Disulphide bonding: This results from electrostatic attrac-
tion between positively and negatively charged spacies.
3. Ionic interactions or salt bridges: These are nonpolar bonds
between hydrocarbon containing compounds.
4. Ester bonding.
5. Hydrophobic interactions: These are the result of mutual
interaction of electron and nuclei of molecules.
6. van der Waals forces.
Quaternary Structure
Proteins containing more than one polypeptide chain display
fourth level of structural organization called quaternary struc-
ture. In quaternary structure of proteins, the individual poly-
peptide chains are arranged in relation to each other so as
to give a single three dimensional structure of the overall
protein molecule. Each polypeptide chain in such a protein
is called a subunit. Depending upon the number of subunits
such proteins are called dimers, tetramers or polymers, etc.
The various examples are hemoglobin, ferritin, etc.
Reactions of Proteins
1. They give biuret test positive.
2. They give blue color with ninhydrin.
Biuret Reaction
The name of the reaction is derived from the organic com-
pound, a biuret, obtained by heating urea at high temperature
which gives this test positive. The compound biuret contains
two peptide linkage.
Biuret test is given by those compounds which contain two
or more peptide bonds. Since proteins are polypeptides hence,
it is a general test for proteins.
When proteins are treated with alkali and minute quantities
of cupric ions, a pink or purple color is obtained.
Precipitation Reactions
Proteins are precipitated from the solution by a large number
of reagents and the process is called deproteinization. Such
precipitation reactions are important in the isolation of
proteins, in the deproteinization of blood and other biological
fluids.
a. Effect of salt concentration: Proteins are precipitated from
the solution by the addition of (NH
4
)
2
SO
4
and Na
2
SO
4
.
Addition of large amounts of ionic salts results in increase
in protein: protein interaction and decrease in protein:
water interaction, the process is called salting out.
b. Effect of positive ions: The positive ions most commonly
used for protein preci- pitations are heavy metal cations
such as Cu
++
, Zn
++
, Fe
+++
etc. These cations precipitate
proteins from alkaline solution by combining with the
negatively charged protein to form an insoluble precipitate
of metal proteinate.
c. Effect of negative ions: Addition of tungstic acid, phosphot-
ungstic acid, trichloroacetic acid, picric acid, sulphosalicylic
acid results in precipitation of protein in acidic solution.
Denaturation
Denaturation is the unfolding of the characteristic native
folded structure of the polypeptide chain of protein. Compara-
tively weak forces responsible for maintaining the secondary,
tertiary and quaternary structure of proteins are rapidly
disrupted during the denaturation. The primary structure held
by covalent peptide bonds however is not disrupted. After
denaturation such proteins acquire the random coil structure
which may renaturate into native form under favorable
conditions. Denaturation of oligomeric protein involves the
(i) dissociation of subunits peptide chains from each other with
or without (ii) the unfolding of individual chains into random
coils. Such proteins usually are unable to renaturate or refold
into the natural form.
There are two conspicuous changes that often result from
denaturation.
1. Loss of (Partial or Complete) biological activity of the
protein.
2. The solubility decrease drastically, i.e. almost the preci-
pitation takes place.
Denaturation results in loss of biological activity caused by
heat, pH changes, by organic solvents, effect of radiation, etc.
In electrophoresis, an ampholyte such as protein, peptide
or amino acid in a solution buffered at a particular pH is placed
in an electric field. Depending on the relationship of the buffer
pH to the pI of the molecule, the molecule will either move
toward the cathode () or the anode (+) or remain stationary
(pH = pI).
For plasma protein separation the solution is buffered at
pH 8.6 which is at a pH substantially above the pI of the major
plasma proteins. The proteins are negatively charged and move
toward the positive pole. The peaks are obtained according
to their rate of migration in order of their pI values are these
of albumin,
1
-,
2
- and -globulins, fibrinogen and
1
- and
2
-globulins.
The different major proteins are designated underneath
the peaks. The direction of migration is from right to left.
Electrophoretic pattern of normal serum
Functions of Proteins in the Body
1. Catalytic proteins: Enzymes
2. Structural proteins: Collagen
3. Contractile proteins: Actin, myosin
4. Natural defence proteins (Immunity): Antibodies
5. Transport proteins: Albumin, Globulin, Hemoglobin, ceru-
loplasmin, apolipoprotein
6. Blood proteins: Fibrinogen
7. Hormonal proteins: Insulin
8. Respiratory proteins: Cytochromes
9. Repressor proteins: Regulate expression of genes of chro-
mosomes
10. Rece ptor proteins: Transport information to cell interior
after interacting with proteins on the outside.
11. Ribosomal proteins: Associated in the proteins synthesis
12. Toxin proteins: Venoms
13. Vision proteins: Rhodopsin
14. Storage: Ferritin.
Plasma Proteins
Normal value of plasma proteins is 6 to 8 gm per 100 ml of
blood.
Plasma protein include albumin, globulin and fibrinogen.
They can be separated.
1. By precipitation method using sodium sulfate, ammo-
nium sulfate, etc.
2. By electrophoresis.
In normal human plasma, 6 fractions have been separated
by electrophoresis. They are:
i. Albumin
ii.
1
-globulin
iii.
2
-globulin
iv.
1
-globulin
v.
2
-globulin
vi. Fibrinogen.
Functions of Plasma Proteins
1. Osmotic pressure: Plasma proteins are important in regu-
lating water between blood and tissues. Small molecules
of plasma and tissue fluid such as glucose, amino acids,
urea, electrolytes, freely diffuse back and forth and hence,
exert the same osmotic pressure in both fluids, i.e. on
both sides of capillary. However, plasma and lymph
protein do not freely diffuse through the capillary walls
and since the prot ein concentration of plasma is much
higher than of lymph by difference in the protein osmotic
pressure of the two fluids. This difference in the osmotic
pressure of lymph and plasma is estimated to average
about 22 mm Hg and represents effective osmotic pres-
sure of plasma.
2. As buffers: Proteins are amphoteric in nature and thus help
in maintaining pH of the body.
3. Reserve proteins: Proteins serve as source of proteins for
the tissues when the need arises.
4. As carrier of certain metabolites: The transport of certain
insoluble substances such as bilirubin, free fatty acids,
steroid hormones and lipids is carried out by various
fraction of serum proteins.
5. As immunoglobulins: The property of antibodies formation
resides in -globulin fraction of the proteins.
Immunoglobulin
Immunoglobulins or antibodies, make up the -globulins
fraction of the plasma. These defensive proteins are synthesized
in response to exposure to a foreign material usually a protein
or complex carbohydrate. The foreign material is called
antigen. The formation of antibodies affords immunity against
the antigen and this response is protective.
Immunoglobulins are composed of four polypeptide chains,
two light chains (L-chains) and two heavy chains (H-chains)
per molecule. These chains are linked by disulphide bonds.
There are two classes of light chains, and thus creating
two series of immunoglobulin molecules. Each class of immuno-
globulin contains a unique type of heavy chain. These are
designated as , , , and chains. These immunoglobulins
and their chemical formulae are represented as follows:
Immunoglobulins H-chains K-type -type
IgG K
2
r
2
2
r
2
IgA K
2
2
IgM K
2
2
IgD K
2
2
IgE K
2
2
Immunoglobulins also called Antibodies, comprises the
gamma-globulin fraction of the plasma. They are synthesized
in the body in response to the exposure (or administration)
to a foreign moiety called Antigen. The foreign material or
antigens are usually proteins or carbohydrates. The formation
of antibodies give rise to immunity against the antigen and
this response is protective.
The immunoglobulins are glycoproteins containings 3% to
12% carbohydrates including D-mannose, D-galactose, L-
fucose, D-glucosamine and a sialic acid.
On ultracentrifugation the immunoglobulins are separated
into three major fractions IgM, IgG and IgA. Two other
immunoglobulins IgD and IgE occur in plasma in small amount.
Most of the antibodies are in IgG fraction which represents
70% of the total r-globulins. Immunoglobulins are made up
of subunit peptide chains called heavy chain (mol wt 40,000)
and a light chain (mol wt 20,000). The three types of
heavy chain are , r and . Two types of lighter chain are
K and .
Both types of light chains contain a segment with a constant
sequence of amino acids comprising about half the chains and
a variable portion of other half. Heavy chains also contain
a variable portion of amino acid sequence (about 110 amino
acids) and 330 amino acids forming the constant portion of
the chains. The variable portion of the light and heavy chains
of immunoglobulins contains the active sites of the molecule.
Light chains and heavy chains are linked together in the
whole immunoglobulin molecule by means of disulphide
linkages.
Type Subunit Mol wt Carbohydrate Serum
composition content level
(%) mg/100 ml
IgG
2
k
2
,
2
2
153,00 3 0.81.6
IgA (
2
k
2
)
n
, n(
2
2
) 180,000- 5-10 0.2-0.4
n = 1 to 4 5000,000
lgM (
2
k
2
)
n
, (
2
2
)
n
900,000 10-10 0.2-0.5
n = 5,6
Each heavy chain has four interchain disulfide bonds; two
between the pair of -chains in the monomer one to the light
chain, and one intersusunit bridge between the monomers.
The light chains are denoted by smaller lines and may be
of the or type.
The solid circles attached to the heavy chains of one of
the monomers represent complex oligosaccharides.
PORPHINS
Porphins are cyclic compounds formed by the linking of four
pyrrole rings through methane bridges (CH=).
Hemoglobin
CHAPTER
5
The four pyrrole rings are labeled as I, II, III and IV and
the bridges as , , and .
Substituents on the rings are labeled as 1, 2, 3, 4, 5, 6, 7,
and 8. Porphins have hydrogens at all 8 substituent positions.
In short, the molecule can be represented as:
HEMOGLOBIN 103
PORPHYRINS
Substituted porphins are called porphyrins.
Porphyrins are of two types, i.e. type I and type III.
A porphyrin with completely symmetrical arrangements
of substituents is called type I porphyrins whereas if the
arrangement of substituents is not symmetric then it is called
type III porphyrins.
In nature both type I and type IlI porphyrins are found
but type III porphyrins are more abundant.
Porphyrins are colored compounds and show characteristic
absorption spectra in both UV and visible regions.
Some of the important porphyrins are:
Porphyrins Nature of the substituents at the
following positions
1,2 3,4 5,6 7,8

1. Mesoporphyrin ME ME MP PM
2. Uroporphyrin AP AP AP PA
3. Coproporphyrin MP MP MP PM
4. Protoporphyrin MV MV MP PM
where M = Methyl group (CH
3
)
E = Ethyl group (C
2
H
5
)
A = Acetate group (CH
2
COOH)
P = Propionate group (CH
2
CH
2
COOH)
V = Vinyl group (CH = CH
2
)
Porphyrins can form complexes with metal ions. This
property is very important in their functioning in biological
system.
Examples: Heme is iron porphyrin, chlorophyll is a
magnesium porphyrin, Vitamin B
12
is a cobalt porphyrin.
HEMOGLOBIN
The red coloring material of blood is because of hemoglobin.
It is present in RBC. Hb is globular in shape.
Hemoglobin belongs to class of conjugated proteins
whereas heme is the prosthetic group and globin, the protein
part:
Hemoglobin = Heme + Globin
Normal adult blood contains 97% HbA
1
, 2% HbA
2
and
1% HbF. Both alpha and beta chains have 75 percent alpha
helical structure. The -chains has 7 and -chains has 8 helical
structure.
Functions of Hemoglobin
1. In the transport of oxygen from lungs to the tissues and the
transport of carbon dioxide from tissues to the lungs. Hem-
oglobin forms a dissociable hemoglobin-oxygen complex.
Hb + O
2
Oxyhemoglobin
2. As buffers. The buffering action of hemoglobin is due to
the amino acid histidine present in the globin part of hemo-
globin. Histidine comprises 8 percent of the total amino acid
make up of the globin.
3. Hemoglobin is required for both carbon dioxide and oxygen
transport because these gases are only sparingly soluble
in water.
The presence of hemoglobin increases the oxygen trans-
porting capacity of a liter of blood from 5 to 250 ml of oxygen.
Hemoglobin plays a vital role in the transport of carbon dioxide
and hydrogen ion. Myoglobin which is located in muscles,
serves as a reserve supply of oxygen and also facilitates the
movement of oxygen within muscle.
Significance of 2,3-Diphosphoglycerate (2,3-DPG)
The stability of deoxy conformation is inceased by 2,3-
diphosphoglycerate in mammals. It binds electrostatically to
143rd histidine and 82th lysine in -chains of deoxy-Hb and
stabilizes T-conformation. During oxygenation 2,3-diphospho
glycerate is released and T form reverts to R-conformation.
Mountain sickness: When an unclemetized subject goes to higher
attitudes (Hill areas/mountains) than the level of 2,3-DPG
increases in the blood. This reduces the affinity of oxygen to
hemoglobin liberating more and more of oxygen to peripheral
tissues.
HEMOGLOBIN 105
Carbon Monoxide Poisoning
Carbon monoxide has the tendency to form coordination
compounds with metals, in particular with hemoglobin iron.
It combines with hemoglobin to form carboxyhemoglobin (Hb
CO). Hemoglobin in this form does not carry oxygen efficiently
since by competiting specifically and effectively with oxygen
for ferrous site0s of hemoglobin, CO can displace oxygen from
hemoglobin in arterial blood.
The affinity of hemoglobin for CO is approximately 210
times greater than for O
2
. In the lungs, hemoglobin combines
with O
2
to form oxyhemoglobin (HbO
2
) which is carried in
this blood stream. O
2
is released at the tissue capillary level.
Since there are four heme groups in hemoglobin which can
combine reversibly with 4CO or 4 O
2
molecule in any
combination. At the physiological pH and temperature, the
combination of CO with human hemoglobin is about 10 times
slower than O
2
. However, once formed the dissociation of
carboxyhemoglobin is 210 times slower than oxyhemoglobin,
which explains why the affinity of CO for hemoglobin is
210 times more than that of O
2
.
In lead, poisoning the RBC refer to as Howells Jolly bodies
and Cabot ring.
Heme
Ferrous protoporphyrin is called heme. Heme is a chelate of
ferrous iron with protoporphyrin. Heme is also called proto-
heme.
Synthesis of Heme
The starting materials of hemoglobin synthesis are glycine
and succinyl CoA.
Structure of Hemoglobin
In hemoglobin, iron is in ferrous form.
When hemoglobin is converted to oxyhemoglobin, one of
the linkage of iron with imidazole group of histidine in globin
is replaced by oxygen. In oxyhemoglobin, iron remains in the
ferrous form.
HEMOGLOBIN 107
About 85 percent of the heme thus formed is used for
hemoglobin synthesis. About 10% is used for myoglobin
synthesis and the remaining 5% for cytochromes and other
heme proteins.
There are about 250,000 hemoglobin molecules in a single
RBC.
Hemoglobin molecule contains 4 heme groups combined
with a globin molecule, i.e. 2 -chains and 2 -chains, i.e. globin
part and four heme groups as prosthetic groups (one with
each chain). The total molecular weight is 64, 540.
globin (ferroheme)
4
+ 4O
2
= globin (ferroheme-O
2
)
4
Deoxyhemoglobin Oxyhemoglobin
Each chain has one-heme group. One hemoglobin molecule
contains four heme groups as subunits.
HEMOGLOBIN 109
Globin
Globin contains 4 polypeptide chains. Two are -chains and
other two are -chains. These four chains are arranged in
tetrahedron configuration.
-chain contains 141 amino acids, whereas -chain contains
146 amino acids. In all there are 574 amino acids in the globin
molecule.
The globin moiety is formed from amino acid pool in
amount of 8 gm per day in the normal adult. Thus, about 14%
of the amino acids from the average daily protein intake are
used for globin formation.
Each -chain has 141 amino acids whereas -chain (also
gamma and delta chains) have 146 amino acids.
There are 38 histidine molecules in hemoglobin molecule.
The 58th residue in -chain is called distal histidine because
it is far away from the iron atom, whereas 87th residue in
alpha chain is called proximal histidine because it lies near to
iron atom. The - and -subunits of Hb are connected by
weak noncovalent bonds like vander Walls forces and
hydrogen bonds.
Each of the four polypeptide chains of hemoglobin has its
own heme prosthetic group and iron atom. Iron contained
in the heme is coordinately linked with each chain by 2
histidine residues at two imidazole nitrogens of histidine at
position 58 and 87 in -chains and 63 and 97 in -chain of
globin.
The structure of oxyhemoglobin is described as R (relaxed)
form and that of deoxyhemoglobin is T (tight) form. The T-
conformation of deoxy Hb is maintained by electrostatic forces
between carboxyl and amine groups.
Methemoglobin
Methemoglobin is a hemoglobin derivative in which iron is
in the ferric form. It is also called ferrihemoglobin. Methemo-
globin is dark brown in color.
Conversion of ferrous to ferric iron in hemoglobin destroys
its capacity to combine with oxygen and to transport oxygen.
Hence, methemoglobin is useless in the transport of oxygen.
Normally the conversion of hemoglobin to methemoglobin
takes place in the blood but reducing substances present in
red cells tend to prevent the accumulation of any appreciable
amount of methemoglobin. The amount of methemoglobin
present in blood is 0.3 g per 100 ml of blood.
Increased amount of methemoglobin in blood gives rise
to a condition called methemoglobinemia. It is caused by the
failure in the normal reconversion of methemoglobin to
hemoglobin or by production of methemoglobin by certain
drugs. The symptoms observed in methemoglobinemia are
cyanosis (blue skin) and dyspnea (labored breathing).
Hemoglobin Cooperativity
The oxygenation process of hemoglobin and myoglobin is very
peculiar. This can be understood in terms of a graph of
fractional saturation of hemoglobin and myoglobin molecules
plotted against the partial pressure of oxygen. As shown in
figure, myoglobin oxygenation curve is hyperbolic whereas
for hemoglobin it is sigmoidal. For myoglobin the half
saturation pressure is quite low which tells us that it is a better
oxygen storage molecule then oxygen carrier. The difference
in the oxygenation curves between hemoglobin and myoglobin
is related to their structural difference. In hemoglobin the
presence of four subunits alter the nature of the oxygenation
curve. As a consequence of the interplay between four
subunits the binding of oxygen is cooperative. The affinity
of a given heme for oxygen increases as the other heme in
the hemoglobin molecule are oxygenated. Consequently the
degree of saturation at first does not respond much to the
pressure, then begins to rise abruptly and finally the curve
levels off at high pressure. This phenomenon is called
HEMOGLOBIN 111
cooperative or allosteric effect. There is an advantage of the
sigmoidal curve.
The structure of hemoglobin differs in the oxygenated and
deoxygenated states. The quaternary structure of oxygenated
state is called the R state (for released), and the conformation
of the deoxygenated state is called the T state (for tense).
The ability of hemoglobin to bind oxygen decreases with
an increase in acidity protons make hemoglobin dump oxygen.
Oxygenation curves for hemoglobin and myoglobin
Hemoglobin Variants
Hemoglobin A
1
HbA
1
contains two -chains and two
2
-chains. HbA
1
constitute-
over 98% of the total hemoglobin of the normal adult hemo-
globin and is designated as
2
A
2
-A, or more simply
2
2
.
Hemoglobin A
2
HbA
2
contains two
2
-chains and two
2
-chains. HbA
2
constitute about 2 percent of the total hemoglobin in the normal
adult and is designated as
2
2
.
Hemoglobin F
Human fetal hemoglobin is designated as HbF and is repre-
sented as
2

2
.
HbF contains two -chains and two -chains. HbF is pre-
dominant form present at birth but is almost totally replaced
by HbA
1
within few months after birth.
Hemoglobin S (Sickle Cell Hemoglobin)
HbS contains two -chains and two -chains in which glutamic
acid at 6 position from the N-terminal end of the -chain is
replaced by valine. HbS is also called sickle hemoglobin due
to the fact the red cells assume the shape of sickle on
deoxygenation. HbS gives rise to sickle cell anemia.
Hemoglobin Gun Hill
It contains only two heme groups instead of four. Five amino
acids are missing from the -chain and this leads to the
interference with the heme binding.
In short hemoglobin variants are represented as:
Chains
HbA
1
2 2
HbA
2
2 2
HbF 2 2
HbS 2 2 glu val
at position 6
Myoglobin
Myoglobin is a single polypeptide chain. Human myoglobin
contains 152 amino acids with a molecular weight of 17,500.
The heme is attached to 92nd histidine residue. One molecule
of myoglobin can combine with one molecule of oxygen.
Myoglobin has higher affinity to oxygen than that of Hb.
Myoglobin has high oxygen affinity while Bohr effect,
cooperative effect and 2,3-diphosphoglycerate effect can
absent. The isoelectric point of myoglobin is 6.5.
Bohr Effect
The increase in acidity of hemoglobin as it binds oxygen is
known as Bohr effect; or Bohr effect is the increase in basicity
of hemoglobin as it releases oxygen.
HEMOGLOBIN 113
The effect is expressed by the equation.
HHb
+
+ O
2
HbO
2
+ H
+
The above equation indicates that increase in hydrogen
ion concentration will favour the formation of free oxygen
from hemoglobin and conversely that oxygenation of hemo-
globin will lower the pH of the solution. This reversible uptake
and release of protons is responsible for the isohydric transport
of carbon dioxide. The term isohydric refers to a lack of
change of pH in the process.
Breakdown of Hemoglobin
Bilirubin in combination with albumin reaches the liver,

where it undergoes conjugation to form bilirubin diglucuronide
which passes with the bile into the intestines. In the intestines,
bilirubin diglucuronide is hydrolyzed, bilirubin is converted
to urobilinogen. A portion of urobilinogen is absorbed from
the intestines into the blood and some of it is excreted in the
urine (4 mg/day). The remainder is re-excreted in the bile.
The unabsorbed urobilinogen is excreted in the stool as fecal
urobilinogen which is oxidized to urobilin.
PORPHYRIA
When the blood levels of coproporphyrins and uroporphyrins
are increased above normal level and excreted in urine or
faeces the condition is known as porphyria.
Additionally reduced catalase activity has been reported
in cases or porphyria.
Classification
Inherited Erythropoietic Porphyria
It is rare inherited disorder and is due to autosomal recessive
pattern. Preponderance of type and prophyrias, both uropor-
phyrin type and coproporphyrin type. This is due to increased
deaminase activity with isomerase deficiency. Affected indi-
viduals exhibit abnormal sensitivity to lightphoto sensitivity
and develop skin lesion. Urine is usually red colored.
Explanation: As uroporphyrinogen III is less formed or absent,
heme formation surffers. Relative deficiency of heme produces
induction of . ALA synthetase leading to massive production
of type I.
HEMOGLOBIN 115
Hepatic Porphyria
In this, there occurs abnormal and excessive production of
prophyrins (chiefly type III), their precursors ALA and
porphobilinogen. There is three types of hepatic porphyrias.
1. Acute intermittent porphyria or paroxysmal porphyria:
It is autosomal dominant partial deficiency of uroporphy-
rinogen and synthetase. Patients present with acute attacks of
abdominal pain, nausea and vomiting, constipation, CV
abnormalities and neuropsychiatric signs. This is due to
increased production of porphyrinogen and d-ALA. The
patients do not have photosensitivity. Freshly passed urine is
often normal in color but on standing in sunlight turns to red
urine color. Both colorless compounds porphobilinogen and
d-ALA in sunlight. Polymerases to form two colored red
compounds porphobilin and porphyrin.
Note: Drugs and steroids requiring cyt P-450 can precipitate
acute case. Reason is excessive utilisation of cyst P-450 for
which heme is utilised. This decrease in heme is associated
with depression of -ALA synthetase.
2. Porphyria cutanea tarda: It is autosomal dominant: This
is due to partial deficiency of uroporphyrinogen decarboxylase
and patients are characterized by photosensitivity. Urine
contains increased quantities of uroporphyrins and
coproporphyrins of both types and also elevated urinary
excretion of d-ALA and PBG occurs and is associated with
use in serum iron.
3. Varicyate porphyria or mixed (combined) porphyria: In
this neurological as well as cutaneous symptoms are seen. This
is autosomal dominant. There is deficiency of portopor-
phyrinogen oxidase and ferrochelatase. Clinically there is
vomiting, acute attacks of abdominal pain and neuropsychiatric
signs and cutaneous photosensitivity.
Bilirubin is of two types:
1. Direct bilirubin: Direct bilirubin is bilirubin diglucuronide.
It is water soluble. It is expressed as conjugated bilirubin
because it can be coupled readily with Diazo Reagent
(diazotized sulphanilic acid). This is the direct van den
Bergh reaction.
2. Indirect bilirubin: Albumin bound bilirubin is called indirect
bilirubin. It is water insoluble. It is expressed as unconju-
gated bilirubin as it will not react until it is released by
the addition of alcohol. The reaction with Diazo reagent
after the addition of alcohol is called the indirect van den
Bergh reaction.
Normal serum bilirubin level is 0.2-0.6 mg %.
Jaundice
Jaundice is due to increase in the concentration of bilirubin
in the blood which imparts yellow color to the skin and
conjunctive. Jaundice may be either due to over production
of bilirubin than what the liver can normally excrete or a
damage in liver, fails to excrete bilirubin in normal amounts.
Jaundice is of three types:
Hemolytic or Pre-hepatic Jaundice
In hemolytic jaundice, there is an increased breakdown of
hemoglobin, the liver cells are unable to conjugate all the
increased bilirubin formed. Increased production of bilirubin
leads to increased production of urobilinogen which appears
in urine in large amounts. Bilirubin will be absent in urine.
Hepatocellular or Hepatic Jaundice
This type of jaundice results from liver damage which cannot
conjugate bilirubin. The indirect serum bilirubin level will be
high. Urine will show the presence of bilirubin and increased
amount of urobilinogen. Stool is light in color.
HEMOGLOBIN 117
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HEMOGLOBIN 119
Obstructive or Post-hepatic Jaundice
This type of jaundice results from the obstruction of common
bile duct. As a result of obstruction, bilirubin does not pass
into the intestine, so no urobilinogen is found in the urine.
Direct serum bilirubin level will be high, urine will show the
presence of bilirubin. Stool is clay colored.
Physiological Jaundice or Neonatal Jaundice
Usually mild form of jaundice appears in some newborn
children on the 2nd and 3rd day of life called neonatal jaundice.
Causes
1. Excessive destruction of RBCs after birth causing increased
in serum bilirubin.
2. Due to hepatic immaturity
During IU life, bilirubin formed is mainly eliminiated by
placenta immediately after birth where has to eliminate all
the bilirubin but it is unable to deal adquately during first
10 days.
Note:
1. In infants, when serum bilirubin rises beyond 5% clinical
jaundice appears.
2. Jaundice is more common and more severe is premature
babies.
Phototherapy
Exposure of skin to white light converts bilirubin to a com-
pound which has shorter life than bilirubin called lumirubin.
Phototherapy is used to treat infants with hemolysis.
ENZYMES
Enzymes are biological catalysts which bring about chemical
reaction in living cells. They are produced by the living
organism and are usually present in only very small amounts
in various cells. They can also exhibit their activity when they
have been extracted from the source. Enzymes are all organic
compounds and a number of them have been obtained in
crystalline form.
General properties of enzymes are:
1. All enzymes are proteins with exception of ribosomes.
2. Enzymes accelerate the rate of reaction by:
a. Not altering the reaction equilibrium
b. Being required in a very small amount
c. By being not consumed in the overall reaction.
3. They have the enormous power for catalysis.
4. Enzymes are highly specific for their substrate.
5. Enzymes possess active sites at which interaction with
substrate takes place.
6. Enzymes catalysis involves the transformation of enzyme-
substrate complex as an important intermediate in their
action.
7. Enzymes lower the activation energy.
8. Some enzymes are regulatory in function.
Some enzymes are purely protein in nature and depend
for activity only on their structure while certain enzymes
require for their function one or more nonprotein component.
They are termed as coenzymes, cofactors or prosthetic groups.
If such a compound is firmly attached to enzyme proteins then
Enzymes
CHAPTER
6
ENZYMES 121
it is called a prosthetic group. If its attachment to protein is
not very firm then it is called coenzyme. Certain coenzymes
exist in free state in solution and contact enzyme protein only
at the times of reaction.
The term apoenzyme refers to the protein part of the
enzyme.
The apoenzyme in combination with its prosthetic group (or
coenzyme) constitute a complete enzyme or holoenzyme
system.
Holoenzyme = Apoenzyme + Coenzyme
= Protein part + Nonprotein part
Coenzymes
Many enzymes in order to perform their catalytic activity
require the presence of small nonprotein molecules. Coen-
zymes are low molecular weight, organic compounds, non-
protein, thermostable and can be separated by dialysis.
Characteristics of Coenzymes
1. They are stable towards heat.
2. Generally derived from vitamins.
3. Function as cosubstrates.
4. They participates in:
a. Hydride (H) and electron transfer reactions, e.g.
NAD
+
, NADH, FMN, FAD, etc.
b. Group transfer reactions, e.g. CoA, TPP, pyridoxal phos-
phate, tetrahydrofolic acid, etc.
Coenzymes Functions performed
NAD
+
, NADP
+
Hydrogen transfer
FAD, FMN Hydrogen transfer
Thiamine pyrophosphate Acetyl group transfer
Pyridoxal phosphate Amino group transfer
Biotin Carboxyl group transfer
Coenzyme A Acyl group transfer
Most of the coenzymes are the members of water soluble
B-complex group of vitamins. Coenzymes function as the
intermediate carrier of functional groups of specific atoms or
of electrons that are transferred in the overall enzymatic
reactions.
Classification of Enzymes
According to the International Union of Biochemist, the
enzymes are classified into six major classes.
1. Oxidoreductases: They catalyze oxidation and reduction
reactions. These enzymes are divided into three groups.
a. Oxidases: Those which use oxygen as hydrogen acceptor,
e.g. tyrosinase, uricase.
b. Anaerobic dehydrogenases: Those which use some other
substances as hydrogen acceptor, e.g. lactic dehydro-
genase, malic dehydrogenase.
c. Hydroperoxidases: Those which use hydrogen peroxide
as substrate, e.g. catalase, peroxidase.
2. Transferases: They catalyze the transfer of some group from
one molecule to another molecule. These enzymes are
important in biological synthesis, e.g. transaminases, hexo-
kinases, transacylase, transaldolase, ketolase, phosphomu-
tases.
3. Hydrolases: They catalyze the hydrolysis of substrate by
addition of water molecule across the bond which is split,
e.g. esterases, peptidases, phosphatases, deamidases.
4. Lyases: They catalyze the addition or removal of groups
from the substrate without hydrolysis, oxidation or reduc-
tion, e.g. decarboxylases, carboxylase, carbonic anhydrase,
aldolase, enolase, etc.
5. Isomerases: They catalyze the conversion of a compound into
an isomer, e.g. racemases, epimerases, isomerases, mutases.
6. Ligases: They catalyze the linking together of molecules
coupled with the breaking of pyrophosphate bound in ATP,
e.g. glutamine synthetase, succinic thiokinases.
Enzyme Specificity
Enzyme specificity is determined by how well the reactant
fit into the enzyme surface. Some enzymes are very specific
and show activity with only one substrate. However, some
other enzymes are much less particular and will catalyze
reaction with similar compounds.
ENZYMES 123
Generally two types of enzymatic specificities are observed
in different reactions.
Zymogens: Several proteins are synthesized in inactive forms.
These are called zymogens eq. proteins digesting enzymes and
blood clotting proteins. To activate zymogens, a small amount
of protein is cleared from one end. This causes the protein to
change shape and activate it. These changes are not reversible.
Stereospecificity
Some enzymes show specificities only with a specific group
of a substrate, e.g. Urease catalyzes the hydrolysis of urea.
Alteration in the structure of urea results in the loss of
activity. For example, N-methyl urea and thiourea are not the
substrate for enzyme urease.
Also some enzymes show specificity towards D- and L-
form of the same substrate, e.g. D-amino acid oxidase acts
only on the D-form of amino acid and not on L-form.
Substrate Specificity
Some enzymes catalyzes similar type of reactions but differ
in their action due to absolute substrate specificity, e.g. Pepsin
hydrolyzes peptide bond involving amino group of aromatic
amino acids as phenylalanine or tyrosine.
Similarly trypsin hydrolyzes peptide bond involving the
carboxyl group of basic amino acids such as lysine or
arginine.
FACTORS INFLUENCING THE RATE OF
ENZYMATIC REACTIONS
Effect of Substrate Concentration
At a low substrate concentration, the initial velocity of an
enzyme catalyzed reaction is proportional to the substrate
concentration. However, as the substrate concentration is
increased, the initial velocity increases less as it is no longer
proportional to the substrate concentration. With a further
increase in the substrate concentration the reaction rate becomes
independent of the substrate concentration and assumes a
constant rate as a result of enzyme being saturated with its
substrate.
It was Michaelis and Menten who suggested an explanation
of these findings by postulating that at low substrate concen-
trations, the enzyme is not saturated with the substrate and
the reaction is not proceeding at maximum velocity whereas
when the enzyme is saturated with substrate, maximum
velocity is observed. They further visualized the combination
of enzyme with the substrate to form an enzyme-substrate
complex and assumed that the rate of decomposition of the
substrate being proportional to the concentration of enzyme-
substrate complex. The velocity of the reaction at this high
ENZYMES 125
substrate concentration is termed as maximum velocity. The
substrate concentration at which the velocity is half of the
maximum velocity is called the Michaelis constant and is termed
as K
m
.
K
m
indicates the affinity of the substrate towards the
enzyme and is inversely proportional to the affinity.
m
1
K
Affinity
Higher the affinity the smaller will be the K

m
and lower
the affinity, the higher will be the K
m
.
The Michaelis-Menten equation is given by the expression
V
0
=
max
m
V [S]
K [S] +
where V
0
= Initial velocity
V
max
= Maximum velocity
K
m
= Michaelis constant
[S] = Substrate concentration
The Michaelis-Menten equation relates the initial velocity,
the maximum velocity and the initial substrate concentration
through Michaelis-Menten constant.
When the initial velocity is exactly half of the maximum
velocity the Michaelis-Menten equation assumes the form
max
1
V
2

=
max
m
V [S]
K [S] +
K
m
+ [S] = 2 [S]
i.e. K
m
= [S]
Thus Michaelis-Menten constant is equal to the substrate
concentration at which the initial velocity is half of the
maximum velocity.
Determination of important physical constants of an enzyme
such as V and K
m
would be difficult from the curve that would
be obtained by plotting [V] against [S]. So the Michaelis-
Menten equation can be transformed into the form which is
useful in plotting experimental data.
Taking the reciprocals of both the sides of Michaelis-Menten
equation.
0
1
V
=
m
max
K [S]
V [S]
+
or
0
1
V
=
m
max max
K [S]
V [S] V [S]
+
0
1
V
=
m
max max
K 1
V [S] V
+
This equation is called Line-weaver Burk equation and is the
equation for a straight line y = mx + c, where m is the slope
of the straight line, c is the intercept on the y-axis and x is
the intercept on x-axis.
When
1
[ ]
0
V
is plotted against
1
[ ] S '
a straight line is obtained,
the slope of which is
max
m
K
V
and has an intercept of
max
1
V
on
the
1
[ ]
0
V
axis and intercept of
1
m
K
on the
1
[ ] S
axis.
ENZYMES 127
Since, Line-weaver-Burk equation is in the form of a
straight line, so it requires few points to define, K
m
. By using
small concentrations of substrate it is possible by this double
reciprocal plot to determine K
m
.
Significance of K
m
and V
max
Values
The Michaelis constant [K
m
] has two meanings:
One is that it is equal to that substrate concentration at
which half of the active sites are filled and so once the K
m
is shown, the fractions of sites filled (fs) at any substrate
concentration can be calculated by:
fs =
max
V
V
=
m
[S]
[S] K +
Second, K
m
is related to rate constant of the individual
steps
K
m
=
1 2
1
K K
K
+
Now, when the K
1
is much more than K
2
, the K
2
becomes
negligible and K
m
is then equal to
1
1
K
K
, which is the disso-
ciation constant of the ES complex, a reversible reaction, i.e.
1
1
K
K
E S ES
+
R
1
= K
1
[E][S]
R
2
= K
1
[ES]
when R
1
= R
2
(at equilibrium)
K
1
[E] [S] = K
1
[ES]
or
[E] [S]
[ES]
=
1
1
K
K
= KSE
(the equilibrium constant of ES)
K
m
=
1
1
K
K

So when this condition is met, K
m
indicates the strength
of ES complex and at such conditions a high K
m
indicates weak
binding and a low K
m
indicates strong binding. But this is
true only when the K
2
is much less then K
-1
.
V
max
V
max
indicates the turn over number of the enzyme if the con-
centration of active sites, i.e. the total enzyme (E
t
) is known
since V
max
= K
2
[E
t
].
Here, in this relation K
2
is called the turn over number
of an enzyme which is defined as number of substrate
molecules converted into product per unit time when the
enzyme is fully saturated with the substrate and the time
required for each round of catalysis is thus given by 1/K
2
.
Method of Determining K
m
K
m
can be determined by double reciprocal Line-weaver-Burk
method. In this the velocity of reaction is noted with different
1
[ ] S
and
1
[ ] V
from the graph, the value of K
m
is determined.
Another advantage of this equation is that it is used to
differentiate certain type of inhibitors of enzyme system.
Effect of Enzyme Concentration
The rate of an enzyme catalyzed reaction is directly pro-
portional to the concentration of the enzyme. The greater the
concentration of enzyme, the faster will be reaction taking
place.
Effect of pH
Most enzymes have a characteristic pH at which their activity
is maximum. Above or below that pH, the enzyme activity
decreases. If a curve is drawn between the activity of an en-
zyme on a given substrate with the pH of the reaction mixture,
it will reveal a maximum activity at a definite pH. This value
is known as optimum pH. See Diagram on Page No. 129.
ENZYMES 129
This is probably due to the changes in the net charge on enzy-
mes, (as they are protein in nature) resulting from changes in
pH. Excessive changes of pH brought on by the addition of
strong acids or bases may completely denature and inactivate
enzymes.
Effect of Temperature
The rate of an enzyme catalyzed reaction generally increases
with temperature, within the temperature range in which the
enzyme is stable and retains its full or maximum activity.
Enzyme catalyzed reactions have an optimum temperature at
which the reaction is most rapid.
Above this temperature the reaction rate decreases as
enzymes being protein in nature are denatured by heat and
becomes inactive.
The increase in rate below optimal temperature results from
increased kinetic energy of the reacting molecules.
ENZYME ACTIVITY
Activity: Amout of substrante converted to products by the
enzyme per unit time (e.g. micromoles/minutes)
Specific activity: Activity per quality of protein (e.g. micromoles/
minute/mg protein)
Catalytic constant: Proportionality constant between the reaction
velocity and the concentration of enzyme catalyzing the reaction.
Unit: Activity/mole enzyme.
Turnover number: Catalytic constant/number of active sites/
mole enzyme.
International unit (IU): Quality of enzyme needed to transform
1.0 micromole of the substrite to product per minute at 30C
of optimal pH.
The activity of an enzyme is expressed in standard units
U = the amount of activity of an enzyme which catalyzes the
transformation of one micromole of substrate per minute. The
specific activity of an enzyme is the number of units of enzyme
activity per mg of protein. The reason for needing this is that
often the enzyme is not pure and there is contamination protein
in the sample.
The catalytic constant is units of enzyme activity per mol
of protein (mmol/min/mol enzyme).
Katab (kat) are the conversion of 1 mol/sec (International
units).
Turnover Number
The number of molecules of substrate converted to products
per enzyme molecule per minute is called turnover number.
ENZYME INHIBITIONS
Since, enzymes are proteins, any agent which denatures
proteins will inactivate the enzyme. Inhibitors are the
substances which lower down the rate of enzyme reactions.
They exert their effect by acting on the apoenzyme, coenzyme,
prosthetic group or activator present in the enzyme system
or by interfering with the binding of the substrate to the
enzyme. Reversible inhibitors bind the enzymes through non-
covalent bonds and dilution of the enzyme-inhibitor complex
results in dissociation of the reversibly bound inhibitor where
as irreversible inhibitors occurs when an inhibited enzyme
does not regain activity on dilution of the enzyme-inhibitor
complex.
Substances that inhibit enzymatic reactions are classified
into three groups:
1. Competitive inhibition
2. Noncompetitive inhibition
3. Uncompetitive inhibition.
ENZYMES 131
This classification depends upon the manner of combination
of the inhibitor with the enzyme.
Competitive Inhibition
As the name implies, the competition is between normal
substrate and the inhibitor molecules for binding at the active
site of the enzyme to form enzyme-substrate or enzyme
inhibitor complex. As a result of structural similarity between
the substrate molecules and inhibitor molecules, they compete
both for active sites of the enzyme molecule and tie up to
the active sites. These sites are then not available to the normal
substrate molecules. The overall rate of inhibition is governed
by the affinities of inhibitor molecules and normal substrate
molecules for the enzyme binding site and by the concentra-
tions of the reactants.
The presence of competitive inhibitor thus increases the
apparent K
m
of the enzyme for the substrate, i.e. causes it
to require a higher substrate concentration to achieve the
maximum velocity. On the other hand, a competitive inhibitor
does not affect the V
max
indicating that it does not interfere
with the rate of breakdown of enzyme substrate complex.
Competitive inhibitors are frequently called antagonists
or antimetabolites of the substrate with which they compete.
The example of competitive inhibitions are:
i. The inhibition of enzyme succinate dehydrogenase by
malonate for succinate.
Both have similar structural resemblance and hence, both
compete for the active site of enzyme succinate dehydrogenase.
ii. Sulphanilamide has structural resemblance with para-
minobenzoic acid and blocks the folic acid synthesis which
results in the deficiency of the vitamin to microorganisms.
ENZYMES 133
In case of competitive inhibition.
Affinity Decreases
Efficiency Remains same
1/K
m
Decreases as K
m
increases
1/V
max
Remains same
Noncompetitive Inhibition
As the name implies there is no competition between the sub
strate and the inhibitor molecules. There is little or no
structural resemblance between the substrate and the inhibitor
molecules and hence they bind to the different sites of the
enzyme. Inhibitors combine with the allosteric site of the
enzyme, this combination results in the distortion of the active
site. In noncompetitive inhibition, the affinity of enzyme
remains same but its efficiency decreases. This inhibition is
also known as allosteric inhibition. The inhibitory action cannot
be overcome by increasing the substrate concentration. The
complex formation between the inhibitor and enzyme is
reversible. Noncompetitive inhibitors lowers the V
max
but does
not effect the K
m
.
Examples of noncompetitive inhibitions are:
There are many enzymes which require free sulphydryl
group (i.e.SH group) for activity, are noncompetitively inhi-
bited by heavy metal ions such as Pb
++
, Hg
++
, etc. Urease is
an example of an enzyme which experiences heavy metal
inhibition. The action of nerve gas poisons on acetylcho-
linesterase, is an example of noncompetitive inhibition.
In case of noncompetitive inhibition.
Affinity Remains same
Efficiency Decreases
1/K
m
Remains same because the substrate
concentration has no effect on the
inhibitory action.
1/V
max
Increases as V has decreased.
Uncompetitive Inhibition
These inhibitors combine only with the enzyme-substrate
forming an irreversible complex. The inhibition is dependent
only on the concentration of the inhibitor.
In case of uncompetitive inhibition
V
max
is lower
Slope is same
Apparent K
m
< K
m
CATALYTIC SITE OR THE ACTIVE SITES
OF THE ENZYMES
The portion of the enzyme protein molecule which actually
takes part in catalysis is called active site or the catalytic site
of the enzyme. Although the enzymes differ widely in
structure specifically and catalysis, there are certain common
features about the active sites.
ENZYMES 135
1. Normally the active sites makes up a small volume of the
total portion of an enzyme.
2. The active site is a three dimensional activity.
3. It is made up of groups that come from the different parts
of the linear amino acid chain. Indeed the residues are far
a part in the linear sequence but may come together to
bring about catalysis.
4. The specificity of the substrate binding depends upon the
precisely defined arrangement of the atoms or groups at
the active site.
Emil Fisher postulated that substrate and enzyme reacted
in a well defined clear cut lock-key fashion signifying the
predominated structure of the active fit complementary to
the substrate molecule structure with which it will bind. This
model implied therefore the rigidity of the catalytic site. But
this hypothesis was soon found unable to explain the possibility
of such a catalytic site reacting with the product to reform
substrate in a reversible manner. Then Koshland proposed
a more flexible hypothesis called induced fit model regarding
the structure of the active site. According to this hypothesis,
enzymes in the inactive state in the absence of substrate and
that various groups in the active site are not correctly oriented
to interact with the complimentary groups on the substrate.
Binding of the specific substrate however, results in a confor-
mational change in the enzyme and thus to the active site and
shifting of those groups or atoms in the site into the correct
position for proper binding with the substrate and catalysis.
Feedback inhibition: In many multienzyme systems, the end
product of the reaction sequence may act as a specific inhibitor
of an enzyme at or near the beginning of the sequence, with
the result that the rate of entire sequence of reactions is deter-
mined by the steady state concentration of the end product.
This type of inhibition is called feed back inhibition.
For example, cholesterol synthesis is regulated, by feed-
back inhibition.
ENZYME INDUCTION
Enzymes are classified according to the condition under
which they are present in a cell. They are of two types.
a. Constitutive enzymes.
b. Inducible enzymes.
Constitutive Enzymes
These enzymes are formed at constant rates and in constant
amounts. Their presence in a cell is not related to the presence
or absence of their substrates. They are considered to be part
of the permanent enzymatic make of the cell.
For example, enzymes of glycolytic pathway.
Inducible Enzymes
Also called adaptive enzymes. They are always present in trace
amounts but their concentrations vary in proportions of their
substrates.
Isoenzymes
They are multiple forms of a given enzyme having different
mobilities on electrophoresis, differently depressed by inhi-
bitors towards different substrates. Isoenzymes catalyze the
same reaction but differ in K
m
, V
max
or both. The relative
amounts of the isoenzymes of a particular enzyme differ in
different organs so that in disease, different isoenzyme
patterns are found according to the organs from which they
have come. These forms are the characteristics of different
organs and tissues of the human body.
Example
1. Lactate dehydrogenase (LDH): This enzyme catalyzes the
dehydrogenation of lactate to pyruvate. This occurs in five
different isoenzymes.
This enzyme is a tetramer having two types of units,
i.e. L and M units. Depending upon the various combi-
nation, five isoenzymes are known, i.e. thest two subunits
can combine in five different ways.
Test Composition Location
LD-1 HHHH Heart and RBC
LD-2 HHHM Heart and RBC
LD-3 HHMM Brain and kidney
LD-4 HMMM
LD-5 MMMM Liver and skeletal muscle
ENZYMES 137
LD-1 is the predominant form in heart and LD-5 in muscles.
LDH is elevated from 12 to 48 hours after initial attack.
2. Alkaline phosphatase: It occurs in two forms.
3. Isocitrate dehydrogenase: It occurs in two forms.
4. Creatine phosphokinase: It occurs in three forms.
CPK is a dimer consisting of one subunit found in the brain
(B) and other in muscle (M). CPK is found in three isoenzymes,
as CPK
1
(BB), CPK
2
(MB) and CPK
3
(MM).
In normal serum 95% of the CPK activity is in CPK
3
.
CPK is, found in three isoenzymes as:
i. CPK (MM), largely found in skeletal muscle tissue.
ii. CPK (BB), predominately found in brain tissue.
iii. CPK (MB), exclusively found in heart tissue.
Blood level of both CPK (total) and CPK (MB) usually
markedly increases following acute myocardial infarction. Only
CPK (MB) elevation is highly specific for the diagnosis of MI.
CPK (MM) increases rapidly following exercise or muscle
trauma.
CPK (BB) is heat labile and rarely detected in serum.
The activitiy of CPK
2
is the cornerstone for the diagnosis
of myocardial infarction because of its abundance in heart and
absence from other cells. It may be elevated after 4 hours and
its activity may increase from two to ten folds after 16-24
weeks.
DIAGNOSTIC VALUE OF PLASMA ENZYMES
A determination of enzyme levels in the serum is often helpful
in pinpointing which, if any, body tissue or organ has been
damaged or malfunctioning. When a tissue is injured some
cells of that tissue are destroyed and their contents, enzymes
included, are released into the blood stream. Therefore, if
an enzyme is normally found predominately in a tissue other
than blood, an increase in its level in the blood indicates that
tissue has been damaged.
Serum acid phosphatase is increased in Pagets disease of
bone, hyperparathyroidism, metastases of bone, Gauchers dis-
eases, chronic renal failure and prostatic carcinoma.
Serum alkaline phosphatase is increased in rickets, hyper-
parathyroidism, obstructive jaundice, osteomalacia.
Serum glutamate oxaloacetate transaminase SGOT/AST
(Aspartate transaminase) is increased in myocardial infarction
and skeletal muscle dystrophies.
Serum glutamate pyruvate transaminase SGPT/ALT (Ala-
nine transaminase) is increased in viral hepatitis, toxic hepatitis
and other forms of liver diseases associated with some degree
of hepatic necrosis.
Typical profiles of serum enzymes following a myocardial
infarction.
SGOT catalyzes the reversible transfer of the amino group
from glutamate to oxaloacetate to form -ketoglutarate and
aspartate. GOT is released from many diseased cells into serum
as SGOT. SGOT is elevated in liver disease and following a
myocardial infarction. The serum level has diagnostic value.
It can be moderately elevated (5-fold) in people with cirrhosis
and obstructive liver disease (a stone blocking bile duct). It
can become very high (25-fold) in viral hepatitis.
Serum lactate dehydrogenase (LDH) is increased in myocar-
dial infarction, acute liver disease, pernicious anemia, progres-
sive muscular dystrophies.
Serum creatine phosphokinase (CPK) is increased in muscu-
lar dystrophy, myocardial infarction.
Serum amylase is increased in various forms of pancreatic
disturbances (Pancreatitis).
Serum isocitrate dehydrogenase is increased in liver
diseases, severe pulmonary infarction.
Serum lipase is increased in acute pancreatitis and carci-
noma.
Enzyme Evidence of rise
Creatine kinase 3-6 hr
(CK-MB)
Aspartate transaminase 6-8 hr
(AST)
Lactate dehydrogenase 12 hr
(LDH)
ENZYMES 139
Typical profiles of serum enzymes following a myocardial
infarction.
BIOLOGICAL OXIDATION
All body reactions require energy which is obtained from
chemical reactions carried out in the living cells. The stepwise
oxidation of various metabolites is the principal mechanism
for the liberation of energy. The utilization of oxygen and
production of carbon dioxide by the tissues in the process
of cellular respiration is the final phase of biological oxidation.
Transfer of electrons are involved in all oxidation-reduction
reactions. Every oxidation must be accompanied by simulta-
neous reduction and the energy required for the removal of
electrons in oxidation is supplied by the reduction.
The electron transport is important for the following reasons:
1. It explains how oxygen finally enter the metabolism.
2. It provides the mechanism for the regeneration of oxida-
tion-reduction coenzymes.
3. It provides the majority of the energy derived from
metabolic processes.
The energy transfers involved in the oxidation-reduction
systems are measured by difference in potential of various
systems.
Oxygen has the highest oxidation potential of the systems
in the living cells and hydrogen atom the lowest.
Biological oxidation is catalyzed by enzymes which func-
tions in combination with coenzymes or electron carriers.
Oxidases
These enzymes catalyze the removal of hydrogen from the
substrate directly to the molecular oxygen, e.g. cytochrome
a
3
(cytochrome oxidase), tyrosinase, uricase, etc.
2H + O
2
H
2
O
Biological Oxidation
CHAPTER
7
BIOLOGICAL OXIDATION 141
Dehydrogenases
They are further divided into:
a. Aerobic dehydrogenases
b. Anaerobic dehydrogenases.
a. Aerobic dehydrogenases: These enzymes remove hydrogen
from the substrate using either O
2
or artificial substance as
hydrogen acceptor. These dehydrogenases are flavoproteins,
e.g. xanthine oxidase, D-amino acid oxidase, catalase, peroxi-
dases.
b. Anaerobic dehydrogenases: These enzymes use substances other
than oxygen as hydrogen acceptor.
These dehydrogenases are classified as:
i. Pyridine nucleotides: Under this group comes nicotinamide
adenine dinucleotide (NAD
+
) and nicotinamide adenine
dinucleotide phosphate (NADP
+
). The effective part
which participates in the reaction is nicotinamide.
ii. Flavonucleotides: They are flavin mono nucleotide (FMN)
and flavin adenine dinucleotide (FAD). The effective part
which participates in the reaction is riboflavin.
FAD + 2H
+
+ 2 e FADH
2
+
iii. Cytochromes: The cytochromes are iron containing hemo-
proteins in which iron oscillates between Fe
++
and Fe
+++
during oxidation-reduction.
Various cytochromes are cytochromes b, c
1
, c, a.
Oxygenases
The enzymes incorporate oxygen into the substrate molecule.
They are divided into two groups:
a. Dioxygenases: These enzymes catalyze the incorporation of
both atoms of oxygen into the substrate.
A + O
2
AO
2
For example, tryptophan dioxygenase and homogentisic
acid dioxygenase.
b. Monoxygenases: They add only one atom of oxygen into the
substrate.
MIXED FUNCTION OXIDASES
These oxidases cause reduction of one atom of O
2
and utili-
zation of other atom for specific oxygenation of hydroxylation
of the substrate.
Two catalytic functions are performed by these enzymes:
i. Reduction of an atom of oxygen to O
ii. Transfer of oxygen to the substrate.
These enzymes not only require O
2
but also a source of
electrons, i.e. reducing agent to reduce an atom of O
2
to O
Mixed function oxidases are metalloproteins with prosthetic
group containing Fe, Cu.
Examples of mixed function oxidases are:
i. Phenylalanine hydroxylase
ii. p-Hydroxy phenylpyruvate oxidase
iii. Imidazole acetic acid oxidase
iv. Phenolase complex (this enzyme is involved in the
formation of melanins from tyrosine).
Hydroperoxidases or Peroxidases
These peroxidases catalyze the transfer of electrons from
donars (substrates) to H
2
O
2
, reducing it to water. The peroxi-
dases are specific in requiring H
2
O
2
as electron acceptor (oxi-
dizing agent) but various substrates may act as substrates or
electron donars.
Catalases
Catalases are specific type of hemin-containing hydroperoxi-
dases which have the property of rapidly catalyzing the
decomposition of H
2
O
2
.
2H
2
O
2
2H
2
O + O
2
Catalase enzymes are simply a specific type of peroxidase
enzymes possessing very high activity toward H
2
O
2
as a sub-
strate but also capable of catalysing regular peroxidate
reactions.
Superoxide anion O
2
is highly reactive. It is generated
by a number of biological reactions including the antioxidation
of quinones, thiols and reactions catalyzed by xanthine oxidase
and flavoprotein dehydrogenases.
Superoxides are very toxic to the cells and consequently
the enzyme superoxide dismutase, present in all the cells, is
responsible for protecting the cell from the harmful effect of
superoxide anion, the cell in turn is protected from H
2
O
2
by
catalase
Superoxide dismutase
2H
+
+ 2O
2
H
2
O
2
+ O
2
Catalase
2H
2
O
2
2H
2
O + O
2
HIGH ENERGY COMPOUNDS
High energy compounds are:
1. Acid phosphates: They are acid anhydrides of an organic
acid (RCOOH) and phosphoric acid. Their general formula
is:
For example 1, 3, diphosphoglyceric acid, acetyl phosphate.
2. Enol phosphates: For example, 2-phosphoenol pyruvic acid.
3. Guanidinophosphates: Two important compounds in this are
creatine phosphate in vertebrate and arginine phosphate
in invertebrate.
4. Organic pyrophosphates: These compounds are ADP and ATP,
GTP, IDP, etc.
5. Coenzyme A: Derivatives of coenzyme A are high energy
compounds.
6. Methionione: As S-adenosyl methionine.
RESPIRATORY CHAIN
Transfer of electrons from substrate to molecular oxygen
through a chain of electronic carriers is called electron transport
chain or respiratory chain.
Mitochondria contains series of catalysts called respiratory
chain which are involved in the transfer of hydrogen and
electrons and their final reaction with oxygen to form water.
The components of respiratory chain are arranged sequen-
tially in the order of increasing redox potential. Electrons flow
through the chain in a stepwise manner from lower redox
potential to higher redox potential. The amount of energy
liberated in transfering electrons from one system to another
is determined by difference in redox potential of the two
systems.
The respiratory chain is given as:
A redox potential of 0.2 volt between the components of
respiratory chain results in the formation of 1 mole of ATP.
The three sites of ATP formation in the respiratory chain
are:
1. Between NAD
+
and flavoprotein
2. Between cytochrome b and c
3. Between cytochrome a and cytochrome a
3
.
If the substrate enters the respiratory chain through NAD
+
than the ATP yield is 3.
If the substrate enters the respiratory chain through flavo-
proteins than the ATP yield is 2.
Coenzyme (Ubiquinone)
It is called ubiquinone because of its ubiquitous occurrence
in nature. It is a lipid soluble hydrogen (electron) carrier found
in mitochondrial membranes and is a benzoquinone derivative.
It contains an isoprene side chain which varies from source
to source. Human coenzyme Q contains 10 isoprene units. It
is lipid soluble electron carrying protein and is reversibly
reduced by 2H
+
from FADH
2
. Reduced coenzyme Q is the
final stage at which oxidation reaction occurs as a process of
transfer of hydrogen atoms. Thereafter it is only the electrons
of the hydrogen atoms which are carried down the electron
transport and the 2H
+
ions liberated into the medium. Other
homologous of coenzyme Q contains 6 to 10 isoprene units
and have been isolated from various microorganisms, e.g.
chloroplasts of green plants and mitochondria of beef and
other animal tissues.
Cytochromes
Cytochromes are electron carrier proteins containing heme.
They contain protein part to which heme is attached as
prosthetic group. The cytochromes undergo oxidation and
reduction as a result of oscillation of iron atom with Fe
++
and
Fe
+++
from which donates the reduced and oxidized form
respectively.
The five different cytochromes that has been identified in
the inner mitochondrial membrane are cytochromes a
1
, a
3
, b,
c, and c
1
. It has been found that cytochrome a and cytochrome
a
3
are combined with the same protein molecule to form
cytochrome aa
3
complex which is also called cytochrome c
oxidase or respiratory enzyme. It contains 2 atoms of copper.
Oxidative Phosphorylation
Oxidative phosphorylation means that oxidation is accom-
panied by phosphorylation. The energy released as a result
of biological oxidation is trapped in the form of high energy
phosphate bonds in ATP by phosphorylation of ADP.
It is divided into two groups:
1. Substrate level phosphorylation: In the substrate level phos-
phorylation the formation of high energy phosphate takes place
on the substrate, without undergoing into the respiratory
chain.
The characteristics of substrate level phosphorylation are:
i. Formation of ATP does not require oxygen
ii. Respiratory chain does not participate
iii. It is dinitrophenol insensitive.
Examples
Substrate level phosphorylation is best described by two
examples:
NAD
+
(1) D-glyceraldehyde-3-PO
4
+ Pi + ADP Phospho-
ATP glyceric acid
(2) Succinyl CoA + Pi + GDP Succinic acid + GTP + CoA
GTP + ADP GDP + ATP
2. Respiratory chain phosphorylation: In this phosphorylation the
formation of high energy phosphate bonds takes place as a
result of transfer of hydrogen and electrons through the
respiratory chain to oxygen.
The characteristics of respiratory chain or electron-oxygen
transport chain are:
1. It is completely inhibited by trace amounts of dinitrophenol
(DNP) or by antimycin A.
2. Oxygen uptake however, is not inhibited by DNP.
When a substrate is oxidized via NAD linked dehydro-
genases, 3 moles of inorganic phosphates are incorporated
into 3 moles of ADP to form 3 moles of ATP per atom of
oxygen consumed.
Similarly when a substrate is oxidized via flavin linked
dehydrogenases only 2 moles of ATP is formed.
Type of phosphorylation Reactions ~P trapped
1. Substrate level D-Glyceraldehyde 3-Phospho
-3-PO
4
Glyceric acid 1
Phosphoenol
Pyruvate Pyruvate 1
Succinyl CoA Succinate 1
2. Respiratory chain
Phosphorylation isocitrate Oxalosuccinate 3
(electron transport chain)
-ketoglutarate Succinyl CoA 3
Succinate Fumarate 2
P/O Ratio
P/O ratio is defined as the number of inorganic phosphate
taken to phosphorylate ADP per atom of oxygen consumed.
Mechanism of Oxidative Phosphorylation
Three hypothesis for the mechanism of oxidative phospho-
rylation has been postulated to account for the transfer of
energy from the oxidation reductions of reactions involving
electron transport chain (respiratory chain) to the synthesis
of ATP.
1. Chemical coupling hypothesis
2. Chemiosmotic hypothesis
3. Conformational coupling hypothesis.
Chemical Coupling Hypothesis
This is the oldest hypothesis and it postulates that the energy
yielding electron transfer process is coupled with energy
requiring oxidative phosphorylation through the formation
of high energy intermediate compound which is generated
by the electron transport system and then subsequently uti-
lized in the ATP formation from the ADP. In effect, it proposes
the existance of specific carrier proteins called C
1
, C
2
and C
3
at each of the three ATP producing sites along with an inter-
mediate I carried by them. At the site of release of energy
sufficient to form ATP, intermediate I is combined with the
carrier to form a high energy carrier and intermediate
complex. This complex then is again linked to combine with
another intermediate X to form I~X again an high energy inter-
mediate. The I component is then finally replaced by inorganic
phosphates and ADP is phosphorylated to form ATP using
the energy contained in I
2
~X complex, which is used up.
A strong objection to this effect is that no such intermediate
has been found even after intensive research.
This hypothesis takes the help of hypothetical carriers and
hypothetical intermediates I and their effects are explained
as:
Effect of inhibitors: Inhibitors arrest respiration by blocking
the respiratory chain at energy site I, II and III.
Inhibitors of site I = Rotenone, amobarbital, piericidin
Inhibitors of site II = Antimycin, BAL
Inhibitors of site III = H
2
S, CO, CN
Uncouplers: Uncouplers are substances which allow elec-
trons to continue but prevent phosphorylation of ADP to ATP.
They are dinitrophenol (DNP). Uncouplers causes the hydro-
lysis of one of the high energy intermediates (car ~ l) resulting
in the release of carrier I and energy as heat.
Chemiosmotic Hypothesis
This hypothesis (Peter Mitchell) assumes two points.
i. The outer mitochondrial membrane is impermeable to
hydrogen ions and hydroxide ions.
ii. The process goes on within matrix.
During electron transport, protons are released to the
outside of the mitochondria. This results in the establishment
of a proton gradient across the membrane, with a high
concentration of protons (H
+
) outside the mitochondria and
low concentration of protons inside the mitochondria creating
an electrochemical potential difference. This electrochemical
potential difference is used to derive a vectorial membrane
located ATP synthetase, or the reversal of a membrane located
ATP synthetase which in the presence of inorganic phosphate
and ADP forms ATP.
Conformational Coupling Hypothesis
According to this hypothesis (PD Bayer) the release of energy
during the electron transport induces some conformational
changes in the carrier protein or the coupling factor. These
changes are due to the energy dependent shift in the number
of location of weak bonds such as hydrogen bonds and
hydrophilic interactions which normally maintain the three
dimensional conformation of the proteins. Then this energy
conserved in this energised conformational state is used to
derive the phosphorylation of ADP by inorganic phosphorous
into ATP. Simultaneously the carrier protein or the factor
returns back to the original low energy conformation.
This theory gets some support from the fact that inner mito-
chondrial membrane undergoes very rapid physical changes
as the electron pass along the respiratory chain. Also this
membrane shows some ultrastructural changes that accompany
the addition of ADP to the respiratory mitochondria.
This theory is in a way similar to the chemical coupling
theory except the fact that it postulates the non-covalent bonds
as the energy intermediates rather than the postulation of true
high energy intermediate in the chemical theory.
Shuttle System
NADH is produced in the cytosol but cannot penetrate the
mitochondria, i.e. the extra mitochondria NADH cannot pene-
trate the mitochondrial inner membrane but electron derived
from it can enter electron transport chain by an indirect route
called shuttles.
Two important shuttle systems are:
1. -glycerophosphate shuttle
2. Malate-aspartate shuttle.
-glycerophosphate shuttle: This shuttle transfers reducing
equivalents from cytosol to the mitochondrial electron trans-
port chain by the following route.
Malate-aspartate shuttle: This shuttle transfer NADH from the
cytosol to mitochondria by the following route.
METABOLISM OF CARBOHYDRATES 151
The major function of carbohydrate in metabolism is as a fuel
to be oxidized and provide energy for other metabolic
processes. In this role, carbohydrate is utilized by cells mainly
in the form of glucose. It has the advantage of being cheap,
easily digested and rapidly metabolized.
Carbohydrate metabolism is basically the metabolism of
glucose and substance related to glucose in their metabolic
processes. Glucose serves as a ready source of chemical energy
for humans.
The sugar of blood is glucose. The digestion of carbo-
hydrates such as starch, sucrose and lactose produces glucose,
fructose and galactose which passes into blood circulation.
Conversion of fructose and galactose into glucose takes place
in the liver.
Carbohydrates supply more than 50 percent of the energy
requirement of the body.
Except for ascorbic acid (vitamin C), carbohydrates are not
essential to the diet, through gluconeogenesis, the body can
synthesize necessary carbohydrates from certain amino acids.
GLYCOLYSIS
The breakdown of glucose to pyruvic acid is called glycolysis.
Under aerobic condition, pyruvic acid enters mitochondria
and is completely oxidized to CO
2
and H
2
O. Whereas, under
anaerobic conditions, pyruvate is converted to lactic acid.
The sequence of reactions from glucose to pyruvic acid is
also called Embden-Meyerhof pathway. Glucose is converted to
pyruvate in 10 steps by glycolysis.
Glycolysis is an extramitochondrial pathway and is carried
by a group of eleven enzymes.
Metabolism of
Carbohydrates
CHAPTER
8
Mutases are enzymes which catalyze the transposition of
functional groups.
Glucokinase is an inducible enzyme and has high km value
for glucose whereas hexokinase is a constitutive enzyme and
has low k
m
value for glucose.
Pyruvic acid has both a ketone or keto group and an acid
group and hence it is a keto acid.
Salient Features of Embden-Meyerhof Pathway
1. The rate limiting step in glycolysis is phosphofructokinase
(PFK). PFK is stimulated by fructose-6-phosphate, AMP
and ADP but is inhibited by ATP and citrate. Since one
of the main object of glycolysis is to produce ATP and since
the presence of excess AMP, ADP or fructose-6-phosphate
means that the cell is deficient in ATP. These molecules
are activator of the enzyme (PFK), stimulating it to degrade
more glucose and hence more production of ATP. Conse-
quently an excess of ATP means that the cell is catabolizing
more glucose than necessary; excess ATP inhibits PFK.
2. All the reactions of glycolysis are reversible except hexo-
kinase, phosphofructokinase and pyruvate kinase catalyzed
reactions because of energy barriers.
3. Enzyme enolase is inhibited by fluoride. Since erythrocytes
do not have mitochondrial enzymes to oxidize glucose
aerobically, they depend on glycolysis only for their energy
requirement. That is why sodium fluoride (NaF) is used
in the collection of blood sugar sample because it prevents
glycolysis by inhibiting the enzyme enolase. Otherwise a
low result will be obtained due to glycolysis.
4. It is the major pathway by which glucose is metabolized
in erythrocytes.
5. Glycolysis gives rise to certain intermediate compounds
which are important for other biochemical processes.
i. Glyceraldehyde-3 PO
4
: For triglycerides and phospho-
lipid biosynthesis.
ii. Acetyl CoA: Fatty acid and cholesterol biosynthesis.
iii. Pyruvate: Alanine biosynthesis by transamination.
Glycolysis has three principal features:
1. It is the degradative pathway whereby D-glucose is
oxidized to pyruvate, which is further metabolized by either
of the two routes.
i. When the supply of oxygen is inadequate for complete
oxidation, the pyruvate is reduced to lactate.
ii. When the supply of oxygen is adequate (aerobic condit-
ions) the pyruvate is oxidatively decarboxylated to acetyl
CoA, which enters the citric acid cycle, where it is oxidized
to carbon dioxide and water.
2. Glycolysis gives rise to certain intermediates which are
common to other pathway such as pentose phosphate path-
way. These intermediate compounds also provide sources
of starting materials for the biosynthesis of substances
such as triglycerides from glyceraldehyde-3-phosphate,
L-alanine from pyruvate and glycogen from glucose-1-
phosphate.
3. Glycolysis is accompanied by the formation of ATP.
Pasteur Effect
Pasteur effect is the inhibition of glycolysis by oxygen. The
rate limiting step in glycolysis, the phosphofructokinase, is
inhibited by citrate and ATP.
Crabttee Effect
Crabttee effect is the inhibition of cellular respiration by high
concentrations of glucose. This is due to the completion of
glycolytic processes for inorganic phosphate.
CITRIC ACID CYCLE
The complete oxidation of acetyl moiety is effected by means
of a cyclic metabolic mechanism called citric acid, also called
tricarboxylic acid (TCA) cycle and Krebs cycle. This cycle takes
place in mitochondria.
The citric acid cycle operates only under aerobic conditions
because it requires a supply of NAD
+
and FAD which are
regenerated when NADH and FADH
2
transfer their electrons
to O
2
through the electron transport chain.
TCA cycle requires the presence of oxygen, i.e. aerobic
metabolism of carbohydrates and is catabolized by enzymes
found in the mitochondrial fraction of the cell.
Before pyruvate gains entry into the TCA cycle, it is
oxidatively decarboxylated to acetyl CoA.
Conversion of Pyruvate to Acetyl CoA
Pyruvate is oxidatively decarboxylated to acetyl CoA by a
multienzyme complex called pyruvate dehydrogenase com-
plex.
This complex enzyme system comprises of three different
enzymes:
i. Pyruvate dehydrogenase (29 molecule)
ii. Dihydrolipoate transacetylase (1 molecule)
iii. Dihydrolipoate dehydrogenase (8 molecule).
which catalyze the five step reactions involved in conversion
of pyruvate to acetyl CoA.
The six cofactors required are (i) Mg
++
ions (ii) Thiamine
pyrophosphate (TPP) (iii) Lipoic acid (iv) Coenzyme A (CoA-
SH) (v) FAD (vi) NAD
+
(see page 156 for reaction).
Salient Features of Citric Acid Cycle
1. Citric acid is the common pathway for the metabolism of
carbohydrates, fats, and proteins; since it provides the
complete oxidation of acetyl CoA to carbon dioxide and
water.
2. Citrate synthetase catalyze a direct bond between the
methyl carbon of acetyl CoA and carbonyl carbon of oxalo-
acetate. It is an irreversible reaction.
3. It defines the step by which citric acid, isocitric acid, -keto-
glutaric acid, succinic acid are synthesized and degraded.
The stepwise mechanism of the reactions is explained below.
4. Many amino acids enter the cycle at several levels either
at acetyl CoA, -ketoglutarate, oxaloacetate, succinyl CoA
and fumarate.
5. The rate limiting step in the TCA cycle is the conversion
of isocitrate to -ketoglutarate. The enzyme is the citrate
synthetase. The availability of acetyl CoA and oxaloacetate
in plenty stimulates this enzyme while succinyl CoA by
competing with acetyl CoA inhibits this enzyme.
Similarly -ketoglutaric acid, an intermediate in citric acid
cycle is oxidatively decarboxylated to succinyl CoA. The en-
zyme involved is -ketoglutarate dehydrogenase complex
like the pyruvate dehydrogenase complex. This is an
irreversible reaction forming succinyl CoA. Aresenite inhibits
this reaction causing the accumulation of the -ketoglutaric
acid. Cofactors required are the same as in the conversion
of pyruvate to acetyl CoA.
The reaction is summed as:Succinyl CoA
Pyruvate can be channelled into TCA cycle as acetyl CoA or
as oxaloacetate. This point is a switch point which controls
the main function of the cycle. If pyruvate is channelled to
acetyl CoA then the cycle will generate mainly energy. If
pyruvate is channelled into oxaloacetate, then its main function
will be to produce carbon skeletons for amino acid or fat
synthesis, i.e. high levels of acetyl CoA inhibit the activity
of pyruvate dehydrogenase, decreasing further synthesis of
acetye CoA and the same time enhance the activity of pyruvate
carboxylase, stimulating the synthesis of oxaloacetate.
ENERGETICS
For each molecule of glucose, 2 pyruvates are formed. These
are converted to 2 acetyl CoAs, each of which is brokendown
to 3 NADH, 1FADH
2
and 1 GTP. Hence, for 1 glucose molecule,
6 NADH, 2 FADH
2
and 2 GTP are produced in the TCA cycle.
Reactions Where ATP is Consumed
Glucose to glucose-6-phosphate 1
Fructose-6-phosphate to fructose-1, 6-diphosphate 1
Reactions Where ATP is Generated
Glyceraldehyde-3-PO
4
to 1,3 diphosphoglycerate 2 3 = 6
1,3 diphosphoglycerate to 3-diphosphoglycerate 2 1 = 2
(Substrate level phosphorylation)
Phosphoenolpyruvate to pyruvate 2 1 = 2
(Substrate level phosphorylation).
Under Anaerobic Condition
The ATP yield is 2
(Two molecules of ATP are generated in the conversion of
glucose to pyruvate because NADH obtained in the glyceral-
dehyde-3-phosphate dehydrogenase reaction is not oxidized
in mitochondria by the respiratory chain).
Under Aerobic Condition
Pyruvate to acetyl CoA 2 3 = 6
Isocitrate to oxalosuccinate 2 3 = 6
-ketoglutarate to succinyl CoA 2 3 = 6
Succinyl CoA to succinate
(The substrate level phosphorylation) 2 1 = 2
Succinate to fumarate 2 2 = 4
Malate to oxaloacetate 2 3 = 6
Total = 30
Total number of ATP molecules formed under aerobic con-

ditions is 38, i.e. 30 from citric acid cycle and 8 from glycolysis.
Two important features of Krebs cycle are:
i. Two carbon atoms enter the cycle as acetyl CoA and two
carbons leave as carbon dioxide, so no net gain of carbon
atom takes place.
ii. The carbon atoms that leave as CO
2
are not the same ones
as those taken up as acetyl CoA.
The tricarboxylic acid cycle or Krebs cycle serves five major
functions:
1. It produces most of the carbon dioxide made in human
tissues.
2. It is the source of much of the reduced coenzymes that
drive the respiratory chain to produce ATP.
3. It converts excess energy and intermediate to the synthesis
of fatty acids.
4. It provides some of the precursors used in the synthesis
of proteins and nucleic acid.
5. Its components control directly (product precursor) or indi-
rectly (allosteric) other enzyme system.
This cycle is described as biochemical traffic circle, material
coming to it from carbohydrate source might leave it to form
fat whereas material coming to it from amino acid might leave
it to form carbohydrate. The only road closed is that leading
from fat to carbohydrate.
Amphibolic Role of Citric Acid or Krebs Cycle
Citric acid cycle is primarily a catabolic process for the final
oxidation of the carbohydrates, fats and proteins into CO
2
and
H
2
O. But this cycle at the same time takes part in the various
anabolic processes such as gluconeogenesis, fatty acid synthesis
and amino acid synthesis by providing substrates which are
the normal intermediate products of this cycle. Thus, this cycle
has the dual or amphibolic role of both catalyzing the substances
for energy and also taking part in synthesis. For example, the
oxaloacetate and -ketoglutarate are utilized for amino acids.
Similarly, the malate and oxaloacetate are also utilized as glu-
cose precursor in a reaction catalyzed by malate dehydrogenase
first and subsequently by phosphoenol pyruvate carboxylase.
Citrate is also utilized for providing acetyl CoA for fatty acid
synthesis by extra mitochondrial pathway. Further succinyl CoA
is utilized in the heme synthesis. Thus, all these examples
establish amphibolic role of citric cycle.
Hexose Monophosphate Shunt Pathway
This pathway is also known as Warburg-Dickens-Lipmann path-
way, pentose phosphate pathway, phosphogluconate pathway
or direct oxidative pathway or reductive pathway.
Though glycolysis is the principal pathway for the con-
version of glucose into pyruvate in most tissues but there exists
an alternative pathway. Since glucose utilization can proceed
when certain reactions in the glycolytic pathway are blocked
by the addition of inhibitors.
Tissues where this pathway is more prominent are liver,
adipose tissue, lactating mammary gland, leukocytes, testes,
and adrenal cortex, etc.
The enzymes of this pathway are found in the extramito-
chondrial cytoplasm.
Importance of HMP Shunt Pathway
1. This pathway generates NADPH, which is required in
the reductive synthesis of fatty acids, triglycerides and
steroids.
2. Pentose sugars (Ribose-5-PO
4
) are formed which are
required in the synthesis of nucleotides and nucleic acids.
3. This pathway is important in plants which synthesize
glucose from CO
2
by photosynthesis.
Energetics
Energy yield of hexose monophosphate shunt pathway.
Although the greatest importance of HMP shunt pathway
is to provide NADPH, for each carbon of glucose oxidized
to CO
2
. Two molecules of NADPH are reduced or 12 molecules
of NADPH per mole of glucose oxidized is produced.
12 moles of NADPH are equivalent to 36 moles of ATP.
Combined aerobic and anaerobic glycolysis of one molecule
of glucose gives 38 ATP, whereas HMP shunt pathway yields
36 ATP. So these two pathways of glucose oxidation are almost
equivalent in energy yield.
GLYCOGENESIS
The formation of glycogen from glucose is called glycogenesis.
Under the combined act of glycogen synthetase and branch-
ing enzyme, glucose units are added to the non-reducing ends
of the pre-existing glycogen by -(1,4) and -(1,6) linkages
to form glycogen.
Glucose is phosphorylated to glucose-6-PO
4
, by hexokinase
reaction, which is then converted to glucose-6-PO
4
, a reaction
catalyzed by the enzyme phosphoglucomutase. Glucose-1-PO
4
reacts with uridine triphosphate (UTP) to form uridine diphos-
phate glucose (UDPG). The reaction is catalyzed by the enzyme
UDPG pyrophosphorylase. Now in the presence of the enzyme
Glycogen synthetase, C-1 of glucose of UDPG forms a glycosidic
linkage -(1,4) with the C-4 of the preexisting glycogen
molecule. The addition of glucose from UDPG to the existing
glycogen molecule takes place from the non-reducing end of
the glycogen molecule, thus, permitting the origin of new
glycogen molecules. When the chain has been lengthened by
6 to 11 glucose molecules, a second enzyme called branching
enzyme, transfers 6 glucose molecule in -(1,4) linkages and
attaches to the nearby chain in -(1,6) linkages, thus creating
a branched point in the molecule.
Branching is important because it increases the solubility
of glycogen and provides a large number of non-reducing
sugar terminals which are the sites of activity for glycogen
phosphatase, the enzyme that breaks glycogen.
Glycogen Synthetase
a. Glycogen synthetase-D. It is the inactive form of the
enzyme.
b. Glycogen synthetase. It is the active form of the enzyme.
Glycogen synthetase-D, is the dephosphorylated form. It
is glucose-6-phosphate dependent, i.e. it is stimulated by
glucose-6-phosphate.
While glycogen synthetase-I is the dephosphorylated form.
It is independent of glucose-6-phosphate.
Glycogen Storage Diseases
These are a group of inborn error of metabolic diseases in
which there is an accumulation of abnormally large amount
of glycogen in the tissue due to the deficiency or absence of
enzymes involved in glycogen metabolism.
Various type of glycogen storage diseases are given below:
The classification of these diseases are based on the name
of the patient first diagnosed of that disease.
Type Name of disease Enzyme deficient
I. Von Geirkes disease Glucose-6-phosphatase
II. Pompes disease -(1,4) glucosidase
III. Coris disease Amylo-1, 6-glucosidase, i.e.
debranching enzyme.
IV. Andersens disease 1,4 1,6 transglucosylase,
i.e. branching enzyme
V. McArdles disease Muscle glycogen phosphorylase
VI. Hers disease Liver phosphorylase
Glycogenolysis
Breakdown of glycogen to glucose is called glycogenolysis.
The breakdown of glycogen takes place in liver and muscle.
In liver, the end product of glycogen breakdown is glucose
whereas in muscle the end product is lactic acid.
Under the joint action of phosphorylase [breaks only -(1,4)
linkages] and debranching enzymes [breaks only -(1,6) linkages]
glycogen is broken down to glucose.
The breakdown of glycogen is initiated by the enzyme Phos-
phorylase, which cleaves -(1,4) glycosidic linkages starting
from non-reducing end of the glycogen molecule to give
glucose 1-PO
4
and this process continues until four glucose
residues remain on either side of the -(1,6) branched point.
Now another enzyme Glucan transferase, transfer three glu-
cose units from one side to another, leaving a single glucose
residue at the branched point followed by debranching enzyme
to break -(1,6)-linkage.
The breakdown of glycogen takes place in liver and muscle.
The action of liver phosphorylase and muscle phosphorylase
are explained as below.
Liver Phosphorylase
It exists in two forms:
a. Phosphorylase: It is the active form of phosphorylase.
b. Dephosphophosphorylase: It is the inactive form of phosphorylase.
Activation of the inactive form involves phosphorylation
of the hydroxyl group of a serine residue by a specific kinase
in the presence of ATP. Inactivation of the active form is
catalyzed by a specific phosphatase. The action of kinase is
stimulated by c-AMP which itself is formed from ATP in the
presence of adenyl cyclase. Glucagon and adrenaline stimulate
glycogenolysis by increasing the activity of adenyl cyclase.
Muscle Phosphorylase
It exists in the following forms:
a. Phosphorylase a. It is the active form of phosphorylase.
It is active only in the absence of 5-AMP. It is a tetramer
containing 4 molecules of pyridoxal phosphate.
b. Phosphorylase b. It is the inactive form of phosphorylase.
It is active only in the presence of 5-AMP. It is a dimer
containing only 2 molecules of pyridoxal phosphate.
Phosphorylase a contains four molecules of pyridoxal phos-
phate. Whereas phosphorylase b contains 2 molecules of pyri-
doxal phosphate.
Phosphorylase in muscle is activated by epinephrine, which
activates adenyl cyclase to form c-AMP, which stimulate
phosphorylase kinase, key enzymes of glycolysis and gluconeo-
genesis in liver.
Cori Cycle
The cyclic process by which lactic acid is converted to glucose
in liver and eventually reappears as muscle glycogen is known
as Cori cycle.
The Cori cycle is the bodys way of recycling lactic acid
Liver Muscle
During vigorous muscle activity, muscle glycogen is
converted to lactic acid. The lactic acid diffuses from the muscle
into the blood stream and transferred to the liver. In liver,
lactic acid is converted to glucose by gluconeogenesis. Glucose
formed in this way returns to the muscle via circulation. This
cycle continues and is called Cori cycle.
GLUCONEOGENESIS
Gluconeogenesis is the process by which glucose or glycogen
is formed from noncarbohydrate substances. The noncarbo-
hydrate substances include glycogenic amino acids, intermediates
of TCA cycle, glycerol, pyruvate, lactate, etc. gluconeogenesis
is an important source for supplying glucose to various tissues
when glucose is otherwise not available. Especially during
fasting/starvation. Continuous supply of glucose is required for
the functioning of brain, RBC, etc. even when food is not taken.
The conversion of amino acids, lactate and glycerol into glucose
takes place mainly in liver and kidney. Thus, liver and kidney
are the major site of gluconeogenesis. Glucose-6-phosphatase
does not exist in brain, adipose tissues or muscle. Therefore,
these tissues are not gluconeogenic.
Gluconeogenesis takes place when the energy requirements
of the cell are at a minimal level and an energy source ATP
is available.
The production of glucose from noncarbohydrate precur-
sors occurs by following pathways.
Gluconeogenesis is regulated by four key enzymes:
1. Pyruvate carboxylase.
This enzymes is stimulated by acetyl CoA and inhibited
by ADP.
2. Phosphoenol pyruvate carboxy kinase.
3. Fructose-1,6-diphosphate-1 phosphatase (FDPase).
This enzyme is inhibited by AMP and ADP.
4. Glucose-6-phosphatase (G6 Pase).
This enzyme is stimulated by inorganic phosphate (Pi) and
glucose.
The conversion of pyruvate to glucose is shown on the
next page 171.
Insulin represses the synthesis of these four enzymes where-
as glucocorticoid hormones induces their de novo synthesis.
GALACTOSE METABOLISM
Galactose is derived mainly from lactose of the diet. Galactose
is important for the formation of glycolipids and glycoprotein
and for the formation of lactose during lactation.
Important points of galactose metabolism:
1. Conversion of galactose into glucose is the main pathway.
The galactose derived from the milk sugar is readily con-
verted into glucose in the liver.
2. Conversion of UDP-galactose into UDP-glucose is a freely
reversible reaction catalyzed by UDP galactose-4-epi-
merase. Hence, the glucose can be readily converted into
galactose in the states of galactose lack, which is the way
of treating alatosemia. This does not interfere with the
growth. So it is not essential in the diet.
3. Galactosemia results from the deficiency of galactose-
I- phosphate-uridyl transferase deficiency than galacto-
kinase which is normal in the RBC of galactosemic
patients.
4. Galactose is needed as it is a constituent of glycolipids
(cerebrosides), chondromucoids and mucoprotein.
5. Galactose is also required for the lactose synthesis in the
mammary gland by the enzyme lactose synthetase.
Galactosemia
Inability to metabolize galactose is called galactosemia.
Galactosemia is an inherited disease, generally encountered
in infants, characterized by inability to metabolize galactose
or lactose. This results in the accumulation of galactose in the
blood and ultimately excreted in the urine.
Galactosemia is due to the deficiency of the enzyme
galactose-1-phosphate uridyl transferase.
Galactosemia gives rise to loss in weight, mental retardation
and development of cataract due to the deposition of galactitol,
a reduced product of galactose, in the lens. A galactose free
diet avoids these difficulties and galactose that is necessary
for the synthesis of cell membranes, cerebrosides, glycolipids
and mucoproteins can be formed from glucose-1 phosphate.
FRUCTOSE METABOLISM
Fructose is an important source of dietary carbohydrate,
accounting for approximately 20% of the total carbo-hydrate
intake.
Fructose is present in significant amounts in seminal fluid.
It is synthesized in the prostate gland by the following
reaction.
In fructosuria, fructose is found in the urine, due to lack
of enzyme fructokinase.
LACTOSE SYNTHESIS
Blood galactose is readily converted into glucose in liver. Here
glucose is first converted into galactose by the pathway as
above and then glucose and galactose combine to form lactose
by the enzyme lactose synthetase.
Failure to absorb dietary lactose is common in adults and
is due to lactose deficiency and the irriability to hydrolyze
lactose. Individuals with lactose deficiency can generally tole-
rate yoghurt (curd), a milk product. Yoghurt contains lactase
that catalyzes the degration of lactose to glucose and galactose.
URONIC ACID PATHWAY
Biosynthesis of D-glucuronic acid takes place from glucose-
1-phosphate.
UDP-glucuronic acid is required in detoxification reactions
forming glucuronides (e.g. bilirubin diglucuronide) and in the
synthesis of proteoglycans. Also this pathway through
L-uronic acid gives rise to the synthesis of L-Ascorbic acid
(Vitamin C) in the animals and other plants.
These reactions occur in animals and higher plants. In man,
guinea pigs and other primates, the enzyme which converts
L- glunolactone to 2-keto-L-gluconate is absent, thus making
ascorbic acid a vitamin for them.
UDP-glucuronic acid is the active glucuronic acid. It parti-
cipates into the incorporation of glucuronic acid into chon-
droitin sulphate and other polysaccharides.
Glucuronic acid conjugates with bilirubin, steroids and cer-
tain drugs for detoxification.
The compound glucose 6-PO
4
is at a pivotal junction to
undergo various metabolic fats such as pyruvate/lactate
(glycolysis), in HMP shuntpathway, in glycogen synthesis in
liver and muscle, give rise to glucuronate, ascorbic acid (uronic
acid pathway).
Glucose is converted to glucose-6-PO
4
by two possible
enzymes depending upon the tissue. One is glucokinase (found
in liver) which is highly specific for the glucose and other is
hexokinase (muscle and fat cells), which catalysis the phos-
phorylation of most hexoses, including glucose.
Pentosuria
Pentosuria is characterized by the increased excretion of one
or more pentoses. The pentoses normally present in urine are
L-xylulose, D-ribose and D-ribulose.
Essential pentosuria: It is characterized by increased excre-
tion of L-xylulose. This is due to the deficiency of enzyme
L-xylulose dehydrogenase.
Other types of pentosuria include alimentary pentosuria
resulting in excretion of L-arabinose and xylose due to intake
of large quantities of fruits and ribosuria (due to increase in
excretion of D-ribose).
The patients with deficiency of glucose-6-phosphate dehy-
drogenase when given antimalarials like primaquine that
precipitates hemolysis because G-6-PD is responsbile for the
maintenance of reduced glutathione level and antimalarials
produces excess of free radicals as free radicals damages the
RBCs cell membrane by oxidative stress mechanism.
REGULATION OF BLOOD GLUCOSE
The concentration of glucose in the blood is the net resultant
of two processes.
1. Rate of glucose entrance into the bloodstream
2. Rate of glucose removal from the bloodstream.
Ways by which sugar is added to the blood
1. By absorption from the intestine
2. Breakdown of liver glycogen
3. By gluconeogenesis. The sources of gluconeogenesis are:
amino acids, propionate, lactate, glycerol, etc.
Ways by which sugar is removed from the blood
1. Conversion to liver glycogen
2. Conversion to muscle glycogen
3. In the synthesis of fats (i.e. triglycerides)
4. In the synthesis of glycoproteins such as nucleic acids (nuc-
leoproteins), lactose, etc.
5. Loss in the urine.
A balance of the above two processes will keep the blood
sugar level within normal limits.
These two processes are influenced by a number of factors
under physiological conditions.
The blood glucose level is most efficiently regulated by
a mechanism in which liver, extrahepatic tissues and several
hor-mones play an important part.
Role of Liver
Liver, being the centre of all metabolic activities is mainly
responsible for the regulation of blood glucose level. In liver,
exists the developed mechanism for uptake of glucose
from the blood, conversion of glucose to glycogen for storage
(glycogenesis), release of glucose from glycogen (glycogeno-
lysis) and de novo synthesis of glucose from non-carbohydrate
precursors (gluconeogenesis).
Glycogenesis in liver can occur from blood glucose or any
substance capable of giving rise to pyruvate. Due to the pre-
sence of glucose-6-phosphatase, liver glycogen can contribute
directly to blood sugar (gluconeogenesis).
Role of Extra-hepatic Tissues
a. Role of muscle: Muscle glycogen does not contribute directly
to the blood sugar due to the absence of the enzyme,
glucose-6-phosphatase. Glycogenolysis in muscle provides
glucose to blood only through the formation of lactic acid
which by Cori cycle is converted to glucose in the liver.
b. Role of kidney: Kidney also exerts a regulatory effect by
reabsorbing glucose by the reabsorptive system of the renal
tubules. When the blood glucose level rises above the renal
threshold, the excess glucose appears in the urine.
Role of Hormones
Several hormones play an important role in the homeostatic
mechanism of blood glucose level. Out of these insulin is the
only hypoglycemic hormone whereas others are hyperglycemic
hormones.
1. Insulin: Insulin plays an important role in the regulation
of blood glucose concentration. It is secreted into the blood
in response to hyperglycemia. Insulin increases the transport
of glucose across the cell membranes. Insulin reduces the blood
sugar level by increasing the glucose utilization by glycolysis,
decreases hepatic glycogenolysis and increases glycogenesis.
Hormones which keep the blood sugar level high are:
1. Epinephrine
2. Glucagon
3. Glucocorticoids
4. Thyroxine
5. Growth hormones.
Mechanism by which these hormones increase the blood
sugar level are:
1. By increasing the absorption of glucose from the intestines
2. By decreasing the oxidation of glucose at the tissue level
3. By preventing the synthesis of glycogen
4. By stimulating glycogenolysis
5. By potentiating gluconeogenesis.
Blood sugar level is kept normal by insulin, by opposing
the action of these hormones.
2. Glucagon: Glucagon is also called Hyperglycemicglyco-
genolytic hormone. Glucagon is secreted by the -cells of the
islets of Langerhans. Glucagon secretion is stimulated by hypo-
glycemia. It causes glycogenolysis by activating liver phos-
phorylase. Glucagon thus counter balances the action of insulin
which is secreted into the blood when the blood glucose level
is high.
Glucagon acts primarily on liver and does not affect gly-
cogen breakdown in muscles. Glucagon enhances gluconeo-
genesis from amino acids and lactate.
3. Epinephrine: Epinephrine stimulates glycogen breakdown
in liver and muscle. The stimulation of glycogenolysis is due
to its ability to activate phosphorylase. Epinephrine also
inhibits muscle glycogen synthesis in liver and thus directs
the production of increased blood glucose.
4. Adrenal cortex hormones: Adrenal cortex secretes glucocor-
ticoids, which lead to gluconeogenesis which is the result of increased
protein breakdown and stimulation of transaminase. It also inhibits
glucose utilization in extra-hepatic tissues.
5. Anterior pituitary hormones: Growth hormones and ACTH
elevate the blood glucose level. Growth hormones decrease
glucose uptake by the tissues, whereas ACTH stimulates the
secretion of hormones of the adrenal cortex.
6. Thyroid hormone: Thyroxine has a diabetogenic action. It
increases blood glucose concentration by increased absorption
of glucose from the intestines.
Glycosuria
The excretion of detectable amounts of reducing sugar in urine
is called Glycosuria. If glucose is excreted, then the condition
is called glucosuria. Glucose is filtered by the glomeruli but
is completely reabsorbed by the renal tubules. The reabsorp-
tion is effected by phosphorylation in the tubular cells. The
maximum rate of reabsorption of glucose by the tubules
(T
m
GTubular maximum of glucose) is 350 mg/minute. When
the blood levels of glucose are elevated, the filtrate contain
more glucose that can be reabsorbed, the excess passes into
the urine and gives rise to glucosuria.
Renal Glucosuria
The blood glucose level is normal, but as a defect in the reab-
sorption system in the tubules, kidney threshold is lowered
and glucose appears in the urine.
Renal glucosuria is an example of benign glucosuria.
Diabetes Mellitus
Diabetes mellitus is a metabolic disorder due to the deficiency
of insulin, resulting in high blood glucose level and the
excretion of glucose in the urine.
The most important features of diabetes mellitus are:
1. The hyperglycemia and glucosuria persist during fasting.
2. Liver glycogen falls to a low level.
3. Excretion of large quantities of ketone bodies due to increased
fatty acid metabolism giving rise to diabetic coma.
Diabetes or diabetes mellitus is a condition where in the body
does not produce enough insulin or does not properly respond
to the insulin that is produced, there by keeping glucose level
in the blood high.
Diabetes affects nervous digestive circulatory, endocrine,
urinary system but all body system are in some way affected.
There is no diabetic sure but it can only be cautted.
Classification
1. Type 1 diabetes: Also called childhood onset diabetes, juv-
enile diabetes and insulin dependent diabetes mellitus
(IDDM).
Type 1 diabetes is a chronic (life long) disease that occurs
when the pancreas does not produce enough insulin to
properly control blood glucose levels. Type 1 diabetes can
occur at any age but it is most after diagnosed in children,
adolescents or young adults, In this form of diabetes, the
body cannot make insulin the immune system mistakenly
attacks the cells in the pancreas that make and release
insulin. As these cells die, blood glucose levels rise. People
with type 1 diabetes need insulin shots.
2. Type 2 diabetes: In characterized by the inability of the body
to make insulin or properly use insulin as a result, cells
can not take up or utilize glucose resulting in high blood
glucose level.
It is a slow onset process and person having diabetes
for years without knowing.
Typically with type 2 diabetes, the body still make insulin,
but the cells cannot use it. This is called insulin resistance.
3. Gestational diabetes: It occurs during pregnancy, labor and
delivery, women who got gestational diabetes are more
likely to develop type 2 diabetes.
Prediabetes
That condition when a person have impaired glucose tolerance
where blood glucose levels are higher than normal but not
high enough to be classified as diabetes.
Latest autoimmune diabetes of adults (LADA) is a condition
in which Type 1 diabetes develops in adults. Adults with LADA
are frequently initially misdiagnosed as having type 2 diabetes
based on age rather than etiology.
Maturity onset diabetes of young (MODY): Condition
because of defects in cell function.
According to the latest WHO guidelines two fasting glucose
blood measurement about 126 mg/dl is considered diagnostic
for diabetes mellitus.
People with testing blood glucose level from 100-125 mg/dl
is considered to have impaired fasting glucose.
HbA
1c
given an idea about average blood glucose control
over the last 120 days.
Glycosylated hemoglobin (HbA
1c
) test is recommended for:
a. checking blood glucose control in pre-diabetes.
b. Monitoring blood glucose control in diabetes mellitus.
The normal value of HbA
1c
is 4-6% correlation between
HbA
1c
blood glucose level.
HbA
1c
Average blood glucose level over past
three months
6% 120 mg%
7% 150 mg%
8% 180 mg%
10% 240 mg%
Higher the value of HbA
1c
poorer the blood glucose control
HbA
1c
>6.5 = Diabetes
<6.0 = Not diabetes
in between 6-6.5 = May be pre-diabetes or risk of diabetes
The extent of glycosylation of hemoglobin can be conviently
monitored and used to assess the control of hyperglycemia.
Analysis of hemoglobin A (the adult form of hemoglobin)
reveals the extreme of minor components called hemoglobin
A
1
. Hemoglobin A
1
forms by nonenzymatic modification of
hemoglobin A by glucose. Glycosylation has only minor effect
on normal hemoglobin function.
Glycosylation occurs continuously within the red cell, and
the extent of glycosylation reflects the average glucose
concentration to which the cell is exposed during its 120 days
life-span. Measurement of glycosylated hemoglobin content
provides a clinically useful means to assess the degree of
hyperglycemia that existed over the previous several weeks.
HbA
1c
in normal individuals (without diabetes mellitus)
makes up about 6% of total hemoglobin. Individuals with
controlled (blood glucose levels <10 mM, or 180 mg/dl) have
levels of hemoglobin A
1
of about 9% of total hemoglobin.
These with less well controlled diabetes have greater than
9%, hemoglobin A
1
.
Glycosylated hemoglobin is formed in erythrocytes by
the reaction of glucose with hemoglobin. The glycosylation
of hemoglobin is nonenzymatic reactions. The reaction is
virtually irreversible. Its removal from the blood proceeds
very slowly.
The prime importance of the glycosylated hemoglobin
estimation in diabetes diagnosis lies in the fact that HbA
1c
Correlation between HbA
1c
blood glucose level
and blood glucose values of diabetics often present a different
picture. Glycosylated hemoglobin fraction is not affected by
the metabolic state at a given moment whereas the blood
glucose level can change very rapidly. HbA
1c
thus makes it
possible to identify hyperglycemic states which would other-
wise go unrecognized.
The main characteristic of the glycosylated hemoglobin
fraction HBA
1
is that it constitutes a kind of long-term retro-
spective indicator of blood glucose concentrations. This is due
to the fact that the stable HbA
1
is not catabolized throughout
the erythrocyte lifespan (100-200 days).
HbA
1C
gives an indication of the averaged blood glucose
levels during the proceeding weeks rather than the metabolic
state of the patient at the time of testing.
HbA
1C
test estimates the portion of hemoglobin which is
glycated. Increased blood glucose levels increase the glycation
of hemoglobin. Glycation of hemoglobin is nonenzymatic,
irreversible addition of glucose to hemoglobin. In normal
individual 5-6% of the hemoglobin molecule is glycated.
However, in uncontrolled diabetic patient the level can go
up to more than 20% which indicates that the patient has
uncontrolled hyperglycemia.
Since, the formation of glycated Hb is essentially reversible
and the blood level of HbA
1c
depend on both the lifespan
of red blood cell (average 120 days) and the blood glucose
concentration. Since the rate of formation of HbA
1c
is directly
proportional to the concentration of glucose in the blood, the
HbA
1c
concentration represents the integrated values for
glucose for the proceeding 6-8 weeks.
HbA
1c
% Degree of glucose control
<6 Nondiabetic level
6-7 Near normal glycemia
<7 Good
7-8 Good control
>8 Action suggested
Glycosylated hemoglobin (HbA
1c
) test gives an overview
of diabetic control over proceeding 2-4 months.
HbA
1C
circulates in the blood for 2-4 months before being
naturally broken down by the body so the level of HbA
1c
at any stage can show how high the blood glucose has been
over the proceedings few months.
HbA
1c
test shows the average amount of glucose in the
blood over the last three months.
PLASMA LIPOPROTEINS
Lipids are water insoluble and are transported in the body
in an aqueous medium in combination with various specific
proteins. This results in lipid: protein complex called lipopro-
teins. These lipoproteins consists of triglycerides or cholesterol
esters and the central core surrounded by a coat of unesterified
cholesterol, phospholipid and protein.
Plasma lipoproteins occur in four major forms which are:
1. High or heavy density lipoproteins (HDL)
2. Low density lipoproteins (LDL)
3. Very low density lipoproteins (VLDL)
4. Chylomicrons.
Plasma lipoproteins are in dynamic state. They are conti-
nuously being synthesized and degraded with rapid exchange
of both lipid and protein among themselves. Two enzymes
Lecithin: cholesterol acyl transferase (LCAT) and lipoprotein
lipase (also called triglyceride lipase) play a significant role
in the catabolism of lipid fraction of lipoproteins.
The components that are necessary for the synthesis of
lipoproteins are triglycerides, cholesterol, cholesterol esters,
phospholipids and apoproteins.
1. High density lipoproteins: Also called -lipoproteins. This
fraction is rich in phospholipids.
2. Low density lipoproteins: Also called -lipoproteins. This
fraction is rich in cholesterol.
3. Very low density lipoproteins: Also called
2
or pre--lipo-
proteins. This fraction is rich in triglycerides.
4. Chylomicrons: It consists of central core of triglycerides,
phospholipids, and cholesterol combined with small amount
of proteins.
Metabolism of Lipids
CHAPTER
9
METABOLISM OF LIPIDS 185
The density of lipoprotein increases with the protein content.
The protein parts of lipoproteins are called apolipoproteins.
Each lipoprotein differ in terms of size, density, the relative
proportions of triglycerides and cholesterol esters in the core
and in the nature of apoproteins on the surface.
Chylomicrons VLDL LDL HDL
Density <0.95 0.951.006 1.0911.063 1.0631.21
Protein % 12 10 25 4555
TG (%) 8095 5565 10 3
PL (%) 36 1520 22 30
Cholesterol (%) 13 10 8 33
Cholesteryl ester (%) 24 5 37 15
Absorption of Fats
Dietary fat is digested by the action of pancreatic lipase, present
in the intestines. The lipase hydrolyses the triglycerides to
40 percent free fatty acids and glycerol, 50-57% mono-and
diglycerides, 3-10% is absorbed unchanged as triglycerides.
The 2-monoglycerides produced as intermediates are con-
verted to 1-monoglycerides by an enzyme isomerase which
is then digested by lipase to glycerol and free fatty acids. Of
the four products of triglycerides hydrolysis, i.e. free fatty
acids, glycerol, monoglycerides and diglyceride, free fatty
acids and glycerol are easily absorbed as they are water soluble
and are then carried away by the blood.
Higher fatty acids, mono and diglycerides are absorbed
with the help of bile salts in the form of water soluble molecular
aggregates called mixed micelles.
Inside the intestinal epithelial cell, 1-monotriglycerides are
hydrolyzed by intracellular lipases to give free fatty acids and
glycerol, whereas 1-monoglycerides are used for triglyceride
synthesis.
Higher fatty acids are largely utilized for the triglycerides
synthesis inside the intestinal epithelial cells and are carried as
chylomicrons which are hydrophobic water soluble triglycerides
covered with a layer of hydrophilic phospholipids, free and
esterified cholesterol and some proteins.
Fatty acids with odd numbers of carbon atoms and
branched-chain fatty acids make up only a small portion of
our total fatty acid intake. A high fat, low carbohydrate diet
results in metabolic acidosis, whereas high protein, low carbo-
hydrate diet results in protein imbalance with a high urinary
nitrogen output, increasing carbohydrate in the diet prevents
this lose of nitrogen.
Oxidation of Fatty Acids
The action of hormonally controlled lipase results in the hydro-
lysis of neutral fats to glycerol and free fatty acids. Glycerol
enters the glycolytic pathway, via formation of glycerol-3-
phosphate by the action of ATP and glycerokinase.
The fatty acids tightly bound with albumin is carried via
the blood to the various tissues for oxidation.
Fatty acids oxidation takes place in mitochondria.
-oxidation
Fatty acids are oxidised mainly by -oxidation. In -oxidation,
the oxidation takes place at the -carbon atom from the
carboxyl end and the -carbon atom is oxidized to carboxyl
group resulting in the formation of acetyl CoA and a fatty
acid shorter by two carbon atoms.
The first step in -oxidation pathway is the activation of
fatty acid to form acyl CoA by combination with coenzyme A.
The enzyme acyl CoA synthetase also known as thiokinase.
Acyl CoA synthesis occurs in the outer mitochondrial mem-
brane. The acyl CoA so formed cannot penetrate the inner
mitochondrial membrane to the site of fatty acid -oxidation
enzyme system. In order to cross this barrier the acyl group
is transferred to carnitine. The reaction is catalyzed by car-
nitine acyl transferase. Acyl carnitine crosses the inner mito-
chondrial membrane. Now another enzyme located on the
inner surface of the inner mitochondrial membrane catalyzes
the reverse reaction and acyl group is transferred to intra-
mitochondrial CoASH (Coenzyme A).
Outside
Inside
Acyl carnitine + CoASH Acyl CoA + carnitine
Once the acyl CoA is inside the mitochondria, it is
metabolized by the steps given below.
Energy Yield of Palmitic Acid Metabolized
Palmitic acid is C
16
acid.
Palmitic acid will undergo seven steps of -oxidation to
give 8 molecules of acetyl CoA.
In each sequence of -oxidation, 5 ATP molecules are gene-
rated, (2 from FADH
2
and 3 from NADH + H
+
, entering the
respiratory chain). Since 7 steps of -oxidation takes place,
a total of 7 5 = 35 ATPs will be formed.
Each acetyl CoA molecule on complete oxidation by citric
acid cycle will give rise to 12 ATPs. So a total of 12 8 =
96 ATPs will be formed by the complete oxidation of 8
molecules of acetyl CoA by citric acid cycle.
In the activation step, i.e. formation of palmitate oxidation
is (35 + 96 2) = 129.
-oxidation of odd chain fatty acids yield many molecules
of acetyl CoA and a terminal three carbon residue as propionyl
CoA. Propionyl CoA enter the TCA cycle by the following
reactions:
In the formation of palmityl CoA from palmitate, 2
molecules of ATP are consumed.
The net ATP yield per molecule of palmitate oxidation is
(35 + 96 2) = 129.
In addition to -oxidation, the other pathways for fatty
acid oxidation are:
-oxidation
-oxidation is a relatively minor pathway for the production
of energy. Dietary fatty acids which are methylated are meta-
bolized by this pathway.
-oxidation is important in the catabolism of branched chain
and odd chain fatty acids.
Phytanic acid, a metabolic product of phytol (occurs as a
constituent of chlorophyll) is metabolized by oxidation. Phy-
tanic acid, a significant constituent of milk lipids and animal
fat, is metabolized by initial -hydroxylation followed by
dehydrogenation, and decarboxylation.
This pathway is operative in brain and plant tissues.
Refsams Disease
Due to the deficiency of enzyme phytate -hydroxylase,
phytanic acid is not metabolized and accumulate in blood and
tissues giving rise to neurological problems. This is an inborn
error of metabolism.
( ( ( ( (Omega)-oxidation
By this pathway, terminus carbon is oxidized to carboxyl
group to form dicarboxylic acids. Further metabolism takes
place by -oxidation.
Fatty Acid Synthesis
Fatty acids are synthesized by three main processes.
Extra-mitochondrial De Novo Fatty Acid Synthesis
This is the de novo pathway for fatty acid synthesis starting
from acetyl CoA. This pathway is active in liver, kidney, brain,
mammary glands, adipose tissues, etc.
The enzyme is fatty acid synthetase complex, which is a
multienzyme complex system containing acyl carrier protein
and six enzymes to which the growing fatty acid chain is
attached during de novo synthesis. This multienzyme complex
molecule is a dimer consisting of two identical subunits or
monomer. The monomer is not active only the dimer is active.
The enzyme complex contains two SH group, i.e. central and
peripheral.
Malonyl CoA is formed from acetyl CoA by carbon dioxide
fixation reaction also called carboxylation reaction. The enzyme
is acetyl CoA carboxylase and it requires biotin as cofactor.
The source of carbon atoms of fatty acid in acetyl CoA.
Palmitic acid is the normal end product of fatty acid
synthetase complex which requires one molecule of acetyl CoA
and seven molecules of malonyl CoA.
Regulation of De Novo Fatty Acid Synthesis
The rate limiting step in fatty acid synthesis is the carboxylation
of acetyl CoA to malonyl CoA and is catalyzed by the enzyme
acetyl CoA carboxylase. The enzyme consists of two compo-
nents. One contains two proteins, a biotin carboxyl carrier
protein (BCCP) and biotin carboxylase. The second component
is transcarboxylase that catalyses the transfer of carbon dioxide
from BCCP to acetyl CoA.
The enzyme acetyl CoA carboxylase is activated by con-
version from a monomer to a polymer. Citrate activates
enzyme by causing aggregation while long chain acyl CoA
inactivates it by causing it to disaggregate.
Mitochondrial Synthesis of Fatty Acids
This is a chain elongation pathway where the addition
of acetyl CoA to the existing long chain fatty acids takes
place.
Palmitic acid which is a normal end product of de novo syn-
thesis is the precursor of long chain saturated and unsaturated
fatty acids. Elongation of palmitic acid to stearic acid is more
abundant.
Microsomal Pathway of Fatty Acid Synthesis
This pathway also provides a means of elongation of both
saturated and unsaturated fatty acids utilizing malonyl CoA
instead of acetyl CoA.
Main difference between synthesis and degradation of fatty
acids:
Synthesis Degradation
1. Occurs in Cytosol Occurs in mitochondria
2. Acylcarrier = Acylcarrier protein Acylcarrier = CoA
3. Cofactors NADP Cofactor NAD, FAD
4. Synthesized in 2C units Degraded in 2C units
5. 2C units added to -C 2C units taken from the -C end
Triglyceride Synthesis
Biosynthesis of triglycerides takes place in liver, adipose tissue,
lactating mammary gland, intestinal mucosa, muscles and
kidney. The enzymes are present in endoplasmic reticulum.
In the tissues liver, kidney, lactating mammary glands,
glycerol is activated by glycerokinase whereas in adipose
tissues and muscles glycerokinase is absent. The formation
of -glycerophosphate comes from dihydroxy acetone phos-
phate, an intermediate in glycolysis, by reduction catalyzed
by -glycerophosphate-dehydrogenase.
Activation of Glycerol
Activation of Fatty Acid
Cholesterol Biosynthesis
Normal adult synthesizes about 1 to 1.5 gm of cholesterol per
day. Whereas diet provides only 0.3 gm of cholesterol per
day.
In normal human adults cholesterol is found in its largest
amounts in the liver (about 0.3%), skin (0.3%), brain and
nervous tissues (2%), intestines (0.2%) and certain endocrine
glands, with the adrenal glands containing some 10%. As
much as 50% of the myelin sheath there surrounds nerves its
cholesterol.
The relatively high content of cholesterol in skin may be
related to vitamin D formation and that in the adrenals and
certain other endocrine glands to steroid hormone biosynthesis.
Liver is the main site of cholesterol biosynthesis but intes-
tines (intestinal mucosa) is also an important site of synthesis
in man. The tissues where cholesterol synthesis also takes place
are skin, adrenal cortex, testes, aorta, etc.
The cholesterol biosynthesis takes place in the extramito-
chondrial compartment of the cell.
The source of all the carbon atoms in cholesterol is acetyl
CoA. Acetyl CoA is the fundamental or building block unit
of cholesterol biosynthesis.
M is derived from methyl carbon of acetyl group.
C is derived from the carbonyl carbon of acetyl group.
Synthesis of Cholesterol Takes Place in Various Stages
1. Formation of mevalonate from acetyl CoA via HMG
CoA.
2. Three successive phosphorylation followed by decarboxy-
lation to give an isoprene unit which is mainly isopentenyl
pyrophosphate.
3. Condensation of six isoprene units to give C
30
terpene, i.e.
Squalene.
4. Cyclization of squalene to lanosterol.
5. Conversion of lanosterol to cholesterol.
Cholesterol is a product of animal origin. Cholesterol occurs
in egg yolk (the richest source of cholesterol), meat, liver,
brain, etc. The normal serum cholesterol level is 150-250 mg
percent, about 2/3rd of this is present in the esterified form.
The esterification of cholesterol takes place in liver.
Cholesterol is transported in the blood as lipoproteins. The
highest proportion of cholesterol is found in the low density
lipoprotein fraction, i.e. -lipoprotein fraction (LDL).
Cholesterol level in the blood is increased in diabetes
mellitus, nephrotic syndrome, obstructive jaundice, hypopara-
thyroidism, myxodema and xanthomatosis. Low levels of
cholesterol are found in hyperthyroidism, pernicious anemia,
hemolytic jaundice, malabsorption syndrome, severe wasting,
in acute infections, etc.
Cholesterol cannot be catabolized to straight chain molecule
or to acetyl CoA. Therefore, cholesterol cannot be used as
an energy source by the cells.
The combined risk factor of coronary heart disease (CHD)
can be determined following the estimations of serum choles-
terol and HDL-cholesterol. The ratio of serum cholesterol to
HDL-cholesterol has predictive value in determining the risk
of CHD more accurately. For normal males the ratio of 5:1
and for normal females the ratio of 4.5:1 are considered as
average risk. Lower ratios significantly reduce the risk,
whereas ratios of 9.5:1 and 7:1 for males and females respec-
tively, are believed to double the risk of CHD. An inverse
relationship has been observed between the risk of CHD and
the concentration of HDL-cholesterol in the test serum.
HDL-cholesterol represents approximately 20-25% of the
total cholesterol in serum. It is also called good cholesterol,
friendly cholesterol and healthy cholesterol.
HDL-cholesterol may work as a scavenger of cholesterol
from the tissues ridding the body of excess cholesterol. HDL-
cholesterol takes cholesterol away from tissues (extrahepatic)
back to liver for excretion. Low HDL-cholesterol may be
predictive of coronary heart diseases risk whereas high HDL-
cholesterol confers protection. People with high HDL levels
are resistant to the development of atherosclerosis. HDL
removes cholesterol from peripheral tissues and the
cardiovascular system. HDL takes cholesterol away from
tissues (extrahepatic) back to liver for excretion.
People with high LDL levels on the other hand are prone
to development of atherosclerosis. LDL is sometimes called
bad cholesterol (enemy cholesterol) because it may serve as
a source for the cholesterol that accumulates in atherosclerotic
plagues.
The various factors that influence the turnover of body
cholesterol can be illustrated as follows.
Cholesterol serves as a precursor of several important
classes of compounds such as bile acids (liver cells), vitamin
D and neutral sterols (by enteric bacteria) (coprostanol and
cholestanal) steroid hormones, etc. (Progesterone, glucocorti-
coids, mineralocorticoids, androgens, estrogens).
Conversion of cholesterol to steroid hormones is essential
for life. The main steroid hormones include cortisol (a gluco-
corticoid produced by adrenal cortex), aldosterone (a mineralo-
corticoid produced by adrenal cortex), estrogen (a sex hormone
produced by the ovary), testosterone (a sex hormone produced
by the testes) and progesterone (a progestational hormone
produced by the ovary).
All steroid hormones are synthesized from pregnenolone,
a C
21
compound.
Regulation of Cholesterol Biosynthesis
The amount of cholesterol synthesized is, in part, directly
related to cholesterol content in the diet. If the diet contains
large amounts of cholesterol, than the organism will synthesize
little if, however, the diet contains only a small amount of
cholesterol, the organism can produce considerable quantities.
Cholesterol biosynthesis is regulated by negative feedback
mechanism. The rate limiting enzyme in cholesterol biosyn-
thesis is HMG CoA-reductase, which catalyze the conversion
of HMG CoA to mevalonic acid. Dietary cholesterol inhibits
the biosynthesis of cholesterol in liver by depressing the
synthesis of HMG CoA reductase in liver. Fasting inhibits
cholesterol biosynthesis by diverting HMG-CoA to ketone
bodies formation whereas high fat diet accelerate the choles-
terol production.
Atherosclerosis
High levels of cholesterol are associated with atherosclerosis
which is characterized by the deposition of cholesterol ester
and other lipids in connective tissues of arterial walls.
Factors which play a leading part in atherosclerosis are
high blood pressure, obesity, smoking, lack of exercise, etc.
Diet rich in saturated fatty acid, increase the plasma choles-
terol concentration. Replacement of saturated fat with a fat
that is rich in polyunsaturated fatty acids such as linoleic acid,
decreases the plasma cholesterol concentration.
Cornoil, peanut oil and cotton seed oil lower blood choles-
terol while butter fat and coconut oil raises it.
Polyunsaturated fatty acid exerts their effects by
a. Stimulating cholesterol excretion into the intestines
b. Stimulating the oxidation of cholesterol to bile acids
c. Increasing the metabolic rate of cholesterol esters.
Bile Acids
Bile acids are cholic, deoxycholic and lithocholic acids. They
are present in the bile in conjugation with glycine and taurine
as glycocholic and taurocholic acids. Bile acids are the deri-
vatives of cholanic acid.
Deoxycholanic acid is 3,12 dihydroxy cholanic acid. Litho-
cholanic acid is 3-hydroxy cholanic acid.
Salts of bile acids lower the surface tension and are good
emulsifying agents and hence play an important role in the
absorption of fats from the intestine.
Bile salts are polar derivatives of cholesterol and are deter-
gents as they contain polar and nonpolar negious. they are
synthesized in the liver and stored in the gallbladder. They
solubilies dietary lipids so that they can be broken down and
absorbed.
Bile salts are divided into two classes: primary and secon-
dary. Primary bile salts are synthesized by humans. Secondary
bile salts results from the action of intestinal bacteria on the
primary bile salts. The physical and physiological propertis
of the bile salts are similar.
Liver synthesizes 500 mg of bile salts daily. About 94%
of the intestinal bile salts is reabsorbed and 6 percent is lost
in feces.
Ketone Bodies
The ketone bodies are:
1. Acetoacetic acid
2. -hydroxybutyric acid
3. Acetone.
The principle ketone body is acetoacetic acid which gives
rise to -hydroxybutyric acid by reduction and acetone by
decarboxylation.
Ketone bodies are the intermediary breakdown products
of fatty acid metabolism. Under normal conditions fatty acids
are oxidized to carbon dioxide and these intermediary pro-
ducts do not appear to any great extent in blood or urine.
Ketosis
Significant accumulation of ketone bodies in the blood (keto-
nemia) and their excretion in urine (ketonuria) give rise to
a condition known as ketosis.
The total blood concentration of ketone bodies in normal
well fed individuals with a daily urinary excretion of less than
1 mg. Higher than normal urinary or blood concentrations
are called ketonuria and ketonemia respectively. The overall
condition is called ketosis.
Under certain metabolic conditions such as starvation, high
fat diet, severe diabetes, more fat is metabolized for energy
purposes giving rise to increased formation of ketone bodies.
Liver is the net producer of ketone bodies. In liver, ketone
bodies are formed by two ways.
a. HMG-CoA pathway
b. Acetoacetyl CoA pathway.
HMG-CoA Pathway
Condensation of acetoacetyl CoA with another molecule of
acetyl CoA form -hydoxy methyl glutaryl CoA (HMG-CoA).
In the presence of enzyme HMG-CoA lyase. HMG-CoA is
split to acetoacetic acid and acetyl CoA.
Acetoacetyl CoA Pathway
Condensation of two molecules of acetyl CoA give rise to
acetoacetyl CoA. In the presence of enzyme acetoacetyl CoA
deacylase, acetoacetic acid is formed.
Though ketone bodies are synthesized in liver but cannot
be utilized by liver because the enzyme required for the
activation of ketone bodies is absent in the liver. Ketone bodies
pass from the liver to the blood and are oxidized by peripheral
tissues. Cardiac muscle, skeletal muscle and brain prefer ketone
bodies for energy utilization. Before the ketone bodies are
utilized by these tissues they must be activated.
Acetoacetic acid is activated by two ways.
a. Kinase activation
b. Transferase reaction.
Kinase Activation
Transferase Reaction (See Below)
Danger of Ketosis
Acetoacetic acid and -hydroxybutyric acid are fairly strong
acids and are buffered when present in blood or tissues. Their
excretion in the urine results in the loss of buffer cations. Since
ketone bodies are negatively charged, their excretion from
the body is accompanied by excretion of positively charged
ions usually sodium as sodium salts, which depletes the alkali
reserve of the body leading to fall in plasma bicarbonate level,
giving rise to fall in pH. This leads to ketoacidosis, which
may be cause of danger in uncontrolled diabetes mellitus.
Fatty Livers
Significant accumulation of triglycerides in the liver leads to
a condition known as fatty liver. Normally, the liver contains
5% of the lipids. Normal liver is rich in glycogen and not in
fat. But under physiological and pathological conditions the
lipid content rises to 2530%. The increased fat in liver may
result from:
a. Factors associated with increased free fatty acid (FFA)
levelsSuch as (i) Starvation, (ii) Diabetes mellitus (severe,
uncontrolled), (iii) Ketosis, (iv) Pregnancy (toxicemia, etc.).
Here the increased FFA mobilization leads to increased
triglyceride synthesis and accumulation.
b. Due to the deficiency of lipotropic factors such as choline,
methionine, Vitamin E, essential fatty acids, vitamin B
6
or
pantothenic acid.
c. Other intoxicating agents such as carbon tetrachloride,
chloroform, phosphorus, arsenic, lead, alcohol.
Fatty liver is associated with uncontrolled diabetics chronic
alcohol intake obesity and protein malnutrition.
Biochemical Basis of Fatty Liver
Fatty liver falls into two groups.
1. In one group there is some primary factor causing an
increase in free fatty acid either due to increased
mobilization from adipose tissue or increased hydrolysis
of lipoproteins or chylomicrons by lipoprotein lipase. This
increased free fatty acid level leads to increased synthesis
of triglycerides. The production of lipoproteins from
triglycerides specifically the chylomicrons and VLDL, does
not keep pace with the triglyceride synthesis allowing their
accumulation to result in fatty liver. This is the mechanism
in starvation, uncontrolled diabetes, ketosis and toxicemia
of pregnancy.
2. In the other group the defect lies somewhere in the
production of plasma lipoprotein; such a block could be
at any one or more of the following sites:
i. Apoprotein synthesis.
ii. Lipoprotein synthesis from lipid and apoproteins.
iii. Synthesis of lipids-specifically the phospholipids.
iv. Secretory mechanism of the lipoproteins.
Substances which prevent or relieve such abnormal accumu-
lation of lipids in the liver are called lipotropic factors. They
are choline, betaine, methionine, ethomolanine, inositol.
Role of Liver in Lipid Metabolism
Though liver is not the sole organ of lipid metabolism yet
it has the complete enzyme systems to carry out the following
major activities.
1. Synthesis and degradation of fatty acid (-oxidation).
2. Synthesis of triglycerides.
3. Synthesis of cholesterol and its derivatives such as bile acid.
4. Phospholipid synthesis.
5. Synthesis of plasma lipoproteins such as VLDL and HDL.
6. Ketone bodies formation.
Twenty amino acids are present in dietary proteins. These
amino acids are present in L-configuration.
L-form of amino acid is the physiological active form of
the amino acid. The transport of L-amino acids is energy
dependent and require ATP, Na
+
, K
+
, Mn
++
and vitamin B
6
.
While D-form of amino acid is physiological inactive and
is transported by diffusion.
DIGESTION AND ABSORPTION
Two important features of digestion are:
1. It breakdown, the nondiffusible bigger molecules into
diffusible smaller molecules (amino acids).
2. During digestion, the biological specificity of a protein is
destroyed, i.e. they are no longer antigenic, thus averting
allergic reactions to food.
Digestion of Protein by Various Enzymes
Proteins are hydrolyzed to their constituent amino acids by
the action of variety of enzymes.
1. Pepsin: It converts proteins to proteoses and peptones.
2. Trypsin: It cleaves peptide bonds involving carboxyl groups
of arginine and lysine.
3. Chymotrypsin: It cleaves peptide bonds involving carboxyl
groups of phenylalanine, tyrosine and tryptophan.
4. Carboxypeptidases: It cleaves proteins and peptides from the
carboxyl end.
5. Aminopeptidases: It cleaves proteins and peptides from the
amino end.
6. Dipeptidases: It cleaves dipeptides.
Metabolism of Proteins
CHAPTER
10
METABOLISM OF PROTEINS 211
Absorption
L-form is absorbed at much faster rate than the D-form. All
amino acids are absorbed by active process. Active process
requires ATP, pyridoxal phosphate, Mn
++
, Na
+
and K
+
.
Sources of amino acids in the body pool are:
1. Dietary proteins.
2. Intracellular synthesis.
3. Tissue protein breakdown.
Dietary proteins serve three broad functions:
1. Their consituent amino acids are used for the synthesis
of the body proteins.
2. The carbon skeletons of the amino acids are oxidized to
yield energy.
3. Their carbon and nitrogen atoms may be used to synthesize
other nitrogen containing cellular constituents as well as
many non-nitrogen containing metabolites.
How the Amino Acids are Metabolized within the Cell?
Metabolism
Anabolic Phase Catabolic Phase
It is a synthetic phase It is a breakdown phase
1. Protein Biosynthesis 1. Transamination.
Tissue proteins, blood 2. Oxidative deamination.
proteins, enzymes, hormones. 3. Decarboxylation.
2. Synthesis of nonprotein 4. Utilization of nitrogen
nitrogen substances takes residue.
place such as creatine, i. Glutamine synthesis
purines, pyrimidines, ii. Urea cycle.
glutathione, choline, etc.
Transamination
It involves the transfer of an amino group from an amino acid
to the keto group of the keto acid forming a new amino acid
and a keto acid.
The reaction is reversible and is catalyzed by transaminase
enzymes. These enzymes are also known as aminotransferases.
The general reaction is represented as:
The transaminases require pyridoxal phosphate as the coen-
zyme. The pyridoxal phosphate acts as a carrier of amino group
from amino acid to keto acid. The reaction involves the
formation of Schiffs base. The mechanism is represented as:
Transaminases are present in practically all the tissues.
The most abundant of these are the glutamate-oxaloacetate
transaminase (GOT) or aspartate transaminase (AST) and
glutamate-pyruvate transaminase (GPT) or alanine transa-
minase (ALT). GPT is predominent in liver whereas, GOT is
predominent in heart. Determination of the concentration of
GOT and GPT in serum is used to assess the degree of cardiac
and liver damages.
Examples
Alanine, aspartic acid and glutamic acid participate most
in transamination.
Lysine and threonine do not participate in transamination.
Transamination, by converting amino acids to keto acids
(which are prominent in TCA cycle), provide an important
link between the protein and carbohydrate or fat metabolism.
By this way amino acids are sources of energy to the body
by keto acids which undergo complete oxidation.
Also transamination reactions appear to play two major
roles in amino acid metabolism.
i. To serve as a means for the interconversion of number
of amino acids to increase the amount of one that may
be in short supply.
ii. To channel the amino groups of amino acids, ultimately
to glutamate and aspartate.
Decarboxylation
Amino acids are decarboxylated to give amines. The enzyme
is decarboxylase. It requires pyridoxal phosphate as cofactor.
Decarboxylation of amino acids give rise to some of the
biologically active amines (biogenic amines such as histamine,
serotonin and -amino butyrate).
Histidine Histamine
Tyrosine Tyramine
Glutamic acid -aminobutyric acid (GABA)
Tryptophan Tryptamine
5-hydroxy tryptophan 5-hydroxy tryptamine (serotonin)
Arginine Agotomin (in bacteria only).
Oxidative Deamination
Oxidative deamination involves the removal of -amino group
of amino acids to their corresponding keto acids. The enzyme
is amino acid oxidase. In the amino acid oxidase reaction, the
amino acid is first dehydrogenated by the flavoprotein of
oxidase forming an amino acid. This spontaneously adds water
to decompose the corresponding -keto acid with the loss
of -amino nitrogen as ammonia.
For L-form of amino acids, the enzyme is L-amino acid
oxidase. It is FMN dependent.
For D-form of amino acids, the enzyme is D-amino acid
oxidase. It is FAD dependent.
UREA CYCLE (KREBS-HENSELEIT CYCLE)
The deamination of amino acids produces ammonia which is
toxic. By Krebs cycle it is converted to urea, a nontoxic com-
pound, which is transported via the blood to the kidneys and
excreted in the urine. Urea formation takes place mainly in
the liver. Two molecules of ammonia and one molecule of
CO
2
are converted to urea for each turn of the cycle. Urea
is synthesized in the liver. One nitrogen of urea is derived
from ammonium ion, and the second is derived from aspartate.
The carbonyl group is derived from carbon dioxide (as
bicarbonate).
Overall Reaction:
NH
3
+ CO
2
+ 3ATP + 3H
2
O Urea + 2ADP + AMP + 2Pi + PPi
Various stages of urea cycle are:
1. Synthesis of carbamoyl phosphate
2. Synthesis of citrulline
3. Synthesis of arginine
This is divided into two parts:
a. Synthesis of argininosuccinic acid
b. Cleavage of argininosuccinic acid.
4. Synthesis of urea.
Synthesis of Carbamoyl Phosphate
The first step in urea synthesis, is the formation of carbamoyl
phosphate. The enzyme is carbamoyl phosphate synthetase.
The enzyme requires biotin and N-acetyl glutamic acid (AGA)
for activity (i.e. cofactors). AGA is the modifier of the enzyme.
It acts on the noncatalytic site and keeps the enzyme in active
configuration.
Synthesis of Citrulline
Carbamoyl phosphate and ornithine combines to form citrulline.
The reaction is catalyzed by ornithine transcarbamylase.
Synthesis of Arginine
a. Synthesis of argininosuccinic acid
b. Cleavage of argininosuccinic acid.
Argininosuccinic acid is formed from citrulline and aspartic
acid. The reaction is catalyzed by enzyme argininosuccinate
synthetase.
Argininosuccinic acid so formed is cleavaged by enzyme
argininosuccinase to arginine and fumaric acid.
Synthesis of Urea
Enzyme arginase splits arginine to urea and ornithine.
Ornithine so formed again enters the cycle at the second
step and hence, the cycle continues.
In urea cycle, everytime a citrulline passes out of the
mitochondria ornithine passes in. This is what happens and
the protein in the mitochondrial membrane that allows this
to happen is called an ornithine citrulline exchanger.
Urea cycle is a route for biosynthesis of arginine, a semi-
essential amino acid.
Link between urea cycle and TCA cycle is shown below:
The urea cycle eliminates excess ammonia. Ammonia is
derived mainly dietary amino acids that are not used promptly
for protein synthesis.
A human consuming 100 g of protein daily excretes about
16.5 g of nitrogen per day. Urea is the chief nitrogen metabolite
of humans, accounts for 80-90%, of excreted nitrogen. Urate
and ammonium ions are other end products.
Hyperammonia
Hyperammonia or hyperammonemic syndrome is because of
increased level of ammonia in the blood. The urea is formed
from ammonia by urea cycle. So any deficiency or defect of
urea cycle enzymes give elevated levels of ammonia. Hyper-
ammonemia gives rise to mental retardation.
Metabolism of Individual Amino Acids
According to metabolic fates of their skeletons amino acids
are classified in following way:
Glucogenic Amino Acids
Those amino acids which give rise to the intermediates of
carbohydrate metabolism. The product may be pyruvic acid,
fumaric acid, oxaloacetic acid, -ketoglutaric acid, etc.
The glucogenic amino acids are glycine, alanine, asparatic
acid, glutamic acid, arginine, cysteine, histidine, proline, serine,
methionine, valine.
All the nonessential amino acids are glucogenic in character.
The amount of glucose derived from protein can be calcu-
lated by estimating nitrogen and glucose excreted in the urine.
The glucose to nitrogen ratio in urine was found to be 3.65
in diabetic animals which were fed proteins only.
Therefore the amount of glucose derived from 100 gm
of protein is 3.65 16 = 58.4 gm.
The factor 16 is because of the average nitrogen content
of proteins is 16%.
This suggests that 58% of protein is glycogenic. The
glycogenic character of proteins is taken into account while
calculating diet schedule of diabetic patients are made.
Ketogenic Amino Acids
Those amino acids which give rise to the intermediates of fat
metabolism. The product may be acetyl CoA and acetoacetyl
CoA, i.e. ketone bodies.
The ketogenic amino acids are leucine, isoleucine, phenyl-
alanine and tyrosine lysine.
Phenylalanine and tyrosine tryptophase are both glucogenic
and ketogenic amino acids. These amino acids yield aceto-
acetic acid and fumaric acid which can be converted to acetic
acid and pyruvic acid respectively.
GLYCINE
It is a nonessential amino acid and is synthesized by the living
cells. Glycine contains no asymmetric carbon atom and hence
does not exist in D or L-form. Glycine is glycogenic amino
acid.
Metabolism
1. Glyoxalate pathway: It is the main pathway by which glycine
is metabolized:
Hyperoxaluria
The metabolic defect in this disease is due to the disorder
of glyoxalate metabolism where glyoxalic acid is not oxidized
to formic acid, but is converted to oxalate, giving rise to
increased excretion of oxalate in the urine.
2. Serine pathway: Glycine picks up one carbon moiety and
is converted to serine.
This signifies the glycogenic character of glycine.
3. Hemoglobin synthesis: Glycine participates in the synthesis
of heme part of hemoglobin.
Glycine + Succinyl CoA -amino--ketoadipic acid
4. Glycine-choline cycle: See page 223.
5. Purine synthesis: The entire molecule of glycine is incorpo-
rated in the synthesis of purines. The source of C-4, C-5 and
N-7 in purine skeleton is glycine.
6. Synthesis of glutathione: Glutathione is tripeptide, i.e. it
contains three amino acids which are glutamic acid, cysteine
and glycine.
-glutamic acidCysteineGlycine
7. Creatine synthesis: Glycine participate in the synthesis of
creatine. The other two amino acids are arginine and
methionine.
8. Conjugation reaction: For the formation of bile acids, glycine
is important.
Glycine + Cholic acid Glycocholic acid
The following diagram signifies that though glycine is non-
essential but it is metabolically most active amino acid.
In detoxification reactions of body.
Glycine + Benzoic acid Hippuric acid.
Compounds which are toxic detoxified by these reactions.
Glycinuria
This disease is due to the decreased reabsorption of glycine
because of defect in renal tubular transport of glycine.
Glycinuria is characterized by high excretion of glycine in the
urine and tendency to form oxalate renal stones.
Sulfur Containing Amino Acids
The sulfur containing amino acids are methionine, cystine and
cysteine. Methionine is an essential amino acid. The deficiency
of it causes negative nitrogen balance and loss in weight in
man, whereas, cystine and cysteine are nonessential or
dispensible amino acids.
Cysteine is synthesized from methionine and cysteine from
cystine. The presence of cystine in the diet decreases methi-
onine requirement in that it relieves the demand for the for-
mation of cystine.
The structure of methionine, cysteine and cystine are:
METHIONINE
Methionine is the principal methyl donor in the body. The
transfer of methyl group of methionine to appropriate donors
is called transmethylation.
The active form of methionine that functions in methylation
reaction is S-adenosyl methionine also called active methio-
nine. It is a high energy compound.
Methionine + ATP S-adenosyl methionine.
The various transmethylation reactions carried by methio-
nine are:
a. Guanidoacetic acid to creatine
b. Norepinephrine to epinephrine
c. Dimethylethanolamine to choline
d. Nicotinic acid to N-methylnicotinic acid
e. Phosphatidyl ethanolamine to phosphatidyl choline.
CYSTEINE AND CYSTINE
Cysteine and cystine are related by oxidation-reduction
reactions.
Mercaptoethanolamine goes in the coenzyme A synthesis
whereas cysteic acid on decarboxylation gives taurine, which
conjugates with bile acids to produce taurocholic acids.
Metabolic Role of Cysteine
1. It is a major constituent of the proteins of hair and hooves
and the keratin of skin.
2. It forms a part of many proteins in which it takes part in
the formation of disulphide bond such as in insulin.
3. It takes part in the synthesis of coenzyme A.
4. It is a part of glutathione.
5. It is a precursor of taurine that conjugates with cholic
acid.
Metabolism of Cystine and Cysteine
Cystinuria
Excessive excreting of cystine in urine takes place. Also there
is defect in renal reabsorption mechanism for lysine, arginine
and ornithine. This is an inherited disease.
Cystinosis
Deposition of cystine crystals in many tissues and organs takes
place. Aminoaciduria is also present.
Active Sulfate
Coenzyme adenosine-3'-phosphate-5'-phosphosulfate or 3'-
phosphoadenosine-5-phosphosulfate (PAPS) is called active
sulfate.
The structure of active sulfate is given below.
Active sulfate sulfating agent
Active sulfate is involved in the sulphonation of phenols
and of hexosamine derivatives as in the formation of chon-
droitin sulfate and in other sulfanaling transfer reactions, i.e.
sulfolipids or sulfatides. They are formed from cerebrosides
by reaction with active sulfates.
Phenol, steroids also react with PAPS giving respective sulf
ate derivatives which are eliminated in the urine. This
detoxification mechanism takes place in liver.
PHENYLALANINE AND TYROSINE
Both phenylalanine and tyrosine are aromatic amino acids.
Tyrosine is an hydroxylated phenylalanine.
The structures of phenylalanine and tyrosine are:
Phenylalanine is an essential amino acids whereas tyrosine
is nonessential amino acid. Phenylalanine can be converted
to tyrosine but tyrosine cannot give rise to phenylalanine.
Hence, the requirement of tyrosine can be met by the adequate
amount of phenylalanine in the diet.
Phenylalanine and tyrosine are ketogenic amino acids.
Metabolism of Phenylalanine and Tyrosine
Phenylalanine and tyrosine are involved in the synthesis of
a number of important compounds which include thyroxine,
melanin, epinephrine and norepinephrine.
Conversion of phenylalanine to tyrosine takes place in the
liver. The enzyme is phenylalanine hydroxylase. The enzyme
requires molecular oxygen. Fe
++
, NADPH and biopterin as
cofactor.
The reaction is explained below:
Major pathway by which phenylalanine and tyrosine are
metabolized are given as follows:
Phenylalanine and tyrosine metabolism give rise to the syn-
thesis of the important hormones, thyroxine and triiodotyrosine.
Synthesis of melanine
Formation of Thyroxine (T
4
) and Triiodotyrosine (T
3
)
The complete metabolism of phenylalanine and tyrosine is
summed as below:
Inborn errors of metabolism associated with phenylalanine
metabolism.
Inborn Error of Metabolism
There are a number of metabolic abnormalities which are con-
genital, present throughout life and hereditary. Such abnor-
malities are represented as in born error of metabolism.
In some of these conditions failure of a metabolic step leads
to the excretion of intermediate products which cannot be
further metabolize along the metabolic path because of specific
enzyme deficiency but which normally readily metabolizes.
Inborn errors Enzyme deficit
Phenylketonuria Phenylanine hydroxylase,
Tyrosinosis p-hydroxy phenylpyruvate-
hydroxylase.
Alkaptonuria Homogentisic acid oxidase
Albinism Tyrosinase.
Various blocks in the metabolism of phenylalanine giving
rise to different inborn errors of metabolism are shown below:
Phenylketonuria
Phenylketonuria is an inborn error of metabolism associated
with phenylalanine metabolism. The enzyme deficit is phenyl-
alanine hydroxylase. This enzyme catalyze the conversion of
phenylalanine to tyrosine. Due to the deficiency of the enzyme
phenylalanine hydroxylase, the main pathway for the meta-
bolism of phenylalanine via tyrosine is blocked and the minor
alternate pathway takes place. The various metabolites that
accumulate in the blood and excreted in the urine by the minor
pathway are explained below.
Phenylketonuria results in severe mental deficiency and
the children suffering from this disease are mentally retarded
because, the metabolites of phenylketonuria, i.e. phenylpyruvic
acid, phenyllactic acid and phenylacetic acid, inhibit the
formation of serotonin, a brain potent metabolite.
Tyrosinosis
Tyrosinosis is an inborn error of metabolism associated with
phenylalanine metabolism. The enzyme deficit is p-hydroxy
phenylpyruvic acid hydroxylase. Due to the deficiency of this enzyme,
p-hydroxy phenylpyruvic acid is not converted to homogentisic
acid, resulting in the accumulation of p-hydroxy phenylpyruvic
acid in blood and the excretion of p-hydroxy phenylpyruvic acid
and its reduction product, p-hydroxyphenyllactic acid in the urine.
Alkaptonuria
Alkaptonuria is an inborn error of metabolism associated with
phenylalanine metabolism. The enzyme deficit is homogentisic
acid oxidase. The deficiency of homogentisic acid oxidase causes
a block in the metabolic pathway at homogentisic acid,
resulting in its accumulation in the blood and excretion in the
urine. Alkaptonuria is characterized by the excretion of urine
which upon standing gradually becomes darker in color and
finally turns black.
Accumulation of homogentisic acid in body fluid and its
deposition in cartilages and other connective tissues give rise
to a condition called ochronosis.
Albinism
Albinism is an inborn error of metabolism associated with
phenylalanine metabolism. The enzyme deficit is tyrosinase.
Due to the deficiency of enzyme tyrosinase, the conversion
of tyrosine to melanin formation is blocked.
TRYPTOPHAN
Tryptophan is an essential amino acid. It is the only amino
acid containing indole ring. Tryptophan has the metabolic
distinction of giving rise to nicotinic acid, a number of vitamin
B-complex group, during its course of metabolism. It is
ketogenic in nature.
Metabolism of Tryptophan
Tryptophan is metabolized by the following way. The meta-
bolism takes place in liver.
Synthesis of niacin
The major pathway by which tryptophan is metabolized to
niacin is given page 238.
This pathway gives rise to the synthesis of niacin. 60 mg
of tryptophan gives rise to 1 mg of nicotinic acid in the human
body. In diet, tryptophan is not in sufficient amount to meet
the requirement of this vitamin.
Synthesis of serotonin
This is another important pathway by which tryptophan is
metabolized. Serotonin is also called 5-hydroxytryptamine
(5HT), Enteramine or Thrombocytin.
Carcinoid Syndrome: Increased excretion of 5-hydroxy indole
acetic acid (5-HIAA) due to increased production of serotonin
give rise to carcinoid syndrome. Normally one percent of
tryptophan is metabolized by this pathway but in the carcinoid
patients 60 percent of the tryptophan is metabolized by this
pathway. Normal excretion of 5-HIAA is 2-10 mg per day
but in carcinoid syndrome patients it may go as high as 50-
1000 mg per day. The patient of these syndrome exhibit chronic
diarrhea.
Synthesis of Indole and skatole
This is the minor pathway by which tryptophan is metabolized.
The synthesis of indole and skatole takes place in the large
intestines due to certain bacteria. The foul smell of the feces
is due to these.
Synthesis of melatonin
Hartnup Disease
This is an inborn error associated with tryptophan metabolism.
The enzyme deficient is tryptophan pyrrolase.
This disease is characterized by three symptoms:
a. Pellagra like dermatitis
b. Intermittent cerebellar ataxia
c. Mental retardation.
Urine of the patient contains large amount of tryptophan,
indole acetic acid and its glutamine conjugate. Feces also contain
large amounts of tryptophan. In Hartnup disease there is a
deficiency of nicotinic acid because tryptophan is not available
for the synthesis of nicotinic acid.
LEUCINE, ISOLEUCINE AND VALINE
These are the branched amino acids.
Metabolism of Branched Chain Amino Acids
Branched chain amino acids are metabolized as given below:
Maple Syrup Urine Disease
This is an inborn error of metabolism, in infants, resulting
in the block in the metabolism of leucine, isoleucine and valine.
Due to the deficiency of enzyme oxidative decarboxylase (i.e.
step 2), oxidative decarboxylation of -keto acids does not
take place and hence branched chain keto acid derivatives of
leucine, isoleucine and valine accumulate in blood and appear
in urine. The odour of urine of such infants resembles that
of maple syrup.
NUCLEIC ACIDCHEMISTRY AND METABOLISM 241
Nucleoprotein belongs to the category of conjugated proteins,
the nucleic acid part is the prosthetic group and the protein
part consists of protamines and histones, which are basic in
nature.
The successive degradation of the nucleoproteins is shown
below:
Nucleic AcidChemistry
and Metabolism
CHAPTER
11
Bases present in nucleic acids are purines and pyrimidines
Pyrimidine Bases
Three main pyrimidine bases are:
1. Uracil = It is 2,4-dioxy pyrimidine.
2. Thymine = It is 2,4-dioxy-5 methyl pyrimidine.
3. Cytosine = It is 2-oxy-4-aminopyrimidine.
The oxypurines and oxyprimidines exist in enol and keto
form. Enol form is called lactim form and keto form is called
lactum form. Lactum form is predominent of the two.
Purine Bases
Two bases are:
1. Adenine = It is 6-amino purine
2. Guanine = It is 2-amino-6-oxypurine.
Their structures are:
Pentose Sugars
The pentose sugars present are D-ribose and D-2-deoxyribose.
Both sugars occur as furanose form in nucleotides.
Nucleoside
Base-sugar unit is called nucleoside.
Nucleoside = Base Sugar
Purine bases are attached at N-9 position to sugar moiety
whereas pyrimidine bases are attached at N-1 position to sugar
moiety. The nature of linkage is -glycosidic linkage.
Base Sugar Nucleoside
Adenine D-ribose Adenosine
Guanine D-ribose Guanosine
Uracil D-ribose Uridine
Cytosine D-ribose Cytidine
Thymine D-deoxyribose Thymidine
Nucleotides
Nucleotides are phosphorylated nucleosides. They are repre-
sented by base-sugar-phosphate unit.
Nucleotides = Base-sugar-phosphoric acid.
Base Sugar Phosphate Nucleotide
Adenine D-ribose Phosphoric acid Adenylic acid.
Adenosine
monophosphate (AMP)
Guanine D-ribose Phosphoric acid Guanylic acid.
Guanosine
monophosphate (GMP)
Contd...
Contd...
Base Sugar Phosphate Nucleotide
Hypoxan- D-ribose Phosphoric acid Inosinic acid.
thine Inosine mono-
phosphate (IMP)
Uracil D-ribose Phosphoric acid Uridylic acid.
Uridine mono-
phosphate (UMP)
Cytosine D-ribose Phosphoric acid Cytidylic acid.
Cytidine mono-
poshphate (CMP)
Thymine 2-deoxy- Phosphoric acid Thymidylic acid.
D-ribose Thymidine
monophosphate (TMP)
NUCLEIC ACIDS
Nucleic acid are polynucleotide and the nucleotides are linked
by means of 3',5'-phosphodiester bonds.
Nucleic acids are of two types:
1. Deoxyribose nucleic acid (DNA)
2. Ribose nucleic acid (RNA).
Hydrolytic products of DNA and RNA
Deoxyribose Nucleic Acid (DNA)
DNA is a polymer of 2-deoxyadenylic acid, 2-deoxyguanylic
acid, 2-deoxycytidylic acid and thymidylic acid. It is repre-
sented as Base-deoxyribose-phosphate.
Structure of DNA
Watson and Crick proposed the helix structure of DNA, in
which the two polynucleotide chains are wound about a com-
mon axis in the form of a double helix. These two polynucleo-
tide chains are antiparallel, i.e. they run in opposite directions.
The backbone of DNA, which is a constant throughout the
molecule, consists of deoxyriboses linked by 3', 5' phospho-
diester bridges. Polynucleotide chains are so oriented that an
adenine is always located in the space opposite a thymine and
a guanine is opposite a cytosine. This positioning of base is
called base pairing or base complementarity. There exists three
hydrogen bonds between guanine and cytosine. The hydrogen
bonding involves the keto and amino groups of purine and
pyrimidine bases. DNA double helix is stabilized by hydrogen
bonding.
In DNA, purine and pyrimidine bases carry genetic infor-
mation whereas sugar and phosphate groups perform a
structural role.
Ribose Nucleic Acids (RNA)
RNA is a polymer of ribonucleotides. RNA is made up of a
ribosephosphate backbone to which the various bases are
attached. RNA, like DNA, exhibits polarity. The 5 hydroxyl
group points towards the 5 end and 3-hydroxyl group points
to the 3 end of the molecule. The sequence of bases along
the sugar-phosphate backbone determines the primary struc-
ture (information content) of RNA, and this is the factor that
distinguish one RNA from another.
RNA is present in three forms. All the three forms are
present as single strand, each has characteristic molecular
weight and sedimentation coefficient.
1. Transfer or soluble RNA
2. Messenger RNA
3. Ribosomal RNA.
Transfer or Soluble RNA
It comprises 10-20% of the total RNA of the cell.
1. It is present in the soluble fraction of the cell.
2. It is involved in the transfer of amino acids. Each amino
acid has a specific t-RNA.
3. It is a small molecule containing 75 to 90 nucleotides.
4. It has a heterogenous base composition.
5. It has a clover leaf structure and possess a specific triplet
nucleotide known as anticodon, which is complimentary
to the 3 bases on m-RNA called codon.
The structure of transfer RNA is
Messenger RNA
Messenger RNA is synthesized in the nucleus during transcrip-
tion in which sequence of bases in one strand of DNA is trans-
cribed in the form of a single strand of m-RNA. The sequence
of bases in, m-RNA strand is complimentary to that of DNA.
After transcription, m-RNA passes into cytoplasm and then
on to ribosomes where it serves as a template for the sequence
of amino acids during biosynthesis of proteins.
Ribosomal RNA
It comprises 50-80% of the total cellular RNA. Ribosomes carry
ribosomal RNA.
It has a homogenous base composition.
Ribosomal RNA is required to bind m-RNA and specific
enzymes utilized for peptide bond synthesis.
Various fractions of ribosomes are 30s and 50s.
Some Biologically Important Nucleotides
1. Adenosine diphosphate (ADP), adenosine triphosphate (ATP).
They are sources of high energy phosphate bonds and take
part in oxidative phosphorylation.
2. Inosine diphosphate (IDP) and inosine triphosphate (ITP)
participates in phosphorylation reactions.
3. Guanosine triphosphate (GTP) and Guanosine diphosphate
(GDP) participates in citric acid cycle.
4. Uridine diphosphate glucose (UDPG) participate in gluco-
neogenesis.
5. NAD
+
and coenzyme A are of biomedical importance and
are synthesized from amphobolic intermediate.
6. Cyclic AMP and cyclic GMP serves the secondary
messenger function.
7. CDP-Acyl glycerol in lipid biosynthesis acts as high energy
intermediates.
Purines and Pyrimidines Metabolism
Precursors of Purine Ring
N-1 is derived form amino nitrogen of aspartate.
N-3 and N-9 derived from amide nitrogen of glutamine.
N-2 and C-8 are derived from format.
C-6 is derived from respiratory carbon dioxide.
C-4, C-5 and N-7 are derived from glycine.
Precursors of Pyrimidine Ring
N-3 is derived from amide nitrogen of glutamine.
C-2 is derived from respiratory carbon dioxide.
N-1, C-4, C-5 and C-6 are derived from aspartate.
Biosynthesis of the Purine Ribonucleotides
The starting material for the de novo synthesis of purine ribo-
nucleotide is an activated form of D-ribose-5-phosphate on
which purine ring is built up step-by-step.
D-ribose-5-phosphate, derived from pentose phosphate
pathway is converted to 1-pyrophosphate ribose-5-phosphate
(PPRP) by the transfer of pyrophosphate group from ATP to
C-1 of ribose.
The first ribonucleotide formed by this pathway is inosinic
acid which is a precursor of adenylic acid and guanylic acid.
Biosynthesis of Pyrimidine Nucleotides
This biosynthetic pathway involves the formation of pyri-
midine ring first from its chain precursors followed by the
attachment of D-ribose-5-phosphate moiety.
The first pyrimidine ribonucleotide formed is uridylic acid
(UMP) which is a precursor of cytidine nucleotides and
thymidine deoxynucleotides.
Orotic Aciduria
It is an inherited metabolic disorder of pyrimidine biosynthesis,
characterized by accumulation of orotic acid in blood and its
excretion in urine due to deficiency of enzyme orotidylic acid
phosphorylase and orotidylic acid decarboxylase.
Salvage Pathway
De novo synthesis of purines is the main pathway by which
purines bases are synthesized in the body. But there exists
another pathway in the body called salvage pathway by which
purine nucleotides are also formed.
Salvage pathway involves the synthesis of purine nucleo-
tides from free purine bases and purine nucleotides which
are salvaged from dietary sources and tissue breakdown. This
pathway is mainly operative in leukocytes, brain, etc. which
are not capable of de novo synthesis of purine nucleotides. Since
90 percent of the free purines formed by man are salvaged
and recycled by this pathway and hence this pathway is
important in purine economy in the vertebrates.
Salvage reactions promoted by the action of two enzymes
which convert free purines into corresponding purine nucleo-
tides for reuse are Adenine phosphoribosyl transferase and
Hypoxanthine-guanine phosphoribosyl transferase (HGPR
Tase).
The reactions performed by these enzymes are as
follows:
Lesch-Nyhan Syndrome
Deficiency of enzyme hypoxanthine-guanine phosphyribosyl
transferase (HGPR Tase) give rise to Lesch-Nyhan syndrome,
a genetic disorder. This enzyme catalyze the transfer of ribose
phosphate group of 5 PRPP to either guanine, xanthine or hypo-
xanthine. When this enzyme is deficient guanine, xanthine and
hypoxanthine are not salvaged and hence degraded to uric
acid.
Catabolism of Purines
This first step in the catabolism of purines is their hydrolytic
deamination to hypoxanthine, i.e. adenine is converted to
hypoxanthine and guanine to xanthine. In the second step both
xanthine and hypoxanthine are oxidized to uric acid.
In man, the end product of purine catabolism is uric acid.
Whereas in lower primates, the enzyme uricase, hydrolyze
uric acid to allantoin.
Catabolism of Pyrimidines
The catabolism of pyrimidines takes place mainly in the liver
and the breakdown pathway is represented as:
The breakdown of pyrimidines gives rise to the formation
of -alanine and -aminoisobutyric acid. -alanine is metabolized
to acetate, carbon dioxide and ammonia, whereas -aminoiso-
butyric acid is metabolized to propionic acid, which in turn
gives rise to succinic acid.
-alanine is an important constituent of pantothenic acid
and therefore, of coenzyme A. Also -alanine is a component
of carnosine and anserine which are synthesized by the muscle
and brain.
Regulation of Purine Synthesis
Purine biosynthesis is regulated by feedback inhibition at the
following sites:
a. Feedback inhibition of 5-phosphoribose pyrophosphate
synthetase by AMP, GMP and IMP.
b. Feedback inhibition of amidotransferase by the final
products of the pathway ATP, ADP, AMP, GTP, GDP, GMP,
IMP, IDP and IMP.
c. Inosinic acid (IMP) is at a branched point in the synthesis
of AMP and GMP. AMP inhibits the conversion of IMP
to adenylosuccinate by inhibiting the enzyme adenylosucci-
nate dehydrogenase. Similarly, GMP inhibits the conversion
of IMP to xanthylic acid by inhibiting the IMP dehydro-
genase.
d. ATP is a substrate in the synthesis of GMP whereas GTP
is a substrate in the synthesis of AMP. This reciprocal
substrate relationship regulates the synthesis of AMP and
GMP, i.e. GMP synthesis is accelerated when AMP levels
are high.
Regulation by Pyrimidine Biosynthesis
The committed step in pyrimidine biosynthesis is the formation
of N-carbamoyl aspartate from aspartate and carbamoyl phos-
phate. The enzyme is aspartate transcarbamylase. This enzyme
is feedback inhibited by CTP, the final product in the pathway.
The second control site is carbamoyl phosphate synthesis which
is feedback inhibited by UMP.
Gout
Gout is a metabolic disease, the salient biochemical feature
of which is hyperuricemia. As a result of hyperuricemia, large
amounts of uric acid (as sodium salt) are deposited in the
joints and tissues (tophi).
Gout is of two types:
1. Metabolic gout
2. Renal gout.
Metabolic Gout
i. Primary metabolic gout
ii. Secondary metabolic gout.
Primary metabolic gout: This is due to inherited metabolic
defect in purine metabolism leading to increased rate of conver-
sion of glycine to uric acid.
Secondary metabolic gout: This is due to increased catabolism
of nucleic acids, i.e. polycythemia, leukemia, etc.
Renal Gout
i. Primary renal gout
ii. Secondary renal gout.
Primary renal gout: In this type of gout, the defect lies in
the kidney where there is faulty enzymatic transport of urates
by the renal tubules. The rate of excretion of uric acid is
lowered.
Secondary renal gout: This is due to glomerulonephritis or
some other destructive process leading to kidney failure.
VITAMINS 259
In addition to oxygen, water, proteins, fats, carbohydrates
and inorganic salts, a number of organic compounds are also
necessary for the life, growth and health of animals including
man. These compounds are known as the accessory dietary
factors or vitamins and are only necessary in very small
amount. Vitamin cannot be produced by the body and hence,
must be supplied.
Vitamins are defined as organic compounds, occurring in
natural food either as such or as utilizable precursors which
are required in minute amounts for normal growth,
maintenance and reproduction. They differ from other organic
foodstuffs in that they do not enter into the tissue structure
and do not undergo degradation for the purpose of providing
energy. The absence of these results in deficiency disease.
Most of the vitamins are supplied by the diet. Very few
vitamins which are synthesized in the intestine belongs to the
vitamin B group.
Vitamins which are synthesized in the intestinal flora are:
vitamin K, thiamine, riboflavin, pyridoxine, folic acid, niacin
and biotin.
But the entire requirement of these vitamins are not met
by the endogenous synthesis.
Vitamin deficiencies are must often the result of consuming
monotonous diet, i.e. diet-based on limited number of food
sources. The requirements for vitamins are usually greatest
during the neonatal period.
The vitamins have been classified into:
1. Fat soluble vitamins: They are soluble in fat solvents.
Vitamins in this group are vitamins A, D, E and K.
Vitamins
CHAPTER
12
2. Water soluble vitamins: They are water soluble and
includes vitamin C and vitamins of B-complex.
Most of the vitamins form the integral part of coenzymes.
Fat soluble vitamins are stored in our fat deposits (liver
and adipose tissue) and water soluble vitamins are constantly
flushed from our bodies. Therefore, we can do without lipid
soluble vitamins for a resonable amount of time but we must
keep replacement the water soluble vitamins. Vitamins acts
as coenzyme, antioxidants, (free radical quenching agents)
as signalling agents in the cells, as regulator of gene expression
and as redox.
Vitamin Like Compounds
Those compounds which are highlighted because of their
known role as coenzymes in prokaryotes eukaryotes or roles
as a probiotic (growth promoting substance) in higher animals
are defined as vitamin like.
Vitamin like substances are taurine, queuosine, coenzyme
Q, pteridines (other then folic acid), such as biopterin and the
molybdenum containing pteridine cofactor, pyrroloquinoline
quinone (PQQ).
Vitamins as Coenzymes
Vitamins Active form Functions performed
Thiamine Thiamine pyrophosphate Aldehyde group
transfer
Riboflavin Flavin mononucleotide Hydrogen group
(FMN) transfer
Flavin adenine Hydrogen group
dinucleotide (FAD) transfer
Pantothenic acid Coenzyme A Acyl group transfer
Nicotinamide Nicotinamide adenine Hydrogen transfer
dinucleotide (NAD)
Nicotinamide adenine Hydrogen transfer
dinucleotide phosphate
(NADP)
Pyridoxine Pyridoxal phosphate Amino group transfer
Contd...
VITAMINS 261
Biotin Biocytin Carboxyl group
transfer, i.e. CO
2
fixing
Folic acid Tetrahydrofolic acid, Methyl, methylene,
i.e. Folacin formyl or formimino
group transfer
Cyanocobalamin Cobamides Alkyl group transfer
Lipoic acid Lipoyl lysine Acyl group transfer
FAT SOLUBLE VITAMINS
VITAMIN A
Retinol, growth promoting vitamin, anti-infective vitamin,
antixerophthalmia.
Structure
Vitamin A occurs in two forms, vitamin A
1
and vitamin A
2
.
The structure of vitamin A
1
is:
Vitamin A
2
contains an additional double bond between
C-3 and C-4.
Vitamin A
2
is 40 percent active to vitamin A
1
.
Certain carotenes called provitamins A are converted into
vitamin A in the body. -carotenes give rise to two molecules
of vitamin A whereas - and -carotenes give rise to one
molecule each of vitamin A.
Functions
1. The most important function of vitamin A is in the visual
cycle (page 262).
Retina contains conjugated protein rhodopsin. Rhodop-
sin consists of protein opsin and vitamin A
1
aldehyde.
Rhodopsin is the major light receptor of rod cells. Under
Contd...
the influence of light rhodopsin is converted to transretinal
and opsin. Transretinal is inactive in the resynthesis of rho-
dopsin. Transretinal is converted to transretinal by reductase
which is also inactive in rhodopsin synthesis, is passed into
blood stream. During resynthesis of rhodopsin, which occurs
in dim light and in the dark, active cis-retinal enters the
retina from the blood and is oxidized to cis-retinal by retinal
action of retinal reductase. Now cis-retinal couples with
opsin to form rhodopsin. The visual process involves the
removal of active retinal isomer from the blood by the retina
which returns the inactive isomer to the circulation.
2. In the maintenance of proper health of epithelium tissues.
3. For the stability and integrity of cellular and subcellular
membranes.
4. Necessary for the synthesis of mucopolysaccharides as it
helps in the incorporation of sulfur in chondroitin sulfate.
5. It is also involved in the nucleic acid metabolism.
6. It is also involved in the election transport chain and in
oxidative phosphorylation.
Sources
Provitamin sources: Food rich in carotenes such as carrot,
papayas, tomatoes, etc.
Readymade or preformed sources: Fish liver oils such as shark,
cod, halibut fish, liver oils, egg yolk, butter, milk products.
Visual cycle
VITAMINS 263
Daily Requirement 5000 IU
Deficiency Disease
Deficiency of vitamin A gives rise to night blindness.
Hypervitaminosis A
Excessive intake of vitamin A gives rise to hypervitaminosis A.
The symptom of this toxicity include anorexia, irritability,
headache, peeling of skin, vomiting.
VITAMIN D
The term vitamin D does not refer to a single dietary
factor but to a number of chemically related compounds, all
of which have the property of preventing or curing rickets.
The two most active substances in this respect are vitamin
D
2
and vitamin D
3
.
Structure
Vitamin D
2
is also known as ergocalciferol and vitamin D
3
as cholecalciferol.
Irradiation of 7-dehydrocholesterol with ultraviolet radia-
tions produces cholecalciferol whereas irradiation of ergosterol
produces ergocalciferol.
Vitamin D
2
differs from vitamin D
3
with respect to the
double bond additionally present in the side chain at position
20 and 21.
The biologically active form of vitamin D are 25-hydroxy
cholecalciferol and 1,25-cholecalciferol.
Liver converts cholecalciferol to 25-hydroxy cholecalciferol
(25 HCC) whereas kidney converts 25-HCC to 1,25 dihydroxy
cholecalciferol (1,25-DHCC).
Another important active form formed by the kidney is
24,25-dihydroxy cholecalciferol (24,25-DHCC) but very little
is known of biological function of this form.
1,25-dihydroxy cholecalciferol perform the following func-
tions:
i. It promotes calcium absorption from the intestine
ii. It promotes mobilization of calcium from bones.
Functions
1. The basic action of vitamin D is to increase the absorption
of calcium and phosphorus from the intestines.
VITAMINS 265
2. Vitamin D promotes resorption of bone and mobilization
of calcium from bones.
3. Vitamin D increases excretion of phosphate by the kidney.
Sources
Fish liver oils (i.e. cod liver oil, shark liver oil, halibut liver
oil), egg yolk, milk.
Daily requirement 400 IU infants and children
100 IU adults
400 IU pregnancy and lactation
Deficiency Disease
Deficiency of vitamin D gives rise to rickets in children and
osteomalacia in adults.
In rickets, there is a fall in intestinal absorption of calcium
and phosphate, increased excretion of urinary phosphate and
loss of calcium from the bones, leading to softness and
deformities of bones.
In rickets and osteomalacia, there is an increase in serum
alkaline phosphatase activity.
Hypervitaminosis D
Doses above 1500 units per day for long period rise to vitamin
D toxicity.
Excessive intake of vitamin D causes loss of appetite,
nausea, irritability, excessive mobilization of calcium from
bones into blood.
VITAMIN E
(Antisterility vitamin, fertility factor)
Vitamin E refers to a group of compounds having vitamin E
activity and are known as tocopherols. Four unsaturated
alcohols, i.e. , , and tocopherols occur in nature. These
tocopherols differ slightly in structure in their side chain,
-tocopherol is most potent of them.
They are thermostable and sensitive to the effects of oxidi-
zing agents and ultraviolet rays.
Functions
1. Tocopherols act as powerful antioxidants:
a. They prevent the autoxidation of vitamin A and caro-
tenes.
b. They prevent the formation of fatty acid peroxidases
in tissues due to the autoxidation of unsaturated fatty
acids with oxygen.
c. They protect the lipids of biological membranes against
oxygen by acting as antioxidants (i.e. prevent the pero-
xidation of polyunsaturated fatty acids that occur in
membranes throughout the body).
2. Vitamin E prevents rancidity.
Sources
Wheat germ oil, corn oil, peanut oil, soyabean oil, sunflower
oil, egg yolk, leaves of spinach, alfalfa, sweet potatoes.
Daily Requirement 15 IU
Vitamin E intake is related to the intake of polyunsaturated
fatty acids, i.e. 0.4 mg per gm of polyunsaturated fatty acids.
Deficiency Disease
Deficiency of vitamin E gives rise to sterility in rats.
VITAMIN K
(Coagulation factor)
Two naturally occurring vitamin K are vitamin K
1
and vitamin
K
2
. Both are naphthaquinone derivatives.
Structure
The structure of -tocopherol is:
VITAMINS 267
Structure
Vitamin K
1
is phylloquinone and is chemically known as
2-methyl-3-phytyl-1, 4-naphthaquinone.
Vitamin K
2
is farnesoquinone and is chemically known as
2-methyl-3 difarnesyl-1, 4-naphthaquinone.
Vitamin K is thermostable can withstand reduction and
is rapidly oxidized both in acidic and alkaline medium. It is
completely destroyed by ultraviolet radiations.
Certain compounds having vitamin K activity are called
vitamines. Vitamines are synthetic compounds possessing
vitamin K activity (Example: Menadione). Menadione is more
potent than vitamin K
1
on weight basis.
Functions
1. In the synthesis of prothrombin.
2. It is involved in oxidative processes taking place in photo-
synthesis of plant kingdom.
3. It is involved in electron transport chain and is involved
in oxidative phosphorylation.
Vitamin K cycle
Sources
Vitamin K
1
, is present in alfalfa, spinach, cabbage, cauliflower,
egg yolk, liver.
Vitamin K
2
is present in putrifying fish. It is also synthesized
by intestinal flora.
Daily Requirement
Sufficient amounts of vitamin K are synthesized by intestinal
bacteria so there is no dietary requirement under physiological
condition.
Deficiency Disease
Vitamin K deficiency gives rise to hypoprothrombinemia which
leads to prolongation of prothrombin time.
WATER SOLUBLE VITAMINS
Water soluble vitamins includes vitamin C and members of
vitamin B complex.
Vitamin C
(Ascorbic acid, anti-scorbutic vitamin)
Structure
VITAMINS 269
Vitamin C, which is hydrophilic, acts as an antioxidant in
solution.
Vitamin C is a powerful reducing agent and is oxidized
to dehydroascorbic acid. Both forms are biologically active.
It is stable in acidic solution at low temperatures but undergoes
destruction in alkaline solution when in contact with air.
Vitamin C is not synthesized by man and its entire
requirement is met by diet.
The most rich sources of vitamin C in the body are adrenal
cortex, corpus luteum, pituitary, pancreas, liver, etc.
Functions
1. Participation in the hydroxylation of proline and lysine
present in collagen, an intracellular cementing substance.
2. Participates in the synthesis of steroid hormones both in
adrenal cortex and corpus luteum.
3. Participates as cofactor in the following reaction:
a. In phenylalanine metabolism.
p-hydroxy phenylpyruvic acid homogentisic acid
b. Dopamine Norepinephrine
c. Folic acid Folinic acid.
4. Vitamin C is necessary for the synthesis of carnitine in the
liver.
5. Necessary for the absorption of iron by reducing ferric
form to ferrous form.
6. In tissue respiration, i.e. oxidation-reduction phenomenon.
7. In bile acid formation, vitamin C is required at the 7--
hydroxylase step.
8. Ascorbic acid may act as water soluble antioxidant and
inhibit the formation of nitrosamine.
Sources
Citrus fruits such as lemon, orange, pineapple, etc. Indian goose-
bury, green pepper, cauliflower, tomatoes, spinach, potato.
Daily requirement: 60-80 mg.
Deficiency Disease
Deficiency of vitamin C gives rise to scurvy. The early mani-
festation in man are swelling of joints, hemorrhage in skin,
muscle, gastrointestinal tract, inflammation of gums, ulce-
ration, etc. In scurvy, vitamin C level in blood falls down.
Vitamin B Complex
The members of this group are:
1. Thiamine (B
1
).
2. Riboflavin (B
2
).
3. Pantothenic acid (B
3
).
4. Choline (B
4
).
5. Niacin (B
5
).
6. Pyridoxine (B
6
).
7. Biotin (B
7
).
8. Folic acid (B
9
).
9. Cyanocobalamin (B
12
).
10. Para amino benzoic acid.
11. Inositol.
12. Lipoic acid.
The vitamins of this family have been grouped together
because of the following fulfilment:
1. Usually present in yeast.
2. Present in the outer covering of seeds and cereals.
3. Synthesized by the microorganisms in the intestines.
4. They are water soluble.
5. Required in minute amounts and their deficiencies give
rise to ordinary manifestations.
6. They usually serve as a coenzyme of various enzyme
systems. Foods that are poor sources of one of the B
vitamins level to be the poor sources of several B vitamins.
THIAMINE
(Vitamin B
1
, anti-neuritic vitamin, anti-beriberi factor,
aneurin)
Structure
VITAMINS 271
Thiamine consists of a substituted pyrimidine ring joined
by a methylene bridge to substituted thiazole ring.
Thiamine is water soluble. It is thermolabile, destroyed
in alkaline medium but thermostable in acidic medium.
Thiamine occurs in the cells largely as its active coenzyme
form, i.e. thiamine pyrophosphate, also called cocarboxylase.
Functions
Thiamine pyrophosphate (TPP) is used mainly as a coenzyme
in the carbohydrate metabolism.
1. In oxidative decarboxylation of -ketoacids.
Pyruvic acid and -ketoglutaric acid which are inter-
mediates in the carbohydrate metabolism are oxidatively
decarboxylated to acetyl CoA and succinyl CoA respectively.
Pyruvic acid Acetyl CoA
-ketoglutaric acid Succinyl CoA
2. In transketolation reaction.
An intermediate step in hexose monophosphate shunt path-
way where transfer of glycolaldehyde group from D-xylulose-
5-phosphate and glyceraldehyde.
D-xylulose-5-PO
4
D-sedoheptulose-PO
4
+ +
D-ribose-5-PO
4
D-glyceraldehyde-PO
4
3. In yeast TPP acts as coenzyme for nonoxidative decar-
boxylation of -keto-acids, i.e. pyruvate to CO
2
and acetal
dehyde.
Sources
Yeast, outer coating of seeds, cereals, legumes, wheat, pork,
eggs.
Vitamin B
1
is also synthesized during germination.
Daily Requirement: 1-1.4 mg.
Thiamine requirement rises with a rise in caloric intake
of food, i.e. 0.5 mg of vitamin B
1
for every 1000 calories in
the form of carbohydrates.
Deficiency Disease
Deficiency of vitamin B
2
gives rise to beriberi in man, and
polyneuritis in birds and animals.
Rise in blood pyruvic acid is the best biochemical test for
vitamin B
1
deficiency.
RIBOFLAVIN
(Vitamin B
2
, lactoflavin)
Structure
Riboflavin is a derivate of isoalloxazine, i.e. dimethyl-iso-
alloxazine attached to ribotyl group.
Riboflavin is thermolabile, destroyed in alkaline medium,
active in acidic medium. Its aqueous solution gives a yellow-
green fluorescence.
Functions
1. Riboflavin is present as such in retina where it plays a part
in light adaptation.
2. Riboflavin exists as component of two coenzymes called
flavin mononucleotide (FMN) and flavin adenine dinu-
cleotide (FAD) which acts as coenzymes or prosthetic group
of many flavoprotein enzymes.
Examples of flavoprotein containing FMN and FAD as
prosthetic groups are:
VITAMINS 273
Flavin Mononucleotide (FMN)
a. Warburg yellow enzyme
b. L-amino acid oxidase
c. Cytochrome C reductase.
Flavin Adenine Dinucleotide (FAD)
a. D-amino acid oxidase
b. Xanthine oxidase
c. Glycine oxidase
d. Thiophorase oxidase
e. Acyl CoA dehydrogenase.
Sources
Yeast, milk, egg, meat, fish, liver, kidney, green leafy vegetables.
Daily requirement: 0.4-1.4 mg
Deficiency Disease
Riboflavin deficiency does not give rise to any clear cut disease
but the important deficiency symptoms are choliosis, fissures
in lips, mouths, inflammation of the tongue, dermatitis.
Riboflavin is excreted mostly in fecal matter. In urine, a
pigment known as urochrome is excreted which is very much
related to riboflavin.
NIACIN
(Pellagra preventive factor)
Structure
Niacin is a pyridine derivative, thermostable and is not
rapidly oxidized.
Functions
Niacinamide is a component of nicotinamide adenine
dinucleotide (NAD
+
) and niacinamide adenine dinucleotide
phosphate (NADH
4
) and acts as coenzyme for many anaerobic
dehydrogenases by accepting hydride ions during oxidation
of their substrates.
Enzymes requiring NAD
+
or NADH coenzymes are:
a. Glyceraldehyde-3-PO
4
dehydrogenase
b. Lactate dehydrogenase
c. Ketose reductase
d. Malic dehydrogenase.
Enzymes requiring NAD
+
or NADPH as coenzymes are:
a. Isocitrate dehydrogenase
b. Glucose-6-PO
4
dehydrogenase
c. Aldolase reductase.
Sources
Yeast, meat, liver, kidney, eggs, fish, legumes.
Nicotinic acid is also synthesized during tryptophan meta-
bolism, 60 mg of tryptophan gives rise to 1 mg of niacin.
Deficiency Disease
Deficiency of niacinamide gives rise to pellagra in man and
black tongue in dogs.
Pellagra affects the skin, central nervous system and the
gastrointestinal tract.
The three important symptoms of pellagra are diarrhea,
dermatitis and dermentia.
Diets such as corn maize give rise to niacin deficiency and
ultimately to pellagra because corn or maize are distinctly
deficient in tryptophan.
The important excretory products of nicotinic acid in human
urine are nicotinic acid as such, nicotinamide, N-methylniacina-
mide, nicotinuric acid and 6-pyridone of nicotinic acid.
VITAMINS 275
PANTOTHENIC ACID
Structure
Pantothenic acid is a peptide like compound formed from
pantoic acid and -alanine.
It is thermostable and resistant to oxidation.
Functions
Pantothenic acid is a part of coenzyme, which serves as carrier
of acyl group in enzymatic reactions.
1. Fatty acid oxidation
2. Fatty acid synthesis
3. Pyruvic acid oxidation
4. Biological acetylations
5. Cholesterol biosynthesis
6. In acyl carrier proteins.
Sources
Yeast, liver, kidney, egg yolk, molasses.
Daily Requirements
Synthesized by the intestinal flora in sufficient amount to meet
the requirement.
PYRIDOXINE
(Antiachrodynia factor)
Structure
Vitamin B
6
refers to a group of pyridoxine, pyridoxal and
pyridoxamine compounds having similar biological activity.
Pyridoxine is most abundant in plants, and pyridoxal and
pyridoxamine are most abundant in animal tissues.
Pyridoxal and pyridoxamine are thermolabile and photo-
labile. Pyridoxine can be converted to pyridoxal and pyridoxa-
mine but pyridoxal and pyridoxamine cannot be converted
back to pyridoxine.
All are biologically active but only the phosphate esters
of pyridoxal and pyridoxamine can function as coenzymes.
Functions
Pyridoxine containing enzymes is important in amino acid and
protein metabolism.
1. Pyridoxine acts as coenzymes for decarboxylation reactions:
a. Histidine Histamine
b. Tyrosine Tyramine
c. 5-hydroxy tryptophan 5-hydroxy tryptamine (5HT)
d. Glutamic acid -amino butyric acid (GABA)
e. -amino--keto adipic acid -amino levulinic acid
2. As coenzyme in transamination reaction.
3. As coenzymes in dehydrases reactions.
Serine Pyruvic acid
Threonine -ketobutyric acid
4. As coenzymes in trans-sulfurases reaction.
Homocysteine Serine
5. As coenzymes for desulfuration reactions.
Cysteine Pyruvic acid
Homocysteine -ketobutyric acid
6. In tryptophan metabolism, the conversion of kynurenine to
anthranilic acid requires pyridoxal phosphate as coenzyme.
VITAMINS 277
In vitamin B
6
deficiency, it is converted to xanthurenic acid.
So measurement of xanthurenic acid is an indication of vitamin
B
6
deficiency.
7. Also helps in the transport of amino acids to the cell
membrane and is also necessary for the absorption of amino
acids from the intestinal mucosa.
8. Also involved in the conversion of linolenic acid to arachi-
donic acid, hence, it is essential for essential fatty acid
synthesis.
Sources
Yeast, liver, egg yolk, rice polishings.
Synthesized by the microflora of the intestines.
Daily requirement: 2-2.5 mg.
BIOTIN
Structure
Biotin contains fused imidazole and thiophene.
Biotin is present in food both in free and in combined form
with proteins. The combined form is liberated by proteolytic
enzymes.
Biotin is also synthesized by intestinal flora.
Functions
Biotin is required in carbon dioxide fixation reactions. Reactions
where biotin is involved are:
1. Acetyl CoA Malonyl CoA (Fatty acid synthesis)
2. Pyruvic acid Oxaloacetic acid (Glucongenesis)
3. Propionyl CoA D-methyl malonyl CoA
4. CO
2
+ NH
3
Carbamyl phosphate (Urea cycle)
5. In purine ring synthesis, i.e. the C-6 position in purine ring
skeleton.
Structure of coenzyme A
Sources
Yeast, egg yolk, milk, molasses, chocolate, tomato, peanuts.
Daily requirement: 200 g.
Deficiency Disease
Raw egg induces biotin deficiency because it contains a protein
avidin which tightly binds biotin and prevents its absorption
from the intestine.
VITAMINS 279
FOLIC ACID
Structure
Folic acid consists of three components:
i. Pteridine nucleus
ii. Para-aminobenzoic acid.
iii. Glutamic acid.
Folic acid occurs in nature in three types:
a. Pteroyl monoglutamate which contains only one
molecule of glutamic acid.
b. Pteroyl triglutamate which contains three molecules of
glutamic acid.
c. Pteroyl heptaglutamate which contains seven molecules
of glutamic acid.
Before folic acid can function as coenzyme in various meta-
bolic reactions it must be reduced to tetrahydrofolic acid.
Folic acidDihydrofolic acidTetrahydrofolic acid (TH
4
).
The various activated forms of folic acid are:
N
5
-formyl TH
4
, N
10
-formyl TH
4
N
5-10
-methenyl TH
4
, N
5-10
-methylene TH
4
.
N
5
-TH
4
contains formyl group at 5-position. It is also called
folinic acid. TH
4
is biologically inactive except that it has only
one role, i.e. in the formylation of glutamic acid in histidine
metabolism whereas N
10
-TH
4
and N
5
10
-TH
4
are the biologically
most active forms of tetrahydrofolic acid.
Folic acid coenzymes are collectively known as folacin.
Functions
Folic acid coenzymes are involved in the transfer and incorpo-
ration of single or one carbon moiety.
One carbon moieties are:
i. Formyl group and formate group,
i.e.CHO and HCOOH
ii. Hydroxymethyl group, i.e. CH
2
OH
iii. Methyl group, i.e. CH
3
iv. Formimino group, i.e. CH=NH
They are inter convertible:
CH
2
OH CHO -COOH
Sources of one carbon moieties are:
i. -carbon of glycine gives rise to CHO group
ii. Histidine gives rise to CH=NH group
iii. Choline donates CH
3
group via betaine (choline as such
cannot donate methyl group)
iv. Biotin gives rise to CH
3
group
v. -Carbon of serine gives rise to CHO group.
Utilization of one carbon moiety:
1. Conversion of ethanolamine to choline.
2. Conversion of glycine to serine.
3. Conversion of norepinephrine to epinephrine.
4. Conversion of guanidoacetic acid to creatine.
5. Conversion of uracil to thymine.
6. Conversion of ribonucleotides to deoxyribonucleotides.
7. In the formation of N-formylmethionine transfer RNA.
8. In purine synthesis, i.e. C-3 and C-8 positions come
through one carbon moiety.
FIGLU excretion test (Formiminoglutamic acid excretion test).
This is a diagnostic test for finding folic acid deficiency.
In the histidine metabolism, the conversion of N-formi-
minoglutamic acid to glutamic acid is a folic acid dependent
step. When folic acid deficient patients are given an increased
load of histidine, there is an increased excretion of formimino-
glutamic acid in urine due to the nonconversion of above step
due to folic acid deficiency. So increased histidine load test
is used to find the folic acid deficiency.
VITAMINS 281
Sources
Yeast, liver, kidney, green vegetables.
Daily requirement: 0.4-0.8 mg.
Deficiency Disease
The main deficiency symptoms are anemia, i.e. the reduced
ability to produce red blood cells.
CYANOCOBALAMIN
(Vitamin B
12
, antipernicious factor,
castles extrinsic factor)
Structure
Cyanocobalamin is made up of two components.
The larger component is corrin ring system, containing four
pyrrole rings. One of the pair of pyrrole rings is joined directly.
Cobalt is coordinated to the four nitrogen of the pyrrole rings,
the second component is a ribonucleotide, 5,6 dimethyl benzi-
midazole joined to corrin by a nitrogen atom of nucleotide
and cobalt atom and by an ester linkage between the 3 phos-
phate group of the ribonucleotide and a side chain of the corrin
ring.
Replacement of cyanide group by hydroxy group, nitro
group, and methyl group forms hydroxy cobalamin, nitro coba-
lamin and methyl cobalamin respectively. All of them possess
vitamin B
12
activity.
But hydroxycobalamin also called vitamin B
12
is more potent
because it is readily absorbed and its concentration in blood
rises very early.
Vitamin B
12
acts in the form of three coenzymes, called
cobamides. The three types of cobamides are:
1. Cobamide I (where CN is replaced by dimethyl benzimi-
dazole group).
2. Cobamide II (where CN is replaced by benzimidazole
group).
3. Cobamide III (where CN is replaced by adenyl group).
Function
Cobamide coenzymes are involved in:
1. Conversion of L-methylmalonyl CoA to succinyl CoA.
2. Conversion of glutamic acid to -methyl aspartic acid.
3. Conversion of ribonucleotides to dexyribonucleotides.
Sources
Liver, kidney, meat, fish, egg yolk.
Also synthesized by intestinal bacteria.
Daily Requirement: 3-4 g.
Deficiency Disease
Deficiency of vitamin B
12
gives rise to pernicious anemia. Perni-
cious anemia is not simply the result of vitamin B
12
deficiency
in the diet but is caused by a lack of specific glycoproteins
in the gastric juice called the intrinsic factor. This protein binds
vitamin B
12
and is transported.
VITAMINS 283
Also, deficiency of vitamin B
12
gives rise to increased excre-
tion of methyl malonic acid in urine.
ANTIVITAMINS
Antivitamins are substances which possess structural similarity
to certain vitamins but behave antagonistically to these
vitamins when introduced into the body, thereby preventing
the normal function of these vitamins. Examples of vitamins
with their antivitamins are Thiamine: Pyrithiamine, Riboflavin:
Isoflavin, Pyridoxine: Isonicotinic acid hydrazide (INH), Folic
acid: Aminopterin, vitamin K: Dicoumarol.
ACID-BASE BALANCE
The acid-base balance of the body is basically the metabolism,
of hydrogen ions. When these hydrogen ions are produced
in the body, there are many ways by which they are handled
and excreted by the body so as to maintain the pH of the
body fluids constant.
Electrolyte Composition of Plasma
Cations mEq/L Anions mEq/L
Na
+
142 Cl 103
K
+
5 HCO
3
27
Ca
++
5 HPO
4
2
Mg
++
3 SO
4
11
Organic acids 6
Total 155 Proteinate 16
Total 165
Three mechanisms for the maintenance of acid-base balance
are:
1. Buffer systems of the body fluids.
2. Lungs (Respiration).
3. Kidneys (Renal mechanism).
Buffer Systems of the Body Fluids
The chief acids produced in the body are H
2
CO
3
, HHb,
HHbO
2
, proteins and various organic acids such as lactic acid,
Acid-base Balance
CHAPTER
13
ACID-BASE BALANCE 285
pyruvic acid, citric acid, are taken by the chemical buffer
systems of the body.
The buffer systems of the body fluids represent the first
mechanism involved in the regulation of pH.
The buffer systems of the various body fluid compartments
are:
The important buffers in the plasma are bicarbonate:
carbonic acid buffer. In the RBC, the hemoglobin buffer system
predominates whereas proteins and phosphate buffer repre-
sent the buffers of intracellular fluids.
When protons are added to the body fluids, they are taken
up by the buffer bases (anions) to form buffer acids, and when
there is a deficit of protons they are given to the body fluids
by the buffer acids.
Bicarbonate Carbonic Acid Buffer
It is the most important buffer system of nonvolatile acids
entering the extracellular fluids because of two reasons.
1. It is present in high concentration than the other buffer
systems.
2. The production of H
2
CO
3
, is effectively buffered and is
disposed by the lungs as CO
2
.
Such acids, e.g. HCl, H
2
SO
4
, lactic acid, etc. react with
NaHCO
3
as follows:
The pH of the buffer system is given by
pH =
+
3
2 3
B HCO
pk + log
H CO

The pH of the body fluids depends on the ratio of BHCO
3
and H
2
CO
3
. As long as the ratio remains 20:1, the pH is normal.
Phosphate Buffer
Phosphate buffer plays a minor part in blood but is more
important in the kidney in regulating pH. It is important in
raising plasma pH through excretion of H
2
PO
4
by the kidney.
4
2 4
HPO
H PO
=
80
20
At pH 7.4, plasma has four parts dihydrogen phosphate
to one part of monohydrogen phosphate.
At pH 5.8, the ratio is ten parts of monohydrogen
phosphate to one part of dihydrogen phosphate. This variation
in pH is made use of in the removal of hydrogen ions by the
kidney.
Protein Buffer
At the pH of the blood, the plasma proteins are anions but
act as weak acids.
H Protein H
+
Protein
In plasma, protein buffer plays a much smaller part than
bicarbonate buffer but in the cells proteins form the most
important buffering system.
Lungs
The level of H
2
CO
3
in the plasma is under the control of lungs.
The respiratory mechanism, compensates for disturbance of
acid-base balance by regulating H
2
CO
3
. Whenever, there is
acidosis (i.e. hydrogen ions in blood increase), pH is decreased,
with a lowered HCO
3
: H
2
CO
3
ratio. The lung ventilation is
increased, alveolar pCO
2
is decreased and H
2
CO
3
is increased
which increases the HCO
3
: H
2
CO
3
ratio and the pH returns
to normal.
In alkalosis, when the pH is increased, the ratio HCO
3
:
H
2
CO
3
is high. Lung ventilation is decreased, the pCO
2
of
the alveolar is increased and H
2
CO
3
in the blood and other
fluids is increased which lowers the HCO
3
: H
2
CO
3
ratio and
thus pH returns towards normal.
Kidney
Kidney plays the important role in regulating both the
electrolyte concentration and the acid-base balance of the body
fluids. The main mechanism by which the kidney maintains
the pH of the body fluids is by regulating secretion of H
+
ions which is linked up with the conservation of base in the
form of sodium bicarbonate, the formation of acid phosphates
and generation of NH
4
+
ions by the kidney tubules.
Various acids such as lactic acids, ketone bodies, sulfuric
acid, phosphoric acid are taken by bicarbonate for neutra-
lization as soon as they are formed.
This shows that the bicarbonate is the alkaline reserve of
the body. These acids buffered with Na
+
are first removed
by glomerular filtration. The cation is then reabsorbed by the
renal tubules in exchange of H
+
which are secreted.
Mechanism of H
+
excretion.
The H
+
secreted by the tubular cells are handled in three
principal ways.
1. Bicarbonate reabsorption
2. Ammonium ion production
3. Acidification of the urine.
Bicarbonate Reabsorption
Mobilization of H
+
for tubular secretion is accomponished by
the ionization of carbonic acid. In the proximal tubule, the
exchange of H
+
against sodium bicarbonate takes place. The
formation of carbonic acid is catalyzed by the enzyme carbonic
anhydrase.
Ammonium Ion Production
Another mechanism of the conservation of cation is the
production of NH
3
. NH
3
formation takes place in the distal
tubules. NH
3
formed enters the tubular filtrate, combines with
H
+
to form NH
4
+
ions. The NH
4
+
then replace Na
+
ions. The
Na
+
ions is reabsorbed by the H
+
-Na
+
exchange and re-enters
the plasma as NaHCO
3
. The NH
4
+
ions are excreted in urine
as NH
4
Cl.
Acidification of the Urine
After all the bicarbonate has been absorbed, the H
+
ion
secretion then proceeds against NaHPO
4
. The exchange of Na
+
ion for secreted H
+
ion changes Na
2
HPO
4
to NaH
2
PO
4
.
Na
+
and HCO
3
return to plasma and H
+
is excreted in urine
to maintain normal acid-base balance and electrolyte concen-
tration.
As long as the ratio of bicarbonate to carbonic acid is 20:1
in blood, pH of blood is normal. Any variation in the ratio,
will disturb the acid-balance of the blood and leads to acidosis
or alkalosis.
Disturbances in acid-base balance can be classified broadly
under two headings:
1. Acidosis
2. Alkalosis.
Acidosis Alkalosis
(a) Respiratory acidosis (a) Respiratory alkalosis
(b) Metabolic acidosis (b) Metabolic alkalosis.
Respiratory Acidosis
Respiratory acidosis is due to increase in H
2
CO
3
in the blood,
resulting in lowering of pH of the blood. This is compensated
by increase reabsorption of HCO
3
in the renal tubules. Such
condition occurs in pneumonia, asthma, etc.
Metabolic Acidosis
This is due to the decrease in HCO
3
in the blood with no
change in H
2
CO
3
. This is compensated by elimination of more
CO
2
(hyperventilation). Such condition occurs in uncontrolled
diabetes with ketosis.
Anion Gap
Anion gap is defined as the difference between the plasma
sodium and potassium concentrations and the sum of the
chloride and bicarbonate. The anion gap is made up of the
difference between the sum of unmeasured cations such as
ionized calcium and magnesium and of unmeasured anions
such as phosphate, urate, organic acids and plasma proteins.
Since the sum of plasma cation and anion concentrations
must be equal to maintain electrochemical neutrality.
The normal value of anion gap is 10-15 mEq/L (average
12 mEq/L).
Anion gap may change because of an alteration in any of
the quantities on the right hand side of the equation. A rise
in anion gap is usually due to a rise in the unmeasured anions
in diabetic ketoacidosis or renal failure. Small increase may
occur in alkalosis due to unmeasured anionic equivalence of
plasma proteins.
A reduced anion gap is caused by a low plasma albumin
concentrations.
Anion Gap
The anion gap is a mathematical approximation of the difference
between the anions and cations routinely measured in serum.
Routine electrolyte measurement include Na
+
, K
+
, Cl and
HCO
3
and unmeasured cations include Ca
+2
, Mg
+2
and
unmeasured anions, i.e. PO
4
3
, SO
4
2
, protein
1
, organic acids.
If the Cl and HCO
3
conc are summed and substracted
from total of Na
+
and K
+
conc the difference comes about
10-15 mmol/L (average 23 mmol/L).
a. If anion gap exceeds 17 mmol/L, i.e. increased concen-
tration of unmeasured anions
Diabetes mellitus
Alcoholism
Starvation
Salicylate
Uremia.
b. If anion gap less than 10 mmol/L, i.e. increase in unmea-
sured cations or a decreases in unmeasured anions
Lithium intoxication
Multiple myeloma
Hypoalbuminemia.
Respiratory Alkalosis
This is due to decrease in H
2
CO
3
in the blood. This is compen-
sated by decreased reabsorption of HCO
3
by the renal tubules.
Such condition occurs in hepatic coma.
Metabolic Alkalosis
This is due to increase in HCO
3
in the blood giving rise to
increase in pH of the blood. This is compensated by retention
of CO
2
in the blood. The increased pH of blood leads to retany.
It can occur in Cushings syndrome.
Urine pH Plasma
[HCO
3
] [H
2
CO
3
] H
2
CO
3
mEq/L mEq/L
[HCO
3
]
Normal 6-65 2.5 51.25 1 : 20
Respiratory acidosis <1 : 20
Respiratory alkalosis >1 : 20
Metabolic acidosis <1 : 20
Metabolic alkalosis >1 : 20
BIOLOGICAL IMPORTANCE OF WATER
1. Water is an essential constituent of cell structures and
provides the media in which the chemical reactions of the
body takes place and substance are transported.
2. It has a high specific heat for which, it can absorb or gives
off heat without any appreciable change in temperature.
3. It has a very high latent heat. Thus, it provides a mechanism
for the regulation of heat loss by sensible or insensible
perspiration on the skin surface.
4. The fluidity of blood is because of water.
Water comprises 70% of the lean body mass of the
adult.
Lean body mass = weight of the bodyfat content of the
body. Water is present in the body both inside and outside
the cells. Strictly speaking there are two water compartments
in the body.
i. Intracellular water: Water present inside the cell.
ii. Extracellular water: Water present outside the cell.
Extracellular fluid is further divided into:
1. Plasma: It comprises 7.5% of the body weight.
2. Interstitial fluid: It comprises 20 percent of the body
weight.
3. Dense connective tissue, i.e. water content in the bones and
cartilages.
It comprises 15% of the body weight.
4. Transcellular fluid (intracellular fluid): It comprises 2.5%
of the body weight.
Water and Mineral
Metabolism
CHAPTER
14
WATER AND MINERAL METABOLISM 293
The volume of fluids in various compartments of the body
can be found out by various methods such as isotopic dilution,
injection of dyes, heavy water, antipyrine, etc.
Total distribution of water in the human body.
percent ml/kg of
body weight
Intracellular water 55 335
Extracellular water 45 270
i. Plasma 7.5 45
ii. Interstitial 20 120
iii. Bones and cartilages 15 90
iv. Transcellular water 2.5 15
The quantity of water in the body depends upon the body
weight.
The daily water requirement is about 1 ml/Kcal, i.e. a requi-
rement of 2000 Kcal necessitates a water intake of 2000 ml.
Infants have proportionately more water loss and should
be allowed about 150 ml water for each 100 Kcal.
Thirst is a good guide for adequate fluid intake.
The source of water in the body are as follows (Figures
in bracket indicate the average water intake).
1. Water by drinking (1200 ml).
2. Water present in food (1000 ml).
3. Metabolic water, i.e. water formed in the body by the
oxidation of food stuffs, i.e. oxidation of carbohydrates,
fats and proteins (Amino acids). The amount of water
produced in the body from metabolism is about 200 to
450 ml daily.
100 gm of carbohydrates, fats and proteins yield 85 ml,
107 ml and 41 ml of water each. Normal diet provides 300
ml of metabolic water daily.
Water is lost from the body by the following routes. Figures
in bracket indicate the average quantity of the water loss.
1. Water lost through skin both as sensible and insensible
perspiration (600 ml).
Insensible perspiration is so called because one is not aware
of it; it evaporates as it is formed.
On the other hand, with vigorous activity especially in hot
weather, we lose much additional water through visible perspi-
ration.
Sensible perspiration is called active sweating. It depends
upon:
i. Habits
ii. Type of activity.
If metabolic rate is high, then water loss will be high.
Greater the respiration rate, the higher will be metabolic rate,
i.e. more loss. Water loss depends upon:
1. Metabolic rate
2. Climate conditions
3. Water lost through lungs in expired water (400 ml).
The loss of water through lungs depends upon:
i. Rate of respiration
ii. Temperature and humidity of atmosphere
iii. Water lost through kidney in urine.
iv. Water lost through intestines in feces (100 ml).
Water loss is proportional to the function of metabolic rate.
Kidney is the most important guardian of the water content
of the body. The water loss through skin, lungs and feces
are not controlable but there is an automatic feedback
mechanism by the kidney.
Certain volume of urine has to be lost by the kidney and
it is called minimum urine volume or obligatory excretion.
Kidney controls the excretion of waste products and to
dissolve them minimum urine volume of 500 ml is needed.
The 500 ml constitutes the 2 percent of body weight which
has to be lost even when the body does not take any water.
Water content depends upon body weight and water loss
depends upon metabolic rate and both are not correlated.
Minimum water excreted by the kidney depends upon:
i. Concentrating power of the kidney
ii. Quantity of water products.
Healthier the kidney, the greater will be concentrating
power of the kidney.
Concentrating power of the kidney decreases in certain
diseased conditions such as:
i. Obstruction to the flow of kidney
ii. Chronic nephritis
iii. Any dehydration
iv. Shock.
Dehydration
Dehydration results:
1. When the water intake is less than the body needs. This
occurs in no food of fluid intake.
2. When the fluid loss from the body is abnormally high,
e.g. excessive perspiration in hot weather, severe diarrhea,
vomiting, fever, with increased loss from the skin, severe
burns with accompanying water losses and in uncontrolled
diabetes with frequent urination.
Dehydration is corrected by electrolytes and water.
Edema
Edema is the accumulations of water in the body. It occurs
when the body is unable to excrete sodium in sufficient
amounts. This is not unusual in diseases of the heart when
the circulation is impaired or when the kidneys are unable
to excrete waste normally. Edema also occurs following
prolonged protein deficiency because tissues are no longer
able to maintain normal water balance.
MINERALS
Minerals are inorganic substances. Minerals are present in all
body tissues and fluids. Unlike carbohydrates, fats and
proteins, mineral elements do not furnish energy.
Unlike vitamins, the minerals are not destroyed in food
preparation. However, they are soluble in water so that some
loss will occur if cooking liquids are discarded.
In contrast to the organic substances, which can be
considered as energy sources, the inorganic substances do
not supply any energy. Their presence is necessary for the
maintenance of certain physiochemical conditions which are
essential for life.
Principal minerals required by the body are sodium, potas-
sium, calcium, magnesium, phosphorus, sulfur and chlorine.
These comprises 70% of the total mineral of the body contents.
In addition, copper, zinc, cobalt, manganese, molybdenum,
iodine, fluorine.
Basic functions performed by the minerals are:
i. As structural components of body tissues.
ii. In the maintenance of acid-base balance.
iii. In the regulation of body fluids.
iv. In transport of gases.
v. In muscle contractions.
Iron
Total iron content in the body is 3.5 gm. 70% of this iron is
present in hemoglobin.
Biologically important compounds of iron are hemoglobin,
myoglobin, cytochromes, catalases, peroxidase. In all these
compounds iron is present as heme form or porphyrin form.
In addition to these iron is present in nonheme form called
nonheme iron. Nonheme iron is present as ferritin (a stored
form of iron) and transferrin (a transport form of iron).
Functions
1. As hemoglobin, in the transport of oxygen.
2. In cellular respiration, where it functions as essential compo-
nent of enzymes involved in biological oxidation such as
cytochromes c, c
1
, a
1
, etc.
Absorption of Iron
The maximum absorption of iron is not more than 10 percent
of the iron content of the diet.
In the food, iron is present in ferric form either as ferric
hydroxides or in combination with ferric organic compounds.
Acidity of gastric juice results in the liberation of ferric form.
Ferric form is reduced to ferrous form by the reducing
substances such as glutathione, vitamin C and cysteine present
in the food absorption.
The regulation of iron absorption is governed by mucosal
block theory. According to this theory, ferrous ions on entering
the mucosal epithelial cell are oxidized to ferric ions which
combines with a protein called apoferritin to form ferritin (also
known as siderophilin). Apoferritin is a glycoprotein containing
sialic acid, galactose, mannose as the carbohydrate moieties.
Each molecule of apoferritin combines with 2 atoms of
ferric, iron to form ferritin. This ferritin is a stored form of
iron. The amount of apoferritin present in the mucosal cells
is the controlling factor.
Factors which affect iron absorption are:
1. Low phosphate diet increases iron absorption whereas high
phosphate diet decreases iron absorption by forming
insoluble iron phosphates.
2. Iron in ferrous form is more soluble and is readily absorbed
than the ferric form.
3. Phytic acid and oxalates decreases iron absorption by
forming iron phytate and iron oxalate.
No absorption of iron takes place under following conditions:
1. Any condition of partial or total gastrectomy
2. Dissertion of small intestine
3. Achlorohydria
4. Profuse diarrhea
5. Malabsorption syndrome.
Iron is transported in the plasma as Fe
+++
form in combi-
nation with -globulin called transferrin also known as
sidoferrin. The iron in this form is called protein bind iron
(PBI). The entire iron in the plasma is in the protein bound
iron.
The protein bound iron in:
Adults : 120-140 g per 100 ml of blood
Females : 90-120 g per 100 ml of blood.
The plasma iron content is the net resultant of the following:
i. Rate of RBC destruction
ii. Rate of iron absorption from intestines
iii. Rate of apoferritin synthesis
iv. Rate of erythropoiesis
v. Extent of blood losses.
Iron is stored in the body as ferritin. Ferritin can bind up
to 4000 iron atoms per molecule. If iron is taken in abnormally
large amounts, the excess is deposited in liver as hemosiderin.
Excessive accumulation of iron in liver, lungs, pancreas,
heart are other tissues results in hemosiderosis, when this is
accompanied by bronze pigmentation of the skin, the condition
is called hemochromatosis.
Sources
Meat, heart, kidney, spleen, egg yolk, fish, dates, nuts,
legumes, molasses, spinach, cooking of food in iron vessels.
Daily requirement: 10-15 mg.
Calcium
Calcium is present in the body in the largest amount of all
the minerals present in the body. Calcium comprises 2% of
the body weight. RBC is devoid of calcium. The normal serum
level is 9-11 mg percent.
Calcium is present in three forms:
1. Ionized form: This form is phy siologically active form.
2. Protein bound fraction: This form is physiologically
inert.
3. In combination with citrates: Protein bound fraction is
nondiffusible whereas other two fractions are diffusible.
Absorption of Calcium
1. Calcium salts are more soluble in acidic media than the
alkaline media. Greater the acidity, the more will be the
absorption.
2. Certain foodstuffs contain phytic acid (present in cereals)
and oxalates (present in spinach) which inhibit calcium
absorption by forming insoluble calcium salts.
3. When fat absorption is not proper, the free fatty acids
present react with calcium to form soaps (calcium salts of
fatty acid) will hinders absorption.
4. On a high protein diet the calcium absorption will be more.
5. Vitamin D is necessary in the diet to promote the absor-
ption of calcium.
6. The optimum calcium phosphorus ratio in the diet should
be 1:1.
Functions
1. Calcium along with phosphorus is essential for bones and
teeth formation.
2. In blood coagulation: Calcium activates the conversion of
prothrombin to thrombin.
3. In milk clotting.
4. In enzyme activation: Calcium activates large number of
enzymes such as adenosine triphosphatase (ATPase), suc-
cinic dehydrogenase, lipase, etc.
5. In muscle contraction.
6. In normal transmission of nerve impulses.
7. In neuromuscular excitability.
Regulation of Blood Calcium Level
1. Indirect factors: Those factors which have an effect on cal-
cium absorption. Under this comes dietary factors which
have been discussed in the absorption of calcium.
2. Direct factors: Those which have direct effect on blood
calcium. These are:
a. Hormones
i. Parathyroid hormone regulates the concentration of
ionized serum calcium.
ii. Calcitonin lowers calcium level by inhibiting bone
absorption and thus decreases the loss of calcium
from bones.
b. Serum proteins
Decrease in serum proteins will result in decrease in
total calcium level as most of the calcium bound to
protein will be less.
c. A reciprocal relationship exists between calcium and
phosphorus in the blood. Increase in serum phosphorus
causes decrease in serum calcium and vice versa.
Sources
Dairy products such as milk, cheese are the best sources.
It is also present in lentils, nuts.
Daily Requirement
Adults 800 mg
In females during 2nd and 3rd semester of pregnancy and
lactation 1200 mg
Infancy 350-540 mg
Children from 1 to 8 years 0.8-1.2 gm.
Rickets
Deficiency of vitamin D gives rise to rickets in children. The
main symptom of rickets is, insufficient calcification by calcium
phosphate of the bones in growing children. The bones,
therefore, remain soft and deformed by the body weight.
Osteoporosis
Osteoporosis is a disease of demineralization or decalcification
of the bones.
It is a condition when calcium is withdrawn from the bones.
The bone becomes week and porous and hence breaks. It is
more prevalent in older women than in men.
Sodium
Sodium is the principle cation of the extracellular fluid.
Functions
1. In the regulation of acid-base balance.
2. In the maintenance of osmotic pressure of the body fluids.
3. In the preservation of normal irritability of muscles and
permeability of the cells.
The normal serum sodium level is 133-146 mEq/L.
Increased level of sodium in the serum is called hyper-
natremia. Hypernatremia occurs in:
i. Cushing disease
ii. Administration of ACTH
iii. Administration of sex hormones
iv. Diabetes insipidous
v. After active sweating.
Low levels of sodium in serum is called hyponatremia.
Hyponatremia occurs in:
i. Acute Addisons disease
ii. Vomiting, diarrhea
iii. Severe burns
iv. Intestinal obstruction
v. Nephrosis
vi. Any situation where there is active sweating and we take
plain water.
Potassium
Potassium is the principal cation of the intracellular fluid.
Functions
1. Intracellular cation in acid-base balance
2. In muscle contraction, particularly in cardiac muscle
3. Conduction of nerve impulse
4. Cell membrane function.
The normal concentration of potassium in the serum is
3.5-5.5 mEq/L.
Increased level of potassium in serum is called hyperkalemia.
Hyperkalemia occurs in:
i. Addisons disease
ii. Advanced chronic renal failure
iii. Dehydration
iv. Shock.
Low levels of potassium in serum give to hypokalemia.
Hypokalemia occurs in:
i. Diarrhea
ii. Metabolic alkalosis
iii. Familial periodic paralysis
Potassium is required during glycogenesis. This potassium
is withdrawn from the extracellular fluid giving rise to
hypokalemia.
Phosphorus
Functions
1. Phosphorus along with calcium is essential for bones and
teeth.
2. Buffering action, i.e. phosphate buffers.
3. In the formation of high energy compounds, i.e. ATP.
4. In the synthesis of RNA and DNA.
5. In the synthesis of phospholipids.
6. In the synthesis of phosphoproteins.
Absorption
The absorption of phosphorus is related to that of calcium.
Normally 1/3rd of the ingested phosphorus is passed in the
feces and 2/3rds in the urine. A high calcium diet, diminishes
the phosphorus absorption by forming insoluble calcium
phosphates.
Phosphorus is present in the blood as:
i. Inorganic phosphorus
ii. Organic phosphorus
iii. Lipid phosphorus.
The normal serum inorganic phosphorus level is 2.5-4 mg%.
It is higher in children, the value being 4-6 mg%.
Increase in serum phosphorus is found in:
i. Chronic nephritis
ii. Hypoparathyroidism
Decrease in serum phosphorus is found in:
i. Rickets.
ii. Hyperparathyroidism
iii. De Toni-Fanconi syndrome.
Sulfur
Sulfur is present in three amino acids. Methionine, cystine and
cysteine and thus it is present in all proteins in the body.
Connective tissue, skin, hair and nails are especially rich in
sulfur. Also thiamine and biotin (member of vitamin B
complex) and coenzyme A contain sulfur in these molecules.
Diet which is adequate in protein meets the daily require-
ment of sulfur.
Copper
Total copper content in the human body is 100-150 mg. It is
present in almost all the tissues of the body. Liver is the richest
source of copper.
Functions
1. Copper is an important constituent of certain enzymes such
as, cytochromes, cytochrome oxidase, catalase, peroxidase,
ascorbic acid oxidase, uricase, tyrosinase, cytosolic super-
oxide dimutase, etc.
2. Necessary for growth and bone formation.
3. Necessary for formation of myelin sheaths in the nervous
systems.
4. Helps in the incorporation of iron in hemoglobin.
5. Helps in the absorption of iron from GI tract.
6. Helps in the transfer of iron from tissues to the plasma.
Copper is present in the plasma as ceruloplasmin. The
concentration of ceruloplasmin in plasma is 23-40 mg %. The
copper containing protein in RBC is erythrocuperin, in liver
it is hepatocuperin and in brain it is cerebrocuperin.
Like iron, copper is conserved and reutilized by the body.
Ceruloplasmin
It is a blue colored copper containing metalloprotein with
2
globulin. It contains 8 atoms of copper bound per molecule.
The reduced form is colorless. It is glycoprotein containing
8-10 units of sialic acid residues per molecule. About 90-95
% of the total copper in the plasma is present in the
ceruloplasmin molecule and remainder is bound to albumin.
Ceruloplasmin has oxidase activity and thereby facilitates
the incorporation of ferric iron into transferrin. Vitamin C is
utilized as hydrogen donor.
Increased levels are seen in acute infections. Chronic condi-
tions such as rheumatoid arthritis, cirrhosis and in post-
operative stages. Malnutrition also has increased levels.
Wilson disease shows decrease serum levels in both copper
and ceruloplasmin.
Sources
Molasses, nuts, legumes, shell fish.
Daily Requirement: 2-5 mg.
Wilson Disease or Hepatolenticular Degeneration
In Wilsons disease, large amount of copper is deposited in
liver, brain, etc. total copper content in the plasma and
ceruloplasmin bound copper content decrease. There is an
increased excretion of copper in the urine.
Some time copper is also deposited in renal tubules giving
rise to renal tubular degeneration. The salient features of which
are glycosuria and amino aciduria.
Zinc
Zinc is an important constituent of pancreas.
Functions
1. Zinc is a constituent of certain enzymes such as carbonic
anhydrase, carboxypeptidase, alkaline phosphatase, lactate
dehydrogenase, alcohol dehydrogenase, superoxide dimu-
tase, retinene reductase, DNA and RNA polymerase.
2. Necessary for taste buds.
3. Necessary for fertility of mice.
4. Necessary for tissue repair and wound healing.
5. Necessary for protein synthesis and digestion.
6. Necessary for optimum insulin action as zinc is the integral
constituent of zinc.
Fluoride
Functions
1. It gives strength to enamel tissues.
2. It prevents the bacterial action to the teeth.
3. Necessary for the health of teeth.
Fluoride ions inhibits all those enzymes which needs Mg
++
also, i.e. inhibition of glycolysis reactions. On enolase, it has
the maximum inhibition activity.
Addition of fluoride salts in water is known as fluoridation.
Fluorosis
Excessive intake of fluorine gives rise to fluorosis. Deficiencies
of fluorine seem to increase the incidence of dental caries
whereas excessive concentrations of fluorine in the drinking
water causes corrosion of the enamel of the teeth, a process
known as mottling.
A xenobiotic is a compound that is foreign to the body. The
principle classes of xenobiotics includes drugs, chemical
carcinogens and other pollutants and insecticides.
Metabolism of Xenobiotics
1. Phase I: The major reaction involved is hydroxylation cata-
lyzed by cytochrome P-450. In addition to hydroxylation,
deamination, dehalogenation, desulphuration are included
in this phase.
Cytochrome P-450 is hemoprotein. Highest concentration
of which is present in water.
Xenobiotics
CHAPTER
15
2. Phase II: The hydroxylated compounds produced in phase
I are converted into various polar metabolites by conju-
gation with glucuronic acid, sulfate, acitate, glutathione or
methylation.
The overall purpose of two phases of metabolism of xeno-
biotics is to increase their water solubility and thus facilitates
their excretion from the body.
Liver is the main site for detoxification process though
kidneys also participate to some extent. Detoxified products
are excreted in urine or feces.
XENOBIOTICS 307
Detoxification mechanism is classified under four main
types:
a. Oxidation
b. Hydrolysis
c. Reduction
d. Conjugation.
Oxidation
A large number of foreign compounds are destroyed in the
body by oxidation.
a. Primary alcohols are oxidized through aldehyde to acids
Secondary alcohols are oxidized to ketones.
Chloral, used as hypnotic, is oxidized to trichloroacetic acid.
Primary aromatic amines undergo oxidation to correspon-
ding acids.
Sulfur of organic sulfur compounds is oxidized to sulfates.
Hydrolysis
Hydrolysis of esters, amide glucosides, etc. brings about
significant changes in the alteration of foreign molecule in the
body.
Reduction
Reduction is less common than oxidation in the body.
Picric acid Picramic acid
Conversion ofSSlinkages to SH groups.
Conversion of azocompounds to amines
Reduction of double bonds.
Conjugation
Conjugation process usually includes oxidation, reduction and
hydrolysis of foreign substances although, some compounds
are conjugated without previous alteration.
a. Bilirubin is conjugated and excreted as the glucuronoids.
Bilirubin + Glucuronic acid Bilirubin mono and
diglucuronides
b. Benzoic acid is conjugated with glycine and excreted as
hippuric acid.
XENOBIOTICS 309
c. Phenylacetic acid is conjugated with glutamine to form
phenylacetyl glutamine. l l
d. Phenolic compounds are conjugated with sulfates.
Food is the prime necessity of life. The purpose of food is
to provide fuel which when broken down by oxidation gives
energy required for performing vital activities. A balanced
diet must provide for the maintenance of the body as well
as energy requirements and where necessary, for growth and
reproduction. Essential elements lost from the body excretion
must be replaced.
Essential nutrients are those that cannot be synthesized
in adequate amounts (if at all) and are required in the diet.
All the calories in the food comes only from the carbohy-
drates, fats, proteins and not from the vitamins, minerals and
water though they are also essential components of food.
The unit of energy is kilocalorie, which is 1000 times the
small calorie. One calorie is defined as the amount of heat
required to raise the temperature of 1 gm of water by 1C.
The calorie value of the foodstuffs can be determined by
the Bomb calorimeter.
Foodstuff Heat of combustion (Kcal/gm)
In Bomb In the Corrected
calorimeter organism value
Carbohydrates 3.8-4.2 3.8-4.2 4
Fats 9.0-9.6 9.0-9.6 9
Proteins 5.0-5.3 4.0-4.5 4
Caloric Value of Food
The food we eat is rarely of pure carbohydrate, pure fat or
pure protein, but a mixture of these.
Nutrition
CHAPTER
16
NUTRITION 311
The caloric value of a mixed food depends on its
composition and digestibility.
The appropriate caloric value of a food can be calculated
by simple formula.
Caloric value = 4 (Carbohydrates + Proteins) + 9 Fats
(in Kcal/gm).
Respiratory Quotient (RQ)
Respiratory quotient is defined as the ratio of the volume of
carbon dioxide produced by the oxidation to the volume of
oxygen consumed for the oxidation.
RQ =
Volume of carbon dioxide produced
Volume of oxygen consumed
RQ depends upon the type of foodstuffs being metabolized.
For carbohydrates:
RQ is 1
For fats:
RQ is low, because fats are deficient in oxygen as compared
to carbohydrates, therefore to oxidize fat, more amount of
oxygen is required, e.g. the oxidation of tristearin.
2C
57
H
110
O
6
+ 163 O
2
114 CO
2
+ 110 H
2
O
RQ =
114
163
= 0.7
For proteins:
RQ of proteins has been found to be 0.80. For proteins,
it is not possible to write equation because the exact structure
is not known in many cases.
On a mixed diet, RQ is found to be 0.85.
Very low values of RQ are found in diabetes, when large
amount of glucose and ketone bodies are excreted in the urine.
High values of RQ are found when carbohydrates are
converted into fats and deposited in the adipose tissue.
Hence, it is clear that RQ gives some indication of the type
of food being metabolized.
The respiratory quotients for various mixtures of fats and
carbohydrates are given as follows:
RQ Percent fats Percent carbohydrates
1 0 100
0.89 20 80
0.83 20 60
0.77 60 40
0.74 80 20
0.71 100 0
Basal Metabolic Rate (BMR)
By basal metabolism, we mean the amount of energy required
just to maintain the body processes when a person is at
complete rest. This lowest amount of energy is called basal
metabolic rate.
BMR is measured in terms of heat production. The higher
the rate of metabolism, the more is the heat production.
To measure the BMR, the following conditions are necessary:
1. He should be in the postabsorptive state
2. He should be physically relaxed
3. He should be awake
4. He should be in an environment having the temperature
20-25C
5. His body temperature should be normal.
The following factors which influence BMR are:
1. Surface area: Larger the surface area, the higher would
be BMR
2. Age: BMR decreases with age
3. Sex: Woman has lower BMR than men
4. State of nutrition: BMR is lowered in starvation and
undernutrition
5. Hormonal action: BMR increases in hyperthyroidism,
decreases in hypothyroidism
Specific Dynamic Action (SDA)
Specific dynamic action of foodstuff is defined as the extra
amount of heat produced over and above the caloric value
of the foodstuff when burnt inside the body.
NUTRITION 313
When protein equivalent to 100
o
C is ingested, on metabo-
lization, its production is 130
o
C. This extra 30
o
C is due to SDA
of protein which is derived at the expense of tissue meta-
bolizing the foodstuff. Thus, the SDA of proteins is 30 percent.
Similarly, the SDA of carbohydrates is 5 percent and that of
fat is 13 percent.
On the mixed diet, the SDA is reduced to about 10-12
percent.
While calculating the energy requirement for daily activities
10 percent of the total calories is added to provide energy
for the expenditure of SDA.
Four basic food values contribute to nutrient essential for
a complete and balanced diet.
1. Milk group: Provides high quality protein, calcium, phos-
phorus, riboflavin and vitamin D.
2. Meat group: Supplies protein of high biologic value, niacin,
thiamine, vitamin B
12
, heme iron and minerals.
3. Vegetable fruit group: Supplies ascorbic acid, carotene, other
water soluble vitamins, minerals and fiber (roughage). No
vegetable protein has a high biologic value but when
properly mixet it is possible for one protein to complement
another.
4. Cereal group: High in carbohydrates for energy needs, also
include vitamins, fiber and iron if the cereals are not
refined. Proteins of this group do not have a high biologic
value as animal protein.
Biological Value of Proteins
The biological value of a protein is a measure of the degree
to which its nitrogen can be used for growth or maintenance
of total body function.
The biologic value of a dietary protein is a measure of the
extent to which it satisfies the amino acid requirement for
growth or the maintenance of total body function.
In general, animal proteins have a high biologic value, a
major exception is gelatin which lacks the essential amino acid,
tryptophan and therefore has no biologic value. Vegetable
proteins have a low biologic value because each one has a
low level of one or more essential amino acid.
The biological value is expressed as:
=
N intake - N loss in feces, sweat, urine
100
Nintake - N loss in feces
=
N retained
100
N absorbed

Biological Value of Protein
It is the ratio between the amount of N retained and N
absorbed during specific internal.
BV =
Retained N
100
Absorbed N
Net Protein Utilization (NPU)
NPU =
Retained N
100
Intake N
NPU is a better index to assess nutritional quality and
availability of a protein.
Net Dietary Protein Value (NDPV)
NDPV = Intake of N 6.25 NPU. This will assess both quality
and quality of protein in the diet.
The biological value of a protein signifies:
1. The presence and amounts of various essential amino
acids
2. The digestibility of protein
3. Availability of digested products.
In general, animal proteins have a high biological value
because of its high essential amino acid contents whereas vege-
table proteins have a low biological value because of low
contents of one or more essential amino acids.
NUTRITION 315
Caloric Requirement
The daily caloric requirement of the body is the sum total
of basal energy demands and energy required for the
additional work of the day. During growth, pregnancy,
convalescence, caloric demand of the body increases and extra-
calories must be provided.
Carbohydrates
Carbohydrates are the main energy source in human nutrition.
Carbohydrates supply about 55-70% of the total requirement
of the human body. The widespread use of carbohydrate rich
food is due to the fact that they are relatively inexpensive.
They are far cheaper than fats or proteins of the same caloric
value.
Carbohydrates are not strictly essential since all carbo-
hydrates can be synthesized from dietary amino acids. The
most important carbohydrate in the food of man is starch.
Starch is the main constituent of all cereals. Another important
carbohydrate of our food is sucrose. Glucose, fructose and
other monosaccharides are present in many foods.
Fats
Fats are the most concentrated source of energy because of
their high caloric values. Fats provide about 20-25% of the
total caloric requirement of the body. Out of this, at least one
percent of the fat should comprise of essential fatty acids.
The purpose of essential fatty acids is:
1. Essential fatty acids lower the serum cholesterol level.
2. Essential fatty acids support good growth.
In general, animal fats are poorer in essential fatty acids.
It is recommended that less than 10% of total calories be
obtained from saturated fatty acids and less than 10 percent
be from polyunsaturated fatty acids.
Proteins
The main purpose of proteins is to provide tissue repair and
synthesis. For energy purpose, its effect is secondary. The
quality of protein depends upon its amino acids make up.
Depending on the essential amino acids content they are
classified as:
1. Complete proteins: It contains all the essential amino acids
in adequate amounts and support good growth in the young
experimental animals.
2. Partially complete proteins: Proteins partially lacking in one
or more essential amino acids. They cannot support growth
in young experimental animals but can maintain nitrogen
balance.
3. Incomplete proteins: They completely lack in essential amino
acids. They do not support growth and nitrogen balance.
All the proteins of animal origin are complete proteins.
On the average the nitrogen content of dietary protein is
16% by weight, thus
100
nitrogen content (g)
16
= protein (g).
In other words 6.25 N (g) = protein (g).
Dietary Fiber
Dietary fiber is defined as those components of food that
cannot be broken down by human digestive enzymes. Vege-
tables, wheat and most grain fibres are the best sources of
water insoluble cellulose, hemicellulose and legnin. Fruits, oats,
and legumes are the best source of water soluble fibers pectins,
gums, etc.
Another form of carbohydrate in the diet is as dietary fiber.
This form of carbohydrate is not digested. They are of two
types; insoluble and soluble. Soluble fiber foms a gel-like
solution when combined with water. Soluble fiber slows down
the passage of food through digestive tract. Oatbran, dried
beans vegetable and pulp of fruits are rich sources of soluble
fiber. Some type of soluble fiber appears to decrease cholesterol
absorption.
Insoluble fiber facilitates the movement of food though
the digestive tract but it tends to bind with certain minerals
during digestion and decrease their absorption. The bran that
NUTRITION 317
covers wheat, rice, coss and other plants whole grain is rich
in insoluble fiber.
Overall view of catabolic pathways of diet.
Almost all dietary carbohydrates is of plant origin. Dietary
carbohydrate can be divided into available (absorbable) and
unavailable (fiber) varities. Some types of fibers have a high
capacity for water absorption and increases the bulk of the
stool.
Protein-Calorie Malnutrition (PCM)
Two forms of protein-calorie malnutrition are kwashiorkor
and marasmus. They are seen in infants and young children
in Africa and Latin America due to severe poverty or as a
result of parental ignorance regarding infant feeding or child
neglect.
Kwashiorkor
It is a disease caused by malnutrition specifically by prolonged
insufficient intake of necessary proteins in infants. The infants
obtain enough calories but the high carbohydrate food does
not supply enough protein. The infant fail to grow. The chief
characteristic of kwashiorkor are lack of appropriate cellular
developments, edema, diarrhea, poor growth, low plasma
protein levels, muscle wasting, edema, diarrhea and increased
susceptibility to infection.
Marasmus
This occurs in infants who are weaned very early and who
are fed diets which are low in calories as well as protein. Since
severe malnutrition has occurred very easily in life, brain cells
develop less giving rise to mental retardation.
Protein calorie malnutrition can be prevented with protein
rich foods.
Marasmus is defined as inadequate intake of both protein
and energy.
Kwashiorkor is defined as inadequate intake of protein
in the presence of adequate energy intake.
Marasmic Kwashiorkor
This occurs when the clinical features of both marasmus and
kwashiorkor are present. Edema is present and body weight
is less than 60 percent of standard weight. Skin and hair
changes and fatty liver, characteristics of kwashiorkor are
found.
Vitamins
Vitamins are organic compounds, essential for health and
necessary for the maintenance of proper activity of the body.
Vitamins are present in the naturally occurring foods and are
required in very small amounts. A complete diet provides all
the necessary vitamin requirements of the body. Deficiency
of vitamins gives rise to various deficiency diseases.
Minerals
Inorganic substances do not supply energy but their presence
is necessary for the maintenance of certain physiochemical
conditions which are essential for life. Minerals cannot be
synthesized and required minerals are therefore dietary
essentials.
NUTRITION 319
Water as an Essential Nutrient
Humans can live without oxygen for only a matter of minutes.
Water is the next most essential requirement for life. Death
from dehydration following within several days without fluid
intake. Death can occur when there is excessive water loss
because of diarrhea, etc. The massive diarrhea of cholera can
kill in 8 hours. Water balance exists when fluid intake is
equivalent to output. Metabolic water is produced when
protons, electrons and oxygen react during metabolism.
FOOD VALUES
Ingredients Qty Protein Fat CHO Calories
calories
gm gm gm gm
(1) (2) (3) (4) (5) (6)
Rice 100 6. 5 0. 4 97. 0 345
Atta 100 12. 1 1. 7 69. 4 341
Dal (Avg) 100 24. 1 1. 2 60. 0 359
Vegetables (Avg) 100 2. 0 0. 2 6. 0 34
Vegetables (Green) 100 2. 0 0. 4 3. 3 25
Potatoes 100 1. 6 0. 1 22. 6 97
Orange 100 0. 6 0. 1 11. 4 48
Banana 100 1. 2 0. 4 27. 2 116
Mutton 100 18. 5 1. 33 194
Fish 100 17. 0 1. 3 1. 8 87
Liver (Goat) 100 20. 0 0. 3 107
Egg 100 13. 3 13. 3 173
Milk (Buffalo) 100 ml 4. 3 8. 8 5. 1 117
Milk (Cows) 100 ml 3. 2 4. 1 4. 4 67
Milk (DMS) 100 ml 3. 2 3. 5 4. 7 63
Milk (Skimmed fresh) 100 ml 2. 5 4. 1 4. 6 48
Cheese (Processed) 100 24. 1 25. 1 6. 3 22
Bread 100 7. 8 0. 7 51. 9 248
Cornflakes 100 6. 8 3. 8 88. 2 367
Soyabeans 100 43. 2 195 22. 9 432
Cream 100 36. 0 324
Butter milk 100 0. 8 1. 1 0. 5 15
Sugar 100 100.0 400
Honey 100 0. 3 79. 5 320
Sago 100 0. 2 0. 2 87. 1 351
In order to make the diet schedule of a person, first the
total caloric requirement is estimated from the table given
on next page. Out of this, one gm per kg body weight should
be provided in the form of proteins and fats each approximately
and rest all are given as carbohydrates.
Caloric Requirement
Activity Calories Protein Calcium Iron
(g) (g) (mg)
Normal man Sedentary 2400
Moderate 2800 55 .4-.5 20
Hard work 3900
Normal woman Sedentary 1900
Moderate 2200 45 .4-.5 03
Hard work 3000
Pregnancy +300 +10 1.0 40
Growing child
1 year 1200 17
2 years 18
3 years 20 16
.4-.5 to
4-6 years 1600 22 80
7-9 years 1800 23
10-12 years 2100 41
The detailed menu can be prepared roughly from the table
given below.
Normal Balanced Diet for Adult Man
Foodstuffs Sedentary work Moderate work Hard work
Veg Nonveg Veg Nonveg Veg Nonveg
(g) (g) (g) (g) (g) (g)
Cereal 400 400 475 475 650 650
Pulse 70 55 80 65 80 65
Leafy vegetables 100 100 125 125 125 125
Other vegetables 75 75 75 75 100 100
Roots and tubers 75 100 75 100 100 100
Fruit 30 30 30 30 30 30
Milk 200 100 200 100 200 100
Fat and oil 35 40 40 40 50 50
Meat/fish 30 30 30
Egg 30 30 30
Sugar/jaggary 30 30 40 40 55 55
Peanuts/fat 50/30 50/30
NUTRITION 321
Normal Balanced Diet for Adult Woman
(g) (g) (g) (g) (g) (g)
Cereal 300 200 250 353 475 475
Pulse 60 45 70 55 70 55
Leafy vegetables 125 125 125 125 125 125
Other vegetables 75 75 75 75 100 100
Roots and tubers 50 50 75 75 100 100
Fruit 30 30 30 30 30 30
Milk 200 100 200 100 200 100
Fats and oil 30 35 35 45 40 45
Meat/fish 30 30 30
Egg 30 30 30
Sugar/jaggary 30 30 30 30 40 40
For Growing Child
Foodstuffs 1 3 yrs 4-6 yrs 7-9 yrs 10-12 yrs
Veg Non- Veg Non- Veg Non- Veg Non-
veg veg veg veg
(g) (g) (g) (g) (g) (g) (g) (g)
Cereal 150 150 200 200 250 250 420 420
Pulse 50 40 60 50 70 60 70 60
Green leafy veg 50 50 75 75 75 85 100 100
Roots and tubers 30 30 50 50 50 50 75 75
Fruit 50 50 50 50 50 50 50 50
Milk 300 200 250 200 250 200 250 200
Meat/fish/egg 30 30 30 30
Sugar and jaggary 30 30 40 40 50 50 50 50
Balanced Diet for Pregnant Lady
(g) (g) (g) (g) (g) (g)
Cereal 350 340 400 400 525 525
Pulse 60 45 70 55 70 55
Roots and tubers 50 50 75 75 100 100
Contd...
Contd...
(g) (g) (g) (g) (g) (g)
Green leafy veg 150 150 150 150 150 150
Other veg 75 75 75 75 100 100
Fruit 30 30 30 30 30 30
Milk 325 225 325 225 225 225
Fats/oil 30 35 35 50 40 45
Sugar/jaggary 40 40 40 50 50 50
Meat/fish 30 30 30
Egg 30 30 30 40
1500 CALORIES DIABETIC DIET CHART
Total Food for Day
Foodstuffs Veg Nonveg Household
gm gm measures
(1) (2) (3) (4)
Wheat flour (unsifted) 125 125 5 small chapattis
Bread 50 50 2 slices
Milk (DMS) 400 400 2 small glasses
Green vegetables 500 500
Fruit 125 125 1 small
Dal/lean meat, 50 150 2 katori cooked
fish, chicken dal 1

serving of
mutton
Cottage cheese/egg 35 1
Salted biscuits 15 15
Ghee/oil 10 10 2 tea spoons
Butter 5 5 1 tea spoon
Protein-65 gm
(approx)
Fiber-11.5 gm
MEAL PLAN
Breakfast
Milk 1 cup Bread : 2 slices
Butter 1 tea spoon Cheese/egg : 1
Contd...
NUTRITION 323
Contd...
Foodstuffs Veg Nonveg Household
gm gm measures
(1) (2) (3) (4)
Mid morning
Fruit 1 Tea: 1 cup
(without sugar)
Lunch
Chapatti 3 (small) Cooked vegetable
and salad
Dal 1 katori Oil/ghee : 1 tea
spoon
Tea
Tea with-
out sugar
1-2 Biscuits
Dinner
Chapattis: 2
Cooked
veg 1 katori
Salad
Curd 1 katori
Dal 1 katori
Bedtime
Milk 1 cup
2000 Calories Diabetic Diet (Vegetarian/Nonvegetarian)
Carbohydrates = 280 gm, Fats = 64 gm, Proteins = 74 gm
Foodstuffs Quantity in gm
Atta 200
Vegetables 450
Milk 400 ml
Curd 200
Cheese 25
Egg 1 No.
Dal 30
Meat/fish/chicken 50/120/100
Bread 100
Ghee 15
Butter 15
Fruit 1 No.
Contd...
Contd...
Foodstuffs Quantity in gm
Condiments 10
Salt 15
Tea leaves 7
Breakfast
Milk 150
Bread 50 (2 slices)
Butter 8
Cheese 25
Egg 1 No.
Mid morning
Tea of coffee milk 50
Lunch
Atta 100
Vegetable 250
Dal 30
Meat/fish/chicken 55/120/100
Curd 100
Ghee 5
Evening
Tea of coffee milk 50
Bread 50 (2 slices)
Butter 7
Dinner
Atta 100
Vegetable 200
Curd 100
Fruit 1 No.
Ghee 5
Milk 150
NUTRITION 325
1800 Calories Low Cholesterol Diet
(Vegetarian/Nonvegetarian)
Carbohydrate = 312 gm, Fat = 30.6 gm, Proteins = 70 gm
Foodstuff Quantity in gm
Skimmed milk 600 ml
Curd (Skimmed) 200
Bread 100
Atta or rice 150
Dal (for veg) 50
(for nonveg) 25
Fruit 2 No.
Vegetables 500
Oil 25
Sugar 25
Salt 10
Tea leaves 7
Fish/chicken (for nonveg) 100/80
Breakfast
Skimmed milk 250 ml + sugar
Fruit 1 No.
Mid morning
Tea or coffee milk 50 ml + sugar
Lunch
Atta or rice 70
Fish/chicken 100/80
Vegetables 250
Curd 100
Fruit 1
Oil
Evening
Tea or coffee milk (50 ml + sugar)
Bread 50 gm
Dinner
Atta or rice 75
Vegetables 250
Curd 100
Oil 100
Bedtime
Skimmed milk 250 ml
LIVER FUNCTION TESTS
The various function tests that are in existence depending upon
the array of activities by the liver are enumerated below:
Metabolic Function
A. Carbohydrate metabolism:
a. Galactose tolerance tests (oral and intravenous).
b. Fructose tolerance tests.
B. Lipid metabolism:
a. Serum cholesterol: Free and esterified form of choles-
terol estimations.
b. Estimation of fecal fats.
C. Protein metabolism:
a. Total proteins, A/G ratio, prothrombin time.
b. Flocculation tests: Thymol turbidity test, zinc sulfate
test, colloidal gold test, cephalin cholesterol flocculation
test, formal gap test.
Detoxification and Protective Functions
A. Conversion of ammonia to urea: Estimation of blood urea
and blood ammonia.
B. Formation of bilirubin diglucuronide: Estimation of serum
bilirubin (direct and indirect), icteric index, urinary estima-
tion of bilirubin and urobilinogen.
C. Hippuric acid test.
Excretory Functions
Bromsulphalein (BSP) test.
Organ Function Tests
CHAPTER
17
ORGAN FUNCTION TESTS 327
Storage Functions
A. Glycogen estimation.
B. Lipid estimation.
C. Estimation of vitamin A, D, and B
12
.
D. Estimation of serum iron and serum iron binding capacity.
Hematologic Function
Estimation of prothrombin time and factors VII, IX and X.
Cellular Structural Studies
Liver biopsy:
The importance of liver function tests are:
1. To assess the severity of liver damage.
2. To differentiate different types of jaundice.
3. To find out the presence of latent liver diseases.
There are hosts of test to evaluate the functions of liver
but those that are commonly employed have got significance
for assessing the conditions of patients.
The first group of tests regarding the secretory, excretory,
and enzymatic functions are: Serum bilirubin estimation,
bilirubin and urobilinogen in urine, BSP excretion test, serum
alkaline phosphatase estimation and SGPT.
The second group meant for assessing the protein synthetic
functions are: Total proteins estimation, A/G ratio and pro-
thrombin time.
The final and the third group that are meant for lipid
metabolic functions are: Estimation of serum cholesterol and
determination of free and esterified cholesterol ratio.
Estimation of Serum Bilirubin
Bilirubin is estimated by van den Berghs reaction involving
Diazo reagent. van den Berghs reaction consists of two parts,
the direct and indirect reactions.
A. Direct reaction
a. Immediate direct reaction: An immediate development
of violent color in 10/30 seconds.
b. Delayed direct reaction: In which color appears from
five to thirty minutes and then develops slowly to a
maximum.
No direct reaction may be observed.
B. Indirect reaction, bilirubin bound to albumin is estimated.
Interpretations
Normal serum bilirubin level is 0.2-0.6 mg %.
Immediate direct reactionin obstructive jaundice.
Indirect/Delayed direct reactionin hepatic jaundice.
Direct reactionin hepatic jaundice.
The three types of jaundice has been discussed in Chapter 5.
Determination of serum bilirubin gives a measure of the
intensity of the jaundice. Higher values are found in obstructive
jaundice than in hemolytic jaundice.
Estimation of Urine Bilirubin
Urine bilirubin is qualitatively estimated by Fouchet reagent
and interpretations.
Estimation of Urine Urobilinogen
Urine urobilinogen is qualitatively estimated by Ehrlich reagent
and interpretations.
Bromsulphalein Excretion Test (BSP Test)
A measured amount of dye is injected intravenously. The liver
rapidly removes the dye and excretes in the bile. If the liver
function is impaired, the excretion is delayed and larger
proportion of dye remains in the serum. It is very sensitive
test and is most useful in liver cell damage without jaundice,
in cirrhosis and chronic hepatitis.
In healthy adults not more than 5% of the dye should
remain in blood, but the bulk of dye is removed in twenty
five minutes.
In hepatic diseases, cirrhosis, 4050% of dye retention takes
place. Also abnormal retention of dye in hepatocellular or
obstructive jaundice takes place.
Estimation of Serum Alkaline Phosphatase
Normal level is 3-12 King-Armstrong units or 3-4 Bodansky
units.
Increased level of alkaline phosphatase is found in post-
necrotic disease, cirrhosis, carcinoma of liver, obstructive
jaundice, hepatocellular jaundice, bone disease and may go
up to 200 KA units.
Serum alkaline phosphatase activity is high in obstructive
jaundice but remains unchanged in hemolytic jaundice. So
estimation of this activity may be of help to identify the type
of jaundice.
Estimation of Serum Glutamic Pyruvic Transaminase (SGPT):
Alanine Transaminase (ALT)
Normal SGPT level is 9-39 IU.
Increase of SGPT activity is a more specific indicator of
cell damage than that of SGOT.
Increased levels of SGPT are common in hepatocellular
damage and obstructive jaundice.
Determination of Serum Proteins and A/G Ratio
Serum proteins estimation yields most useful information in
chronic liver disease. The liver is the site of albumin, fibrinogen
and some of - and -globulins synthesis.
The normal serum protein level is 6.0-8.0 g%
Albumin level is 3.5-5.5 g%
Globulin level is 2.0-3.5 g%
The normal A/G ratio is 1.2:1.
In advanced liver disease, the albumin is decreased and
globulin is increased so that albumin-globulin ratio is reversed.
Serum proteins are decreased in malnutrition, liver damage.
Low serum albumin is found in severe liver damage due
to the impaired ability of the liver to form albumin.
Prothrombin Time
Prothrombin time is the time required for clotting to take place
in citrated plasma to which optimum amounts of thrombo-
plastin and calcium has been added.
Prothrombin is formed by the liver cells, vitamin K being
required. When bile salts are not present in the intestine, the
absorption of vitamin K from the intestine is impaired.
Prothrombin time is used in liver disease and jaundice.
In jaundice and liver disease, the prothrombin time is pro-
longed.
The normal prothrombin time is 16-18 seconds.
Estimation of Total and Esterified Cholesterol
Liver synthesizes, esterifies and excrete cholesterol into bile.
So cholesterol level is affected in liver disease.
Normal cholesterol level is 150-250 mg%. Esterified form
is 60-70% of the total.
Cholesterol level is decreased in hepatitis, cirrhosis, hyper-
thyroidism, malabsorption syndrome in severe wasting in
acute infections, pernicious anemia, etc.
Cholesterol level is increased in obstructive jaundice, intra-
hepatic obstruction, myxedema, lipid storage disease, athero-
sclerosis, nephrotic syndrome, diabetes mellitus, xanthomatosis.
Liver Biopsy
Histophathological studies of liver biopsy reveals various
pathological states of liver cells.
RENAL FUNCTION TESTS
Kidney plays an important role in the maintenance of acid
base-balance and volume of water in the body. It serves an
important function of excretion of products of metabolism and
other harmful substances.
Renal function tests are done to assess the functional capa-
city of kidney. The aims of renal function tests in clinical bio-
chemistry are to detect impairment of renal function as early
as possible.
The kidney regulates the chemical composition of body
fluids by selective filtration of blood through the glomerular
basement membrane.
The movement of molecules through the membrane is
dependent upon their size, plasma concentration and electrical
change. In healthy kidneys, most proteins are too large to
cross the basement membrane.
Damage to glomerular basement membrane in the kidney
can alter its permeability. This enables large protein molecules
such as albumin to pass through the membrane into the urine
resulting in proteinuria.
The size of protein molecule detected in the urine may
give an indication of underlying kidney dysfunction causing
proteinuria.
Following tests are generally done to assess the renal
function:
1. Concentration test (specific gravity test)
2. Dilution test
3. Phenolsulfonphthalein (PSP) test
4. Urea clearance test
5. Inulin clearance test
6. Creatinine clearance test
7. Renal blood flow.
Microalbumin
Detection of sustained elevations of albumin (>20 g/min) in
the urine indicates kidney damage and if left untreated can
result in irreversible damage. Microalbuminuria (20-200 g/
min) is an early marker of renal damage and enables preventive
measures to be taken. Measurement of urinary albumin levels
is an important diagnostic test in conditions associated with
increased risk of renal failures, e.g. diabetes, essential hyper-
tension, nondiabetic renal disease and pregnancy.
Concentration Test
This is designed to test the concentrating power of the kidneys.
The capacity of the kidney to concentrate urine is one of the
most sensitive tests for early loss of function. It is also the
simplest test to perform since it does not require any laboratory
facilities.
When the kidney loses its capacity to do osmotic work,
the urinary solids must be excreted in more dilute solution,
amount of solids to be excreted, greater volume is required
to accommodate the same.
The specific gravity of urine, which in the normal individual
fluctuates widely in response to fluid intake becomes confined
within normal limits until it is fixed at approximately 1010.
These changes are usually expressed as polyuria. The day night
ratio of urine volume, normally 3-4 to 1 tends towards 1:1
ratio.
The advantage of this test is that it is useful for the detection
of renal defect where the blood urea is normal.
Dilution Test
In addition to the loss in the power of the kidneys to produce
concentrated urine, there is also an impairment in its ability
to excrete dilute urines. This later fact has been used in dilution
test.
In this test no water is taken after midnight, the bladder
is emptied at 7 AM and the patient is given 120 ml of water
to drink in 30 minutes. Urine samples are collected hourly
for next four hours, i.e. at 8, 9, 10 and 11 AM. The volume
and specific gravity of each specimens are measured.
In the normal individual, approximately 1200 ml of urine
will be excreted during this time and the specific gravity of
at least one specimen should fall to 1003 or below. With
impaired renal function such a low specific gravity is not
reached and small volumes may be less than 100 ml and the
specific gravity of urine may not fall below 1010.
Phenolsulfonphthalein (PSP) Test
PSP test indicates a general loss of nephron function. This test
consists of intramuscular injection of solution of PSP, a dye
that is eliminated only by the kidneys, the amount of this dye
in urine can be estimated colorimetrically. The time of its first
disappearance in the urine and the quantity eliminated within
a definite period are taken as a measure of the functional
capacity of the kidneys. The test is harmless, simple and for
general purposes the most satisfactory of the functional tests.
In the normal individuals 25% or more of the dye will be
excreted during the first 15 minutes after intravenous injection
and not less than 70% will be excreted during the two-hour
period. The rate at which the dye appears in the urine depends
both on the renal blood flow and the action of tubular
epithelium in removing the dye from the blood.
Percentage of impairment of PSP excretion is as follows:
a. Slight: 52-40% excretion of injected dye
b. Moderate: 39-25% excretion of injected dye
c. Marked: 20-11% excretion of injected dye.
Urea Clearance Test
Urea clearance is defined as the number of ml of blood which
contains the urea excreted in a minute by the kidneys.
Urea clearance =
mg urea excreted per minute
mg urea per ml of blood
=
UV
B
where U = mg urea per ml of urine
B = mg urea per ml of blood
V = ml urine excreted per minute.
The clearance is a ratio of the urinary excretion to the
average blood level determinated simultaneously. Such a ratio
is correlated more directly with the progress of kidney disease
and shows a deviation from normal in early renal damage.
Normal urea clearance on average is 75 ml per minute when
rate of excretion of urine is 2 ml or more per minute. This
is maximum urea clearance.
Maximum urea clearance
=
observed urea clearance
100
average normal standard urea clearance

=
UV
100
B
75
=
100 UV
75 B
If the quantity of urine excreted is less than 2 ml per minute
then the clearance is found to be 54 ml per minute and this
clearance is called standard urea clearance.
Standard urea clearance
=
observed urea clearance
100
average normal standard urea clearance

=
UV
B
100
54 V
=
100 U V
54 B
Grading renal function on the basis of urea clearance value
is as follows:
Urea clearance Renal function
Over 70 Normal
70-40 Mild deficit
40-20 Moderate
Below 20 Severe deficit
Below 5 Coma
Inulin Clearance Test
This test is done to find the glomerulus filtration rate. Inulin
is filtrated by the glomerulus but it is neither secreted nor
absorbed by tubules. Inulin is given subcutaneously or by
intravenous infusion. The amount of inulin excreted in each
minute (U in V) is equal to the amount filtered by the glomeruli.
The concentration of inulin in the glomerular filtrate is equal
to that in the plasma. So the clearance value of inulin is same
as glomerular filtration rate.
Normal rate is 110 to 150 ml per minute.
Glomerular filtration rate (ml per minute)
= Inulin clearance C =
UV
C
B

=

=
mg inulin per 100 ml urine
ml urine passed per minute
mg inulin per 100 ml plasma
Creatinine Clearance Test
Creatinine clearance test is also done to find out the glomerular
filtration rate but the situation is complicated in human beings
because a portion of creatinine is secreted by tubules. This
portion rises unpredictably when failure of filtration occurs
in renal disease.
Normal value for creatinine clearance is 95-105 ml per
minute.
Renal Blood Flow
Renal blood flow can be determined by using para-amino
hippuric acid, which at low blood concentration is removed
almost completely by tubular excretion in a single circulation
through kidney.
Effective renal blood flow as calculated from this type of
clearance procedure is about 1000 to 1150 ml per minute or
expressed as plasma flow about 600 to 700 ml per minute.
PANCREATIC FUNCTION TEST
The important constituents of pancreatic juice are:
1. Enzymes
a. Carbohydrates splitting enzymes such as - and -amy-
lases.
b. Proteolytic enzymes consist of trypsinogen, chymo-
trypsinogen and peptidase. They are converted to
trypsin, chymotrypsin and carboxypeptidase respec-
tively. Other proteolytic enzymes include deoxyri-
bonuclease (DNAase) and ribonuclease (RNAase).
c. Fat splitting lipase acts on neutral fats and phospholipids
liberating fatty acids and glycerol.
2. Bicarbonate.
3. Water.
Bicarbonate and water are the major components of
pancreatic juice. Daily volume varies from 1500-3000 ml. The
bicarbonate and the fluid is dependent on the hormone
secretion. This hormone is secreted mainly as a result of
stimulation by HCl.
Enzyme secretion in the pancreatic juice is not under the
control of secretion but of another hormone pancreozymin.
The test most commonly employed in pancreatic dysfunction
are:
1. Determination of enzymes in serum and urine. The
enzyme studies are amylase and lipase.
2. Examination of stool.
Determination of Serum Amylase
Serum amylase is estimated by two methods.
Sacchrometric Method
Serum is incubated with a starch solution and the amount of
reducing substances present is determined before and after
the incubation. The difference gives a measure of amylolytic
activity of the serum.
Somogyis Iodine Test
The time required for complete digestion of a certain amount
of starch is determined by periodic testing with iodine.
Normal serum amylase value is 80-200 Somogyi units per
100 ml.
It is increased in acute pancreatitis, as high as 1000 Somogyi
units per 1,000 ml and even more.
Low serum amylase has been found in abscess of liver,
acute hepatocellular damage, cirrhosis of liver, cholecystitis.
Amylase is usually absent in newborn.
Urinary Amylase
Variation in urinary amylase reflects alteration in serum
amylase so long as kidneys are functioning normal. In renal
disease, serum amylase may be increased and urine amylase
is low.
Normal value is 1-3 ml/minutes.
It is elevated in acute pancreatitis, obstruction of pancreatic
duct and in cases of pancreatic carcinoma.
Determination of Serum Lipase
Serum lipase hydrolyzes the esters of long chain fatty acids
containing 818 carbon atoms.
Serum lipase parallels change in amylase but rises later and
lasts longer. The increase is more pronounced. Serum lipase
value is elevated in all conditions in which amylase is elevated.
Serum lipase value is more informative than amylase in
pancreatic cancer.
Urinary Lipase
Lipase is excreted by the kidneys and can be demonstrated
in the urine.
It is elevated in:
1. Hemorrhagic pancreatitis.
2. Some cases of renal impairment.
Stool Examination
i. Fat in stool
ii. Nitrogen in stool.
Fat in Stool
Ingested fat is normally split by pancreatic lipase into fatty
acids and glycerol and the products of hydrolysis are absorbed
by intestinal tract. Therefore, in stool, the neutral fat, free
fatty acids and soaps are relatively 6 gm/24 hr.
Increase in neutral fat (steatorrhea) to 11% represents
deficiency of fat splitting enzyme.
Nitrogen (Protein in Stool)
a. Clastro colic fistula
b. Obstructive jaundice
Nitrogen (protein in stool)
Total fecal nitrogen is 0.25-2 g/day.
Increased nitrogen content is found in pancreatic insuffi-
ciency.
In addition to these, various other tests such as provocative
test, secretion test. Pancreozymin test, vitamin A tolerance
test and fat absorption test are also helpful in pancreatic
dysfunction.
GASTROINTESTINAL (GIT) FUNCTION TEST
D-xylose Excretion Test
Function test for: GIT
Condition and Limitations
Gives true results only when kidneys are normal.
Method
The patient is given 5 gm of D-xylose orally, urine samples
are collected for the next 5 hours and a blood sample is
collected after 2 hours.
Xylose is absorbed in the intestine, reaches liver and
excreted by kidneys. The blood level reaches around 35 mg%
after excretes arount 1.5 gm of xylose in urine with in five
hours.
Result
Lower values of blood and urine indicate malabsorption due
to mucosal damage.
IMMUNOLOGY 339
INTRODUCTION
The main function of immune system is to prevent or limit
infections by microorganisms such as bacteria, viruses, fungi
and parasites.
Protection is provided primarily by cellmediated and
antibody mediated (humoral) arms of immune system. Two
other major components of immune system are complement
and phagocytosis.
Cell mediated arm consists of T lymphocytes (e.g. helper
T cell and cytotoxic T cell) and humoral arm consits of B
lymphocytes. B lymphocytes when activated convert into
plasma cells which in turn produce antibodies. The main
functions of antibodies are to:
1. Opsonize bacteria
2. Neutralize toxins and virus cell mediated immunity:
(i) inhibits organisms sich as fungi, parasites and intercel-
lular bacteria, it also kills virus infected cells and tumor
cells, (ii) regulates antibody response.
Natural and Acquired Immunity
Natural immunue is resistance not acquired through contact
with an antigen. It is nonspecific this immunity does not
improve after exposure to organism in contrast to acquired
immunity, also natural immune response have no memory in
contrast to long-term memory of acquired immunity.
Active and Passive Immunity
Active immunity is resistance induced after contact with
foreign antigens, e.g. microorganism. In this, the host actively
Immunology
CHAPTER
18
produces immune response consisting of antibodies and
activatedd helper and cytotoxic lymphocytes. Main advantage
of acute immunity is that response is long-term but major
disadvantage is its slow onset.
Passive immunity is resistance-based on antibodies per-
formed in another host. Performed antibodies against certain
viruses (e.g. rabies and hepatitis A and B) can be injected to
limit viral multiplication other forms of passive immunity are
IgG passed from mother to fetus during pregnancy and IgA
passed from mother to newborn during breastfeeding.
Antigens
Antigens are molecules that react with antibodies whereas
immunogens are molecules that induces an immune response.
In most cases, antigens are immunogens, but there are certain
exceptions, e.g. haptens.
Haptens are molecules that are not immunogenic but can
reach with specific antibody. Haptens are usually small
molecules and are not protein in nature, e.g. penicillins,
catechol.
IMMUNOLOGY 341
Features of molecules that determine immunogenicity are
as follows:
A. Molecular weight
B. Complexity of chemical structure
C. Foreigness
D. Genetic constitution of host.
Origin of Immune Cells
During postnatal life, stem cells reside in bone marrow and
differentiate into erythroid, myeloid and lymphoid series. The
latter give rise to 2 populations in ratio of 3:1 as T and B-
lymphocytes.
Thymic Selection
Negative Selection
CD-4+, CD-8+ cells, bearing antigen receptors for self-proteins
are killed by programmed cell death called apoptosis. This
refer to clonal deletion.
Positive Selection
CD-4+, CD-8+ cells, bearing antigen receptors that do not react
with self MHC proteins are also killed.
These two processes produce cells that are selected for
their ability to react both with foreign antigens via antigen
receptors and with self MHC proteins.
T Cells
Within the thymus, T cells differentiates under the influence
of thymic hormones (thymosine) to express surface glyco-
proteins, e.g. CD-3, CD-4, CD-8 (CD-cluster of differentiation).
CD-3 is present on all T cells. But CD-4 and CD-8 are present
on different populations of T cells.
Activation of T Cells
Activation of helper T cells requires that they recognize a
complex on the surface of antigen presenting cells (APC) like
macrophages. B cells dendritic cells, Langerhans cells within
the cytoplasm of APCs let say, macrophage, foreign protein
is cleaved into small peptic. These small peptides are then
associated wtih MHC II proteins. This complex of small
peptides and MHC II molecules are transported to cell surface
of macrophage, where this complex is presented to receptors
on CD-4+ helper T cells.
Similar events occur within a virus infected cell, cleaved
viral peptide associates with a class I MHC molecule and
complex is transported to the surface and viral antigen is
presented to the receptor on CD-8+ cytotoxic cell.
Further steps in activation include the following:
1. Interaction of antigen with TCR (that is present on T cells)
specific for that antigen.
2. IL-1 produces by macrophages is also necessary for
activation.
3. For full activation of helper T cells an additional co-
stimulatory signal is required, i.e. B protein present on
surface of APC must interact with CD-28 protein on helper
T cell.
IMMUNOLOGY 343
Generally speaking class I MHC proteins present endo-
genously synthesized antigens, e.g. viral proteins whereas
class II MHC proteins present the antigens of extracellular
microorganisms that have been phagocytowed, e.g. bacterial
proteins.
There are two subpopulations of helper T cells. These are
named as Th
1
and Th
2
. These two types of cells are orginated
from Th-O cells also known as native helper T cell. This
gives use to different types under influence of different
interleukins.
FUNCTIONS OF T CELLS
Effector Functions of T Cells
1. Delayed hypersensitivity against intracellular bacteria like
mycobacterium tuberculosis. It is shown by T
H
cells.
2. Cytotoxicity: By it body destroys virus infected cells, tumor
cells and allograft rejection. T cells take part in this by
releasing performs.
3. Antibody dependent cellular cytotoxicity (ADCC).
Antibody bound to surface of infected cell is recognized
by IgG receptors on the surface of macrophages and NK cells.
IMMUNOLOGY 345
Regulatory Function of T cells
1. Antibody production: Helper T cells secrete IL-4 (B cell
growth factor) and IL-5 (B cell differentiation factor) which
are responsible for production of antibodies.
2. Cell mediated immunity: Helper T cells also secrete IL-2 (T
cell growth factor) which acts on the same cell at IL-2
receptor, thus increasing the cell mediated immunity.
B Cells
B cells constitute about 30% of circulating pool of small
lymphocytes and their life-span is short. B cells precursors
after originating from fetal cover migrate to bone marrow
and they do not require thymus for maturation. There are
two stages of development of B cells:
1. Antigen independent
2. Antigen dependent.
Antigen independent phase include: Stem cells pre B
cells B cells.
Antigen depedent phase includes: Activates B cells
plasma cells.
Activation of B Cells
1. When antigen binds to surface IgM present on B cell surface,
endocytosis of that takes place this antigen is processed and
appear on the surface again in conjugation with class II MHC
proteins. This complex is recognized by helper T cells and
this produces IL-4, IL-5 which are B cell growth factor and
differentiation factor respectively.
2. Costimulatory signal is necessary
CD-28 on T cell must interact with B-7 on B cell.
This signal stimulate T cell to produce IL-2.
3. CD-40 L on T cell must interact with CD-40 on B cell. This
interaction is required for class switching from IgM to IgG.
Macrophages
These are derived from bone marrow and exist as both free
and fixed.
a. Free macrophages: Wandering macrophages, e.g. mono-
cytes.
b. Fixed macrophages, e.g.
Kupffer cells (liver)
Langerhans cells (skin)
Neurological cells (brain)
Dust cells (lung).
Macrophages migrate to the site of inflammation under
the influence of Csa, Anaphylatoxin.
There are three main functions of macrophages:
1. Phagocytosis
2. Antigen presentation
3. Cytokine production, e.g. IL-1, TNF.
Natural Killer Cells
1. These are called so because they are active without prior
exposure to virus.
2. These are nonspecific.
3. These kill without antibodies but presence of antibodies
enhance its efficiency.
4. They kill virus infected cells and tumor cells by secreting
performs and these are similar to cytotoxic T cells.
5. IL-12 and gamma interferon are potent activator of NK
cells.
6. NK cells are 5-10% of peripheral lymphocytes.
7. NK cells have no memory. No T cell receptor (TCR), no
requirement of MHC proteins and no passage through
thymus for maturation.
Differences between T cells and B cells
Characters T cells B cells
1. IgM on surface
2. CD-3 on surface
3. Immunoglobulin synthesis
4. Regular of antibody synethsis
5. IL-2, 4, 5, gamma interferon synthesis
6. Effector of cell mediated immunity
7. Maturation in thymus
8. Maturation in bursa equivalent
IMMUNOLOGY 347
Brief Description about Cytokines
1. Interleukin 1
Source: Macrophages
Function:
activator of Th cells
endogenous pyrogen
2. Interleukin 2
Source: Th
1
subset of helper T cell
Function: T cell growth factor (TCGF)
3. Interleukin 4
Source: Th
2
subset of helper T cells
Function: B cell growth factor (BCGF)
4. Interleukin 5
Source: Th
2
Function: B cell differentiation factor (BCDF)
5. Gamma interferon
Source: Th
1
Function: Stimulate phagocytosis and killing by macro-
phages and NK cells
6. Tumor necrosis factor (TNF)
Source: Macrophages
Function: Causes necrosis of tumors
7. Transforming growth factor (TGF-)
Source: T cells, B cells, macrophages
Function: Anticytokine actions.
Antibodies
Antibodies are globulin proteins (immunoglobulins) that
react specifically with the antigen that stimulated their
production.
Antibodies are gamma globulins.
Structure
Immunoglobulins are glycoproteins made up of light (L) and
heavy (H) polypeptide chains. Molecular weight of H and L
chains are respectively 50,000 and 25,000.
Shape: Y shaped
Consists of:
Four polypeptide chains
Two heavy chains
Two light chains
Linked by: Disulphide bond.
Specialties
1. An individual antibody molecule always consists of
identical H and identical L chains.
2. L and H chains are subdivided into variable and constant
regions:
L chain: Constant region CL
Variable region VL
H chain: Constant region CH
Variable region VH
3. L chains are of two types kappa () and lambda ().
4. H chains are of five types gamma (), alpha (), mu (),
delta () and epsilon ().
5. Variable regions are responsible for the binding of antigens
whereas constant regions are responsible for:
a. Complement activation
b. Various biologic functions.
6. IgG and IgA have three Ch domains and IgM and IgE have
four.
Effect of Proteolytic Enzymes
1. Papain: It breaks the immunoglobulin molecules in hinge
region, above disulphide interchain bond. Thus producing
two identical Fab fragments and one Fc fragments.
2. Pepsin: It breaks the immunoglobulin molecules at hinge
region below the interchain disulphide bond, thus, produ-
cing one Fab fragment and many Fc fragment.
IMMUNOLOGY 349
Isotype, Allotype, Idiotype
Isotype
Isotypes are defined by antigenic differences in the their cons-
tant regions like IgG, IgA, IgD, IgM, IgE are different isotype,
also IgG
1
, G
2
, G
3
, G
4
and IgA
1
, A
2
are different isotypes.
Allotype
Allotypes are additional antigenic features immunoglobulins
that vary among individuals.
e.g. H chain contains an
allotype called Gm
k L chain contains an
allotype called Inv.
Idiotypes
Idiotypes due to variation in variable region of H and L chain
individual CDR which differ with each other are known as
idiotypes.
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IMMUNOLOGY 351
About idiotypes, immunological network are made by Jerne,
which consists of idiotype, anti-idiotype.
Antibody Diversity
This is due to:
1. Multigene organization
2. Combinatorial joining
3. Junctional flexibility
4. Somatic hypermutation.
Antibody Class Switch (IL-4)
Mature B cells 1st express both IgM and IgD following
stimulation. Other isotypes are also produced so, it is clear
that there is switch from IgM and IgD to other classes.
Eight types of genes are present in definite sequence in
C chain from 5-3,
Each of these C genes, have a switch site towards 5 end
which are 1-2 k base pair length but not present at 5 end
of C
So, this leads to expression of IgM and IgD together rest
are switched from this.
Hybridoma and Monoclonal Antibodies
Hybridoma is a hybrid cell capable of producing monoclonal
antibodies. When one clone of antibody producing cells secrete
a particular type of antibody against a particular antigenic
determinant. It is called as monoclonal antibodies.
Kohler and Milstein in 1975, first produced monoclonal
antibodies from hybridoma cells. They showed that
cultured splenic cells from mouse immunized with specific
antigen can be fused with that of cultured mouse myeloma
cells in 5:1 ratio. The hybrid cells so produced will remain
immortal in culture like myeloma cell and produce
monoclonal antibodies like immunized splenic cells.
Uses
1. Diagnostic purpose:
Bacterial viral diseases
Blood grouping.
2. Therapeutics:
Anticancer therapy
Immunosuppression in organ transplantation.
MHC Molecules
Also known as HLA complex
Discovered by Dausset
Present in all nucleated cells
Coded by genes present on chromosome 6
3 groups
Class I 2000 kb ABC
Class II 1000 kb DP, DQ, DR
Class III 1000 kb
Class I codes protein for antigen recognition and binding
by T cells
Class II codes protein for antigen recognition and binding
by Th

cells
Class III: Do not participate in major histocompatibility but
some other products like tumor necrosis factor, heat shock
protein, complement component, etc.
Class I Molecule
Long glycoprotein polypeptide chain. 3 regions in chain
1

2

3
and associated with other molecule
2
micro-
globulin, i.e. non MHC molecule
Ag binding site is present between
1
and
2
.
This codes protein for antigen recognition and binding by
cytotoxic T cells
This only expresses endogenous antigen like that of viral
antigens.
IMMUNOLOGY 353
Class II Molecule
It is heterodimer consisting of
1

2

1

2
Ag binding site is present between
1
and
1
This codes protein for antigen recognition and binding by
Helper T cells
This only expresses exogeneous antigen like that of bacterial
antigens.
*Class I includes 3 multiple gene loci A, B, C while
Class II includes DP, DQ, DR.
MHC Polymorphism
Need: Large variety of antigens to be present so wide range
of antigens binding sites on these molecules
This is due to following reasons:
Multiple gene loci
Multiple alleles for a locus
Codominant expression.
Complement System
The complement system consists of approximately 20
proteins that are present in normal human serum. The
complement refers to ability to those proteins to comple-
ment, i.e. augment, the effecsts of other components of
immune system. So, this is important component of our
innate host defences.
There are three main effects of component of complement:
1. Lysis of bacteria, tumors
2. Generation of mediators
3. Opsonization.
Complement system is heat labile while antibodies are heat
stable.
This consists of C
1
+ Ca
C
1
is formed by C
1q
C
1r
C
1s
.
Activation of Complement
There are two pathways of activation of complement:
1. Classical pathway
2. Alternate pathway.
Classical Pathway
This is due to Ag-Ab complex CH
2
domain of Fc portion of
Ab remains hidden when it is exposed due to binding of Ag.
This activate complement
Ag-Ab complex
activates C
1q
C
1r
C
1s
Due to active esterase

activing of C
1S
Activation of C
4
C
4a
+ C
4b
then of C
2
C
2a
+ C
2b
Now this C
4b, 2b
acts as
C
3
convertase
Which activate C
3
C
3a
+ C
3b
Chemotactic Opsonization
and anaphylactic
C
3b
attach to C
3
convertase, to form C
5
convertase
which activate C
5
C
5a
+ C
5b
Chemotactic
and anaphylactic
C
5b
joins C
5
convertase
then sequential attachment of C
6
C
9
Membrane attach complex lysis

IMMUNOLOGY 355
Alternate Pathway
This may include properdin, cobra-venom, IgA, IgE,
bacterial endotoxin, yeast cell wall, etc.
Properdin: protein in serum
combines with zymosan

Mg
2+
properdin-zymosan complex
This activates C
3
directly rest sequences are repeated to
form membrane attack complex (MAC) with the help of
factor B and factor D.
Biologic Effects
1. Opsonization C
3b
2. Chemotaxis C
5a
3. Anaphylatoxin C
3a
C
4a
C
5a
4. Cytolysis
5. Enhancement of antibody production.
Cancer cells are characterized by three properties:
1. Diminished control of growth, i.e. loss of contact inhibition.
2. Invasion of local tissues.
3. Spread metastasis to other body parts cancer specially
refers to malignant tumor for benign tumor properties (2)
and (3) are absent.
Certain genes controlling growth and interactions with
other normal cells are apparently abnormal in structure of
regulation in cancer, e.g. isolation of BRCA-1 gene which
increases susceptibility to breast and ovarian cancer.
Agents Causing Cancer
Cancer is second most common cause of death in USA after
cardiovascular diseae.
Agents causing cancer fall into three main categories:
1. Radiant energy
2. Chemical compounds
3. Some virus.
Radiant Energy
UV rays, X-rays, -rays are mutagenic and carcinogenic. UV
rays cause pyridine dimers to form. Also X-rays and -rays
causes free radicals to form in tissues. These free radicals
damage the DNA and macromolecules.
Chemical Compounds
Approximately 80% of human cancers are caused by environ-
mental factor, principally chemicals. These include polycyclic
Cancer
CHAPTER
19
CANCER 357
aromatic hydrocarbons, aromatic amines, nitrosam-ines,
arsenic, cadmium, chromium, D-actinomycin, aflatoxin B
1
.
Viruses
Viral oncogenesis is also important aspect to study. These
viruses include adenovirus. Herpes virus, retrovirus, Epstein-
Barr virus (EB-virus).
Changes during Malignant Transformation
1. Alteration of morphology
2. Loss of contact inhibition of growth
3. Loss of anchorage dependence
4. Loss of contact inhibition of movement
5. Increased rate of glycolysis
6. Diminished requirement for growth factors.
Oncogenes of Rous Sarcoma Virus
Various Mechanisms of Activation of
Proto-oncogenes to Oncogenes
1. Promotor insertion: Certain retroviruses lack oncogenes but
may cause cancer over a longer period of time than those
containing oncogenes. Promotor is inserted upstream to
myc gene. The integrated ds-cDNA is called provirus.
2. Enhancer insertion: In this case, provirus is inserted down
stream from the myc gene or upstream from it but oriented
in reverse direction.
3. Chromosomal translocation:
a. Philadelphia chromosome: Involves translocation in
chromosome 9 and 22 and causes chronic myelogenous
leukemia.
b. Burkitt lymphoma: Involves translocation in chromosome
8 and 14. This is fast growing cancer of human B-lymp-
hocytes.
4. Gene amplification: Administration of anticancer drug metho-
trexate, inhibitor of enzyme dihydrofolate reductase.
Resistance to this drug implies the amplification of gene
for dihydrofolate reductase.
5. Point mutation: Point mutation is also able to convert proto-
oncogene to oncogene.
Tumor Suppressor Genes
RB
1
It is involved in genesis of retinoblastoma
Gene is located on chromosome 13 and 14
pRB is nuclear phosphoprotein
Unphosphorylated species of pRB binds certain viral
proteins, forming complexes that inactive them.
p53
Acts as guardian of genome or gurdian of tissue
p53 is nuclear phosphoprotein
p53 binds various viral proteins forming inactive complexes.
CANCER 359
Mechanism of Action of Oncogenes
Cancer Chemotherapy
Compound Treatment use
1. Vincristine and vinblastine Kaposis sarcoma
(from chrysanthemum)
2. Cisplatin Carcinoma of lung
(metallo-organic compound of
platinum)
3. Alkylating agents Myeloma
4. Antimetabolites Leukemia
5. Antitumor antibiotics Hodgkins disease
Hormones are defined as the chemical substances formed in
one part of the body, carried in the blood stream to the other
organs or tissues where they exert the action.
The main function of hormones is to catalyze and control
various metabolic reactions.
They resemble enzymes in two ways:
1. They are catalyst and needed in very small amount.
2. They are not used in the reaction and hence, resemble
enzymes.
They differ from enzymes in several aspects.
1. Enzymes are utilized at the site where they are
produced, while in case of hormones the site of origin
is far from the site of action (target organ).
2. Hormones have to be discharged into blood stream
whereas enzymes show their action in the cell.
3. Hormones are not only proteins but also have diverse
structure. Hormones can be proteins, small peptides,
single amino acids or steroids whereas enzymes are only
protein in nature.
Hormone Action
General features of hormone classes:
Group I Group II
Types Steroids, iodothyronines Polypeptides proteins
calcitriol glycoproteins
Solubility Lipophilic Hydrophilic
Receptor Intracellular Plasma membrane
Transport proteins Yes No
Hormones
CHAPTER
20
HORMONES 361
Group I (Steroid) Hormones Action
These hormones diffuse through plasma membrane of all cells
and encounter specific receptors in target cells.
This hormone receptor complex undergo activation process.
Now this binds to hormone response element (HRE) of DNA
and activate the inactivated specific genes. To the 3 end of
HRE there is promotor element (PE), which is to the 5 end
of structural gene. In this way, information is transferred and
there is formation of specific proteins to elicit metabolic
response.
Group II (Peptide) Hormones
These have membrane receptors and information is carried
in cell by secondary messengers.
For example, we are considering adenyl cyclase system
here the interaction of hormone with its receptor results in
activation/inactivation of adenyl cyclase. This is coupled to
a protein which is having intrinsic GTPase activity. By this
activation of adenylyl cyclase. c-AMP formation takes place
which activate protein kinase, which in turn phosphorylate
proteins to phosphoproteins, which elicit physiological effects.
Cyclic AMP (3',5' Cyclic Adenylic Acid)
Cyclic AMP is formed from ATP by the enzyme adenyl cyclase.
Hormones which activates adenyl cyclase are:
1. Epinephrine, (more in muscle than liver)
2. Norepinephrine
3. Glucagon, (brings about a greater increase of c-AMP
in liver than in muscle)
4. Thyroid.
Thus, by activating the enzyme adenyl cyclase, they increase
the level of c-AMP.
c-AMP is destroyed by the enzyme phosphodiesterase to
5'-AMP.
Hormone which activates phosphodiesterase is insulin.
Thus the level of c-AMP is the result of these two enzymes.
Functions
1. c-AMP stimulates glycogenolysis and inhibits glycogenesis.
2. c-AMP acts like a second messenger.
3. To stimulate specific kinase enzymes.
Lipolysis is controlled by the amount of c-AMP present
in the tissues. The process that destroy or preserve c-AMP
has an effect on lipolysis.
The enzyme phosphodiesterase is inhibited by caffeine, xan-
thine theophylline, etc. giving rise to accumulation of c-AMP
in tissues. This leads to increased lipolysis giving rise to more
FFA in the plasma.
The enzyme adenyl cyclase is inhibited by insulin, nicotinic
acid and prostaglandins.
Hormones are classified structurally into three groups:
1. Amino acid derivatives: Those hormones derived from amino
acid thyrosine such as epinephrine, norepinephrine and
thyroid hormones.
2. Peptide: Protein hormones: Those hormones containing large
proteins or medium size peptides such as insulin, glucagon,
parathormone, calcitonin, pituitary hormones.
3. Steroid hormones: They are all derived from cholesterol
and contain steroid nucleus, i.e. cyclopentanoperhydro-
phenenthrene nucleus. Progestrogen, estrogen, androgen,
mineralocorticoid and glucocorticoid.
HORMONES 363
Glucocorticoids (cortisol), mineralocorticoids (aldosterone), and
progestrogens (progesterone) all contain 21C carbon atoms and
are referred as C-21 steroids. The androgens (testosterone) are
C-19 steroids while estrogens (estradiot) are C-18 steroids.
Androgens and estrogens contain a potential keto group
at C-17 position and hence are called 17-ketosteroid.
Steroids, hormones are divided into three types:
i. Glucocorticoids: They primarily affect metabolism of
carbohydrates, fats and proteins but are most important
in adaptation to stressful situations, examples: cortisol,
cortisone and corticosterone.
ii. Mineralocorticoids: Those hormones which are important
for the regulation of salt balance, i.e. reabsorption of Na
+
and excretion of K
+
and water distribution in tissues.
Examples: Aldosterone, II-deoxycortisol and II-deoxy-
corticosterone (DOC).
iii. The adrenal androgens: Those which are important in
the development of sexual hair and libido in females.
INSULIN
Insulin is an anabolic protein hormone. It is isolated from
pancreas. Its crystalization requires traces of zinc.
The major metabolic actions of insulin are centered in liver,
muscle and adipose tissue.
Liver
Glucose is freely permeable to liver cells.
Insulin induces some of the enzymes of gluconeogenesis.
Insulin stimulates glycolysis by stimulating the synthesis
of following enzymes:
1. Glucokinase
2. Phosphofructokinase
3. Pyruvate kinase.
Insulin depresses gluconeogenesis by depressing the
synthesis of the following enzymes:
1. Pyruvate carboxylase
2. Phosphoenol pyruvate carboxykinase
HORMONES 365
3. Fructose-1, 6-diphosphatase
4. Glucose-6-phosphatase.
Muscle and Adipose Tissue
Insulin stimulates the metabolism giving rise to:
1. Increased glycogen deposition
2. Increased glycolysis
3. HMP shunt pathway is stimulated
4. Increased fatty acid synthesis.
In adipose tissues, insulin increases, fatty acid synthesis
from Acetyl CoA (glycolysis ) and NADPH (HMP Shunt
pathway ) and triglyceride synthesis from glycerophosphate.
In adipose tissues, insulin inhibits the release of free fatty
acids. Since the liberation of fatty acids from adipose tissues
is stimulated by c-AMP, insulin depresses c-AMP level and
hence, inhibits fatty acid release giving rise to enhanced
lipogenesis and triglyceride synthesis.
Insulin Receptors
Insulin receptors has been studied in great detail using bio-
chemical and recombinant DNA techniques. These are
glycoprotein in nature. It is heterodimer consisting of two
subunits called as and . This is represented as
2
2
. The
two subunits are linked by disulphide bonds.
-subunit is extracellular and it binds insulin.
-subunit is transmembrane protein and serves the purpose
of signal transduction cytoplasmic portion of -subunit has
tyrosine kinase activity and an autophosphorylation site.
The human insulin receptor gene is located on chromo-
some 19
Insulin receptor density is 20,000 per cell
Both subunits are extensively glycosylated
Effect of binding of insulin to insulin receptor:
1. There is conformational change in receptor
2. The receptors make cross links
3. Receptors are internalized
4. One or more signals are generated (See on page 366).
Diabetes
Insulin deficiency results in diabetes mellitus. As a result of
insulin lack, glucose transport is impaired and hence, hyper-
glycemia occurs.
1. Key enzymes of glycolysis are depressed, whereas the
enzymes of gluconeogenesis are activated which contri-
butes to hyperglycemia.
2. Uptake of amino acids is depressed, the level of amino
acid in blood increases, glycogenolysis takes place which
add glucose to the blood.
3. Protein synthesis is decreased. Since, protein synthesis
requires ATP consumption, ATP production is decreased
because of glycolysis.
4. Fatty acid synthesis and triglyceride synthesis is depressed
because of decrease in acetyl CoA, ATP, NADPH and
glycerophosphate. Increased lipolysis of stored lipids give
rise to free fatty acids which interfere with several steps
of carbohydrate phosphorylation in muscle.
HORMONES 367
5. Glycogen synthetase is depressed because of depression
of glycogen synthetase by the activation of phosphorylase.
Fatty acids in high concentrations inhibit fatty acid synthesis
by feedback inhibition of acetyl CoA carboxylase-step. Increa-
sed level of acetyl CoA from fatty acids activate pyruvate, and
hence, stimulate gluconeogenesis.
Fatty acids also stimulate gluconeogenesis by entering TCA
cycle, which produces citrates. Citrates inhibit glycolysis at
phosphofructokinase step.
Fatty acids inhibit the citrate synthetase and pyruvate
dehydrogenase and hence, inhibit TCA cycle.
The level of ketone bodies and cholesterol increases.
GLUCAGON
Glucagon also called hyperglycemic, glycogenolytic horm-
one. Glucagon is secreted by the -cells of the islets of Lang-
erhans.
1. Glucagon increases blood glucose by accelerating glyco-
genolysis in the liver. This action is mediated through
c-AMP. Glucagon increases c-AMP level, which in turn acti-
vates phosphorylase kinase which results in glycogenolysis.
2. Glucagon stimulates gluconeogenesis in the liver via c-AMP
which stimulates the enzyme pyruvate carboxykinase.
3. Glucagon inhibits synthesis of fatty acids and cholesterol
in the liver. It activates lipase in the liver, which results
in increased free acid liberation from liver triglycerides.
4. Glucagon stimulates the release of glycerol and free fatty
acid from adipose tissue.
Glucagon does not stimulate glycogen breakdown in the
muscles.
TRIIODOTHYRONINE (T
3
) AND THYROXINE (T
4
)
Thyroid gland contains iodized glycoprotein thyroglobulin,
hydrolysis of which gives triiodothyronine and tetraiodo-
thyronine (thyroxine). They are also abbreviated as T
3
and T
4
.
T
3
is 5-10 times biologically active than T
4
.
Metabolic Effects
1. Calorigenic effect: Increases rate of energy exchange and oxy-
gen consumption of all tissues. BMR increases.
2. Protein metabolism: Protein metabolism which leads to positive
nitrogen balance.
3. Carbohydrate metabolism:
a. Increases the rate of intestinal absorption of glucose
b. Hyperglycemia may also be associated with increased
degradation of insulin
c. Thyroxine enhances gluconeogenesis
d. Glycolysis, Krebs cycle and HMP pathway are enhanced.
4. Lipid metabolism: Stimulates fat breakdown. Release of
unesterified FFA from adiposes tissues with consequent
increase in their concentration in blood.
CALCITONIN
Calcitonin secretion into the blood is regulated by the high
level of serum calcium.
In plasma, thyroxine (T
4
) is transported as thyroxine binding
globulin (TBG) and thyroxine binding prealbumin (TBPA)
whereas triiodothyronine (T
3
) is poorly binded to plasma.
The structures of these hormones are:
HORMONES 369
Calcitonin lowers calcium level.
The main effect of calcitonin is to decrease the loss of
Ca
++
from the bones and hence, it opposes the action of
parathyroid.
Parathyroid
Parathyroid hormonal secretion maintains the concentration
of ionized calcium in the plasma. Secretion of parathyroid is
regulated by the concentration of ionized serum calcium and
it varies inversely with the concentration of serum Ca
++
.
Administration of parathyroid results in:
1. Increased serum calcium concentration. This results from:
(a) Increased absorption of Ca
++
from the intestine
(b) Increased rate of mobilization of Ca
++
from the bones
(c) Increased renal reabsorption of calcium.
2. Increased phosphorus excretion in the urine. The meta-
bolism of Ca and P are interrelated. As the level of one
rises the excretion of other is increased.
PARATHORMONE
Parathormone regulates Ca and P metabolism. The control is
exerted by negative feedback mechanism whereby hypocalcemia
stimulates and hypercalcemia inhibits the release of the hormone.
Metabolic action:
1. Increases serum Ca
2. Decreases serum Pi
3. Increases urinary PO
4
4. Increases citrate content of blood plasma, kidney and bone.
Ionized calcium:
1. Increased absorption of Ca
++
from intestines (in presence
of adequate amount of vitamin D)
2. Increased renal tubular absorption of Ca
++
from bones
3. Increased renal tubular absorption of Ca
++
Effect on Skeleton
In the presence excess parathormone, reabsorption of bone
occurs and Ca
++
increases in blood.
THYROID GLAND
Antithyroid Drugs
Reduction of hypersecretion of thyroid hormones in hyper-
thyroidism can be achieved by drugs which acts in different
ways on hormone synthesis and release.
1. Drugs which inhibit trapping of iodide by thyroid
By metabolic poisons, e.g. cyanide dinitrate
By monovalent anions, e.g. chlorate, perchlorate, pertech-
nitate, thiocyanate, periodate, nitrate.
2. Drugs inhibiting oxidation and organic binding of iodine
and formation of T
3
and T
4
, e.g. thiouracil, carbamizole,
propyl thiouracil.
3. Iodine/iodide: Acts mainly by reducing release of thyroid
hormones.
4. -adrenergic blocking drugs like propanolol, atenolol,
reduced symptoms of hyperthyroidism.
5. Radioactive I
131
: It is given to destroy overactive thyroid tissue.
6. Inhibiting oxidation, e.g. PABA, sulphonamides.
PROTEIN BIOSYNTHESIS 371
It is now established that DNA is the macromolecule that
ultimately controls every aspect of cellular function primarily
through protein synthesis.
DNA RNA Protein
The flow of biological information is clearly from one class
of nucleic acid to another from DNA to RNA and from their
to protein.
The process of protein biosynthesis is also called translation
because the language consisting of four base pairing letters
of the nucleic acid is converted into that comprising the twenty
letters of amino acids in the proteins. It is a very complex
process which requires more than 100 macromolecules.
Transfer RNA molecules, activating enzymes, soluble factors
and m-RNA are required, in addition to ribosomes.
Proteins are synthesized in the amino to carboxyl direction
by the sequential addition of amino acids to the carboxyl end
of the growing peptide chain.
Site of protein synthesis is ribosomes.
Protein biosynthesis takes place in four major steps. Each
step requires specific enzymes and cofactors.
1. Activation In the cytosol requiring t-RNAs, amino
acids, ATP and Mg
++
ions.
2. Initiation In ribosomes m-RNAs, initiation fac-
tors. IF
1
, IF
2
and IF
3
as well as GTP
and Mg
++
ions.
3. Elongation Two elongation factors EF-T and EF-G
are required. GTP provides the energy.
Protein Biosynthesis
CHAPTER
21
4. Termination Requiring some releasing factors for
releasing the synthesized proteins from
ribosomes in the cytoplasm.
ACTIVATION STEP
The formation of a peptide bond between the amino group
of one amino acid and the carboxylic group of the other amino
acid is not favorable thermodynamically as such. This barrier
is overcome by the activation of the carboxylic group of the
amino acid molecule. The activated intermediates in protein
synthesis are amino acid esters in which the carboxylic group
of an amino acid is linked to either the 2' or 3'-hydroxyl group
of the ribose moiety at the 3' end of t-RNA. This amino group
can migrate rapidly between 2' and 3'-hydroxyl group. This
is called acyl t-RNA.
This activation, besides facilitating the peptide bond forma-
tion, is also important because only the t-RNAs can recognize
the codon message carried by the m-RNA. The amino acid
themselves are not able to do such decoding.
The activation is catalyzed by a class of enzyme called
aminoacyl-t-RNA synthetase, each of which is highly specific
for one amino acid and its corresponding t-RNA.
In this reaction, pyrophosphate cleavage of ATP takes place
to yield AMP and pyrophosphate. The transfer of an amino
acid to t-RNA takes place in two steps. In the first step, ATP
reacts with amino acid to form amino adenylic acid in which
the 5'-phosphate group is linked in an acid anhydride bond
with the carboxyl group of the amino acid. This high energy
anhydride bond activates the carboxyl group. In the next step,
the amino acyl group is transferred to the t-RNA to give amino-
acyl-t-RNA and adenylic acid (AMP).
The new ester linkage between the t-RNA and the amino
acid formed at the expense of ATP is a high energy bond.
The pyrophosphate so formed may undergo subsequent
hydrolysis to orthophosphate, thus, utilizing ultimately two
high energy phosphate bonds. This overall activation reaction
is essentially reversible.
The specificity of the aminoacyl t-RNA synthetase indicates
that this enzyme must possess two different very specific sites
for binding amino acid and its corresponding t-RNA. There
is also a third site for binding ATP. These enzymes are so
specific that there is 1 in 10,000 chance of an error under
intracellular condition, however these enzymes can still be
fooled by certain nonbiological amino acid analogs such as
p-fluorophenylanine and ethionine which are incorporated in
place of phenylalanine and methionine.
INITIATION OF POLYPEPTIDE CHAIN (IN RIBOSOMES)
In E. coli and other prokaryotes, the polypeptide synthesis
begins with the methionine. This enters after its free amino
group has been formylated (i.e. blocked) and activated as
formylmethionine t-RNA. This is enzyme catalyzed reaction
in which N
10
formyl tetrahydrofolate acts as a formyl group
donar. The free methionine does not take part in the initiation.
The reaction takes place is:
Methionine + t-RNA Methylene t-RNA
N
10
formyltetrahydrofolate Formyl-met-t-RNA +
+ Met-t-RNA Tetrahydrofolate
The t-RNA carrying methionine occurs in two forms. Only
one form designated as met-t-RNA, is capable of accepting
N-formyl group. The other one cannot accept formylmethio-
nine-t-RNA but only methionyl-t-RNA.
The enzyme catalyzing this reactiontransformylase is also
specific and does not formylate methionine residue attached
to the other species of t-RNA i.e. t-RNA
met
.
The blocking of free amino group has significance, i.e. does
not allow the amino acid to be inserted into the chain during
the process of elongation.
So, it can only be used to start the protein synthesis.
However, it has been found that the initiating residue in the
cytoplasmic protein synthesis of eukaryotic cells is unacety-
lated-methionine residue bound to one specific t-RNA
-met
, the
initiator t-RNA.
As studied and described for the bacteria E. coli the initi-
ation process takes place in the following steps (as mentioned
on page 375).
The ribosomes separates into 50s and 30s subunits. The
30s subunits reacts with the initiation factor-3 (IF-3) to form
a complex, the complex then binds with m-RNA to which is
then added a molecule of initiation factor-1 (IF-1).
The f met-t-RNA and GTP binds to initiation factor-2 (IF-2)
to form a complex of f-met-t-RNA-GTR-IF-2. This complex
then binds with the complex formed of 30s subunit IF-3,
m-RNA and IF-1 to form what is called the initiation complex.
To this is then combined the 50s subunit to form a complex
70S functional ribosome. In this process, GTP is hydrolyzed
to GDP + Pi and the three initiation factors IF-1, IF-2 and IF-3
are dissociated from the ribosome.
The binding of the specific m-RNA molecule on small 30s
subunit is also very accurate. It is believed that each m-RNA
contains one or more ribosomal binding site. Each site contains
a specific nucleotide sequence which helps in the correct
positioning of the m-RNA molecule on the 30s subunit. Each
m-RNA has at least one ribosomal site for the production of
one polypeptide chain. A m-RNA coding for 3 polypeptide
chain will, therefore, contain 3 sites.
This process of initiation ensures that the initiating amino-
acyl m-RNA is correctly placed on the P site and positioned
at the initiation codon AUG so that the ribosome starts
translating the correct point on m-RNA.
The P and A sites are the two carriers on the 70s ribosome
into which t-RNA molecules can be inserted. The sites
(P-peptidyl site and A-aminoacyl site) are bounded partially
by the 50s and 30s subunits and a specific m-RNA codon. It
is this m-RNA codon in relation to these sites which determines
the correct binding of the specific aminoacyl t-RNA molecule.
The sites themselves, however, can allow the attachment of
any aminoacyl t-RNA.
It has been established that translation of codons on
m-RNA begins in 5'-3' direction.
The initiation factors are proteins which can be extracted
with strong salt solution. They have a molecular weight of
about 9,000, 55,00 and 21,000 respectively.
As seen, these factors keep on undergoing attachment and
release reaction on the 30s subunits.
ELONGATION
It begins when:
The initiating t-RNA is bound on the P site, with its
anticodon pairing with the triplet codon on the m-RNA.
A site is free.
This process takes place in steps:
Binding of the Incoming Aminoacyl-t-RNA on the A-
Site
It occurs on the A-site of the functional 70s ribosomal complex.
As studied in prokaryotes, the incoming aminoacyl-t-RNA
binds to a specific protein present in the cytoplasm called as
elongation factor T (EF-T). This consists of two subunits, EF-
T
u
and EF-T
3
. The combination of EF-T and GTP in the next
step results in the formation of EF-T
s
free. This now combines
with t-RNA carrying the new amino acid to form a ternary
complex EF-T
u
-GTP-aminoacyl-t-RNA.
This complex then binds to the ribosome in such a way
that aminoacyl-t-RNA is positioned correctly on A-site with
its anticodon bound to codon on the m-RNA in the site A.
The energy required for the positioning of the new aminoacyl-
t-RNA is provided by the GTP which undergoes. hydrolysis
and then leaves the ribosomes as EF-T
u
-GTP complex after
the aminoacyl-t-RNA has been properly placed on the A-
site.
It is stated that the carrier protein EF-T does not bind with
the initiating f-met-t-RNA. The process is blocked by tetra-
cylines.
Peptide Bond Formation
With the f-met-t-RNA on the P-site and new aminoacyl-t-RNA
on the A-site, peptide bond is formed by the nucleophilic
attack of the amino group of the incoming amino acid on the
carboxylic group of the f-met-t-RNA present in the P-site. This
reaction is catalyzed by the enzyme peptidyl transferase
resulting into formation of dipeptidyl-t-RNA on the A-site,
i.e. the amino acid on the initiating t-RNA is transferred on
the A-site of t-RNA leaving an empty t-RNA on the P-site.
The energy provided is given by the high energy ester bond
between f-methionine and t-RNA.
TERMINATION
After the complete synthesis of the polypeptide chain, the
termination of the chain is signalled by one of the three special
termination codons on m-RNA. After the attachment or the
incorporation of last amino acid into the polypeptide chain,
the chain is still attached to t-RNA by its carboxyl terminal
on the A-site. The release of chain from here is mediated by
the releasing factors symbolized as R
1
, R
2
and R
3
. They bind
to the ribosome to cause a shift of the polypeptidyl-t-RNA
from A-site to P-site; the ester bond between the polypeptide
chain and the last t-RNA is then hydrolyzed apparently by
the action of peptidyl transferase enzyme. Once, the polypep-
tide chain is released the last t-RNA and m-RNA also leave
the ribosome which then dissociates into 50S subunits the new
polypeptide chain is again started.
The exact details of this step are not yet clearly known.
The chain probably leaves the ribosome as a folded molecule
with tertiary structure because the ribosome is found to contain
several enzymes, in addition to protein synthesizing agents.
Post-translational Processing of Polypeptide Chain
After the polypeptide chain has been synthesized completely
it undergoes certain changes to yield its biologically active
form.
Most of the proteins do not have a formyl group at the
amino terminal. So, it is thought that formyl group is removed
by the action of the enzyme deformylase. Some proteins do
not have methionine as the amino terminal residue. It is
believed to be removed afterwards by the enzyme methionine
aminopeptidase. Still further some proteins are acylated at
their N-terminal residue after they are synthesized. The disul-
phide bonds are similarly formed in reactions catalyzed in
microsomes to allow them the tertiary structural modifications.
Energy Requirements of Protein Synthesis
2 ATP bonds are required in the activation of amino acid.
i. GTP is hydrolyzed in the binding of aminoacyl-t-RNA
to A-site.
ii. GTP is hydrolyzed in the translocation ribosomes.
Total of 4 high energy bond = 4 7.3 = 29.2 Kcal, for each
peptide bond synthesized. And each bond gives about 50 Kcal
on hydrolysis. Hence, it is highly energy consuming process
to generate peptide bond. Infact, it is the most expensive
process in cells which is biosynthetic.
Inhibitors of Protein Synthesis
Many inhibitors used in human beings for treating infections
act by inhibiting the protein synthesis in the prokaryotes.
Examples are: tetracycline, chloramphenicol, puromycin, strep-
tomycin, neomycin, etc.
Inhibitor Site of action
Chloramphenicol Block peptidyl transfer in
70s ribosome.
Streptomycin Binds to 30s subunit to
affect initiation.
Tetracycline Inhibits binding of incoming
aminoacyl-t-RNA to A-site.
Puromycin Reacts with peptidyl-t-RNA
to give puromycin-peptidyl-
t-RNA.
Fusidic acid Inhibits translocation.
CODON
Genetic experiments have shown that a group of three bases
in fact, codes for one amino acid. This group of bases is called
codon.
General Characteristic of Genetic Code
1. Each code word consists of three nucleotide bases arranged
in a definite sequence, i.e. it is a triplet.
2. There are in all 64 code words out of which 61 code for
various amino acids. These are called nonsense codons.
The other three codons do not code for any particular amino
acid which are signals of chain termination. They are UAG,
UAA and UGA.
3. There are more than one codon for many amino acids such
as 4 codons each for glycine and alanine and 6 codons each
for arginine, leucine and serine. This property is called as
degeneracy of genetic code. Only tryptophan and methio-
nine have only one codon for them.
4. Out of the triplet codon, the first two bases are very specific
as far as the sequence is concerned. But third one is not
that specific. For exampleGCU, GCA and GCG, all are
specific codons for the alanine. Each codon has two same
bases at first and second position but the third one is
different. Thus the third base tends to be loose and wobbles
about. Third base wobbles.
Further when the two amino acids have two codons
each of which have first two bases common, the third
becomes the determining one and in such case the third
position is filled by purine base in one and a pyrimidine
base in the other.
For examplefor histidine and glutamic acid, there are
two codons for each.
Histidine CAU, CAC
Glutamic acid CAA, CAG.
In those, the third base is purine base in both the codons
for glutamic acid and third base in both the codons are
a pyrimidine base for histidine. The nucleotide sequence
is from 5' to 3' end and as the third base lies on the 3'
end.
5. In the codon messages, no signals are required to indicate
the end of one codon and the beginning of the other codon,
i.e. they are identical in all species of life right from virus
to man. This has been tested in more than one way. The
codon specific for serine, i.e. is identical in virus, bacteria,
lower animal and even in man. This refers to the universality
of codon, i.e. many amino acids are designated by more
than one triplet.
Only tryptophan and methionine are coded by one triplet
only.
REGULATION OF GENE EXPRESSION
Gene, carrying the genetic information, expresses itself by
leading to the formation of a specific protein through the
process of transcription and translation. Hence, the regulation
of gene expression in effect means the regulation of protein
synthesis. This regulation can therefore, takes place at the
following two levels.
1. Transcription control, i.e. the formation of m-RNA from
the gene is the point of regulation.
2. Translation control, i.e. the synthesis of the proteins
from m-RNA is the point of regulation.
However, in most bacteria and prokaryotes transcriptional
control is the main regulatory method.
Transcription Control
The basic process of protein synthesis is regulated by induction
and or repression. Depending upon the metabolic state of the
organism, certain enzymes, i.e. the proteins are either induced
or repressed.
Jacob and Monard proposed the concept of operon to explain
the phenomenon of induction and repression as the means
of transcription control of protein synthesis. Initially, this
hypothesis named as operon model was postulated with
particular reference to lactose metabolism regulation in E. coli
by the genetic pathway.
This hypothesis postulates the existance of an operon as the
group of functionally related structural genes lying contigious
to each other in the chromosomes which can be turned off
and on coordinately by the same regulatory gene. Since they
explained the protein synthesis with particular reference to
lactose. They proposed the lactose operon (Lac operon) as
the model for regulatory mechanism.
This explained the induction of three protein brought about
by the lactose and the repression of these proteins by the
presence of glucose in the following way.
There are present structural genes which are responsible
for transcription m-RNA for three proteins, i.e. -galacto-
sidase, permease and transacetylase. These three functional
genes are under the regulation of an inhibitory locusnow
called as regulatory gene. This is then the operator locus on
the chromosome of DNA adjacent to the structural genes. The
regulatory gene normally excerts an inhibitory influence on
the structural genes preventing them from transcripting the
various m-RNAs by means of a protein molecule called repressor.
This represser then binds with the operator to inhibit the trans-
cription under normal circumstances.
The binding of the repressor, which is reversible to the
operator interferes with the bindings of RNA polymerase on
to the promotor, another locus present in the vicinity of
operator. There appears to be some overlappings in the limits
of the promotor and operator. To initiate the protein synthesis
and when the repressor molecule is not bound to the operator,
the transcription is carried out uninterrupted by the structural
gene. Thus according to this operon model, the repressors
control the rate of transcription of DNA into RNA.
DNA Repair
The maintainance of integrity of the information in DNA
molecules is of great importance. The mechanisms responsible
for this monitoring mechanism in E. coli includes 3 to 5 exo-
nuclease activity. Repair is of four types:
1. Mismatch repair
2. Base excision repair
3. Nucleotide excision repair
4. Double strand break repair.
1. Mismatch repair
Problem: Mismatching refers to copying errors.
Solution: 1. Methyl directed strand cutting
2. Exonuclease digestion.
2. Base excision repair
Problem: Chemical/radiation damage to a single base
Solution: Base removal by N-glycosylase, abasic sugar
removal replacement.
3. Nucleotide excision repair
Problem: Chemical/radiation damage to DNA segment
Solution: Removal of an approximate 30-nucleotide base
pairs and replacement.
4. Double strand break repair
Problem: Chemotherapy, oxidative free radicals, ionizing
radiation
Solution: Synapses, unwinding alignment, ligation.
Applied
Pyrimidine dimers can be formed in the skin cells of human
exposed to unfiltered ultraviolet sunlight in the rare genetic
disease xeroderma pigmentosum, the cells cannot repair the
damaged DNA resulting in extensive accumulation of
mutations that leads to skin cancers.
The most common form of this disease is caused by the
absence of UV-specific endonuclease.
DNA replication
The biosynthesis of a duplicate copy of DNA prior to cell
division (DNA DNA).
DNA repair
The removal and synthesis of short segments of DNA damaged
by chemical or physical agents or of DNA synthesized with
errors during replication.
DNA recombination
The exchange of gene segments between different DNA mole-
cules.
DNA transposition
A nonclassical type of genetic recombination involving the
movement of a gene from one location to another on the same
chromosome or to a different chromosome.
INSTRUMENTATION 385
COLORIMETRY
Colorimetry depends upon the measurement of the amount
of color, i.e. intensity of color, produced during a chemical
reaction in which the substance being estimated takes part
quantitatively. The intensity of color produced is proportional
to the concentration of the reacting substances and it is possible
to measure the concentration of the substance by determining
the depth of the color.
Laws governing the absorption of light are governed by
Lamberts and Beers which are as follows:
Lamberts Law
Proportion of light absorbed by the substance is independent
of the intensity of the incident light.
Beers Law
Absorption depends only on the number of absorbing molecules
through which the light passes.
Mathematical derivation is given as:
0
I
log
I
= KCL
where C = Concentration
K = Constant
L = Thickness
Optical density = KCL I
0
= Incident light
I = Emergent light
Instrumentation
CHAPTER
22
Optical Density
It is defined as the logarithmic ratio of the incident light to
that of emergent light.
OD =
0
I
log
I
Transmission
It is defined as the ratio of the intensity of transmitted light
to that of incident light.
T =
0
I
I
Relationship between optical density and transmission:
OD =
0
I
log
I
=
I
log
T
=
100
log
%T
= 2 log T
When transmission is 100%, the optical density is 0.
Colorimeter comes under the visible range. Actually what
we are measuring in the colorimeter is the maximum absorp-
tion of the light. We select a particular filter for a particular
color, so that the maximum absorption of the light should be
their.
ELECTROPHORESIS
Electrophoresis is defined as the migration of charged particles
in the solution under the influence of electric field. The rate
of migration is directly proportional to the number of charges
present on the component. Proteins are colloidal particles and
charged either positive or negative which depends on the pH
of the solution. In acidic medium, it acts as cation and in alka-
line medium as anions. If uncharged particles are charged,
then they can be separated. If a potential difference is applied
INSTRUMENTATION 387
across them, current will flow and cations move towards
cathode and anion towards anode.
Migration depends upon:
i. pH of buffer
ii. Net charge of amphoteric particle
iii. Temperature
iv. Voltage and current.
ISOTOPES AND THEIR APPLICATION
Isotopes are atomic species having similar atomic numbers
but different atomic weights due to the difference in the nucleus
of atom. As the atomic number is same, isotopes have the
same chemical properties but different physical properties.
Isotopes are of two types:
1. Stable or nonradioactive isotopes
2. Unstable or radioactive isotopes
Stable or nonradioactive isotopes: They do not emit any
radioactive radiations.
Unstable or radioactive isotopes: They emit radioactive
radiations, i.e. , or -rays.
Radioactivity
It is the phenomenon where radioactive substances emit ,
or -rays on disintegration.
-rays: They consists of doubly charged helium atoms.
They have least penetration power of all the three particles
with greatest ionization power, because they have heavy
particles they cannot penetrate much.
-rays: They are fast moving electrons having ionization
power less than -particles and less than -rays.
-rays: They are electromagnetic waves with maximum pene-
tration power but less ionization power of all the three particles.
Measurement of Radioactivity
1. Geiger-Mller counter: The radiations entering the gas mixture
produce ions. When , or -rays collide with gas atoms or
molecules the cation and anion move to cathode and anode
respectively and produce electric impulse which is proportional
to the activity of the radioactive substance.
2. Proportional counter: It is same as Geiger-Mller counter
except the gas is not a mixture but a single monoatomic gas.
It can differentiate between , and -rays. However, it is
less sensitive.
3. Scintillation counter: In this method, no gas or mixture of
gas is used. The radiations are allowed to full over fluorescent
substance, e.g. crystals or on a liquid organic solvent (liquid
scintillation counter). The photons emitted are allowed to pass
through a photo multiple tube that converts this light into
electricity and amplified using 10 to 15 diodes whose strength
is proportional to the radioactivity of the substance.
Units of Radioactivity
Radiation absorption dose: Since the radioactive rays can produce
ions inside the tissues also, long exposure to these rays can
be dangerous.
1 rd = 100 ergs of energy/gm of tissue.
1 roentgen = 1 rd (practically).
Application of Isotopes
a. In biochemistry, isotopes are used in working out metabolic
pathways, i.e. cholesterol synthesis from acetic acid, purine
synthesis from glycine. Also some commonly used stable
isotopes are used in the determination of turnover of
different metabolic activities in the body.
b. Determination of total plasma volume and total blood
volume in the body.
c. Determination of average life of RBC.
d. In iodine metabolism.
ELECTROMETRIC DETERMINATION OF pH
The pH of solutions can be determined more accurately by
potential measurements of certain electrodes than by the use
of indicators. They give rapid and accurate results. Common
INSTRUMENTATION 389
electrical methods for pH determination depend upon the use
of hydrogen or glass electrode.
Hydrogen Electrode
It consists of a small platinum strip coated with platinum block
and absorbs hydrogen gas. A platinum wire welded to the
electrode makes contact with the outer circuit, the P
t
strip is
surrounding by glass tube with inlets and outlets for H
2
which
is admitted at 1 atmosphere.
H
2
2H 2e + 2H
+
(On P
t
surface) (Remain on P
t
) (Pass into solution
from electrode)
Since H
2
is admitted at constant pressure, the solution
tension of hydrogen atoms has a constant value. If electrode
1 is maintained constant by immersion in 1 N H
+
ions, then
potential will vary depending on the H
+
ions concentration
around electrode 2.
The electrode with H
2
at 1 atmosphere in a 1 N H
+
solution
is called hydrogen electrode and is arbitrarily assigned a
potential, E
no
of Zero under all conditions and is used as a
standard reference for other electrodes.
EMF = E
n
+ E
no
If potential difference between the normal H
2
electrode
and the electrode in the unknown solution is known for a
given temperature, pH of the solution can be calculated from
the formula:
pH =
EMF
,
0.00019837T
where T is absolute temperature.
Calomel Electrode
It consists of metallic mercury in contact with Hg
2
Cl
2
in KCl
solution. Potential varies with the saturation of KCl solution
but for a given temperature and KCl concentration, the
potential of the calomel electrode against the normal hydrogen
electrode is constant, i.e. at 25C, the potential of saturated
calomel electrode is 0.2458 Volts. Now, if a hydrogen electrode
is placed in the solution of unknown pH and connected with
a saturated calomel electrode, the potential difference regis-
tered will be more than that would have been obtained against
a normal hydrogen electrode by 0.2458 Volts. The pH of un-
known solution can, therefore, be calculated as:
pH =
EMF Encalomel
0.00019837T
Glass Electrode
This method of determining pH is rapidly replacing the
hydrogen electrode procedure. It is not affected by oxidizing
or reducing agent. It is based on the principle that when a
glass membrane separates two different solutions differing
in pH, a potential difference is found to exist between the
two surfaces of the glass. It consists of a thin walled glass
bulb made out of a special type of low melting point glass.
It is filled with normal HCl solution in contact with Ag/AgCl
electrode. The platinum wire dipping in the electrolyte passes
out of the glass tube and the bulb is placed in the solution,
whose pH is to be measured. The potential is measured against
a standard calomel electrodes.
pH Meter
A glass electrode is made up of a bulb containing solution
of known pH into which is dipping an Ag/AgCl electrode.
The bulb is fragile as it is made up of a thin layer of glass.
The glass electrode and calomel electrode both are dipped
in a solution of unknown pH. The electrodes are connected
by potentiometer.
ESTIMATION OF NITROGEN CONTENT BY MICRO-
KJELDAHL METHOD
Any nitrogen containing substance on digestion with concen-
trated H
2
SO
4
is converted into ammonium sulfate. From the
ammonium sulfate so formed, the ammonia is distilled off,
by treating with strong alkali. The evolved NH
3
is trapped
INSTRUMENTATION 391
in a suitable indicator, which on titration with standard H
2
SO
4
gives the amount of ammonia trapped and thus by back
calculation, the nitrogen content is found out.
Reaction
The whole procedure for nitrogen estimation is divided
into following three parts:
a. Digestion
b. Distillation
c. Estimation.
Digestion
A known weight or the volume of the organic compound is
taken in a small micro-kjeldahl flask, followed by 2 ml of
concentrated H
2
SO
4
. To this, a pinch of CuSO
4
(acts as catalyst)
and K
2
SO
4
(raises boiling point and prevents bumping) are
added. The mixture now looks black. The flask is placed on
a microburner and is heated slowly over a small flame. In
the initial stage, a low flame is used and later on a strong
flame is used till the solution is completely digested and
acquires a slight blue tinge. The time of digestion depends
upon the nature of nitrogen in the unknown substance (i.e.
complexity of the nitrogen). Under similar conditions, run a
blank which contains all the reagents except test material.
Distillation
The contents of the digested material are transferred quanti-
tatively into a distillation jacket. The digested flask is washed
with distilled water and the contents poured into the
distillation jacket followed by 10 ml of 40 percent NaOH. A
steady stream of steam is bubbled into the distillation jacket.
The ammonia evolved is trapped in boric acid: Tashiros
indicator. In acidic pH, the indicator is violet in color. In alkaline
solution, it is green in color.
Estimation
The trapped ammonia is estimated by titrating against N/70
H
2
SO
4
.
The color of the indicator becomes green after the ammonia
has been absorbed into it. This is titrated against N/70 H
2
SO
4
until the solution becomes violet again.
Reaction
Indicator
Calculation
Proteins contain 16% nitrogen, i.e. 16 mg of nitrogen is present
in 100 mg of protein.
1 mg of nitrogen is present in 6.25 mg of protein
1 liter of 1N H
2
SO
4
= 1 liter of 1N-NH
3
1 liter of 1N H
2
SO
4
= 14 gm of nitrogen
1 ml of 1N H
2
SO
4
= 14 mg of nitrogen
1 ml of N/70 H
2
SO
4
= 0.2 mg of nitrogen.
If x be the difference between the titre value for test
solution and blank.
INSTRUMENTATION 393
If 0.2 ml of serum is digested initially than 0.2 ml of serum
produces 0.2 x mg of nitrogen.
1 ml of serum produces
x
0.2 mg
0.2
of nitrogen.
100 ml of serum produces
x
0.2 mg
2
of nitrogen.
The results can be converted into proteins by multiplying
by factor 6.25.
Chromatography is defined as the analytical technique for
separating compounds on the basis of differences in affinity
for a stationary and a mobile phase. The difference in affinity
involves the process of either adsorption or partition. In
adsorption chromatography, the binding of a compound to
the surface of the solid phase takes place whereas in partition
chromatography, relative solubility of a compound in two
phases results in the partition of the compound in two phases.
Thus, all types of chromatography known so far have been
grouped in either of the two mentioned form.
As all the experimental techniques have got their own way
of representations, chromatographic method has also entirely
different notation by which results are represented: They are
known as R
f
.
R
f
expresses the relative rate of movement of solutes and
solvents. R
f
is defined as the ratio of the distance travelled
by the compound at its point of maximum concentration to
the distance travelled by the solvent. Both the distances are
measured from the point of application of the sample:
R
f
=
Distance travelled by the solute
Distance travelled by the solvent
R
f
value has no unit.
R
f
is always less than one.
R
f
value of different compounds are entirely different. Just
as melting point, boiling point and other physical constants
are different for different compounds, so is the case with R
f
values. The R
f
values vary with the solvent used, i.e. two
solvents will give two values. Thus, R
f
value is always quoted
with reference to the solvents used.
In paper chromatography, the analysis of an unknown sub-
stance is mainly done by the flow of solvents on specially
designed filter paper. One of the two solvents is imiscible or
partially miscible in the other solvent. The solvent rises up
by the capillary action and by adsorption on the paper, the
separation is effected by differential migration of the mixture
of substances. This occurs due to difference in partition co-
efficients.
When the solvent moves over the spot, two type of forces
are involved. They are:
1. Propelling force: This assists in the propagation of the
substances in the direction of the flow of the solvent.
2. Retarding force: This tries to drag the substances behind
towards their point of application.
The R
f
value, the distance through which the substances
move on the paper under the influence of the solvent is due
to the net resultant of these two types of forces.
INSTRUMENTATION 395
In biological mixture separation:
1. The mixture is available in very small quantity.
2. Mixture is usually made up of proteins, cannot be sub-
jected to high temperature, high or low pH because they
get denatured.
3. Substances having melting points or boiling points very
close to each other. Their solubilities are also very closely
related.
All these difficulties can be overcome by using the chroma-
tographic techniques.
INDEX 397
Index
A
Absorption 18
calcium 299
fats 185
iron 296
Acetoacetyl CoA pathway 206
Acetyl number 62
Acid
phosphates 143
base balance 284
Acidification of urine 289
Acrolein formation 60
Action of
acids 27
amylases starch 47
dilute alkali 34
Activation of
B cells 345
fatty acid 196
glycerol 195
T cells 342
Active
and passive immunity 339
sulfate 229
sulfate sulfating agent 229
Adenosine
diphosphate 249
triphosphate 249
Adenylic acid 373
Adrenal
androgens 364
cortex hormones 178
Aerobic dehydrogenases 141
Agents causing cancer 356
-glycerophosphate shuttle 150
Alanine transaminase 213, 329
Albinism 237
Aldaric or saccharic acid 35
Aldonic acid 34
Aldoses 20
Alkaline phosphatase 137
Alkaptonuria 237
Amino
acid
derivatives 362
molecule 372
sugars 26
Ammonium ion production 288
Amphibolic role citric acid or Krebs
cycle 160
Amylopectin 46
Amylose 46
Anaerobic dehydrogenases 122,
141
Andersens disease 165
Anion gap 290
Anterior pituitary hormones 178
Antiachrodynia factor 275
Antibody
class switch 351
diversity 351
Antigens 100, 340
Antioxidant system 71
Antipernicious factor 281
Antisterility vitamin 265
Antithyroid drugs 370
Antivitamins 283
-oxidation 191
Application of isotopes 388
Arachidonic acid 57
-rays 387
Aspartate transaminase 213
Atherosclerosis 203
B
B cells 345
Balanced diet pregnant lady 321
Banana 319
Barfords test 38
Basal metabolic rate 312
Base excision repair 383
Beers law 385
Benedicts
qualitative reagent 30
reagent 34
Bicarbonate 335
carbonic acid buffer 285
reabsorption 287
Bile acids 204
Binding of incoming aminoacyl-
t-RNA 377
Biochemical
basis of fatty liver 208
changes in jaundice 117
Biological
importance of water 292
oxidation 140
value of proteins 313, 314
Biophysics 1
Biosynthesis of
purine ribonucleotides 250
pyrimidine nucleotides 252
Biotin 277
Biuret reaction 95
Blood
buffers 9
group substances 52
Bohr effect 112
-oxidation 188
-rays 387
Bread 322
Breakdown of hemoglobin 113
Brief description cytokines 347
Bromsulphalein
excretion test 328
Brownian motion 17
Buffer 4
systems of body fluids
284
Burkitt lymphoma 358
Butter 322
C
Calcitonin 368
Calcium 298
Calomel electrode 389
Caloric
requirement 315, 320
value of food 310
Calories diabetic diet 323
chart 322
Calories low cholesterol diet 325
Calorigenic effect 368
Cancer 356
cells 356
chemotherapy 359
Carbohydrate 19, 315
metabolism 326, 368
Carbon monoxide poisoning 105
Carcinoid syndrome 239
Carcinoma of lung 359
Cardiolipin 65
Castles extrinsic factor 281
Catabolism of
purines 254
pyrimidines 255
Catalases 143
Catalytic site oractive sites of
enzymes 134
Cell mediated immunity 345
Cellular structural studies 327
Cellulose 47
Cephalins 64
Cereal group 313
Cerebrosides or glycolipids 66
Ceruloplasmin 304
Characteristics of
coenzymes 121
Characterization of fats 61
Chemical
compounds 356
coupling hypothesis 147
Chemiosmotic hypothesis 148
Chemistry of
amino acids 74
and proteins 74
carbohydrates 19
lipids 53
Cholesterol biosynthesis 197
Chondroitin sulfates 51
Chylomicrons 184
Citric acid cycle 155
Classification and functions of
lipids 54
Classification of
amino acids 75
carbohydrates 19
enzymes 122
proteins 85
Coagulation factor 266
Codon 380
INDEX 399
Coenzyme
ubiquinone 145
Coenzymes 121
Colloids 16
Colorimetry 385
Competitive inhibition 131
Complete proteins 316
Compound lipids 62
Concentration test 331
Conformational coupling hypothesis
149
Conjugated proteins 86
Conjugation reaction 224
Constitutive enzymes 136
Conversion of pyruvate to acetyl
CoA 155
Copper 303
Cori cycle 168
Coris disease 165
Cottage cheese/egg 322
Crabttee effect 154
Creatine
phosphokinase 137
synthesis 224
Creatinine clearance test 334
Crystalloids 16
Curd 323
Cushings syndrome 291
Cyanocobalamin 281
Cyclic amp 361
Cysteine
pyruvic acid 276
and cystine 227
Cystinosis 228
Cystinuria 228
Cytochromes 145
D
Daily requirement 300
Dal 323
Dal/lean meat 322
Danger of ketosis 207
De Toni-Fanconi syndrome 303
Decarboxylation 213
Deficiency disease 265
Degree of glucose control 182
Dehydration 295
results 295
Dehydrogenases 141
Denaturation 96
Dense connective tissue 292
Deoxyribose nucleic acid 244
Derived
lipid 68
proteins 86
Determination of serum
amylase 336
lipase 336
Detoxification and protective
functions 326
Dextrins 48
Diabetes 366
mellitus 179
Diagnostic value of plasma
enzymes 137
Dialysis 16
Dietary fiber 316
Differences between T cells and B
cells 346
Digestion
and absorption 210
of protein various enzymes 210
Dihydrofolic acid 279
Dilution test 332
Distillation 392
Disulphide bonding 94
DNA
RNA protein 371
recombination 384
repair 383
replication 384
transposition 384
Double strand break repair 383
Dust cells 346
D-xylose excretion test 338
E
E. coli 382
Edema 295
Effect of
enzyme concentration 128
negative ions 96
pH 128
positive ions 96
proteolytic enzymes 348
salt concentration 95
skeleton 369
substrate concentration 124
temperature 129
Effector functions of T cells 343
Egg 319, 322
Eicosanoids 73
fatty acid derivatives 59
Electrolyte composition of plasma
284
Electrometric determination of pH
388
Electrophoresis 88, 386
pattern of normal serum 97
Embden-Meyerhof pathway 153
Energetics 158
Energy
requirements of protein synthesis
379
yield of palmitic acid
metabolized 190
Enhancer insertion 358
Enolphosphates 143
Enzyme
activity 129
induction 135
inhibitions 130
specificity 122
Enzymes 120, 335
Epinephrine 178
Essential
amino acids 80
fatty acids 57
pentosuria 175
Esterified cholesterol 330
Estimation of
Serum
alkaline phosphatase 329
bilirubin 327
glutamic pyruvic
transaminase 329
urine
bilirubin 328
urobilinogen 328
Excretory functions 326
Extra-mitochondrial de novo fatty
acid synthesis 191
F
Factors influencing rate of
enzymatic reaction 124
Fat
in stool 337
soluble vitamins 259, 261
Fats 315
energy source 56
oil 322
Fatty
acid 55
synthesis 191
livers 208
Ferrous protoporphyrin 105
Fertility factor 265
Fibrous proteins 85
Figlu excretion test 280
Fish 319
chicken 322
Flavin
adenine dinucleotide 273
mononucleotide 273
Flavonucleotides 141
Fluoride 305
Fluorosis 305
Folic acid 279
Food values 319
Formal titration 82
Formiminoglutamic acid excretion
test 280
Free radicals 72
Fructose metabolism 172
Fruit 322, 323
Functions of
amino acids 79
carbohydrates 19
hemoglobin 104
iron 296
plasma proteins 98
proteins in body 97
T cells 343
G
Galactose metabolism 169
Galactosemia 170
Gamma interferon 347
Gangliosides 67
Gastrointestinal function test 338
Geiger-Mller counter 387
Gene amplification 358
General characteristic of genetic
code 380
Gestational diabetes 180
Ghee/oil 322
Gibbs donnan equilibrium 15
INDEX 401
Glass electrode 390
Globin 109
Globular proteins 85
Glucagon 178,361, 367
Glucocorticoids 364
Glucogenic amino acids 219
Gluconeogenesis 168, 176
Glucose oxidase 35
Glutamate
oxaloacetate transaminase 213
pyruvate transaminase 213
Glutamic acid 279
Glycine 221
choline cycle 222
Glycinuria 226
Glycogen 48
storage diseases 165
synthetase 164
Glycogenesis 163
Glycogenolysis 165
Glycolysis 151
Glycosides formation 40
Glycosuria 178
Glyoxalate pathway 221
Gout 257
-rays 387
Green vegetables 322
Growing child 321
Guanidinophosphates 143
Guanosinediphosphate 249
Guardian of
genome 358
tissue 358
H
Hartnup disease 239
H-chains 99
Hematologic function 327
Heme 105
Hemochromatosis 298
Hemoglobin 102, 103
cooperativity 110
gun hill 112
synthesis 222
variants 111
Hemolytic or pre-hepatic
jaundice 116
Henderson-Hasselbalch equation
5, 6
Heparin 50
Hepatic porphyria 115
Hepatocellular or hepatic jaundice
116
Hers disease 165
Heteropolysaccharides 49
Hexose monophosphate shunt
pathway 160
High
density lipoproteins 184
energy compounds 143
or heavy density lipoproteins
184
HMG-CoA pathway 206
Hodgkins disease 359
Homogentisic acid oxidase 237
Homopolysaccharides 45
Hormone
action 360
classes 360
response element 361
Hormones 360
Hyaluronic acid 49
Hybridoma and monoclonal
antibodies 351
Hydrogen
bonding 94
electrode 389
ion concentration 1
Hydrogenation 60
Hydrolysis 307
Hydroperoxidases or peroxidases
142
Hydrophobic interactions 94
Hyperammonia 219
Hyperoxaluria 222
Hyperparathyroidism 303
Hypertonic solutions 12
Hypervitaminosis
A 263
D 265
Hypotonic solutions 12
Hypoxanthine-guanine
phosphoribosyl transferase 254
I
Immunoglobulin 99
Immunology 339
Importance of HMP shunt pathway
160
Inborn error of metabolism 235
Incomplete proteins 316
Inducible enzymes 136
Inherited erythropoietic porphyria
114
Inhibitors of protein synthesis
380
Initiation of polypeptide chain
374
Inosine
diphosphate 249
triphosphate 249
Instrumentation 385
Insulin 364
receptors 365
Intensity of color 385
Interstitial fluid 292
Intracellular fluid 292
Inulin clearance test 334
Invert sugar 44
Iodine number 61
Ionized calcium 369
Iron 296
Isocitrate dehydrogenase 137
Isoelectric point of amino acids 83
Isoenzymes 136
Isotonic solutions 12
Isotopes and application 387
Isotype, allotype, idiotype 349
J
Jaundice 116
K
Kaposis sarcoma 359
Ketogenic amino acids 219
Ketone bodies 205
Ketoses 21
Ketosis 205
Kidney 284, 287
Kinase activation 207
Krebs-Henseleit cycle 214
Kupffercells 346
Kwashiorkor 317
L
Lactate dehydrogenase 136
Lactose 42
synthesis 173
Lamberts law 385
Langerhans cells 346
Latest autoimmune diabetes of
adults 180
Lecithins 63
Lesch-Nyhansyndrome 254
Leucine, isoleucine and valine
240
Line-weaver burk equation 126
Linoleic acid 57
Lipid metabolism 326, 368
Liver 364
biopsy 327, 330
function tests 326
goat 319
phosphorylase 166
Low density lipoproteins 184
Lungs 286
respiration 284
M
Macrophages 345
Malate-aspartate shuttle 150
Malignant transformation 357
Maltose 41
Maple syrup urine disease 240
Marasmickwashiorkor 318
Marasmus 318
Maturation in
bursa equivalent 346
thymus 346
Maximum urea clearance 333
Mcardles disease 165
Meal plan 322
Meat
group 313
fish 322
Mechanism of
action oncogenes 359
H+ excretion 287
oxidative phosphorylation 147
Messenger RNA 248
INDEX 403
Metabolic
acidosis 290, 291
action 369
alkalosis 291
effects 368
function 326
gout 257
role of cysteine 227
Metabolism of
branched chain amino acids 240
carbohydrates 151
cystine and cysteine 228
individual amino acids 219
lipids 184
phenylalanine and tyrosine 230
proteins 210
tryptophan 237
xenobiotics 306
Methemoglobin 110
Methionine 226
Methionione 144
Method of determining km 128
MHC
molecules 352
polymorphism 353
Microalbumin 331
Microsomal pathway of fatty acid
synthesis 195
Mid morning 323
Milk 322
buffalo 319
cows 319
DMS 322
group 313
Milliequivalent 14
Mineralocorticoids 364
Minerals 295, 318
Mismatch repair 383
Mitochondrial synthesis of fatty
acids 194
Mixed function oxidases 142
Monosaccharides 19
Mountain sickness 104
Mucoproteins and glycoproteins 52
Muscle
adipose tissue 365
fat cells 175
phosphorylase 166
Mutarotation 28
Mutton 319
Myoglobin 112
N
Natural
and acquired immunity 339
killer cells 346
Net
dietary protein value 314
protein utilization 314
Neurological cells 346
Niacin 273
Ninhydrin reaction 82
Nitrogen 337
balance 80
Noncompetitive inhibition 133
Nonessential amino acids 80
Nonrepetitive secondary structure
94
Normal balanced diet for adult
man 320
woman 321
Nucleic acid 244
chemistry and metabolism 241
Nucleoside 243
Nucleotide excision repair 383
Nucleotides 243
Nutrition 310
O
Obstructive or post-hepatic
jaundice 119
Oligosaccharides 40
Opsonize bacteria 339
Optical density 386
Orange 319
Organ function tests 326
Organic pyrophosphates 144
Origin of immune cells 341
Orotic aciduria 252
Osazone formation 30
Osmosis and osmotic pressure 12
Osmotic pressure 12, 98
Osteoporosis 301
Oxidases 140
Oxidation 307
of fatty acids 188
Oxidative
deamination 214
phosphorylation 146
Oxidoreductases 122
Oxygenases 142
Oxygenation curves for hemoglobin
and myoglobin 111
P
Pancreatic function test 335
Pantothenic acid 275
Para-aminobenzoic acid 279
Parathormone 369
Parathyroid 369
Partially complete proteins 316
Pasteur effect 154
Peanuts/fat 322
Pellagra preventive factor 273
Pentose sugars 242
Pentosuria 175
Peptide 361
bond formation 378
pH
meter 390
of buffer 387
Phenolsulfonphthalein test 332
Phenylalanine
and tyrosine 229
hydroxylase 235
Phenylketonuria 235
Phenylthiohydantoin derivative 90
Philadelphia chromosome 358
Phosphate buffer 286
Phosphatidic acid 63
Phosphatidyl inositol 64
Phospholipids 63
Phosphorus 302
Physiological jaundice or neonatal
jaundice 119
Plasma 292
lipoproteins 184
proteins 98
Plasmalogens 65
Polenske number 62
Polysaccharides 45
Pompes disease 165
Porphins 102
Porphyria 114
Porphyrins 103
Post-translational processing of
polypeptide chain 379
Potassium 302
Potatoes 319
Precipitation reactions 95
Precursors
of purine ring 249
pyrimidine ring 250
Prediabetes 180
Primary
antioxidants 71
metabolic gout 257
renal gout 258
Process of lipid peroxidation
72
Promotor
element 361
insertion 358
Propelling force 394
Properties of
colloidal solutions 16
fats 60
Proportional counter 388
Prostaglandins 58
Protein
biosynthesis 212, 371
buffer 286
in stool 337
metabolism 326, 368
calorie malnutrition 317
Proteins 85, 315
Prothrombin time 329
Pteridine nucleus 279
Purine
bases 242
synthesis 222
Purines and pyrimidines
metabolism 249
Pyridine nucleotides 141
Pyridoxine 275
Pyrimidine
bases 242
dimers 384
Pyrroloquinoline quinone 260
R
Radiant energy 356
Radiation absorption dose 388
Rancidity 60
Reactions
monosaccharides 26
proteins 94
with nitrous acid 82
Refsams disease 191
INDEX 405
Regulation
Blood
calcium level 300
glucose 175
by pyrimidine biosynthesis 257
cholesterol biosynthesis 203
de novo fatty acid synthesis
194
gene expression 381
purine synthesis 256
Regulatory function of T cells 345
Reichert meissel number 62
Renal
blood flow 335
function tests 330
glucosuria 179
gout 258
mechanism 284
Repressor 383
Respiratory
acidosis 289, 291
alkalosis 291
chain 144
phosphorylation 146
quotient 311
Retarding force 394
Riboflavin 272
Ribose nucleic acids 247
Ribosomal RNA 249
Rickets 300,303
Rochelle salt 30
Role of
extra-hepatic tissues 177
hormones 177
kidney 177
liver 176
liver lipid metabolism 209
muscle 177
surface tension 17
S
Sacchrometric method 336
Salad 323
Salted biscuits 322
Salvage pathway 252
Sangers method 89, 90
Saponification 60
Schiffs base 212
Scintillation counter 388
Secondary
antioxidants 71
metabolic gout 257
renal gout 258
Semiessential amino acids 80
Serine pathway 222
Serum
creatine phosphokinase 138
glutamate
oxaloacetate transaminase
138
pyruvate transaminase 138
lactate dehydrogenase 138
Shuttle system 149
Sialicacids 51
Sickle cell hemoglobin 112
Simple
lipids 54
proteins 85
Sodium 301
Somogyis iodine test 336
Specific dynamic action 312
Sphingomyelins 66
Starch 45
Stereochemistry 21
Stereospecificity 123
Steroids 69, 361
Stool examination 337
Storage functions 327
Structure of
coenzyme 278
DNA 246
hemoglobin 106
proteins 88
Substrate
level phosphorylation 146
specificity 123
Sugar/jaggary 322
Sulfatides (sulpholipids) 68
Sulfur 303
containing amino acids 226
Supersecondary structures 94
Surface tension 17
Synthesis of
arginine 217
carbamoyl phosphate 215
citrulline 217
glutathione 222
heme 105
indole and skatole 239
melanine 234
melatonin 239
niacin 237
nonprotein 212
serotonin 238
urea 218
T
T cells 342
Tea 323
Terpenes 69
Tertiary antioxidants 71
Tetrahydrofolic acid 279
Thiamine 270
pyrophosphate 271
Thymic selection 341
Thyroid 361
gland 369
hormone 178
Timnodonic acid 57
Total food for day 322
Transamination 212
Transcellular fluid 292
Transcription control 382
Transfer or soluble RNA 247
Transferase reaction 207
Triglyceride synthesis 195
Tryptophan 237
Tumor suppressor genes 358
Turnover number 130
Tyndall effect 17
Tyrosinosis 236
U
Uncompetitive inhibition 134
Under aerobic condition 159
Units of radioactivity 388
Urea
clearance test 333
cycle 214
Uridine diphosphate glucose 249
Urinary
amylase 336
lipase 337
Uronic acid 34
pathway 174
V
van Der Waals forces 94
Vanthoffs law 12
Varicyate porphyria or mixed 115
Vegetable fruit group 313
Vegetables 319
Vegetarian/nonvegetarian 323
Very low density lipoproteins 184
Viscosity 18
Vitamin
A 261
B complex 270
B
12
281
B
2
, lactoflavin 272
C 268
D 264
E 265
K 266
like compounds 260
Vitamins 259, 318
coenzymes 260
Voltage and current 387
vonGeirkes disease 165
W
Water 335
and mineral metabolism 292
essential nutrient 319
soluble vitamins 260, 268
Wheat flour 322
Wilson disease or
hepatolenticular degeneration
304
X
Xenobiotics 306
Z
Zinc 305

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VK Malhotra - Biochemistry For Students, 12th Edition

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BIOCHEMISTRY

) in the solution. Substances that dissociate in water

From the Law of Mass action, the dissociation of water

] ions in solution. As hydrogen ion concentration incr-

are added to a solution of such

Where HB denotes a weak acid and B

its conjugate base.

Weak acid Proton + Conjugate base

) is the ionized form of a weak acid

ions. So that pH is approximately

ions and each ion will exert the

= 5 milliosmol per liter

ions. Each ion, i.e. Na

will exert an independent osmotic pressure. Also the

In the beginning (A) At equilibrium (B)

) on one side of membrane

Bilirubin in combination with albumin reaches the liver,

Higher the affinity the smaller will be the K

128 BIOCHEMISTRY FOR STUDENTS

Total number of ATP molecules formed under aerobic con-

Due to active esterase

Membrane attach complex lysis

combines with zymosan

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