Ghost Biochemistry

1
TEXTBOOK F BIOCHEMISTRY
for
MEDIC L STUDENTS
Prasad R Manjeshwar
Faculty of Med~cine,
Department of Jiochemistry,
A. J. Institute o~ Medical & Dental Sciences
Kuntikana, Ma galore 575004, India
Ph: 0998644957 , 09141949575

E-mail: prasad_ ext@yahoo.com
www.faceboo .com/prasad.r.m.7
WhatsApp@ 9 86449575
Revised F urth Edition 2016

2
Textbook of Biochemistry for Medical Students

- PRASAD R.M
Revised Fourth Edition: 2016, January
Revised International Edition: 2014, September
Fourth Edition: 2014, September
Reprint Revised Third Edition: 2014, May
Revised Third Edition: 2014, January
International Edition: 2013, September
Third Edition: 2013, August
Revised Second edition: 2012, July
Second edition: 2011, August
First edition: 2009
Published by RM publications, Mangalore, India

Phone: 9986449575, 9900406317
Printers: Prakash Offset Printers, Mangalore, India
Copyrights: @ The Author All rights reserved.
ISBN: 978-81-8956-022-5
For orders Call 09986449575, 9141949575

For quick transactions:
Acc. Name: Prasad Book house
Acc. No.: 115711001576
Bank: Dena bank, Kankanady branch
Code : BKDN 0611157
Place : Mangalore
This book is also available Online@ www.parasredkart.com
Follow this book on Facebook at www.facebook.com/prasadbiochem,

and if you like the book, you can also share this page, so that it can reach
many readers. Book can also be reached at authors homepage
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3
PREFACE TO THE FOURTH EDITION - 20 14

What is the need of a Biochemistr textbook exclusively for medical course, while there
are already a large number of text ooks available in the book stores?
Answer is simple. Most of these extbooks are not written in view of undergraduate
medical curriculum. These books are out of the purview of medical students, both in
terms of content and form. Most f it is not required a t the undergraduate level and
what is required by them may no be there in these textbooks. Simply, these textbooks
do not meet the requirement of u dergraduate medical students.
This Biochemistry textbook is written for undergraduate medical students. Unwanted
details are carefully eliminated ithout compromising the clarity of expression and
exam requirement. Redundant che ·cal reactions, analytical aspects, needless structural
details have been carefully omitte , keeping in mind the target readers.
Questions given at the end of each hapter will give a complete insight into the question
patterns, and also provide a last inute preparation manual for students.
Main features of the book:
• Simple language, easy to read d recollect.
• Hand-drawn diagrams, which a e easy to reproduce.
• Tables and flow charts, which ar easy to replicate.
• Contents are selected to suite the requirement of Medical students throughout world.
What's new in this edition?

• All the chapters are thoroughly evised and updated. The suggestions given by the
readers (students and staff) are in
luded wherever required.
• Anew section of Multiple Choi e Questions (MCQ) has been included. MCQ's are
increasingly gaining importance 1various University examinations.
• A new section of exam tips ha~ been included. It gives an insight into answering
questions asked in University exaf inations.
• A new section of clinical case stldy and data interpretation has been included.
['ALKTOME
This textbook has been dedicatec to students and your opinion is valuable. In order
to continue this objective, I reqm st you to give me a feedback, which will form the
basis for preparing future editions.
You can contact me at 099864495'; 5 or by e-mail at prasad_text@yahoo.com.
You can also comment in faceboo, page www.facebook.com/prasadbiochem, and if
you like the book, you may also 1 hare this page, so that it can reach many readers.
I can also be reached at my homeoage www.facebook.com/prasad.r.m.7
Readers can also communicate~ ith me through whatsApp @ 9986449575.
4
This book is dedicated to
All the students who are willing to learn .. .
ACKNOWLEDGEMENTS
I appreciate the response and feedback from my students that inspired me to write this book
and come out with the Fourth edition.
My sincere gratitude to Dr. Raghuveer, Registrar and Director, Post graduate studies, Yenepoya
University, Mangalore for having accepted to w rite the foreword for this book.
I thank my friend Dr. Ranjana Acharya, for having added life to this book through her diagrams
and illustrations and for final proof reading of the book
I extend my gratitude to Mrs. Shantala Baliga and Dr. Bhagyajyothi for having first hand
proof-read the manuscript.
I extend my gratitude to Dr. Ullas Karnath, Dean & Professor of Biochemistry at MMMC, Manipal;
Dr. Nivedita Rao, Faculty, Department of Biochemistry, Yenepoya Medical College,
Mangalore; Dr. R eshma Kumarchandra, Faculty, Department of Biochemistry, Kasturba
Medical College (KMC), Mangalore and Dr. Shivananda Baliga, Faculty, Department of
Biochemistry, Kasturba Medical College (KMC), Manipal for the help rendered in revision of
key chapters and valuable suggestions.
I thank my sister, Mrs. Prathibha Sreekumar, for having helped me w ith the computer graphics.
and my niece Sheetal Sreekumar, for having helped me w ith template plate preparation.
I thank Miss. Aafreen Vaz for having helped me with the cover page design.
I extend my gratitude to my teachers Dr. Urban D'Souza and Dr. Shankar Bhat for their constant
support and blessings.
I thank my friend Mr. Anand Prabhu, Prakash Offset Printers, for printing this book on time.
I extend my gratitude to my friends Dr. Vinaya, Dr.Girish, Dr. Sindhu, Mr. Suren, Dr. Anjana,
Dr. Roshan, Dr. Nishith, Dr. Shabari, Dr. Veena for their constant support.
I thank Mr. Vinayasheela and Mr. Raghavendra Prabl1u, Prakash Offset Printers, for helping
me with the computer graphics and cover page design.
Last, but not the least, I acknowledge all those w ho wished well for this book and felt free to
extend their views and opinions.
5
OREWORD
It is unfortunate that Biochemistry is being taught in Indian Medical Schools as a purely
non-clinical subject. The young edical Student at that stage is not in a position to
learn the subject let alone learning ts relevance and importance. In the West, the Clinical
aspect of the subject is taught mu h later either as Chemical Pathology in the UK or as
Clinical Chemistry in the US. But · Indian Medical Schools even the Clinical relevance
is thrust on the student in the firs year itself when the student hardly has any clinical
exposure. This is a real tragedy d has resulted in virtual detriment to the reputation
of such an important subject. Our Biochemistry teachers in Medical colleges therefore
are forced to teach mere concepts at underlie the biochemical basis of cellular function.
Thus, there is an acute need for good book in this subject which will introduce the
subject to the young student eno h for him to build on his basic understanding.
This book written by Prasad, I hop will go a long way in attempting to infuse confidence
in the minds of the students and a y their apprehensions that Biochemistry is a difficult
subject. Prasad has dealt with th subject in a very systematic way which makes the
book readable and enhances und rstanding even by the average student. The step-by-
step approach combined with £lo charts, simple diagrams and formulas have made
the book a good learning tool. I m sure this painstaking effort by Prasad will bring
laurels to this young author. Hop that the book will progress from strength to strength
in the years to come.
Prof. C V Rag uveer. MD, DCP, FICPath,

The Registrar, also The o· ector of Post Graduate Studies & Research
Yenepo a University, Mangalore
Formerly
Dean & Professor of athology, KMC, Manipal University
6
Co-Authors and Contributors
Author would like to acknowledge the role of various contributors in the

preparation of this edition. These professionals have contributed in various
topics by their valuable suggestions and also by writing some portions of
these chapters. Their contribution and assistance have been most helpful
in the formulation of this edition. - Prasad R.M
1. Dr. Urban D'Souza, Professor of Physiology, KVG Medical College,

Sullia, has contributed in the Hormones & Hormone actions, muscle, and
Connective Tissue chapter.
2. Dr. Shankar Bhat, Professor & Head, Department of Physiology,

Yenepoya Medical College, Mangalore, has contributed in the Plasma
proteins and Haemoglobin chapter.
3. Dr. Reshma Kumarchandra, Faculty, Department of Biochemistry,

Kasturba Medical College, Mangalore, has contributed in Acid base balance
chapter.
4. Dr. Shivanand Baliga, Faculty, Department of Biochemistry, Kasturba

Medical College, Manipal, has contributed in Enzyme chapter.
7
I NOTE TO THE STUDENTS !

This book is based on the new revised syllabus prescribed by Medical Council
of India (MCI) .
Format of the book:

This textbook contains 31 chapters. While including all the topics of new revised syllabus
prescribed by Medical Council of India (MCI), order and organization of chapters are
a matter of personal preference keeping in mind the continuity and flow of the subject.
Each chapter in this book is discussed under following sections.
1) Contents of the chapter

This section contains the key objectives that will be discussed in the concerned chapter.
Contents are selected based on the new revised syllabus prescribed by Medical Council
of India (MCI) and is intended for use by medical students throughout India.
2) Explanation
This section contains detailed explanation of the chapter. A simple lucid language is
used, which is easy to read and recollect. While the presentation of the subject is concept
based (and not in question and answer format), efforts are made to ensure that all the
University exam questions are covered. Exam tips with answer keys have been included
wherever required to help the students to know what to write for different types of
questions asked. Diagrams are hand-drawn so that they are easy to reproduce.
3) Question Bank including Multiple Choice Questions (MCQ)

This section contains the entire pool of questions possible from that chapter. All the
previous years University questions are included.
Note: This section also contains Multiple Choice Questions (MCQ). Questions that
were previously asked in All India, AIIMS, MAHE, COMEDK entrance examinations
are included to get a first hand experience of MCQ's.
ISpecial N ote I
Use of Italics :
Some segments of the chapters are written in 'italics', which generally contain
additional information and may not be needed for the basic understanding of the chapter.
So, students who want to omit these segments in first reading, can do so without
affecting the continuity of the chapter.
But it is highly recommended to read these segments for a comprehensive knowledge
of the subject. However, these italic segments can be asked in MCQ, Viva, Short
answers or Give reason type of questions.
For queries and suggestions, contact the author at prasad_text@yahoo.com or 9986449575

8
CONTENTS
Cell, Chemistry of Biomolecules and Enzymes :

l. Introduction to Biochemistry ............................................................................................ 13
2. Cell Biochemistry .................................................................................................................. 15
3. Carbohydrate Chemistry ..................................................................................................... 32
4. Lipid Chemistry ......... ................................................................................. ................... ........ 58
5. Amino acid and Protein Chemistry .......................................................................... ........ 80
6. Nucleotide and Nucleic acid Chemistry ........................................................................... 107
7. Enzymes ................................................................................................................................... 117
Metabolism:
8. Basic Aspects and Overview of Metabolism ... ................. ..... .......................................... 150
9. Digestion and Absorption ..................................................................................................... 154
10. Carbohydrate Metabolism ................................................................................................ 172
11. Lipid Metabolism ................................................................................................................ 212
12. Amino Acid and Protein Metabolism ................................................. .............................. 246
13. Intermediary Metabolism (Biochemsitry of Starvation and Absorptive State) ............. 287
14. Electron Transport Chain and Oxidative Phosphorylation .................................. 292
Nutrition, Vitamins, Minerals and Water:

15. Nutrition ................................................................................................................................... 304
16. Vitamins ................................................................................................................................. 331
17. Minerals .................................................................................................................................... 362
18. Water and Electrolyte Balance.............................................................................................. 389
Molecular Biology, Genetics, Recombinanat DNA, Techniques, Cancer:

19. Nucleotide Metabolism ....................................................................................................... 405
20. Molecular Genetics Part 1 (Replication, Trasncription, Translation) ....................... 421
21. Molecular Genetics Part 2 (DNA damage, DNA repair, Mutation, Caner)............ 444
22. Molecular Genetics Part 3 (Probes, Blotting, RFLP, Finger printing, PCR) ................ 456
23. Regulation of gene expression ..................................................................................... ··•.··· 468
24. Recombinant DNA technology (Genetic engineering) ............................................... 474
Applied and Clinical Biochemistry):

25. Techniques (Electrophoresis, Chromatography, RIA, ELISA) ....................................... 488
26. Radioisotopes in Biochemistry ................. .. .. ..... .. .. .. ............. .. .. .......... .. .. ............. ........ .. .... ... 499
27. Hormones ...................................................................... ...................................................... 503
28. Biotransformation (Detoxification) ....................................................................................... 515
29. Free radicals and Reactive oxygen Species ....................................................................... 521
30. Biochemistry of Connective Tissues and Muscle .................................... ,................. 529
31. Plasma Proteins and Electrophoresis . .. . . . . .. . . . .............................................................. 537
32. Acid - Base Balance ......... .................................................................................................. 545
33. Hemoglobin and Heme (Synthesis, Porphyria, Degradation, Jaundice) .................. 558
34. Clinical Biochemistry (LFT, RFT, TFT) .................................................................................. 572
35. Clinical Cases ................................................................................................................................... 595
36. University Question Bank with Answer keys ................................................................ 606
37. Index ... ....... .. .. ...... .......... .. ... .. .. ... .. .. ... ... .. ..... ..... ... .. ....... ...... .. ........ ..... .. ..... ..... .. .. ... ...... .. .. ....... ... .. . 626
9
Biochemistry Syllabus
(As per the new revised syllabus of MCI)
All the contents of MCI syllabus have been included in the book. But, order & organization of
chapters are a matter of personal preference keeping in mind the continuity and flow of tile subject.
I. Introduction and Scope of Biochemistry
II. Cell and Sub Cellular Structures (3 hours)

A) Cell membrane-composition
B) Functions of sulH:ellular structure.
C) Transport across the cell membrane.
a) Active Transport
b) Facilitated diffusion
c) Receptor mediation
d) Endocytosis
III. Hydrogen Ion Concentration, Acids, Bases, Buffers, Henderson Hasselbalch Equation
(2 Hours)
IV. Isotopes and their applications in Medicine (1 hou r)
V. Chemistry of Carbohydrates (4 hours)

a) Definition, Classification and Biological importance
b) Monosaccharides: structures, Classification and properties
c) Isomerism and Stereoisomerism
d) Oligosaccharides, Disaccharide's-structure and their importance
e) Polysaccharides: Homo & heteropolysaccharides: structure & their functions
VI. Chemistry of Proteins, Peptides and Amino acids (6 Hours)

a) Proteins: Definitions, Classifications and functions.
b) Amino acids: Classification, properties, side chains of amino acids, charge properties
c) Peptides: Biologically active peptides - GSH, Insulin - its structure
d) Proteins:
i) Definition
ii) Biological importance of proteins
iii) C lassification and properties, charge properties
iv) Structural organization, Conformation and denaturation.
v) Biosynthesis of creatine phosphate and formation of creatinine
e) Plasma Proteins
VII. Chemistry of Lipids (4 Hours)

a) Definition, Classification and Biological importance
b) Simple lipids: Triacylglycerols and waxes- structure and composition
c) Compound lipids: Phospholipids- Sphingolipids, Glycolipids & their properties
d) Derived lipids: Fatty acids -saturated, unsaturated, steroid & their properties, Eicosanoids,
Terpenes.
10
VIII. Chemistry of Nucleic Acids (4 Hours)

a) Definition & biological importance
b) Classification, composition
c) Purines and Pyrimidine - bases nucleosides, nucleotides, biological important
nucleotides.
d ) DNA: Structure and Function
e) RNA: Types of RNA- Structure and function.
IX. Enzymes & Clinical Enzymology (7 Hours)

a) Definition, Classification, Specificity, Co-enzymes, cofactor activators.
b) Mechanism of Enzyme action.
c) Factors affecting enzym e activity- Km value and its importance.
d ) Enzyme inhibition: reversible and irreversible competitive and other types: its clinical
application.
e) Regulator en zymes: Proenzym es Zymogens Isoenzymes, allosteric en zymes and feed
back control.
f) Diagnostic and therapeutic importance of enzymes.
g) ELISA & RIA
X. Vitamins (9 Hours)
a) Definition and Classification
b) A brief account of chemistry, source, deficiency diseases and bioch emical role,
Recommended Dietary allowances (RDA)
c) Vitamin antagonists
d ) Hypervitaminosis.
e) A brief account of role of antioxidants and free radicals.
XI. Biological Oxidation (3 Hours)

Mitochondrial electron transport chain, oxidative phosphorylation, mechanism Uncouplers and
inhibitors
XII. Digestion & Absorption (3 Hours)

a) Digestion and absorption of carbohydrates, lipids, proteins and nucleic acids.
b) Malabsorption syndromes.
XIII. Carbohydrates Metabolism (9 Hours)

a. Glycogenesis, Glycogenolysis and Glycogen storage diseases.
b. Glucose transporters, Glycolysis, Rapaport Leubering cycle, pyruvate oxidation, citric
acid cycle.
c. Pentose phosphate pathway
d. Uronic acid pathway
e. Gluconeogenesis and Cori's cycle.
f. Metabolism of Fructose and GaJactose.
g. Regulation of metabolic pathways.
h. Disorders of carbohydrate metabolism.
i. Regulation of blood Glucose, GIT, Diagnostic and Prognostic importance of glycated
h emoglobin and Diabetes Mellitus.
11
XIV. Lipid Metabolism (10 Hours)

a. Oxidation of fatty acids, propionate metabolism, formation & utilization of ketone bodies,
ketosis, outline of synthesis of cholesterol (reaction up to mevalonate in detail) and
breakdown of cholesterol, metabolic disorders of lipids.
b. Lipogenesis, Denovo synthesis of fatty acids, chain elongation, desaturation,
phospholipid biosynthesis (for example: Lecithjns and Cephalin) and their breakdown.
c. Fatty livers and lipotropic factors.
d. Prostaglandins and their biological functions
e. Plasma Lipoproteins - Classification functions and disorders.
XV. Protein and amino acid metabolism (10 hours)

a. Breakdown of tissue proteins and amino acid pool, general reactions of amino acid.
b. Disposal of ammonia: Urea cycle, glutamate and glutamine formation.
c. Metabolism of individual amino acids (Glycine, Serine, sulphur containing amino acids
aromatic amino acids, Histidine & Arginine)
d. Metabolic disorders of amino acids, aminoacidurias.
e. Synthesis of creatinine, Phospho-creatine, formation of creatinine and clinical significance
of creatinine clearance.
XVI. Purine and pyrimidine metabolism (3 hours)

a. Source of atoms of Purine and Pyrimidine ring, biosynthesis of purine and pyrimidine
nucleotides and their breakdown.
b. Salvage pathway.
c. Disorders of purine and pyrimidine metabolism.
XVII. Intermediary metabolism (2 hours)

Introduction, Methods of study of Intermediary metabolism.
XVIII. Minerals (4 hours)

Calcium, Phosphorous, Iron, Copper, Iodine, Zinc, Fluoride, Magnesium, Manganese, Selenium
XIX. Molecular genetics & proteins biosynthesis (lOhours)

a. DNA, RNA metabolism
b. Replications, transcription, reverse transcription and post transcriptional modification.
c. Translation: Amino acid activation, initiation, elongation and termination, Post
translational modification.
d. Regulation of Gene expression.
e. Mutation.
f. Recombinant DNA technology, PCR and gene therapy
12
XX. Tissue biochemistry (12 hours)
a. Heme metabolism: outline of heme biosynthesis, degradation of heme and functions of
normal hemoglobin
b. Abnormal hemoglobins.
c. Jaundice
d. Porphyrias
e. Plasma proteins: Separation, functions and importance.
f. Immunoglobulins: structure and functions.
g. pH of blood and its regulation - acidosis and alkalosis, Principals of estimation of
body fluids. Role of kidneys and lungs in blood pH maintenance.
h. Water and electrolyte balance and disorders
XXI. Liver functions & kidney functions (3 Hours)

a. Liver Function Tests
b. Detoxication mechanisms and metabolism of Xenobiotics
XXII. Nutrition and energy metabolism (5 Hours)

a) BMR and its importance,
b) Calorific values of food, RQ SDA, balanced Diet.
c) Protein energy malnutrition, biological value of proteins, Nitrogen equilibrium,
d) Dietary fibers,
e) Biochemistry of starvation.
XXIII. Biochemistry of cancer (2 Hours)

a. Oncogenes
b. Growth factors
c. Tumors markers
• Definition
• Clinically important tumors markers-CEA, Alfa fetoprotein (AFP)
• Human chronic gonadotropin (HCG), Calcitonin, Prostate specific Antigen (PSA).
XXIV. Endocrine function: (3 hrs)

a) Hormone actions: Mechanism of actions of insulin, glucagon, epinephrine, steroids.
b) Thyroid function tests
XXV. Biochemical tests for atherosclerosis & myocardial infarction

a) Lipid profile, apoproteins, homocysteine and C-reactive protein
b) CKivIB, Troponins
XXVI. SI units, Quality control and standardization (1 hour)

a) Clinical chemistry- interpretation & reference values of Blood glucose, Urea, Creatinine,
Uric acid, Cholesterol, Calcium, Proteins, Albumin & A/G ratio.
b) Instrumentation including autoanalyser.
This textbook contains 31 ch apters. All the contents of new revised syllabus prescribed
by Medical Council of India (MCI) are included. order & organization of chapters are
a matter of personal preference keeping in mind the continuity and flow of the subject.
Introduction to Biochemistry 13
INTRODUCTION TO BIOCHEMISTRY
Contents:
• Definition
• Biomolecules
• Aim of Biochemistry
• Scope of Biochemistry and Nutrition in Medicine
For queries and suggestions, contactthe author at prasad_text@yahoo.com or 9986449575

Introduction to Biochemistry 14
INTRODUCTION TO BIOCHEMISTRY
Definition: Biochemistry can be defined as "the science concerned with the
chemical basis of life". The term 'Biochemistry' was coined by Neuberg in 1903.
Biochemistry deals with the structure, properties and chemical reactions of various
biomolecules that are present in the living systems.
Biomolecules :
Living systems are composed of various biomolecules like, Carbohydrates, Proteins,
Lipids, Vitamins, Minerals, Water and Nucleic acids etc.
Life depends on a fine balance of various biochemical reactions of these biomolecules
in the body. Any abnormalities in this harmonious balance lead to various diseases.
Hence biochemistry can also be termed as 'the chemical language of life'.
Aim of Biochemistry :
The aim of biochemistry is to study the chemical nature and reactions of biomolecules
in health and diseases.
Importance and Scope of Biochemistry and Nutrition in Medicine:

• Diagnosis, Therapy, Prognosis:
Importance and scope of Biochemistry are fundamental to medicine & health science. Knowledge
of chemical nature and reactions of biomolecules are essential to understand health & diseases.
It should be noted that all diseases have biochemical basis. For instance, diseases like Diabetes,
Jaundice, Rickets etc, all have biochemical basis. Scope of Biochemistry in m edicine often involves
biochemical approach to understand the basis of these diseases in order to diagnose and effective
treatment (therapy). Procedures of treating Atherosclerosis, Jaundice, Cancer e tc, offer means for
biochemical approach. Biochemical approach also helps in the prognosis of various diseases.
• Nutrition, one of the integral part of health care, is a branch of Biochemistry:

A proper understanding of nutrition requires an accurate knowledge of Biochemistry. Normal
development of the body requires proper nutrition. A number of nutrients (like Carbohydrates,
Proteins, Lipids, Vitamins and Minerals etc) are required for the proper metabolism of body.
Deficiencies or excess of any of these nutrients lead to various health abnormalities. A
comprehensive knowledge of nutritional biochemistry is very essential to give proper dietary
advice for the treatment of patients with various health problems. Biochemical perspective of
these nutrients is given in this textbook as and where required.
• Biotechnology, which is a branch of Biochemistry, has created wonders in the field of

Medicine and Health. DNA recombinant technology is an important tool in gene therapy
(introduction of functional genes into individuals having genetic diseases in place of defective
mutated genes), DNA finger printing and PCR methods (which are used in forensic medicine for
identification of criminals and for gene amplification in case of insufficient sample size), gene
transfer (Many genes like human insulin have already been transferred to microorganisms for
mass production of human proteins) etc. These technologies have opened up new outlook in the
treatment of various diseases especially cancer and AIDS.

Cell Biochemistry 15
CELL BIOCHEMISTRY
Contents:
• Introduction
• Cell structure: Structural features and general structure of a typical cell
• Cell membrane:
• Composition, Fluid mosaic model of plasma membrane.
• Functions of cell membrane and various transport mechanisms - Simple diffusion,
Facilitated diffusion, Active transport, Osmosis etc.
• Cytoplasm and subcellular organelles:

• Structure and functions of, Nucleus, Endoplasmic reticulum, Mitochondria,
Golgi apparatus, Ribosome, Peroxisomes, Lysosomes.
• Cytoskeleton
[ Use of Italics )
Some segments of the chapters are written in 'italics', w hich generally
contain additional information and may not be needed for the basic
understanding of the chapter. So, students who want to omit these segments
in first reading, can do so without affecting the continuity of the chapter.
But it is highly recommended to read these segments for a comprehen sive
knowledge of the subject. The segments given in 'italics' can be asked in
MCQ, Viva. Short answers or Give reason type of questions.

CELL BIOCHEMISTRY
Introduction:
Cells are the basic structural and functional unit of all living organisms. Cells aggregate
to form tissues or organs wh ich are further organized to form the whole system.
Functions:
• Cell is the basic structural unit of all organisms:
Cells are the building blocks of all organisms. Cells aggregate to form tissues or organs
which are further organized to form the system of organism.
• Cell is the basic functional unit of all organisms:
Cells are responsible for all the metabolic activities of an organism. Life of an organism
is the outcom e of the coordinated action of its constituent cells. Cells carry the
hereditary materials and are responsible for transmission of characters.
Microscopy:
Microscopy is the science of using microscopes. Study of cells and other objects,
which cannot be seen with the naked eye, is made possible by microscope.
• Microscope was first b uilt by Anton van Leeuwenhoek.
• Robert Hooke in 1665 first discovered cells in cork, then in living tissues using an early microscope.
• Ernst Ruska in 1932 built an electron microscope, which revolutionized the study of cells.
Prokaryotic and Eukaryotic cell organization:

• Prokaryotic cells:
They have no definite nucleus. Their nucleus is not enclosed by a nuclear membrane.
They also do not contain any membrane bound subcellular organelles.
Examples: Bacteria & blue green alga have prokaryotic cells (hence called prokaryotes).
• Eukaryotic cells:
They h ave nucleus which is covered by nuclear membrane.
Eukaryotes have variety of membrane bound subcellular organelles like Mitochondria,
Endoplasmic reticulum, Lysosomes, Golgi b_od ies, Microsomes etc.
Examples: Animals, plants & fungi have eukaryotic cells (hence called eukaryotes).
Characteristics Prokaryotic cells Eukaryotic cells
Nucleus Not well defined Well defined
Subcellular organelles Distinct Membrane Distinct Membrane bound
bound organelles are not organelles are found (eg, Golgi
found aooaratus, mitochondria etc).
Cell division Fission Mitosis
Size Small (1-10 µm) Bi2: (10-100 µm)
Examoles Bacteria, blue green afa:a Animals, plants, fungi.

Composition of cells:
Main constituent of a cell is water (i.e. about 70% of total weight of a cell). 1% of cell is
made up of inorganic compounds (Cations like Na+, K+, Ca+2, Fe+2 / Fe+3, Mg+2 etc. and
anions like chloride, bicarbonate). Rest of the weight is made up of organic compounds
like protein, lipids, nucleic acid and carbohydrates etc.
Structural or morphological features of a cell:

Human body contains at least 1014 cells. Different cells have different shapes, sizes
and functions. Despite these differences, all cells share certain structural similarities.
Each cell is bounded by plasma or cell membrane. All structures inside the plasma
membrane are protoplasm. Nucleus is present in center. Protoplasm includes cytoplasm
and nucleus. Cytoplasm contains organelles like mitochondria, endoplasmic reticulum,
lysosomes, Golgi bodies, microsomes, ribosomes etc., which are dispersed in cytosol.
• Cell membrane: All the cells are bounded by a membrane called the plasma membrane.
• Protoplasm: All the contents inside the cell membrane are called protoplasm. It
constitutes cytoplasm and nucleus (i.e. Cytoplasm is protoplasm minus the nucleus).
Protoplasm= Cytoplasm+ Nucleus
• Nucleus is present in the centre of the cell.
• Cytoplasm: The space between the cell membrane and nucleus is filled by cytoplasm.
Cytoplasm contains various subcellular organelles like Endoplasmic reticulum,
Mitochondria, Ribosomes, Golgi apparatus, Lysosomes and Peroxisomes, etc., which
are dispersed in cytosol.
Cytoplasm = Cytosol + Intracellular organelles
• Cytosol: It is the fluid portion of cell, in which all the organelles are suspended. It
mainly contains water and dissolved organic and inorganic substances. It is the
supernatant obtained by final centrifugation.
Marker enzymes:
Marker enzymes are enzymes that present in only one particular organelle. After
centrifugation, the separated organelles are identified by detection of marker enzymes
in the sample.
Organelle / cell component Marker enzyme
Mitochondria ATP synthetase
Lysosome Cathepsin
Peroxisome Catalase
Endoplasmic reticulum Glucose-6-phospha tase
Golgi complex Galactosyl transferase
Cytoplasm Lactate dehydrogcnase
Nucleus DNA polymerase

Diagrammatic representation of a typical mammalian cell
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Answer hint for the question - Give the composition of cell.

Add a note on the structure (with diagram) of a cell. Write
the functions of sub-cellular organelles (10 Marks):
a) Composition of cell - 1 Mark
b) Diagrammatic representation of a typical cell - 3 Marks
c) Function s o f eac h organelles (like Mitochondria, Endoplasmic
reticulum, Golgi apparatus, ribosome, Peroxisome, Lysosom es - 6 Marks

Cell membrane or Plasma membrane

Each cell is covered by a membrane called the cell membrane or plasma membrane,
which separates the cell from the external environment. It is about 8-10 run in thickness.
Structure of cell membrane (Fluid mosaic model):

Singer and Nicholson proposed the 'Fluid Mosaic Model' to explain the structural
composition of cell membrane. According to the fluid mosaic model, the cell membrane
is made up of bilayers of phospholipids (and small amounts of glycolipids and
cholesterol) along with proteins and carbohydrates.
a) Lipids:
Phospholipid is the principal lipid present in the cell membrane. Small amount of
glycolipids and cholesterol are also present. All the membrane lipids are amphipathic
in nature (as they contain both hydrophobic and hydrophiJic regions). These amphipathic
lipids are arranged with their polar head groups oriented towards the extracellular and
the cytoplasmic side and the non-polar hydrophobic tail oriented towards each other
(in the direction of the hydrophobic core of bilayer).
b) Proteins:
The proteins present in the membrane are categorized into 2 types.
a) Peripheral proteins b) Integral proteins
i) Peripheral proteins: These proteins are loosely attached to the outer or inner surface
of the membrane by weak ionic and polar bonds. They can be easily removed without
disrupting the membrane.
ii) Integral proteins: These proteins are firmly embedded in the bilayer and tightly
bound to the lipid bilayer by hydrophobic bonds. They cannot be removed without
disrupting the membrane (They can be removed by the use of detergents).
Some of these integral proteins span the whole bilayer and are called transmembrane
proteins. Transmembrane proteins serve as transport or carrier proteins.
c) Carbohydrates:
The carbohydrates exist as glycolipids and glycoproteins. They are always found
attached to the external surface of the cell membrane. The carbohydrate coat on the
outer surface of cell membrane is called glycocalyx.
Membrane fluidity: Membrane fluidity refers to the viscosity of the cell membrane.
• The length, nature of fatty acids & nature of the polar h ead groups and ch olesterol content
of the membrane, determine the membrane fluidity .
The membrane fluidity decreases with increase in length of hydrocarbon chain. Unsaturated
fatty acids increase membrane fluidity. More the degree of unsaturation of fatty acids, more
the membrane fluidity. When the cholesterol concenh·ation increases, the membrane fluidity
decreases on the outer surface and increases in the hydrophobic core of the membrane.
• Membrane fluidity affects the activity of the receptors and ion channels.

f'ER1P~ERAL fRGfEIN
(ARBo>t,o,.,E Ruoou<s
. . INTEC..IAL f'•ore,,a
( Fluid mosaic model of cell membrane)
Functions of cell membrane:

1. Provision of shape and size to the cell. Cell membrane maintains a de.finite sh ape
and size of the cell.
2. Maintenance of integrity of the cell: The cell membrane maintains the integrity of
cell by forming a selective barrier between the cell and its surroundings. It allows
only selected compounds to be transported across the membrane.
3. Protective function: It protects the cytoplasm and cell organelles.
4. Absorptive function: The nutrients are absorbed into the cells through the cell
membrane.
5. Exchange of gases: Exchange of gases takes place through cell membrane.
6. Excretory function: Waste products are thown out of cells through cell membranes.
Answer hint for the question - Give the composition of cell.

Add a note on the structure (with diagram) and functions of
cell membrane (10 Marks):
a) Composition of cell - 1 Mark
b) Diagrammatic representation of cell membrane - 3 Marks
c) Explanation of structure of cell membrane - 3 Marks
b) Functions of Cell membrane - 3 Marks

Transport across the membrane:

Cell membrane regulates the transport of substances across the cell by processes like,
A. Transport of small molecules:
1) Simple diffusion } .
2) Facilitated diffusion Passive transport
3) Active transport
B. Transport of large molecules:
1) Endocytosis (Phagocytosis and Pinocytosis)
2) Exocytosis
C. Osmosis: Transport of solvent molecules.
1) Simple diffusion:
Simple diffusion is defined as the process of transport of molecules across the membrane
from the region of higher concentration to the region of lower concentration (i.e down
the concentration gradient).
Simple diffusion neither requires a carrier molecule nor energy.
Examples: Transport of CO 2, 0 2, N 2 , Ions like Na+, K+, Ca H etc.
Simple diffusion can take place in two ways,

a) Through the lipid bilayer: Transport of CO2, 0 2, N 2, water & small lipid soluble
molecules, Pentoses take place across the membrane by passive diffusion.
b) Through the ion channels or pores: Ions like Na+, K+, Ca++ and water are transported
through protein or ion channels and pores.
Ion channels and pores:

Ion channels or pores are transmembrane proteins presentin cell membranes, which permit the
diffusion of substances (mainly ions) across the membrane. They do so by creating a central
aqueous channel (hole) in the channel protein that permits the passage of substances. There is
no binding or physical interaction between the channels and the substances carried.
Ion channels are of 2 types; Passive channels and Active channels.
i) Passive channels (leakage channels): These are always open and ions pass through them
continuously. They are responsible for maintaining resting membrane potential.
ii)Active channels: These have gates that can open or close the channel. In these, channels are
generally closed, but can be opened by a variety of mechanisms. 3 most studied gates are,
• Voltage gated channels: These respond to a change in transmembrane electrical potential.
E.g., Calcium channels in sarcotubular system, Voltage gated sodium & potassium channels.
• Lig and gated channels: These respond to binding of a ligand.
E.g., Opening ofsodium channels by acetyl choline in post-synaptic membrane.
• Mechanically gated channels: These respond to some mechanical factors .
E.g., Sodium channels in pacinian corpuscles open on pressure.

2) Facilitated diffusion:
Facilitated diffusion is defined as the process of transport of molecules across the
membrane down the concentration gr·adien t (i.e. from the region of highe r
concentration to the region of lower concentration) with the help of a carrier molecule.
Facilitated diffusion requires only carrier molecule, but doen not require energy.
E.g.: Absorption of fructose, glucose and galactose by intestinal cells. Speci fic carrier
proteins for the transport of leucine and isoleucine are also found.
Passive transport: Passive transport does not require energy. There are 2 types of passive
transport, Simple diffusion & facilitated diffusion. Facilitated diffusion is similar to simple diffusion
in that both are dependent on concentration gradient and the substances are transported down
the concentration gradient. But facilitated diffusion differs from simple diffusion in that the carrier
protein is required only by facilitated diffusion but not by simple diffusion.
3) Active transport:
Active transport is defined as the process of transport of molecules across the
membrane with the help of a carrier molecule and energy. The transport is mostly
against the concentration gradient (i.e. from the region of lower concentration to the
region of higher concentration)
Active transport requires both energy and a carrier protein molecule.
E.g.: Transport of sodium and potassium by Na/ K ATPase (or Na/ K pump)
( Comparison of three transport systems )
Channel proteins Carrier proteins
·/\·
• • • • "' ATP
Simple Facilitated Active

diffusion diffusion Transport

Comparison table of three transport systems
Requirement Requirement
PROCESS Movement of carrier of energy
protein (ATP)
Down the
Simple diffusion concentration NO NO
gradient
Down the
Facilitated diffusion concentration YES NO
gradient
Against the
Active trans port concentration YES YES
gradient
Additional information regarding Active transport :-
Active transports are of two types; primary and secondary :-
a) Primary active transport:

If the energy is utilized directly for the transport of molecules, then such active transport
system are termed as primary active transport.
E.g.: Sodium & potassium transport by sodium potassium ATPase, calcium transport.
b) Secondary active transport:

If the energy is utilized indirectly for the transport of molecules, then such active transport
system are termed as secondary active transport.
Here the energy by electrochemical or membrane potential gradient generated by the
transport of a compound by primary active transport is utilized for the transport of
another compound by secondary active transport.
E.g.: Intestinal absorption of glucose (Sodium dependant glucose cotransport).
Receptor mediated transport:

Sometimes, the signal molecules (like hormones or growth factors) cannot pass through the cell
membrane. So, they bind to specific cell membrane receptors, which are integral proteins exposed
to outside of the cell membrane. Binding of signal molecules stimulate the production of a new
molecule inside the cell that function as intracellular messenger (by a mechanism called transduction).
Signal transduction leads to the internalization of the signal via membrane receptors.
This type of transport is receptor mediated. It is explained in detail under hormone chapter.
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Carrier mediated transport:

Definition: Transport systems that require carrier protein molecules for their transport
across the membrane are called carrier mediated transport. Generally, molecules that
cannot pass freely through the lipid bilayer membrane are transported in this method.
Facilitated diffusion and Active transport are carrier mediated transport system. Both
require carrier molecules. Difference being that the facilitated diffusion does not require
energy, whereas the active transport requires energy.
Carrier mediated transport systems can be divided into 2 categories.

i) Uniport: The transport mechanisms which involve the movement of a single molecule
through the membrane are called Uniport.
E.g.: transport of glucose, calcium transport.
ii) Co-transport: If transport of one compound leads to the transport of another
molecule, i t is called co-transport. Co-transport is of two types,
a) Symport: Co-transport mechanisms, where the two molecules are transported in
same directions .. E.g.: Sodium dependant glucose transport.
b) Antiport: Co-transport mechanisms, where the two molecules are transported in
opposite directions .. E.g.: Sodium potassium pump, HC03• and Cl" transport
Carrier proteins
0 0 D 0
Uniport Symport Antiport
• Osmosis:
Osmosis is defined as the diffusion of solvents across a semi-permeable membrane
down the concentration gradient. In cells, plasma membrane acts as a semi-permeable
membrane (or selectively permeable membrane).
Water is transported across the cell membrane by osmosis.

Cell Biochemist ry 25
• Sodium pump (also called Na•-K• ATPase):

Sodium pump or sodium potassium pump is a primary active transport process that
pumps Na ions out of the cell and at the same time pumps K ions into the cell.
The carrier protein of Na-K pump has 3 binding sites for Na• on the surface facing the
inside of the cell and 2 binding sites for K• on the surface facing outside the cell. Na•-K•
pump also has a portion for ATPase activity located near Na• binding site.
Mechanism:
• Three Na• ions bind to the binding site on the inner surface of protein.
• Two K• ions bind to the binding site on the outer surface of protein.
• Now, ATPase function of carreir protein is activated, which hydrolyses the ATP into
ADP and Pi, liberating energy.
• The energy liberated causes a conformati onal change in carrier protein of sodium
potassium pump, which pumps Na.. ions outside and K• ions inside.
Functions of sodium potassium pump:

1. Maintenance of resting membrane potential (RMP):
Sodium potassium pump helps in maintainin g the concentrations of Na• and K• ions
across the membrane. This is essential for the maintenan ce of RMP. In case of nerve
and muscle tissues, the passage of nerve impulses cause the diffusion of Na ions into
the cells and K ions out side the cells, causing an imbalance of Na and K concentrations.
Na-K pump bring back the normal levels by pumping sodium ions out of the cell and
at the same time pumping potassium ions into the cells.
2. Required for absorption of glucose and amino acids:
Sodium pump participate in the secondary active transport of glucose and amino acids
through co-transport using the sodium gradient. (Refer digestion chapater for diagrams).
• Filtration:
Filtration is the process of movement ofparticles through a membrane with the help ofhydrostntic
pressure gradient created by a fluid. Pressure gradient rather tlzan concentration gradients
drives the movement & pushes tlze particles through a membrane. Here, when afluid is forced
through a membrane (due to hydrostatic pressure), it also carries various particles along with
it. Size of the pores of membrane determines the size of particles that passes through them.
Significance:
Filtration takes place across various biological membranes. For example,
1) CapillanJ walls: Hydrostatic pressure ofblood vessels pushes the nutrients and small ions
through the capillary wall to the tissues. The blood cells nnd large protein molecules are unable
to pass through the pores in capillary walls.
2) Glomemlo capillary membrane: In glomerular filtration, hydrostatic pressure ofthe blood
pushes the water and small molecules and ions through tile filtration membrane, while blood
molecules and blood cells remain in the blood.

Cell Biochemi stry 26
Transport of large molecules:
1) Endocy tosis:
The process of intake of large macromolecules into the cell is called endocytosis.
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There are 2 types of endocytosis,

a) Phagocy tosis: Endocytosis of solid particles is called phagocytosis.
b) Pinocyto sis: Endocytosis of fluid particles is called pinocyto sis.
a) Phagocy tosis (Cell eating):

In this process, solid particles are internali zed as such. The macromo lecule to be
transport ed makes a contact with the outer surface cell membran e. This segment of
plasma membran e is then invaginat ed finally enclosing the molecule to be endocytosed,
forming an endocytic vesicle. This vesicle is then pinched off ins ide the celJ along
with the matter.
E.g.: Phagocytosis of bacteria, viruses by WBC's.
b) Pinocytosis (Cell drinking):

Similar process, but endocyto sed matter is in solution.
E.g.: Uptake of thyroglob ulin by follicular cells of thyroid.
, 2) Exocyto sis:
It is the process of release of intracellu lar macromo lecule out of the cell. Exocytosis
involves the binding of molecule to the inner surface of cell membran e. At this place,
plasma membran e evaginate s (exvaginates) forming a vesicle that is later pinched
off outside the cells.
E.g.: Release of neurotra nsmitters from nerve endings and release of hormone s from
glands.

• Cytosol
Cytosol is the fluid portion of the cytoplasm in which the organelles are suspended .
Function (Reactions that take place in cytosol):
Glycolysis, HMP shunt pathway, glycogenolysis, glycogenesis, uronic acid pathway,
de novo synthesis of fatty acids, transamination, purine synthesis, part of heme synthesis,
part of urea synthesis, part of gluconeoge nesis and part of pyrimidine synthesis occur
in cytosol.
• Nucleus
Nucleus is the control centre of the cell. All the cells, except mature RBC's have nucleus.
Structure:
The nucleus consists of two membrane s, which are seperated by the intermemb ranous
cisternae. The inner membrane is called the perinuclea r membrane which has numerous
pores. Outer membrane is continuous with membrane s of endoplasm ic reticulum.
In some cells, a portion of nucleus may be seen as dense area known as nucleolus.
Nucleolus contains RNA.
Functions:
• The nucleus contains the master molecule DNA, the genetic material which governs
all the functions of the cell. DNAs are organized in the form of chromosom es. Human
cells contain 46 (23 pairs of) chromosom es. DNA synthesis (Replication) and RNA
synthesis (Transcription) take place inside the nucleus.
• RNA processing and ribosome synthesis occur in nucleolus.
• Subcellu lar Organel les

Cell contains many subcellular organelles that are suspended in the cytoplasm.
• Endoplasmic reticulum
Structure:
This is a membrano us channel system connecting the nuclear membrane and plasma
membrane . They have network like system.
Types: There are 2 types of endoplasm ic reticulum, namely,
a) Smooth endoplasm ic reticulum
Has smooth appearance , which has no attached ribosomes.
b) Rough endoplasm ic reticulum
Has rough appearance because of presence of ribosomes on their structure.
For queries and suggestions, contact the author at prasad_text @yahoo.co m or 9986449575
Functions:
• Endoplasm ic reticulum gives mechanical support to the cell. It also helps in the
transport of synthetic products (like glycoprote ins and lipoproteins) out of the cell.
• Lipid synthesis (Triglyceride, Cholestero l and Phospholi pid synthesis), steroid
hydroxylation & detoxification of various drugs take place in endoplasm ic reticulum.
• Rough endoplasm ic reticulum also helps in protein synthesis because they are
associated with ribosomes.
• Endoplasm ic reticulum acts as intracellular reservoir of calcium.
• Ribosome
Structure:
• Ribosomes are made up of r RNA and proteins ( ucleoproteins).
• A ribosome has 2 sub units, a larger and a smaller sub unit. The subunits are in
dissociated form and the association takes place only during protein synthesis.
• The eukaryotic ribosomes are 80 S ribosomes made up of 60 S larger and 40 S smallel
sub units.
• The prokaryoti c ribosomes are 70 S ribosomes made up of 50 S larger and 30 S smaller
sub units.
Functions:
• Ribosome s are factories of protein synthesis. On ribosomes the mRNA, tRNA
interacts to synthesize proteins.
• Ribosomes present on rough endoplasm ic reticulum are associated with the synthesis
of proteins for their export out of the cell. On the other hand, free ribosomes present in
the cytosol are associated with the synthesis of proteins for use within the cell.
• Golgi apparatu s (or Golgi complex)
Structure:
These are flattened, membrane surrounde d vesicles.
Functions:
• Protein modification, sorting, packaging & secretion takes place in Golgi complex.
The newly synthesized proteins are handed over to Golgi complexes, which are modified
here. For example, addition of carbohydra tes and lipids in the formation of glycoproteins
or lipoprotein s takes place in Golgi apparatus.
• It also helps in the formation of lysosomes and peroxisomes.

• Mitochondria
Mitochondria are called the 'Powerhouse of the cell'. Ma ttue erythrocytes do not contain
mitochondria.
Structure:Mitochondria have two membranes, the outer membrane is smooth and the
inner membrane is convoluted into folds called cristae. The cristae contain a number of
knob like projections. The space between 2 membranes is called intermebrane space.
The space inside the inner membrane is called the mitochondrial matrix. Mitochondrial
matrix contains several enzymes of various metabolisms. It also contains specific circular
DNA (m tDNA), RNA and ribosomes. Mitochondrial DNA produces certain inner
mitochondrial membranes proteins. mtDNA is inherited from mother, so, mitochondrial
DNA disorders (like mitochondrial myopathy) are not transmitted from father to the children.
Functions:
• Mitochondria is called the powerhouse of the cell, where ATP is synthesized. Inner
mitochondrial membrane contains the components of electron transport chain (ETC)
and oxidative phosphorylation, where the energy released from the oxidation of food
molecules (glucose, fatty acids and amino acids) is trapped in the form of ATP.
• TCA cycle, ~-oxidation of fatty acids, pyruvate dehydrogenase (PDH), ketogenesis,
ketolysis, part of heme synthesis, part of urea synthesis, part of gluconeogenesis and
part of pyrimidine synthesis also take place in mitochondrial matrix.
• Peroxisomes
Structure: They are single membrane structures enclosin[ a granular matrix.
Fun ctions:
• Peroxisornes contain catalase and peroxidase enzymes, which destroy peroxides &
other free radicals, thus protecting the cells from their toxic effect.
• Peroxisomes are involved in the synthesis of plasmalogens and glycolipids.
• Peroxisomes are also involved in the oxidation of long chain fatty acids (> C1s).
Zellweger syndrome occurs due to an inherited absence offunctional peroxisomes in alI tissues.
It is characterized by neurological impairment. Child dies in 1st year of life.

• Lysosomes
Structure: Lysosomes are small vesicles surrounded by a single membrane. The pH
inside the Lysosomes is lower (acidic) than that of cytosol (pH around 5).
Functions:
1) Lysomes are responsible for degradation and recycling of intracellular biomolecules.
Lysosomes contain hydrolytic enzymes (hydrolases), which are involved in the
degradation of intracellular biomolecules (digestion of intracellular proteins, lipids,
carbohydrates etc). Hydrolyzed products can then be reutilized & recycled by the cell.
2) Lysosomes also digests the substances brought into the cell by phagocytosis.
Additional information:
Hydrolases of lysosomes are,
• Carbohydrate hydrolyzing enzymes: a-Glucosidase, ~-Galactosidase, a-Fucosidase,
Mucopolysaccaridases, aryl sulfatase, a.-Mannosidase etc.
• Protein hydrolyzing enzymes: Cathepsin, Collagenase, Elastase etc.
• Lipid hydrolyzing enzymes: Fatty acyl esterase, Phospholipase, Arylsuljatase etc.
• Nucleic acid hydrolyzing enzymes: DNAase, RNAase, Phosphodiesterase etc.
• If lysosomal membrane is disrupted, released enzymes can hydrolyze the intracellular materials
leading to death of the cell. Therefore, lysosomes are also referred as 'suicidal bags' of the cell.
• Cat11epsins are the marker enzymes for lysosomes. These proteases are involved in the
intracellular digestion of proteins.
• Lysosomal storage diseases: These are group of diseases that occur due to the absence of specific
lysosomal enzymes resulting in abnormal storage of related compounds in lysosomes.
E.g., Pompe's disease (Type II glycogenosis), Sphingolipidoses (like Niemann-Pick disease, Tay-
sachs disease etc.), mucopolysaccharidoses (like Hurler's disease, Hunter's disease etc.).
• Cytoskeleton:
Cells contain intracellular network of filamentous s tructure called as cytoskeletons.
These include actin filaments (m.icrofilaments), microtubules & intermediate filaments.
a) Microfilaments (Actin filaments): Non muscle cells contain actin molecules that
polymerise to form microfilaments.
Functions: In non-muscle cells, actin interact with myosin to cause cellular movements.
b) Microtubules: These are elongated tubu lar structures. Each microtubule contains 13
longitudinally arranged profilarnents, each consisting of dimers of a and Ptubulin.
Functions: They are involved in the formation of mitotic spindle during cell division.
Microtubules also participate in transport system of the cell (axonal transport).
c) Intermediate filaments: These are elongated, fibrous molecules. Four types of these
filaments are identified. These are lamins, keratins, vimentins neurofilaments.
Functions: They form the important structural components of the cells.

Question Bank on Cell:

Short essays (5 Marks):
1) Describe the structure of a typical cell and its organelles with a neat, labeled diagram.
2) Explain the structure (Fluid mosaic model) of plasma membrane. Add a note on functions.
3) Explain the different transport mechanisms across the cell membranes
4) Structure and functions of mitochondria
5) What are the different types of absorption? Describe the features briefly
Short Answers (2 - 3 Marks):

1) Explain the structure and functions of any organelle,
2) Reactions that take place in cytosol (or cytoplasm)
3) Carrier mediated transport
4) Difference between Simple diffusion, Facilitated diffusion and Active transport
5) Osmosis
Multiple Choice Questions (1 Mark):

1) All of the following occur in mitochondria except (Al)
a) Kreb's cycle b) Glycogenolysis c) Fatty acid oxidation d) ETC
2) Proteins are sorted by (AI)
a) Golgi bodies b) Mitochondria c) Ribosomes d) Nucleus
3) Mechanical support is given by (AI)
a) Endoplasmic reticulum b) Ribosomes c) Lysosomes d) Mitochondria
4) Glycolysis occurs in (A IIMS}
a) Cytosol b) Mitochondria c) Nucleus d) Lysosome
5) Rough endoplasmic reticulum is site of synthesis for (AIIMS)
a) Protein b) Cholesterol c) Carbohydrate d) Fat
6) The subcellular organelle rich in catalase is
a) Lysosomes b) Mitochondria c) Peroxisome d) Ribosome
7) Cathepsin is the marker enzyme for
a) Lysosome b) Peroxisome c) Nucleus d) Cytoplasm
8) In Zellweger syndrome, which of the following will be absent in all types of human cells?
a) ucleosomes b) Peroxisomes c) Lysosomes d) Ribosomes
9) Ribosomes in cell are responsible for

a) Protein synthesis b) Glycolysis c) Lipid synthesis d) Oxidation of fat
10) The fluidity of the membrane is maintained by
a) Length of hydrocarbon chain b) degree of unsaturation c) ature of the polar head groups d) All
Answers for MCQ: l )b 2)a 3)a 4)a 5)a 6)c 7)a 8)b 9)a IO)d

Carbohydrate Chemistry 32
Chemistry of Carbohydrates
Contents:
• Definition
• General classification with examples
• Biomedical importance of Carbohydrates
• Glycosidic bonds
• Examples, Structures, Sources, Properties and Physiological importance of,
• Monosaccharides
• Disaccharides
• Oligosaccharides
• Polysaccharides
• Detailed accounts
• Isomerism, Sugar derivatives
• Starch, Glycogen, Cellulose, Mucopolysaccharides
Use of Italics :
additional infonuation and may not be needed for the basic understanding of the chapter.
So, s tudents who want to omit these segments in first reading, can do so w ithout
of the subject. However, these italic segments can be asked in MCQ Viva. Short

Carbohydrate Chemistry
Carbohydrates are the most abundant biomolecules present in nature. They are w idely
distribu ted in plants & animals. E.g.: Glucose, Fructose, Lactose, Sucrose, Starch etc.
Definition: Carbohydrates ar e defined as polyhydroxy aldehydes or ketones or

compounds which give them on hydrolysis.
Classification of Carbohydrates:
Carbohydrates are classified into 4 classes based on the number of monomeric units.
I) Monosaccharides (Made up of a single monosaccharide unit)
II) Disaccharides (Made up of 2 monosaccharide units)
111) Oligosaccharides (Made up of 3 to 10 monosaccharide units)
IV) Polysaccharides (Made up of more than 10 monosaccharide units)
I. Monosaccharides:
Monosaccharides are the simplest of carbohydrates. These cannot be further hydrolyzed
to smaller units. The empirical formula of monosaccharide is Cn (H20)n.
Monosaccharides can be further classified in 2 ways:
a) Based on the functional (aldehyde or ketone) group present:

Monosaccharides are categorized into aldoses or ketoses based on whether aldehy de
or ketone group is present,
• Aldoses: Monosaccharides containing aldehyde group (CHO) are called aldoses.
E.g.: Glucose, Galactose, Ribose etc.
• Ketoses: Monosaccharides containing keto group (C=O) are called ketoses.
E.g.: Fructose, Ribulose, Xylulose etc.
b) Based on the number of carbon atoms present:

• Trioses (3 carbon) Eg, Glyceraldehyde, Dihydroxyacetone
• Tetroses (4 carbon) Eg, Erythrose, Erythrulose
• Pentoses (5 carbon ) Eg, Rib ose, Ribulose, Xylulose
• Hexoses (6 carbon) Eg, G lucose, Fructose, Galactose, Mannose
• Heptoses (7 carbon) Eg, Aldoheptose, Sed oheptulose
Note: Glucose contains six carbon atoms and aldehyde group, so glucose is n11 aldo-lzexose.
Similarly, Fructose contains six carbon atoms and ketone group, so fructose is a keto-l1exose.

II. Disaccharides:
Disaccharides contain 2 monosaccharide units bonded by glycosidic bonds.
• Sucrose: Made up of one molecule of glucose and one molecule of fructose.
• Maltose: Made up of two molecules of glucose.
• Lactose: Made up of one molecule of glucose and one molecule of galactose.
• Trehalose, Isomaltose, and Cellobiose are some of the other examples.
III. Oligosaccharides:
Oligosaccharides contain 3 to 10 monomeric units bonded by glycosidic bonds.
• Maltotriose (trisaccharides)
• Raffinose (trisaccharides)
• Stachyose (tetrasaccharide)
• Verbascose (pentasaccharide)
• Oligosaccharides present in cell membranes
Blood group antigens are also oligosaccharides
IV. Polysaccharides:
Polysaccharides contain more than 10 monomeric units bonded by glycosidic bonds.
Based on the type of monomeric units, polysaccharides are further classified into 2
groups, homopolysaccharides and heteropolysaccharides.
a) Homopolysaccharides (Homoglyca:ns):
Polysaccharides, which are made up of only one type of monosaccharide units are
called homopolysaccharides.
Examples are,
• Starch: Plant storage homopolysaccharide made up of only glucose molecules.
• Glycogen: Animal storage homopolysaccharide made up of only glucose.
• Cellulose: Plants structural homopolysaccharide made up of only glucose.
• Inulin: Plant homopolysaccharide made up of only fructose molecules.
b) Heteropolysaccharides (Heteroglycans):
Polysaccharides, which are made up of more than one type of monosaccharide units
are called heteropolysaccharides.
Examples are,
• Agar
• Glycosaminoglycans (mucopolysaccharides) like hyaluronic acid, heparin, heparan
sulphate, dermatan sulphate, chondroitin sulfate and keratan sulfate.

Schematic representation of Carbohydrate classification
D isacc h a r id es 0 ligosacc h ari d es

(2 mo nomer ic unit s) (3- 1 0 monomeric unit s)
E.g. Sucrose E.g. Raffinose
M a llo se Ma Ito trio sc
M onosac ch a ri des Polysacc h a rid es

(S ingle m ono m er ic unit ) ( More than IO m onome ric units)
I I
A ldose s K etoses Hom opo lys a cc ha ri d es
i
H eteropo lysaccbari d es
E.g. Glucose e.g. Fru c t ose E .g. Starch E.g. H yal uron ic acid
G a lactose Ribulose Glycogen H epa rin
Answer hint for Carbohy drates classification question (5 Marks):

a. Definition (0.5 Marks)
b. Classification with Basis (1 Marks)
c. Sub-classification with Basis and examples (3 Marks)
8 Exam tip
d. Schematic representation (0.5 Marks)

N ote: Some authors include disaccharides under oligosacharides and classify carbohydrates
into 3 classes, namely, mono, oligo (2-10 units) & polysaccharides (> 10 units).
Biomedical importance of Carbohydrates (Functions of Carbohydrates):

1. Energy source:
Carbohydrates (especially glucose) are the major source of energy in the body. Erythrocytes
and brain cells are almost completely dependant on glucose for energy.
2. Energy storage:
Glycogen in animals and starch in plants are storage form of energy.
3. Structural components:
• Carbohydrates (as glycoproteins / glycolipid s) are component of cell membranes.
• Matrix of connective tissues con tains glycosaminoglycans along with proteins.
4. Miscellaneous:
• Dietary fibers like cellulose prevent constipation and colon cancer, diverticulosis etc.
• Glucuronic acid is involved in the detoxification process.
• Glucose is the precursor for the synthesis of fats and some amino acids.
• Inulin is u sed in inulin clearance test (A kidney function test).
• Heparin is used as an anticoagulant.
• Ribose and deoxy ribose are components of nucleic acids,

I. Monosaccharides:
Monosaccharides are simplest of carbohydrates. These cannot be further hydrolyzed to
smaller units. Empirical formula of monosaccharide is Cn (l\O)n .
Monosaccharides are the building blocks of higher carbohydrates (like disaccharides,
oligosaccharides, polysaccharides).
Monosaccharides may be sub-classified depending on the functional group and the
number of carbon atoms.
No. of
Carbon Aldoses Ketoses
atoms
Trioses 3 Aldotrioses Ketotrioses
E.g.: Glyceraldehyde E.g.: Dihydroxy acetone
Tetroses 4 Aldotetroses Ketotetroses

E.g.: Erythrose E.g.: Erythrulose.
Pentoses 5 Aldopentoses Ketopentoses

E.g.: Ribose E.g.: Ribulose, Xylulose
Hexoses 6 Aldohexoses Ketohexoses

E.g.: Glucose, Galactose E.g.: Frnctose
Heptoses 7 Aldoheptoses Ketobeptose

E.g.: Glucoheptose E.g.: Sedoheptulose.
Structural aspects of monosaccharides:

The structure of monosaccharides can be represented in three ways,%
1) Straight chain (Open chain projections):
Monosaccharides generally represented in straight chain form . Straight chain representation
accounts for some of the properties of them.
2) Ring structures (Fischer's fonnula or Haworth projection):
In aqueous solution, monosaccharides (which contain five or more carbon atoms), exist mainly in
closed ringed structures.
Ring formation (cyclization) of monosaccharides is due to formation of internal hemiacetal linkage
(as in a/doses) or hemiketal linkage (as in ketoses). This hemiacetal or hemiketal linkages are formed
between the carbonyl atom (i.e. C-1 in aldoses & C-2 in ketoses) and a OH group of another carbon
atom of the same molecule.
The closed ring structure can be represented by Fischer's formu la or Haworth projection. Closed
ring formation was firs t proposed by Fischer and called Fischer's formula . Later Haworth shown
that monosaccharides exists as pyranose ring (1st oxygen & 5th carbon atoms) or fu ranose ring
(1s t oxygen & 4th carbon atoms). This is called the Haworth projection.
Glucose mostly exists as pyranose form and fructose as furanose form.
Closed ring structure explains some of the properties of monosaccharides.
3) Boat or chair form:
Pyranose ring structure might again exist either as a boat or chair form. The chair form is
thermodynamically more stable than boat form.

Structure of glucose:
1) Open chain form (Straight chain form):
Glucose is an adohexose having 6 carbon atoms and aldehyde group.
2) Ring structure (Fischer or Haworth projections):
Glucose forms the ring structures due to the formation of hemiacetal linkage. Hemiacetal
bond is formed between the OH of C-1 and OH group of either C-4 or C-5 of the
glucose. Consequetly, glucose can form furanose ring or pyranose ring structures.
Pyranose: The OH of 1st carbon atom reacts with OH of 5th carbon atom to form the 6
membered pyranose ring. Pyranose structure is predominant (99%).
Furanose: The OH of pt carbon atom reacts with OH of 4 th carbon atom to form the 5
membered furanose ring (less than 1 %).
3) Boat or chair form: Pyranose ring might again exist either as a boat or chair form.
The chair form is thermodynamically more stable than boat form.
CHO
I
H"' /OH CH,OH
2 H-C-OH
I H-f-OHI H H H
3 HO-C-H
I HO-C-H 0 I
4 H-C-OH
I 11-t-OH I OH OH
5 H-C-OH H-C__J II OH
I I
6 CH20H CH,OH
Open chain form Fischer's formula Haworth projection

(Straight chain form) (Pyranose form) (Pyranose form)
Boat form Chair form
Structure of Fructose: Fructose exists as 5 membered furanose form in solution. The C=O of
second carbon atom reacts with OH of 5 th carbon atom to form hemiketal to form furanose structure.
CH,OH
I
C=O
I
HJ.CH
(Open chain form I (Furanose form
H-C-CH of fructose)
of fructose) I
H-C-CH
I
CH,OH OH H

Isomerism in carbohydrates
Compounds having same molecular formula but differerent structure or different spatial
arrangement of atmoms are called Isomers.
Carbohydrates exhibit mainly two types of isomerism:

I. Structural isomerism 11. Stereoisomerism
I. Structural isomerism:
Structural isomers have same molecular formula, but different structures.
They are of 3 types;
a) Functional isomerism: Isomers with different functional groups.

E.g. Glucose (contains aldehyde group) and Fructose (contains ketone group)
b) Chain isomerism: Isomers with different arrangement of carbon atoms.

E.g. Maltose and Isomaltose
c) Positional isomerism: Isomers with same carbon chain but differ in position of
substituted group.
E.g. Glucose 1-phosphate and Glucose 6-phosphate
II. Stereoisomerism:
Isom ers with same molecular formula and same structural formula, but different spatial
configuration of atoms (i.e. arrangement of atoms in space) are called stereoisomers
and isomerism is called stereoisomerism.
Stereoisomerism is of 2 types; a) Geometric isomerism b) Optical isomerism
a) Geometric isomerism (cis-trans isomerism):

Definition: Stereoisomerism due to the arrangement of a toms around the double bond
is termed as geometric isomerism).
Geometric isomers have a double bond. If the isomers has the functional groups on the
same side of the double bond, it is called cis form and if the isomer has the functional
group on opposite side of the double bond, then its called the trans form.
E.g. Maleic acid (cis form) and Fumaric acid (trans form)
HC-COOH HCXJC-CH
II II
HC-COOH HC-CCXJH

b) Optical isomerism:
Definition: Stereoisomerism due the capacity of the optically active compounds to
rotate the plane of polarized light to right or left are termed as optical isomerism.
Optical isomers exhibit optical activity, i.e. when a beam of plane-polarized light is
passed through a solution of optical isomer; it rotates either to the right or to the left.
The presence of asymmetric (chiral) carbon atom in a compound confers optical activity.
(Asymmetric carbon is the carbon atom that is attached to 4 different groups.)
Optical isomers are either dextrorotatory (+) or laevorotatory(-), based on that whether
they rotate the beam of plane-polarized light to the right or to the left. If the light turns
right, then the optical isomer is called dextrorotatory, denoted as(+) or (d) and if the
light turns left, then the optical isomer is called levorotatory, denoted as(-) or (1).
E.g. D-glucose is dextrorotatory, whereas D-fructose is laevorotatory.
(Note that D and L notation has no bearing with the optical activity.)
Carbohydrates show different types of optical isomerism. They are,
i) Enantiomerism (D- L isomerism).
ii) Epimerism
iii) Anomerism
Note: Penultimate carbon atom (one before the last carbon atom) is the reference carbon
atom in monosaccharides. It is also the last asymmetric carbon atom in the configuration.
i) Enantiomerism [D and L isomerism]:

Definition: Stereoisomerism due to the difference around the penultimate carbon atom
(i.e. the reference carbon atom) is referred to as Enantiomerism (D - L isomerism).
D and L isomers are mirror images of each other.
D & L stereoisomers are referred to as enantiomers. The enantiomers are termed Dor L
form depending on the spatial arrangement of -H & -OH on the penultimate carbon
atom. If the -OH group is on the right side of this carbon atom, the sugar is O-isorner
and if the -OH is on the left side, the sugar is L-isomer.
E.g.: D- glucose and L- glucose
1 C HO CHO
I I
2 H-C-OH HO-C-H
I I
3 HO-C-H H-C-OH
I I
4 H-C-OH HO-C-H
5
6 CH2OH
$ CH20H
D-GLUCOSE L-GLUCOSE

Additional points:
1) Most of the monosaccharides occurring in mammals are D-stereoisomers. Very few
monosaccharides belong to L series. L-iduronic acid (a Glycosaminoglycan), L-xylulose (a11
intermediate of uronic acid pathway), and L-fucose (found in glycoproteins) are some examples
of naturally occurring L-sugar.
2) D and L notation is not the indication of the optical activity. Optical activities are indicated
by d or (+) if dextrorotatory and l or (-) if levorotatory.
For example, D-glucose is dextrorotatory (+52.5°), whereas O-fructose is levorotatory (-92°).
(So, D-glucose is also called Dextrose; similarly O-fructose is also called laevulose).
Dextrose is given intravenously in bedside medicine as dextrose drip.
3) Designation: An optical isomer may be designated as D (+), D (-), L (+), or L (-) based on
its position of OH & Fi around the penultimate carbon atom & its optical activity.
4) Racemic mixture: If d and I isomers are present in equal concentrations, the mixture is known
as racemic mixture or di mixture. di mixture does not exhibit optical activity, because the dextro
& levorotatory activities cancel out each other.
ii) Epimerism and Diastereoisomerism :
Epimerism: Stereoisomerism due to difference in the configuration around a single

asymmetric carbon atom other than the penultimate carbon atom is referred to as
Epimerism. They are not related to each other as object mirror images. Examples are,
a) Glucose and Mannose are C-2 epimers, i.e. they differ in the configuration at 2nd carbon
atom (orientation of Hand OH groups differ at the C-2).
b) Glucose and Galactose are C-4 epimers i.e. they differ in the configuration at 4th carbon
atom (orientation of Hand OH groups differ at the C-4).
1 CHO CHO CIIO
3
I
H-C-OH
I
11O-C-11
I
H-C-OH
I
HO-C-H
$ HO-C-H
I I
4
5
H-C-OH
I
H-C-O11
$ H-C-OH
ll-C-O11
I
H-C-OH
I I I
6 CHiOH CH2OH CHeOH
GLUCOSE GALACTOSE MAN 'OSE
Diastereoisomerism: Stcrcoisornerism due to difference in the configuration around

one or more asymmetric carbon atom other than the penultimate carbon atom is referred
to as diastereoisomerism. They are also not related to each other as object mirror images.
Note: All epimers are diastereoisorners, but all diastereoisomers are not epimers.
• Galactose & mannose are diastereoisomers, but are not epimers, because they differ
around 2 asymmetric carbon atoms (Epimers differ around 1 asymmetric carbon atom).

iii) Anomerism:
Definition: Stereoisomerism due to difference in the configuration around anomeric
carbon atom is referred to as Anomerism .
Anomerism is caused due to the ring formation (cyclization) of monosaccharides in

solutions. As a result of ring formation, a new asymmetric carbon atom (at carbon 1
of aldoses or carbon 2 of ketoses) is produced. This carbon atom is called Anomeric
carbon atom.
The orientation of OH and H around this anomeric carbon atom results in 2 isomers,
a and P forms, which are called anomers. In a-forms His above the plan e of the ring
and OH below the plane of the ring. In P-forms the positions are reversed.
II II
OH
OH OH
H OH H OH
a form of glucose (36%) 13 form of glucose (63%)

In equilibrium, 36% of glucose exists in a fonn and 63% in {3 form and only less than 1% in open
dwinfonn.
Isomerism in Carbohydrates
j
Structural Isomerism
a) Functional isomerism
l
Stereoisomerism
b) Chain isomerism
c) Positional isomerism
Optical Isomerism Geometric isomerism
Enantiomerism Epimerism Anomerism
Note that this is just a diagra111111atic representation ofdifferent isomerism for t/ie sake ofconvenience
of understanding of isomerism and it is not the classification of isomerism in 111011osacclwrides.
For queries and suggestions, contact the author at prasad_text@yahoo.com or 998644~575

Fonnation of anomeric carbon atom in monosaccharides:

• In aqueous solution, monosaccharides (which contain five or more carbon atoms), exist mainly in
closed ringed structures.
• This ring formation (ci;clization) ofmonosaccharides is due to formation ofinternal hemiacetal linkage
(as in aldoses) or hemiketal linkage (as in ketoses). This hemiacetal or hemiketal linkages are formed
between the carbonyl atom (i.e. C-1 in aldoses and C-2 in ketoses) and a hydroxyl group of another
carbon atom of the same molecule.
• As a result of ring formation, a new asymmetric carbon atom (at C-1 of aldoses or C-2 of
ketoses) is formed. This carbon atom is called Anomeric carbon atom.
Glucose in solution:
In solution, Glucose exists predominantly (63 %) in /3-D-glucopyranose form and 36% in
a-D-glucopyranose form and only less than 1 % in glucofuranose & open chain form.
Mutarotation:
Definition: The spontaneous change of specific optical rotation of optical isomers

(optically active compounds), with time is called mutarotation.
Explanation:
Mutarotation is due to the existence of reducing sugars in a and p anomeric form.
For example, D glucose exists in 2 anomeric forms, a-D- glucose and P-D- glucose.
Freshly prepared a-D-glucose solution has a specific rotation of+112.5°, which gradually
decreases with time and attains an equilibrium constant value of +52.5°. Similarly,
freshly prepared P-D-glucose solution has a specific rotation of +19°, which gradually
increases with time and attains an equilibrium constant value of +52.5°.
Reason: Mutarotation is due to the conversion of some a-D-glucose molecule to P-

D-glucose and vice versa to attain equilibrium. The a and pforms are inter convertible
through an open chain form. When dissolved in water both the forms convert
themselves to an equilibrium mixture of both the isomers a and p.
Equilibrium mixture has 36 % of a form, 63 % of p form of glucose and less than 1 %
open chain form.
a-D glucopyranose - - • Equi1ibrium mixture of J3-D glucopyranose
a and J3 glucopyranose
(+52.50) (+19°)
• Since the solution contains 36% a-form and 63% {3-form, the final value of optical rotation is
+52.5° and not the average of individual specific rotations.
• Only reducing sugars slww mutarotation, because to exhibit mutarotation, the sugar should have a
free anomeric carbon atom. Non-reducing like sucrose does not have a free anorneric carbon and
therefore, does not exhibit mutarotation.

Sugar derivatives of monosaccharides:

Various derivatives of monosaccharides are formed in the body. Some of them have
bio logical im portance. Uronic acids, Sugar alcohols, Sugar phosphates, Amino
sugars, Deoxy sugars are some of the important derivatives of monosaccharides.
1) Uronic Acids (By oxidation of monosaccharides)

Uronic acids are formed when only the terminal alcoholic group of rnonosaccharides
is oxidized to carboxylic group (and carbonyl group is protected).
This reaction is d ifficult chemically, but possible enzyma tically invivo.
Examples: Glucuronic acid, galacturonic acid and L-iduronic acid s are the sugar
acids formed in the body.
CHO CHO
H-C-OH 11-C-OH
I
HO-C-H HO-C-H
H-C-OH 11-C-OH
I I
11-C-OH 11-C-OH
I
CH2OH COOH
D-glucose Gluc uro nic acid

Significance:
• Gluc uron ic ac id , ga lac tu ronic acid, L-iduronic acid s ar e comp o nent of
glycosaminoglycans.
• Glucuronic acid is used in the conjugation and detoxification of toxic substances
like biliru bin.
Not e: In the laboratory, different sugar acids (Aldonic acid, Sacchnric acid, Uronic acid) cn11 be
formed when monosaccliarides are oxidized under different conditions.
i) Aldonic acids: Under mild oxidation conditions (bromine water) aldoses give aldo11ic acids,
where only aldehyde groups are oxidized to carboxylic group.
E.g. glucose is oxidized to gluconic acid; Ga/actose is oxidized to galactonic acid.
ii) A ldaric acids (or Saccl,aric acids): Under strong oxidation conditions (Concentrated HNO),
a/doses form a dicarboxylic acid called aldaric acid (or sacclzaric acid), where both aldehyde & ter111i1Lal
nlcohol groups nre oxidized to carboxylic groups.
E.g. Glucose produces glucosaccharic acid; Galactose gives galctosaccharic acid.
iii) Uronic acids: Under controlled oxidation conditions (platinum- carbon catalyst), a/doses
give uronic acids, where the aldehyde group is protected and 011 /y the terminal primary alcohol
group is oxidized to carboxylic group.
E.~. Glucose produces glucuronic acid; Galactose gives ~alctouronic acid.

2) Sugar alcohols (polyols) or Reduction of monos accharides

Sugar alcohols are formed when the carbonyl group is reduced to hydroxyl group.
These are poly hydroxy alcohols (or polyols).
Examples: • Glucose forms Sorbitol
CHO C H, O H
I I
11 -C-OH H-C-OH
I I
HO-C-H 110-C-H
I
11-C-OH H-C-OH
I I
H-C-OH H-C-OH
I I
CH 1 0H CH ,OH
0-glucose Sorbitol
Similarly, fructose forms sorbitol & mannitol, glyceraldehyde gives glycerol, mannose
forms mannitol, galactose forms dulcitol, ribose forms ribitol, xylulose forms xylitol
Significance:
• Glycerol is a component of triacylglycerols and phospholipids.
• Ribitol is a component of vitamin riboflavin and coenzymes like FMN and FAD.
Clinica l Significance:
• Sorbitol (produced from glucose) accumulation in diabetes causes cataract.
• Mannitol is used as a diuretic. It is used parentarely to reduce intracranial pressure.
• Xylitol is a plant derived polyol. lt is used as a sweetening agent.
• Sorbitol, mannitol, dulcitol are used to identify bacterial colony as these sugar alcohols are used
as energy sources by specific bacterial colonies.
3) Deoxy Sugars
Deoxy sugars are formed when the oxygen of the hydroxy group of sugar is removed.
(Due to the deoxygenation, CH2OH becomes C~ or CHOH become CH2).
Examples: Deoxyribose, L-fucose (L-deoxygalactose), L-rharnnose (L-deoxymannose).
.p
, ,
CH , OH OH
OH H
Significance: Deoxy dbo,.
• Deoxyribose is a structural component of nucleic acids.

• L-fucose is an important constituent of blood group antigens and certain glycoproteins.
• L-rharnnose is presen t in many glycosides.

4) Sugar Phosphates
Alcoholic (OH) groups of monosaccharide are esterified by acids like phosphoric
acids to form sugar phosphates.
Examples: Glucose-1-phosphate, glucose-6-phosphate, fructose 1,6-bisphosphate etc.
CH,00
H OH
Glucose 6-phosphate
Significance:Sugar phosphates are important intermediates of carbohydrate metabolism
5) Amino sugars
Amino sugars are formed when hydroxyl groups of monosaccharides are substituted
by amino group. Generally the amino group is present in the second carbon.
Examples: Glucosamine, galactosamine, mannosamine etc
CH,OH H
O
Oli
OH
H
II
NH,
Glucosaminc
011
Significance:
• Glucosamine and galactosamine are important constituents of glycosaminoglycans.
• Glucosamine is seen in blood group substance.
• Mannosamine is present in glycoproteins.
Acetyl derivatives of amino sugars: Sometimes amino sugars are acetylated.

For e.g., N- acetyl-glucosamine and N- acetyl-galactosamine
Significance: Acetylated amino sugars (N-acetyl-glucosamine & N-acetylgalactosamine)
are present in glycosaminoglycan s, proteins & cell membrane antigens.
Neuraminic acid, NANA (N-Acett1l-neurami11ic Acid), Sialic acids:
These are derivatives of amino sugars.
• Condensation of pyruvic acid and marmosamine gives neuraminic acid.
• Acetylation of neuraminic acid gives NANA (N-AcehJl-neuraminic Acid).
• NANA and its derivatives are called Sialic acid.
Significance: Sialic acids, Neuraminic acids and NANA are found in glycoprotei11s & glycolipids.

Glycosidic bond or glycosidic linkage:

Definition:
• Glycosidic bond is defined as the bond formed between the OH group of an anomeric
carbon atom of a carbohydrate and OH group of another carbohydrate or a non-
carbohydrate (referred to as aglycone), with the loss of water molecule.
• Glycosidic linkage is represented by C-O-C.
Significance:
• Glycosidic bonds are responsible for the formation of higher carboh ydrates
(disaccharides, oligosaccharides and polysaccharides).
(Glycosides:]
Definition:
Glycosides are compounds having both carbohydra te & a non-carbohydrate residue.
The non-carbohydrate residue is referred to as aglycone.
Glycosides are formed by the condensation of OH group of anomeric carbon atom of

a carbohydrate with OH group of a non-carbohydrate by glycosidic bond.
Glycosides are non reducing as their anomeric carbon atoms are not free.
Most of these glycosides have pharmacological importance.
Some physiologically important glycosides are,
• Phlorhizin:Glucose combined with Phloretin (aglycone). Phlorhizin is an inhibitor

of transport of glucose across the mucosal cells of small intestine and renal tubular
epithelium . It causes renal glycosuria and renal damage. Its obtained from Rose bark.
• Digitonin (Cardiac stimulant): Galactose combined with Digitogenin (aglycone).

Digitoni.n obtained from leaves of foxglove.
• Ouabain is a sodium pump inhibitor.
• Streptomycin is an antibiotic.
• Plant indican (Stain): Glucose combined with indoxyl.
• Glucovanillin (Vanillin D- glucoside): present in vanilla.

Disaccharides:
Definition:
Disaccharides are made up of two monosaccharides joined by glycosidic bond.
Examples: Maltose, Lactose, Sucrose, trehalose etc.
1) Maltose (Malt sugar):

• Constituents: Maltose consists of two glucose molecules joined by a- 1, 4 glycosidic
bonds. Anomeric carbon of second glucose is free. Hence, maltose is a reducing sugar.
Maltose forms sunflower shaped maltasazone crystals in osazone test.
• Sources: Germinating cereals and malt. Maltose is an intermediate product of starch
digestion by a -amylase enzyme in the intestine.
Intestinal enzyme maltase hydrolyzes maltose to two molecules of glucose.
2) Lactose (Milk sugar):

• Constituents: Lactose consists of a molecule of galactose and a molecule of glucose
linked by ~-1,4 glycosidic bonds. An omeric carbon of glucose is free. So, lactose is a
reducing sugar. Lactose forms hedge hog shaped lactasazone crystals in osazone test.
• Sources: Milk and milk products. Lactose is produced in lactating mammary glands.
Intestinal enzyme lactase hydrolyzes lactose to glucose and galactose.
3) Sucrose (Table Sugar, Common sugar, Cane Sugar, Invert Sugar):

• Constituents: Sucrose consists of a molecule of glucose and a molecule of fructose
joined by a-~ (1 • 2) glycosidic bonds. Anomeric carbon of glucose and fructose are
involved in bond formation and are not free. So, sucrose is a non-reducing sugar and
does not form osazones.
• Sources: It is present in sugarcane, honey, sorghum, some fruits and vegetables.
Intestinal enzyme sucrase hydrolyzes sucrose to glucose and fructose.
Clinical significance: Sucrose is implicated in dental caries.Dental caries results from
bacteria, which derive their energy from sucrose. People who consume sugars and
sweets or chocolates (made up of sucrose) are more susceptible to dental caries.
Maltose Lactose Sucrose

Common n ame Malt sugar Milk sugar Common sugar, Table
sugar, Cane sugar,
Invert sug.r
Constituents Glucose & Glucose Galactose & Glucose Glucose & Fructose
S ources Malt, C,erm,natn~ Mi lk and milk Sugar c.:ine, Fruits,

cereals, baby foods products Honey
(Summary) Hydrolysis Maltase breaks Lactase breaks lactose Sucrase breaks Sucrose
Maltose into 2 0110 a molecules of into a molecules of
molecules of galactose and a glucose and a m>lecule
glucose molecule of glucose of fructose
Reducrng property Red ucmg sugar Reducmg sugar Non- reducing sugar
Osazone formation Sunflower shaped I ledgehogs haped Does not form Osazone
maltasazone lactasazone crystals

a- 1, 4 glycosidic bond
OH
H Otl OH
Glucose Glucose
~- 1, 4 glycosidic bond
H OH
Galactose Glucose
a- ~ (1 • 2) glycosidic bond
H OH OH H
Glucose Fructose
Some minor disaccharides:
1) Trehalose:
Trehalose is present in yeast, fungi. It is the main sugar in insect hemolymph.
Trehalose contains 2 a-glucose joined by a (1 • 1) glycosidic bond. It is a non-reducing
sugar as anomeric carbons of both the glucose molecules are involved in bond formation.
2) Isomaltose:
Isomaltose is produced during the digestion of starch.
It is made up of 2 a - glucose m olecules, joined by ex (1• 6) glycosidic bond.
Answer hint for Disaccharides (5 Marks):

a. Definition (1 Marks)
b. Examples (1 Marks)
c. Explan ation of individual disaccharides under
common name, composition, sources, property (3 Marks)

Additional Points:
1. Sugars:
Mono and Disaccharides are referred to as 'sugars' as they are sweet in taste.
2. Invert sugar, Invertase, Inversion:

• The change in specific rotation of sucrose when it is hydrolyzed is called Inversion.
• Sucrase enzyme is called invertase.
• Hydrolyzed mixture of sucrose (glucose and fructose) is called Invert sugar.
(Note that some authors consider sucrose it self as the invert sugar).
Explanation: Sucrose is dextrorotatory (+66.5°) in nature. When sucrase enzyme acts on
sucrose, it is converted into an equimolar mixtures of glucose (+52.5°) and fructose (-92°). So,
the resultant mixture of glucose and fructose exhibit a net laevorotation (-29°).
The phenomenon by which dextrorotatory sucrose is converted to laevorotatory mixture of
glucose and fructose is known as 'Inversion'. Enzyme sucrase is also known as 'invertase'
and hydrolyzed mixture of sucrose (glucose & fructose) is called Invert sugar.
3. Reducing and non-reducing sugars:

Based on the reducing property, sugars are termed as reducing s ugars or non-reducing sugars.
Free aldehyde or keto group of anomeric carbon are responsible for reducing properties of
sugars.
All the monosaccharides are reducing sugars. In disaccharides lactose and maltose are
reducing sugars, whereas sucrose and trehalose are non-reducing sugars.
Explanation:
• All monosaccharides are reducing sugars, because they have free anomeric carbon atoms.
• Lactose and maltose are reducing sugars": Because they have free anomeric carbon atom.
In lactose, anomeric ca rbon of second glucose molecule is free and in maltose, anomeric carbon
of second glucose molecule is free.
• Sucrose & trehalose are non-reducing sugars: Because anomeric carbon atoms of sucrose
& trehalose are not free as they are involved in bond formation. In sucrose, aldehyde group of
C-1 of glucose is bonded with keto group of C-2 of fructose; & in trehalose, aldehyde group of
C-1 of glucose is bonded with aldehyde group of C-1 of second glucose).
Osazone test:
Reducing sugars when heated with phenylhydrazine forms characteristic osazone crystals.
This test requires first two carbon atoms of reducing sugars.
• Glucose fructose and mannose forms needle shaped osazone crystals (Glucose, fructose &
mannose give same osazone crystals, as these 3 monosaccharides differ only in first two carbon
atoms & this difference is masked by binding with phenylhydrazine in osazone test).
• Maltose forms sunflower shaped osazone crystals
• Lactose forms hedge hog (Puff) shaped osazone crystals
Note that sucrose doesn't answer osazone test, as it is a non-reducing sugar.
Osazones may be used to identification sugars in biological fluids like urine.

Benedict's Test (Test for reducing sugars):

This test is used to detect the presence of reducing sugar (Glucose, fructose, lactose,
maltose etc). Note that non-reducing sugars like sucrose does not answer this test.
Procedure:
Add 8 drops of reducing sugar solution to 5 ml of Benedict's reagent and boil for
exactly two minutes.
Observation:
Brick red precipitate is obtained.
Significance of Benedict's test:
Benedict's test is commonly used to detect the presence of glu cose (reducing s ugars) in
urine. The color obtained is suggestive of approximate amount of glucose in urine.
Color Blue Green Yellow Orange Brick Red
Amount of Ntl 0.5 % 1% 1. 5 % 2 % or more

Glucose (gm "ii>) + (trace) (+ +) ( + + +) (+ + + +)
Note: Benedict's test is answered by all other reducing sugars (like fructose, galactose, lactose,
rnaltose) and reducing substances (like vitamin C, homogentisic acid etc) in urine, giving a
fal se positive test for diabetes. So, benedict's test should be interpreted with care.
0 ligosaccharides
Definition:
Oligosaccharides are made up of 3-10 monomeric units bonded by glycosidic bonds.
Examples are,
i) Maltotriose: It is a trisaccharide made up of 3 molecules of glucose molecules.
ii) Raffinose: It is trisaccharide made up of fructose, galactose & glucose molecules.
iii) Stachyose: It is a tetrasaccharide made up of 2 molecules of galactose and one
molecu le each of glucose and fructose.
iv) Verbascose: It is a pentasaccharide made up of 3 molecules of galactose and one
molecule each of glucose and fructose.
v) Cell membrane oligosaccharides: Cell membranes have oligosaccharides, w hich are
usually found as glycoproteins or glycolipids.
vi) Blood group antigens: These are also oligosaccharides.

Polysaccharides
Definition: Polysaccharides are made up of more than 10 monomeric units, w hich are
bonded by glycosidic linkages.
Polysaccharides are two types, homopolysaccharides and heterpolysaccharides.
I) Homopolysaccharides:
Definition: These are polysaccharides, which are made up of only one type of monomeric
units.
Examples: Starch, Glycogen, Cellulose, Inulin etc.
II) Heteropolysaccharides:
These are polysaccharides, which are made up of more than 1 type of monomeric units.
Examples: Agar, Mucopolysaccharides like hyaluronic acid, chondroitin sulfate, keratan
sulfate, dermatan sulfate, heparin & heparan sulfate.
I. Homopolysaccharides (In detail) :

Polysaccharides which are made up of only one type of monomeric units are called
homopolysaccharide.
Some important homopolysaccharides are explained below;
Starch
Starch is a plant storage homopolysaccharide made up of only glucose molecules.
a) Source:
Cereals (wheat and rice) and root tubers (like potatoes) are rich sources of starch.
b) Importance:
Starch is the main dietary source of glucose for human beings.
c) Structure:
Starch has two components: i) Amylose (13-20%) ii) Amylopectin (80-87%)
i) Amylose:
Amylase is a straigh t chain, non-branched helical structure. In Amylase, successive
glucose molecules are bonded by cx-1, 4 glycosidic bon ds. Amylase has around 60 to
600 glucose molecules in the chain.
ii) Amylopectin:
Amylopectin is a branched chain structure. Amylopectin has both a-1, 4 and a-1, 6
glycosidic bond s, a-1, 4 glycosidic linkages in straight chains and a -1, 6 linkages at
branch points. In between two branch points, there are about 24 to 30 glucose residues.

Glycogen
Glycogen is an animal storage homopolysaccharide consisting of glucose molecules.
Importance :
Glycogen is stored in liver and muscle. Liver glycogen is utilized for maintaining
blood glucose homeostasis. Muscle glycogen is used by muscles during prolonged
contraction.
Structure:
• Glycogen is a branched structure. Glycogen also has a-1, 4 glycosidic linkages in
straight chain and a-1, 6 linkages at branch points.
• Glycogen is structurally similar to arnylopectin of starch, but comp aratively it is
highly branched (has more branches) than amylopectin.
• In between branch points, there are about 11 to 14 glucose residues (as compared to
amylopectin, which have 24 to 30 glucose molecules between the branch points).
So, glycogen is more extensively branched than amylopectin of starch.
Structure of Starch and Glycogen
a ( 1• 4) glycosidic bond
Amylase •
a ( I • 4) glycosidic bond
Amylopectin •
24 -30 glucose between the branch points
Glycogen •
---v----
11 -14 glucose molecules between the branch points
Answer hint for the question - Compare and contrast the structures
of starch and glycogen OR write the difference between starch
and glycogen (5 Marks):
Firs t draw a line in the centre of the page to divide the page vertically
and write details of starch in the left side and glycogen in the right side.

Cellulose
Cellulose is the main cell wall polysaccharide of plants.
Structure: Cellulose is homopolysaccharide made up of P-D-glucose molecules, linked

by P-1, 4 glycosidic bonds. It is a linear non branched structure.
Cellulose cannot be digested by humans due to the absence of enzyme cellulase that
digests P bonds of cellulose.
Source: Cellulose occurs only in plants, mainly in cell walls.

Importance:
Cellulose is the most common dietary fiber present in the food. Dietary fibers like
cellulose are not digestible in the GIT.
Cellulose and other dietary fibers are have roughage action. Dietary fibers stimulate
perista lsis, help in formation of soft stools which is easier to eliminate and hence
prevents constipation. They also prevent colon cancer and diverticulosis.
Refer nutrition chapter for the detailed explanation of dietary fibers.
Inulin
Inulin is a homopolysaccharide made up of fructose molecules.
Source: Tubers and roots of dahlias, artichokes and dandelions
Clinical significance: Inulin is used in inulin clearance test to determine glomerular
filtration rate. (In assessing the function of kidney).
Chitin
Chitin is present in exoskeletons of crustaceans & insects. Chitin is a homopolysaccharide
made up of N-acetyl glucosamine linked by P -1, 4 glycosidic bonds.
Dextran
Dextrans are hjgh molecular weight carbohydrates (one to four millions) produced
by bacterias and yeast. They are highly branched homopolymers of glucose having
a-1, 6, a-1, 4 and a -1, 3 glycosidic bonds. The linear straight chain has a-1, 6 glycosidic
linkages and branching is brought about by a-1, 3 or a-1, 4 linkages.
Clinical significance:
Dextrans will not easily go out of vascular compartments because they have high
molecular weight and highly network like structure. So, they are used intravenously
as plasma volume expanders for treatment of hypovolumic shock.

Dextrin (Partial hydrolysis products of starch):

Dextrins are intermediates in the hydrolysis of starch (either by amylase enzyme or
d ilute acids). Various d extrins formed during starch hydrolysis are _amylodextrin,
erythrodextrin and achrodextrin. These are identified by iodine test.
Starch is successively hydrolyzed through different dextrins to maltose & finally glucose.
Starch (Blue color with iodine solution)
i
Amylodextrin (Violet color with iodine solution)
i
Erythrodextrin (Red color with iodine solution)
i
Achrodextrin (No color with iodine solution) Maltose
Significance: Dextrins are often included in baby foods as they are better digestible
than starch . Dextrins are also used as paste (m ucilages).
Difference between Dextrose, Dextrin and Dextran :

• Dextrose is the other name of D-glucose.
• Dextrin is the partial hydrolyzed product of starch.
• Dextran is highly branched homopolymer ofglucose produced in bacteria.
Heteropolysaccharides [Heteroglye ans]

Definition :
They are mad e up of more than one type of m onomeric units.
Examples:
Agar and Glycosaminoglycans (Mucopolysaccharides) like hyaluronic acid, heparin,
heparan sulphate, dermatan sulphate, chondroitin sulfate and keratan sulfa te.
1) Agar:
• Source: Agar is obtained from seaweeds.
• Structure: Agar is a heteropolymer, made up of sulafetd galactose & glucose molecules.
• Significance: Agar is used as a culture medium for bacteria. Agar is also used as a
laxative in the treatment of constipation. (Agar is not digested hence add bulk to the
intestinal contents, thus helps in bowel movement). Agar is also used as a sup porting
matrix in gel electrophoresis. Agar is used in th e preparation of Agarose.
Agarose: Agarose is obtained from agar. Agarose is made up of galactose and 3,6-
anhydrogalactose. It is used as a su pporting matrix in agarose gel electrophoresis.

2) Mucopolysaccharides (Glycosaminoglycans or GAG)
Definition :
Glycosaminoglycans (GAG) or mucopolysaccharides are heteropolysaccharides
containing amino sugars and uronic acids. They may be attched to a protein molecule
to form a Proteoglycan. (Proteoglycan is explained in detail in connective tissue chapter).
Examples:
Hyaluronic acid, Chondroitin sulfate, Keratan sulfate (I and II), Dermatan sulfate,
Heparin and Heparan sulfate. Chondroitin sulfate is the most abundant GAG.
Structure:
Glycosaminoglycans are heteropolysaccharides built up of repeating disaccharide unit,
generally composed of an amino sugar and an uronic acid. With the exception of
hyaluronic acid, all the GAG's contain sulfate group. They may contain acetyl groups.
Properties:
GAGs are polyanionic due to the presence of large number of carboxyl and sulfate
groups. Becuase of the presence of anions, they have the property to holding high
amount of cations and water and form viscous solutions.
Significance:
Glycosaminoglycans (GAGs) perform various physiological functions.
a) Shock absorber (Lubricant effect): Most of the GAGs are structural components of
connective tissues (ECM) and found in the ground matrix of connective tissues. These
GAG's have the property to hold water and form viscous solution. So, they act as good
lubricants and shock absorbers to give cushioning effect against mechanical shock.
b) Anti-coagulant: Heparin produced by mast cells of lungs is a nah1ral anticoagulant.
c) Prevent bacterial attack: Hyaluronic acid present in synovial fluid of joints, vitreous
humor of eye etc prevents bacterial attack.
d) Elasticity: GAGs provide elasticity to the connective tissues they are present. When
the viscous solution of GAG is compressed water is squeezed out and GAG occupies a
smaller place. When compression is removed, the GAG again becomes hydrated to
form viscous solutions and occupy the original volume.
Answer hint for Glycosaminoglycans (5 Marks):

b. Examples (1 Marks)
c. Structure (1 Mark)
d. Functions (2 Marks)

Composition and Sources of Ind ivid ual glycosaminoglycans
G lyco sa m i noglyca n C ompo s iti o n Occu rren ce
0-gl uc u ronic aci d and Vitreous humor, synovial

H ya l uronic aci d N -ace tyl glu cosam ine fluid, connective tiss u e, skin,
(Only GAG that is not sulfated) umbilical cord, cartilage e tc.
0-glu cu ro n ic acid and Cartilage, bon e, cornea , skin ,
Cho n d ro i tin s ul fa te N-ace tyl g alacto samine arterial wall, heart va lves,
(Bo th are s ul fa ted) tendons etc.
N-acetyl ga lactosa m ine and Ma inly in s kin ,
De rm a ta n sul fate l -iduronic acid Also in heart va lves,
(Both a re sulfated) te ndons, arterial wall e tc.
0-galactose and Cornea, car tila ge,
K e ratan s ulfa te N -acety l g lu cosam in e in te rv e rtebral di sc,
(S ul fa te d) connective tiss u es e tc.
(No uronic acids)
0 -gl u cosam in e and Prod u ced by ma s t cells.
H e p a rin L-id u ro n i c a ci d I P rese nt in lung, liver, spleen ,
0-glucuro n ic acid sk in and intes tinal mu cosa.
(Both are sulfated)
0-g l ucosami n e and Lung, liver, kidney ,
H epa ra n su lfa te 0-glucu ro n ic acid / A rte rial wall.
L-id u ro n ic aci d It is present in most ce ll
(Both are s u lfated) surfaces
Some glucosamine is acet vlated
1) Hyaluronic acid:
Occurence: Hyaluronic acid is the most important GAG in the ECM, found in synovial fluid
of joint, vitreous humor of the eye, umbilical cord, cartilage, cell membrane, skin etc.
Compositi on: It consists of repeating disaccharide unit of glucuronic acid & -acetyl
glucosamine. It's the 011ly GAG that is not sulfated and covalently linked to proteins.
Sig nificance: Hyaluronic acid along with other GAG in ground substances hold large amount
of water & space, thus cushioning & lubricating other substances. Hyaluronic acid forms a
firm gel like structure and prevents bacterial a ttack to a great extent. (Many bacteria contain
an enzyme hyaluronidase, which cleaves hyaluronic acid and permits the infection to spread. For
this reason, hyaluronidase is referred to as spreading factor). Hyaluronic acid forms a protective
gel around the ovum. Hyaluronidase present in semen facilitates penetration of ovum by sperm by
hydrolyzing the hyaluronic acid layer of ovum during fertilization.
2) Chondroitin sulfate: They contribute to its compressibility and weight bearing capacity.
3) Heparin: Heparin is an anticoagulant.
4) D ermatan sulfate: Have a structural role in sclera, responsible for the shape of eye ball.
5) Keratn sulfate: Plays a critical role in corneal transparency.
Answer hint for Polysaccharides (5 Marks):

b. Subclassification with examples (2 Marks)
c. Details of Starch, glycogen, GAG's etc (2 Marks)
Exam tip
Question bank on Carbohydrate Chemistry:
Long essays (10 Marks)

1) How are Carbohydrates classified? Give examples for each class?
Short essays (5 Marks)

1) Explain the isomerism in monosaccharides
2) Compare and contrast the structures of starch and glycogen
3) Chemjstry of polysaccharides ·
4) Give a brief account of mucopolysaccharides (or Glycosaminoglycans)
Short answers (2 - 3 Marks):

1) Glycosidic bond
2) Write composition of a) Sucrose b) Maltose c) Lactose indicating their linkages,
3) Give reasons why lactose & maltose are reducing sugars, but sucrose is a no n- reducing

1) Whlch of the following has no free aldehydes or ketone group (AIIMS)
a) Fructose b) Maltose c) Sucrose d) Galactose
2) Which of the following is non reducing sugar (AI)
a) Sucrose b) Maltose c) Glucose d) Lactose
3) Chitin is a (AIIMS)
a) Polypeptide b) Polysaccharide c) Fatty ester d) None
4) Sorbitol is (AIIMS)
a) A reducing sugar b) A sugar ester c) Sugar alcohol d) An O-glycoside
5) Regarding proteoglycans, false is (AI)
a) Chondroitin sulfate is a Proteoglycan b) They hold less amount of water
c) They are made up of sugar and amino acids d) They carry charge
6) The glycosidic bond present in amylose molecule is
a) a-1-4 b) a 1-6 c) 1-6 d) 1-4
7) The predominant form of glucose in solution is

a) a-D-glucofuranose b) ~-D-glucopyranose c) a-D-glucopyranose d) ~-D glucofuranose
8) A pair of sugars differing from each other at the anomeric carbon
a) Anomers b) Epimers c) Racemers d) Stereoisomers
9) Glucose contains how many carbon atoms
a) Six b) twelve c) Twenty seven d) Thirty
10) -acetyl neuraminic acid is an example of
a) Sialic acid b) Mucic acid c) Glucuronic acid d) Hippuric acid
Answers for MCQ: 1) c 2) a 3) b 4) c 5) b 6) a 7) b 8) a 9) a 10) a

Lipid Chemistry 58
Chemistry of Lipids
Contents:
• Definition
• General classification with examples
• Fatty acids including Essential Fatty acid and Eicosanoids

• Definition, Classification, Examples, Structure, omenclature, Importance.
• Triacylglycerols
• Definition, Structure, Properties, Importance.
• Phospholipids
• Definition, Classification, Examples, Structure, Importance.
• Glycolipids
• Definition, Classification, Examples, Structure, Importance.
• Cholesterol
• Structure, Functions.
• Detailed accounts:
• Hydrophobic and Hydrophilic lipids, Micelles, Bimolecular leaflet

Lipid Chemistry 59
Lipid Chemistry
Definition:
Lipids are heterogene ous group of organic compound s, which are relatively insoluble
in water (polar solvents) and soluble in organic solvents (non polar solvents) like
ether, benzene, alcohol, chloroform etc.
Classification of lipids:
Lipids are classified into 3 major classes, based on their composition.
1) Simple lipids
2) Compound lipids
3) Derived lipids and Precursor lipids
I) Simple lipids:
Simple lipids are esters of fatty acids with alcohols (i.e. they contain onl y fatty acids
and alcohol). They do not contain any conjugate groups.
Simple lipids are of two types, based on the alcohol present in them.
a) Fats / Oils
b) Waxes
a) Fats/Oils (Acylglyc erols):

They are the esters of fatty acids w ith glycerol (i.e. Acylglycerols). Monoacylglycerols,
diacylglycerols, and triacylglycerols are the three acylglycerols.
Naturally occurring fats and oils are mixtures of acylglycerols, triacylglyce rol being
the most abundant componen t.
Fats are solid at room temperatur e and oils are liquid at room temperatur e.
E.g.: Vegetable oils, Fish liver oils, Butter, Ghee etc.
b) Waxes:
Wax s are esters of fatty acids with higher (monohydr ic long chain) alcohols.
E.g.: Bees wax, Carnauba wax, Wool fat (lanolin), Spermaceti wax etc.
For queries and suggestions , contact the author at prasad_text@yahoo.com or 9986449575

Lipid Chemistry 60
II) Complex (or compound) lipids:

These lipids contain conjugate compound s in addition to fatty acids & alcohols. The
conjugate group can be a phosphate group, carbohydra tes, proteins etc.
Compound lipids include phospholip ids, glycolipid s, sulpholipi ds, aminolipid s etc.
a) Phosphol ipids :
These are compound s lipids containing phosphoric acid residues (phosphate groups)
in addition to fatty acids and alcohol. They also contain a nitrogenou s base.
Phospholip ids are of 2 types depending upon the nature of alcohols present in them.
i) Glyceroph ospholipid s: These contain glycerol as alcohol.
E.g.: Lecithin, Cephalin, Plasmalog en, Lung surfacta nt etc
ii) Sphingoph ospholipid s: These contain sphingosin e as alcohol.
E.g.: Sphingomyelin
b) Glycolipi ds:
These are compound s lipids containing carbohydr ate residues in addition to fatty
acids & alcohol.
Glycolipids are two types based on the type of carbohydra te residues,
i) Cerebrosides: These contain glucose or galactose as carbohydra te residues.
E.g.: Kerasin, cerebron, nervon, & oxynervon
ii) Gangliosid es: These contain complex oligosaccharides as carbohydra te residues.
E.g.: GM1, GM2, GM3, GM4 etc
c) Other compoun d lipids :

i) Sulpholipi ds : These contain sulphate groups as conjugate group
E.g.: Globosides
ii) Aminolipi ds : These contain amino acids as conjugate group
• Liporotein s may also placed under this categonJ,

Lipoprotei ns : These contain proteins in addition to lipids.
E.g.: Chylomicrons, VLDL, LDL, and HDL
III) Derived lipids and Precursor lipids:

Derived lipids and precursor lipids are the products or precursors of simple lipids and
compound lipids.
Examples: Fatty acids, cholesterol, steroid hormones, fatty alcohols, ketone bodies,
prostaglan dins, fat soluble vitamins {A,D,E, K) etc.

Lipid Chemistry 61
Schematic representation lipid classification

LIPIDS
l
Simple Lipids
t
Compound Lipids
l
Derived Lipids and
I Precursor lipids
i l
Fats/oil Waxes
Phospholipids Other Compound lipids

I Lipoproteins.
l
Glycerophospholipids
1
Sphingophospholipids
Amino lipids
Sulpholipids
Glycolipids
I
l
Cerebrosides Ganglios.ides
1) Answer hint for Lipid classification question (5 Marks):

b. Classification with basis (1 Marks)
c. Sub-classification with basis and examples (3 Marks)
d . Schematic representation (0.5 Marks)
2) Answer hint for Sphingolipid question (3 Marks):
Sphingilipids are lipids containing sphingosine as alcohol, that includes
sphingophospholipids & glycolipids (So, students need to write about these two).
3) Functions of lipids is given in page no. 76
Note:
1) Glycerol is water-soluble; hence it is not a lipid by definition. But, some authors include glycerol
under derived and precursor lipids.
2) Lipoproteins: Some authors do not consider lipoproteins under conjugate lipids, because,
liporoteins are soluble in water (By definition, lipids are water insoluble compounds).
3) Neutral lipids: Uncharged lipids are referred to as neutral lipids. These include mono, di and
triacylglycerol, cholesterol and cholesteryl esters. Triaet;lglycerol is also called neutral fat.
4) Amphipathic lipids: These have both polar and non-polar ends. These include fatty acids,
phospholipids, glycolipids, cholesterol etc

Lipid Chemistry 62
Fatty acids
Definition:
Fatty acids are aliphatic carboxylic acids.
General formula:
R-COOH (where R is hydrocarbon chain and COOH is carboxyl group).
Classification of fatty acids:

Fatty acids are classified into two groups based on the nature of hydrocarbon chain,
i) saturated fatty acids ii) unsaturated fatty acid
i) Saturated fatty acids:

They do not contain any double bond in their chain.
Fatty acids Carbon atoms Structure

• Acetic acid 2 carbon atoms C~COOH
• Propionic acid 3 carbon atoms CH3Cf\COOH
• Bu tyric acid 4 carbon atoms CH3 [C}\] 2COOH
• Valerie acid 5 carbon atoms CH3 [CH2] 3COOH
• Caproic acid 6 carbon a toms Cf\ [C~] 4 COOH
• Caprylic acid 8 carbon atoms [C~] 6COOH
• Capric acid 10 carbon a toms CH3 [C~] BCOOH
• Laurie acid 12 carbon atoms CHi [C}\] 10 COOH
• Myristic acid 14 carbon atoms C~ [C}\] 12COOH
• Palrnitic acid 16 carbon atoms Cf\ [C~] 14COOH
• Stearic acid 18 carbon atoms C~ [CJ-\] 16COOH
• Arachidic acid 20 carbon atoms C~ [CI-4] 18COOH
• Behenic acid 22 carbon atoms C~ [C}\] 20COOH
• Lignoceric acid 24 carbon atoms Cf\ [CJ-\] 22COOH
Based o n the number of carbon atoms, there are 2 types of saturated fatty acid,
a) Even Chain Fatty Acids:

These contain even number of carbon atoms.
Examples: Acetic acid (2 C), butyric acid (4 C), palmitic acid (16 C) etc.
b) Odd Chain Fatty Acids:

These contain odd number of carbon atoms.
Examples: Propionic acid (3 C) and Valerie acid (5 C) etc.

Lipid Chemistry 63
ii) Unsaturated fatty acids:

They have double bonds in their hydrocarbon chain.
These may be subdivided into two types based on the number of double bonds.
a) Monounsaturated fatty acids [MUFA]:

These contain only one double bond.
Examples are palmitoleic acid, oleic acids etc.
• Palmitoleic acid
• Oleic acids
b) Polyunsaturated fatty acids [PUFA):

These contain more than one double bond.
Examples are linoleic acid, a -linolenic acid, arachidonic acid e tc.
• Linoleic acid - 2 double bonds
• a- Linolenic acid - 3 double bonds
• Arachidonic acid - 4 double bonds
• Tirnnodonic acid - 5 double bonds
• Cervonic acid - 6 double bonds
Some minor fatty acids:

• Branched chain fatty acids: E.g. lsovaleric acids, Phytanic acid (present in butter).
• Hydroxy fatty acids: E.g. Cerebronic acid
• Cyclic fatty acids: E.g. Hydrocapric acid present in chaulmoogric oil.
Nomenclature of fatty acids:
Numbering and naming of carbon atoms in fatty acids:

• Numbeing: The carbon atoms of fatty acids are numbered from COOH carbon,
which is numbered 1 (C-1). The carbon atom adjacent to the carboxyl carbon
atom is 2 (C-2) and so on.
• Naming: The carbon 2 is referred to as a-carbon. Carbon number 3 is the ~-carbon,
Carbon number 4 is the a-carbon and so on.
• The terminal methyl carbon is termed as (t)-carbon (omega-carbon) or n-carbon atom.
• Alternatively, starting from methyl end, the carbon atoms may be also is numbered
as co-1, co-2, co-3 (or n- 1, n-2, n -3) and so on.
5 4 3 2 1
ro y p a
CH3 - CH2- CH2- CH2 - CH2- CH2 - CH2 - CH2 - COOH
rol ro2 ro3

Lipid Chemistry 64
Number and position of double bonds:

There are two methods to indicate the number and position of double bonds.
i) From carboxyl end (denoted by .'.1)
ii) From ro -carbon atom end (denoted by co or n).
Example:
Palmitoleic acid has one double bond, which can be denoted as L1 9 or ro 7.
10 9
CH3 - (CH2) 5 - CH = CH - (CH2) 7 - COOH
co7 co8
i) L1 9 denotes that the double bond is present between carbon 9th & 10th carbon atom
from carboxyl end of oleic acid.
ii) ro 7 (or n 7) denote that the double bond is present between 7th and 8th carbon
atom counting from the ro end or n end of palmitoleic acid.
Shorthand representation of fatty acids:

Fatty acids are long structures, hence difficult to write. So, the shorthand notations
are used to represent fatty acids.
• The total number of carbon atoms is written first
• The total number of double bonds (if any) is written next
• Finally, the position of double bonds are written (only if double bonds are present)
Examples:
• Acetic acid is written as 2: 0 (2 carbon atoms and no double bonds)
• Palmitic acid as 16: 0 (16 carbon and no double bonds)
• Palmitoleic acid as 16: 1; 9 (16 carbon, 1 double bond, position is C9)
• Oleic acid as 18: 1; 9 (18 carbon, 1 double bond, position is C9)
• Linoleic acid as 18: 2; 9, 12 (18 carbon, 2 double bonds, positions are C9, C12)
• a- Linolenic acid as 18: 3; 9, 12, 15 {18 carbon, 3 double bonds, position are C9, CJ 2, C15)
• Arachidonic acid as 20: 4; 5, 8, 11, 14 (20 carbon, 4 double bond, positions are CS, C8, Cl 1, Cl 4)
Note:
• In unsaturated fatty acids having two or more double bonds, the bonds are always found at
three carbon intervals. For example, ifafathJ acid has 3 double bonds and the first double bond
is at C-9, then second double bond is found after three carbon atoms, i.e. at C-12 alld third one
at C-15.
• Three poly unsaturated fathJ acids i.e. linoleic acid ((J)-6), a -linolenic acid ((J)-3) and
arachidonic acid ((J)-6) are not synthesized in tl1e body; hence they have to be supplied in the
diet. So, these 3 fatty acids are referred to as essential fatty acids.

Lipid Chemistry 65
Omega (ro) - Series Fatty acids (also called n-Series Fatty acids):
Unsaturated fatty acids are designated as ro - Series fatty acids (or n- Series fatty
acids), depending on the position of first double bond &om the ro (CH,, methyl) end.
Next double bonds occur at intervals of 3 carbon atoms in case of polyunsaturated
fa tty acids. Based on this, th ere are 3 families of unsaturated fatty acids.
a) Omega - 3 fatty acids (n-3 family):

First double bond is 3 carbons from Cf\ end.
Example: a linolenic acid, Timnodonic acid, Cervonic acid.
b) Omega - 6 fatty acids (n-6 family):

First double bond is 6 carbons from CH3 end.
Examples: Linoleic acid and Arachidonic acid.
c) Omega - 9 fatty acids (n-9 family):

First double bond is 9 carbons from Cf\ end.
Example: Oleic acid.
Short chain, medium chain and long chain fatty acids:

Based on the length of hydrocarbon chains fatty acids are divided into 3 groups.
• Short chain fatty acids contain 2 to 6 carbon atoms
• Medium chain fatty acids contain 8 to 14 carbon atoms
• Long chain fatty acids contain 16 or more carbon atoms
Most fatty acid in foods are long chain fatty acids. (Exception is palm oil and coconut
oil, which are predominantly made up of medium chain saturated fatty acids).
Cis- trans fatty acids:

Depending on the orientation of h ydrogen atoms at double bonds, there are two
types of unsaturated fatty acids; cis and trans. 1f the hydrogen atoms are on the
same side of the double bond, it is called a 'cis' form, and if the hydrogen atoms are
on opposite sides then it is called a 'trans' form.
Nearly all the naturally occurring fatty acids are in cis form. Trans fatty acids are
present in certain foods (beef and dairy products). They are generally the by products
of hydrogenation process. 'Trans' forms are more stable than 'cis' form.
Examples: Olcic acid is in cis form and elaidic acid is in trans form.
CH, (CH1} - C- H CH~ (CH~)- C- B
I I
I
HOOC {CB:),- C-H H-C-(CB1) 1 COOH
Oleic acid (cis form) Ebidic add (trans form)

Lipid Chemistry 66
Essential fatty acids:

Definition : Fatty acids, which are not synthesize d in the body, hence have to be
supplied in the diet are called essential fatty acids.
Examples: Linoleic acid, a-Linolenic acid and Arachidon ic acid.
1. All essential fatty acids are polyunsaturated fatty acids (PUFA).
2. Arachidonic acid can be synthesized from linoleic acid (and not vice versa).
Functions:
1) Essential fatty acids serve as precursors of Eicosanoids:
Essential fatty acids serve as precursors of eicosanoid s (Prostaglandins, Prostacyclins,
thromboxanes, leukotrien s, lipoxins). Eicosanoids are local hormones.
2) Essential fatty acids help in lipid transport:
Essential fatty acids are required for the synthesis of phospholip ids, w hich are required
for the formation of lipoprotein s. So, it can be concluded that the essential fatty acids
help in lipid transport by forming phospholip ids.
3) Essential fatty acids are required for the formation & function of cell membrane s:
They are required for the formation of phospholip ids, which are integral componen ts
of cell membrane s. Essential fatt acids, being PUFA, increase membrane fluidity.
4) Lowering serum cholesterol:
Essential fatty acids lower serum cholesterol levels, hence have anti-athero genic effect.
5) Lipotropic effect:
Essential fatty acids promote fat mobilizatio n from liver. So, they prevent fatty liver.
6) Normal reproductive function:
Essential fatty acids are required for the normal reproducti ve fu nction. PUFA are the
important constituen ts of lipids of gonads.
7) Normal epidermal growth:
Essential fatty acids are required for the normal epidermal growth. PUFA are the
important constituen ts of epidermal lipids.
Deficienc y:
Deficiency of essential fatty acids in the diet is associated with skin lesion (rough and
dry skin) condition called Phrynoder ma (Toad skin). It is characterized by horny papular
eruptions on the posterior and lateral parts of thigh and on the back and buttocks.
Deficiency of EFA may also cause reproducti ve failure and fatty liver.
Answer hint for Essential fatty acids (5 Marks):
b . Examples (1 Marks)
c. Functions (2 Marks)
d. Deficiency (lMarks)

Lipid Chemistry 67
Eicosanoids (Including prostaglandinds):

Eicosanoids are compounds derived from eicosa (20 -carbon) polyenoic fatty acids
(C20 polyunsaturated fatty acids). There are three different eicosanoids,
• Prostanoids: (Includes Prostaglandins, Prostacyclins and Thromboxanes).
• Leucotriens
• Lipoxins
Eicosanoids
+
Prostanoicb
i
l.eukotrien.s +
Llpaxins
+
j l
(LTs) (IXs)
Pt ostaglandins Prostacyclins Tlnamboxanes

(PGs) (PGls) (IXs)
Synthesis:
Eicosanoids are formed from C 20 polyunsaturated fatty acids by cycloxygenase or
lipoxygenase pathways.
Prostanoids (prostaglandins, prostacyclins, thromboxanes) are formed by
cycloxygenase pathway, whereas leucotriens and lipoxins are formed by lipoxygenase
pathway.
Role of Eicosanoids:
Eicosanoids are biologically and pharmacologically active compounds. Physiologically,
they are considered to act as local hormones.
1. Prostanoids:
Prostanoids include prostaglandins, prostacyclins and thromboxanes. All have distinct
biological functions.
a) Prostaglandins (PGs):
• Prostaglandins are mainly formed from C 20 polyunsaturated fatty acids (Mainly
arachidonic acids) by cycloxygenase pathway. Aspirin is the inhibitor of this
pathway.
• Prostaglandins are first isolated from prostates (hence the name prostaglandins).
• There are different types of prostaglandins that have been identified, such as
PGA, PGB, PGC, PGD, PGE, PGF, PGG, PGH, PGI etc.
Lipid Chemistry 68
Significan ce:
Prostaglan dins act as local hormones. They have diverse physiologi cal functions.
• Regulation of blood pressure: Prostaglan dins (PGE) are vasodilator s. Hence they
lower the blood pressure. Prostaglan dins are used in the treatment of hypertensi on.
• Effect on respiratory function: Prostaglan dins (PGE) are bronchodil ators. So, PGE
are used in the treatment of asthma.
Note that PGF are bronchoco nstrictors (Thus, PGE and PGF have opposite effects).
• Regulation of gastric secretion: Prostaglan dins inhibit gastric secretion and increase
gastric motility. They are used in the treatment of gastric acidity and gastric ulcers.
• Role in reproduction: Prostaglan dins induce labor and terminate pregnancy .
• Role in inflammation: Prostaglan dins induce the symptoms of inflammati on.
• Influence on renal system: Prostaglan dins increase GFR and hence causes diuresis.
b) Prostacyclins (PGis):
• Prostacyclins found in the blood vessel (arterial and capillary) walls.
• Prostacyclins are vasodilato rs and inhibitors of platelet aggregation.
c) Thrombo xanes (TXs):

• Thrombox anes are produced mainly in platelets.
• Thrombox anes are vasoconstrictors. They promote platelet aggregation.
2. Leukotriens (LTs):
• Leukotrien s are synthesize d in leucocytes, mast cells, lung, heart, spleen etc. by
lipoxygenase pathway.
• Role in anaphy Iaxis: Leu kotriens are potent proinflama tory agents and have a role in
anaphylactic reactions. Leucotriens are component s of SRS-A (Slow-reacting substances
of anaphylaxis), which causes bronchoconstriction.
• Leu kotriens are implicated in asthma, allergy and heart attacks.
3. Lipoxins (LXs):
• Lipoxins are involved in vasoactive and immunore gulatory reactions e.g. as
counterreg ulatory compound s of immune response.
Aspirin is used as an anti-inflam atory drug:

Anspirin inhibits the cyclooxygennse pathway, which produces prostaglandins. Prostaglandins
are the inducers of injlamation.
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Lipid Chemistry 69
Acylglycerols (Fats/ Oils):

Definition :
Acylglycerols are the esters of fatty acids with glycerol. One, two or all three hydroxyl
groups of glycerol can be esterified with fatty acids producing monoacylglycerols,
diacylglycerols and triacylglycerols respectively.
CH20-H CH20-FA1 CH2 0-FA I CH20-FA1

I I I I
CHO-H CHO-H C H O-FA2 CH O-FA2
I I I I
CH20-H CH20-11 C H 2 0H CH20-FA3
Glycerol Monoacylglycerol Diacylglycerol Triacylglycerol
Fats and oils:

1) Naturally occurring fats and oils in diet are mixtures of three acylglycerols
(monoacylglycerols, diacylglycerols and triacylglycerols), triacylglycerol being the
most abundant component. 95 % of lipids consumed in the diet are triacylglycerols.
So, even though all the acylglycerols are termed as fat/ oils, triacylglycerols is generally
used synonymous to fat/ oil, as they are found in highest concentration.
Triacylglycerols are composed of one glycerol molecule and three fatty acids.
2) Acylglycerols (mainly triacylglycerols), which are solid at room temperature are
called fats and acylglycerols, which are liquid at room temperature are called oils. In
other word s, fats are solid a t room temperature and oils are liquid at room
temperature. But chemically both are same.
Based on the ttJpe of fatty acids, there are two types of triacylglycerols,
a) Simple triae1Jlglycerols:
They have 3 identical fatty acid residues at all three carbons ofglycerol. They occur rarely.
b) Mixed triacylglycerols:
They have more than one type offatty acids. Ninety-eight percent of dietanJ triglycerides are
mixed triglycerides. They contain a mix tu re of saturated, monounsaturated, and
polyunsaturated fatty acids. In general, fatty acid attached to C-1 ofglycerol is saturated, C-
2 is unsaturated (mono or poly) and C-3 can be either saturated or unsaturated.

Lipid Chemistry 70
Triacylglycerol (Neutral fat):

Triacylglycerol are commonly referred to as 'neutral fat' or simply 'fat'.
Definition : Triacylglycerols are esters of fatty acids with glycerol. They carry three
fatty acids, which are esterified to glycerol.
Functions:
The major bulk of body lipid is triacylglycerols, which is distributed in all organs
particularly in adipose tissue subcutaneously.
1) Fats are storage form of energy :

Whenever body requires energy from fatty acids, (as in starvation or diabetes mellitus),
the body stored fats (triacylglycerols) are hydrolyzed by enzyme lipase to release fatty
acids & glycerol, which are used for energy. So, stored fats serve as reservoir of energy.
2) Fats are concentrated form of energy :
One gram of fat gives more energy (9 Kcals) as compared to one gram of carbohydrate
or protein (4 Kcals). Furthermore, since fats are non-polar hydrophobic, they are
stored in anhydrous form, whereas proteins & carbohydrates are polar and
hydrophilic & therefore are stored in hydrated form which occupies more volume.
Consequently, a gram of fat (stored in anhydrous form) stores six times more energy
than a gram of glycogen stored in hydrated form.
3) Fats act as heat insulators:
The subcutaneously fat serves as an insulator against heat loss from body.
4) Fats act as shock absorbers:
The subcutaneous fat deposits also insulate against mechanical trauma. Fat pad
around internal organs also prevent injury to internal organs.
Lipolysis (Hydrolysis of triacylglycerols):

On hydrolysis triacylglycerols give one molecule of glycerol and 3 molecules of fa tty
acids. Hydrolysis of triacylglycerols occurs during two conditions.
i) In the intestine: During digestion of dietary triacylglycerols.
ii) In adipose tissue: During mobilization of triacylglycerols from adipose tissues during
restricted nutrition. This process releases fatty acids from fat depots.
Lipolysis (lipases)
Triacylglycerol Glycerol + 3 Fatty acids
Details of lipolysis is explained in lipid metabolism and lipid digestion.

Lipid Chemistry 71
Rancidity of Fats/oils:
Fats/ oils have a tendency to become rancid (stale). The term rancidity refers to the
appearance of unpleasant smell & taste for fats/ oils.
Rancidity is mainly of 2 types,

i) Hydrolytic rancidity: It is due to partial h ydrolysis of triacylglycerols by the enzyme
lipases present in na turally occurring fats/ oils.
ii) Oxidative rancidity: It is a result of oxidation of unsaturated fatty acids present in
oils to form lipid peroxides, epoxides and aldehydes (This is called lipid peroxidation).
These products have an unpleasant taste and odor.
Vegetable oils contain vitamin E, phenols, hydroquinones, tannins, etc, which function
as anti oxidants and prevent rancidity.
Vitamin E and artificial anti-oxidants like propylgallate, butyrated hydroxy anisole etc.
are used as food additives to prevent rancidity.
Characterisation of Fat/ Oils:
1) Saponification number:
Definition: Saponification number of fats/ oils is defined as the number of milligrams
of alkali (like KOH) required to saponify one gram of fat/ oil completely.
Significance : Saponification number is an indication of molecular weight of oil.
It is inversely proportional to the average molecular weight of fat/ oil. Higher the
saponification number, lower the m olecular weight of fat/ oil & vice versa.
Examples: Saponification number of coconut Oil= 253 to 262; butter= 210 to 230
This indicates coconut oil has lower molecular weight than butter.
2) Iodine number:
Definition : Iodine Number of fat/ oil is defined as the number of grams of Iodine (1i)
taken up by 100 grams of fat/ oil.
Signifance : Iodine number is an index of degree of unsaturation of fats and is directly

proportional to the content of unsaturated fatty acids. Higher the Iodine number of fat/
oil, higher the degree of unsaturation.
Iodine number is useful in the detection of adulteration of oils.
Examples: Iodine number of Coconut oil = 6 to 10; Sunflower oil = 124 to 136
So this indicates sunflower oil has higher degree of unsaturation.
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Lipid Chemistry 72
Structures of some important Phospholipids and Glycolipids:
[ Phosphatiditic add ) ( Lecithin (Phosphatidyl choline)]
CI-LO-F& CH20-FA1
.I I
CHO-FA2 CHO-FAJ
I I
CHiO-V CH20 - V Clioline
(cephalin (Phosphatidyl ethanolamine)) [ Phosphatidyl serine ]
CH20-FA1 CH10-FA1
I I
CHO-FAJ CHO-F~
I I
CI-LO -0- Ethanolamine CI-L. 0 - 0- Serine
( Sphing omyel in ]
(C eran.tlde]
Sp bingosine - Fatty acid
I
Sphingosine - Fatty acid
®I
Choline
( Cerebrosides ) ( Gangliosides ]
Sphingos.ine - Fatty acid Sph.ingosine - Fatty acid

I I
Galado se / Glucose Glu -Gal - NANA (sialic acid)

Lipid Chemistry 73
Phospholipids (Phosphatides)
Definition: Phospholipids are compound lipids containing phosphoric acid residues
in addition to fatty acids and alcohol. They also contain a nitrogenous base.
Types: Phospholipids are of 2 types depending upon the nature of alcohols they contain,
i) Glycerophospholipids: They contain glycerol as alcohol. Examples are,
• Lecithin (Phosphatidyl choline),
• Cephalin (Phosphatidyl ethanolarnine)
• Other examples are, Plasmalogen (Phosphatidal ethanolamine), Cardiolipin (Diphosphatidyl
glycerol), Lung surfactant (Dipalmitoyl lecithin) etc.
ii) Sphingophospholipids: They contain sphingosine as alcohol.

Example: Sphingomyelin.
Functions of phospholipids:
1) Constituents of cell membrane: Phospholipids (along with proteins) are major
constituents of plasma membrane. They regulate the permeability of cell membranes.
2) Participate in lipid transport: Phospholipids are required for the formation of
lipoproteins (transport form of lipids). So, phospholipids help in lipid transport.
3) Participate in eicosanoids synthesis: Membrane phospholipids are sources of
Arachidonic acid. Arachidonic acids are required for the synthesis of eicosanoids.
4) Phospholipids are required for the action of lipoprotein lipase & TAG lipase .
5) The lung surfactant (Dipalmitoyl lecithin) is a phospholipid. Lung surfactant
prevents the collapsing of lungs.
6) Participate in blood coagulation: A phospholipid called platelet activating factor
(PAF) participate in blood clotting.
Plwspholipids are integral components of cell membranes: Cell t11ernbra11es form a barrier
between 2 adjacent aqueous phases. Amphipat/tic nature of phospholipids makes them the ideal
components of cell membranes. Polar heads of phospholipids are directed towards the aqueous
phase, whereas the hydroplwbic tails face the interior).
Answer hint for Phospholipids (5 Marks):
b. Types with examples (2 Marks)

Lipid Chemistry 74
Phosphatidic acid: Phospholipids are regarded as de rivatives of phosphatidic acid.

Phosphatidic acid are diacylglycerols containing a phosphate group at C3 . Generally, C1 of
glycerol is attached to saturated fatty acids (mainly palmitic acid or stearic acid); C 2 of
glycerol is attached to unsaturated fatty acids (mainly PUFA); and C3 of glycerol is attached
to a phosphate group to form Phosphatiditic acid.
Phosphatidic acids are precursors in synthesis of triacylglycerols and
glycerophospholipids like lecithin, cephalin etc.
Ceramide (Fatty acyl sphingosine): Fatty acid combined with sphingosine is ceramide.
Ceramide is the precursor of Sphingomyelin (and also glycolipids).
1. Lecithin (Phosphatidyl choline): These are glycerophospholipids containing choline.

Structure: They contain choline attached with Phosphatidic acid.
Sources: Lecithins are the most abundant phospholipids, present in brain, nervous tissue etc.
They are very rich in egg yolk.
Functions:
• Lecithins are the principle constituents of cell membranes.
• Lecithins acts as the storage form of choline in the body.
• They are sources of arachidonic acid (C2 of lecithins generally contain arachidonic acid).
• They are required for the formation of lipoproteins (hence they help in lipid transport).
• They help in esterification of cholesterol to form chosteryl ester. (Required in HDL formation).
• They help in the emulsification and helps in the digestion and absorption of lipids.
2.Cephalin (Phosphatidyl ethanolamine): These contain ethanolamine.
Structure: They contain ethanolamine attached with Phosphatidic acid.
Significance: It is a component of cell membranes. It plays a role in blood coagulation.
3. Phosphatidyl serine: These are glycerophospholipids containing serine.
4. Phosphatidyl inositol: These are glycerophospholipids containing inositol.

Significance: Phosphatidyl inositols are important precursors of second messengers.
5. Lung surfactant (Dipalmitoyl lecithin): These are glycerophospholipids present in lungs.
Significance: Lung surfactants prevent the collapsing of lungs. Res piratory Dis tress
Syndrome (RDS) in infants is a disorder characterized by the absence of lung surfactant.
6. Plasmalogen (Phosphatidaethanolmine): In plasmalogens, fatty acid is a ttached with C1
of glycerol with ether linkage, instead of ester linkage. Mainly present in muscle, myelin etc.
7. Cardiolipin (Dipalrnitoyl glycerol): It is composed of 2 molecules of phosphatidate linked

by glycerol. Cardiolipin is a major phospholipid present in mitochondrial membrane in heart.
8. Platelet activating factor (PAF): It is an alkyl phospholipid. It activates platelet aggregation
and thus helps in blood coagulation.
9. Sphingomyelin:
Structure: In sphingomyelin, ceramide is combined with phosphoryl choline.
It is present in myelin sheath of nerves and brain tissues.

Lipid Chemistry 75
Glycolipids :
Definition : Glycolipids are compound lipids containing carbohydrate residues in
addition to fatty acids and alcohol.
Types : Glycolipids are 2 types; i) Cerebrosides ii) Gangliosides

i) Cerebrosides:
These contain glucose or galactose as carbohydrate residues
E.g.: Kerasin, cerebron, nervon and oxynervon.
ii) Gangliosides :
These contain oligosaccharides and sialic acids (like NANA) as carbohydrate residues.
E.g.: GM1, GM2, GM3, GM4, GD and GT.
Globosides: When oligosaccharides (most often glucose, galactose and N-acetyl galactosamine)
attached to ceramides (fatty acyl sphingosine), globosides are formed. They are tile precursors of
gangliosides. They do not contain sialic aids. (Some consider them the 3rd type of glycolipids).
E.g.: Lactosyl ceramide
Functions of glycolipids:
1) Glycolipids are the major constituents of cell membranes, especially nervous tissues.
2) Glycolipids usually present in outer layer of plasma membrane and they p lay a key
role in cell-to-cell interaction, growth and development.
3) Blood group antigens are cell surface globosides. Globosides present on RBC
membranes are determinants of blood group A, B and 0 .
4) Some hormone receptors are gangliosides.
Cerebros ides: These glycolipids contain glucose or galactose as carbohydrate residues.

• Glucocereramides (Glucocerebrosides) contain Galactose attached with ceramide.
• Galactoceramides (Ga lactocerebrosides) contain Galactose attached with ceramide.
Type of cerebrosides depends on fatty acids present in tltem.
• Kerasin contain lignoceric acid.
• Cerebron contain cerebronic acid.
• Nervon contain nervonic acid.
• Oxynervon con tain hydroxy derivative of nervonic acid.
Sulfoga lactosylceramide (sulfatide) is formed when sulfate group is added to galactosylceramide.
Gangliosides: These glycolipids contain oligosaccharides & sialic acids as carbohydrate residues.
E.g.: GMl, GM2, GM3, GM4, GD and GT, where, G represents Gangliosides, the letters M,
D, Tare used to denote number of sialic acid content (mono, di, tri etc) & subscript is a number
assigned on the basis of chromatographic migration (1, 2, 3 etc).
Gangliosides are present in ganglions. Some hormone receptors are gangliosides in nature.

Lipid Chemistry 76
Cholesterol :
Cholesterol is an animal sterol. Its chemical name is 3 -hydroxy 5,6 cholestene.
Structure:
u
Cholesterol contains 27 carbon atoms.
Y~: It contains a 'cyclopentano perhydro

phenanthrene ring' system (17 carbon),
which has -
• An -OH group at C3
• A double bond between CS 1111d C6
• Methyl group at ClO and C13
• Eight-carbon side chain at C17
Functions of cholesterol (Compounds derived from cholesterol):

1) Constituents of cell membranes:
Cholesterol is an important constituent of cell membranes.
2) Synthesis of bile acids and bile salts:
Cholesterol (50- 80% of cholesterol) is required for the synthesis of bile acids/bile salts
in liver. Bile salts are required for the digestion and absorption of lipids in the intestine.
3) Formation of vitamin D 3 :
7-dehydrocholesterol (a derivative of cholesterol) can be converted to vitamin 0 3 by
sunlight in skin.
4) Synthesis of steroid hormones:
Four classes of steroid hormones are derived from cholesterol
i) Glucocorticoids E.g.: Cortisol
ii) Mineralocorticoids E.g.: Aldosterone
iii) Male sex hormones E.g.: Testosterone
iv) Female sex hormones E.g.: Progesterone and Estradiol etc.
Cholesterol ester:
Cholesterol can combine with various fatty acids to form cholesteryl ester. Hydrolysis
of cholesterol ester gives one molecule of cholesterol and a molecule of fatty acid.
Answer hint for functions of lipids (S Marks):

Lipids include triacylglycerols (fat), phospholipids, glycolipids, cholesterol,
fatty acids etc. So, if functions of lipids is asked, write the functions of essential
fatty acids (Page 66), triacylglycerols (Page 70), phospholipids (Page 73),
glycolipids (Page 75) , cholesterol (Page 76).

Lipid Chemistr y 77
Amphipathic lipids:
Lipids in general, are water insoluble compoun ds as they contain predomi nant
hydropho bic hydrocar bon groups (nonpola r). But, some of the lipids also contain
hydroph ilic (polar) groups. Such lipids which contain both hyd rophobic and
hydrophi lic groups are referred to as amphipa thic lipids.
E.g.: Phospho lipids, fatty acids, sphingol ipids, bile salts, cholester ol (to a lesser extent).
Arrangements of amphipathic lipids at oil water interfaces:

In aqueous solutions , amphipa thic lipids become oriented with their hydrophi lic
(polar) groups oriented towards the water phase and hydropho bic (non polar) groups
in opposite direction.
There arc two types of arrangem ents of amphipa thic at oil water interface;
1) Monomolecular film (Monolayer) :

When the amphipa thic lipids come in contact with water surface, the monomo lecular
films (Monolayers) are formed with their polar groups orient towards the water surface
and non polar groups orient towards opposite direction.
Example: Micelle formatio n
Ampliip athic lipids in 1nicelles:

·when amphipathic lipids are mixed with water, their hydrophilic polar groups orient
towards water and hydropl10bic groups faci11g opposite direction forming molecular
aggregates called micelles. Bile salts help in the formation of micelles in the intestine.
This process is called emulsification.
Micellar formation helps in the digestion and absorption of lipids in the intestine.
Aqueous phase Aqueous phase
Non polar phase
[ Micelle )
75
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Lipid Chemistry 78
2) Bimolecular leaflet (Bilayer):

When the amphipathic lipids are in contact with water at both the sides, they form
two monolayer s and the non polar ends of the two monolayer s orient towards each
other to form bimolecula r leaflet (or Bilayer).
Example: Plasma membrane structure
Atnphipat hic lipids in plasma membrane:

Orientation of amplzipatltic lipids (mainly plwsplwlipids) in bilayers forms the basis of
plasma membrane structure. Hydrophilic groups (polar ltead) of phospholipids orient
towards the extracellular surface and hydrophobic groups (non-polar tails) orient towards
each other in the direction of the hydrophobic core of bilayer.
Refer plasma membrane structure in cell chapter for complete explanation.
Aqueous phase
Non polar phase
Aqueous phase
[ Bimolecular leaflet in plasma membran e ]
Note: Amphipathic lipids form monolayers where there is a water surface contact witl1 air (e.g.
micelles), but when there is water in contact with both sides, bilayers (e.g. plasma membrane)
are formed.
Liposomes:
Liposomes are artificially formed spherical lipid vesicles. They are made up of
amphipath ic lipid bilayers enclosing small amount of water in the core.
Liposomes are formed when amphipath ic lipids in an aqueous medium are subjected
to 'sonication ' by agitating them with high frequency sound waves.
Significanc e: Liposomcs can be u sed to encapsulat e drugs, proteins, enzymes, genes
etc & deliver to target organs. Liposomes have proven to have important role in cancer
chemother apy, gene therapy, vaccine, enzyme therapy, antimicrobial therapy etc.
Terpenes:
Te171e11es are large and varied class of hydrocarbon compounds containing froprene units (Isoprene
unit is composed of 5-cnrbon structure).
Vitamin A & Carotenes are the examples of terpenes. Other examples are Isope11te11yl pyrophosphate,
Dimetlzylallyl pyrophosphate, Sq11ale11e (intermediates of cholesterol synthesis pathway).
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Lipid Chemistry 79
Question Bank on Lipid chemistry :
Long essays (10 Marks):

1) What are lipids? Classify lipids giving examples?

1) Essential fatty acids-definition, names, sources and functions
2) Compound lipids- Definition, classification with examples, sources, functions
3) Phospholipids -Definition, sub-classification with examples, sources, functions
4) What are the compounds formed from Cholesterol (Cholesterol functions)
Short Answers (2 Marks):

1) Write the products formed from hydrolysis of a) Lecithin b) Cephalin c) Sphingomyelin
2) Write short notes on a) Lecithin b) Lung surfactant
3) What are Sphingolipids? (Explain both Sphingophospholipids & Glycolipids)
4) Write short notes on a) Saponification number b) Iodine number

1) Bile acids are derived from (AI)
a) Fatty acids b) Amino acids c) Bilirubin d) Cholesterol
2) A major lipid of mitochondrial membrane is (COMEDK)
a) Lecithin b) Inositol c) Plasmalogen d) Cardiolipin
3) The precursor of steroid is (AI)
a) Amino acids b) Fatty acids c) Cholesterol d) None
4) Which one of the following undergoes auto-oxidation (AIIMS}
a) FFA b} PUFA c) Chylomicrons d) Cholesterol
5) Which one of the following is not a phospholipid (AIIMS)
a) Cerebrosides b) Sphingomyelin c) Lecithin d) Cephalin
6) The major constituent of lung surfactant is
a) Cardiolipin b) Diplamitoyl lecithin c) Plasmalogen d) Sphingomyelin
7) Thromboxanes are synthesized in
a) Platelets b) Lcucocytes c) Erythrocytes d) None of these
8) The principle Phospholipid Present in membrane is
a) Phosphatidyl choline b) Phosphatidyl ethanolamine c) Phosphatidyl serine d) Phosphatidyl inositol
9) Which of the following is PUF A?
a) Oleic acid b) Stearic acid c) Arachidonic acid d) Palmitic acid
10) Choline is one of the constituent of
a) Lecithin b) Cephalin c) Plasmalogen d) Sphingomyelin
AnswersforMCQ: l)d 2)d 3)c 4}b S)a 6)b 7)a 8)a 9)c lO)a

Amino acid and Protein Chemistry 80
Chemistry of Amino acids, Peptides and Proteins
Contents:
• Amino acids chemistry

• Definition
• Classification
• Charge properties
• Buffer action
• Optical activity
• Peptide bonds
• Peptide chemistry
• Definition
• Physiologically active peptides
• Protein chemistry
• Definition
• Classification
• Structural organization and denaturation
• Charge properties
• Practical Amino acid and Protein chemistry

Terminology :
Amino acids :
Amino acids are compounds having both amino group (NH2) and carboxyl (COOH)
groups.
Amino acids are the building blocks (monomeric units) of peptides and proteins, which
are bonded by peptide bonds.
Peptides and proteins:

Peptides and proteins are polymers of amino acids bonded by peptide bonds.
Difference between peptides and proteins:

Difference between peptides (oligopeptides, polypeptides) and proteins is based 'on
the number of amino acid residues p resent in the chain.
Peptide: If the chain contains 2-50 amino acids, then it is called a peptide.
Peptides are 2 types.
• Oligopeptide: If the chain contains 2-10 amino acids, then it is called a oligopeptide.
(Which includes dipeptides, tripeptides .......... up to decapeptides).
• Polypeptide: If the chain con tains 11-50 amino acids, then it is called a polypeptide.
Protein: If the chain contains more than 50 amino acids, then it is called a protein.
Note:
This categorization of peptides an d proteins is arbitrary. Some authors label the
peptide chains having more than 100 amino acids as proteins.
For example insulin has 51 amino acids. By general definition, insulin is a protein,
but some authors refer insulin as a polypeptide.
Peptide bonds:
Peptide bond is responsible for the formation of peptides and proteins. Peptide bond
is formed between two amino acids. It is formed between a carboxyl group of first
amino acid and an amino group of the next amino acid.
Number of peptide bonds are one lesser than the number of amino acid residues in the
chain. For example, dipeptides have 2 amino acids and 1 peptide bond; tripeptides
have 3 amino acids and 2 peptide bonds and so on.
Amino acids / Amino acid residues:

Amino acid molecules, once included in the peptide or protein chain, are no longer
called amino acids; instead they are called amino acid residues or amino acid moiety.

Chemistry of amino acids

Amino acids are compounds having both amino group (-NH2) and carboxylic acid
groups (-COOH). Amino acids are the monomeric units (Building blocks) of proteins.
More than 300 amino acids are found in nature. Of these, only few (20 primary
amino acids and some secondary amino acids) are present in body proteins. These
amino acids are called protein amino acids. Other amino acids, which are not found
in the protein, are called non-protein amino acids.
I. Protein amino acids :

The amino acids that are found in the proteins are called protein amino acids.
There are two types.

a) Primary protein amino acids (Standard amino acids)
b) Secondary protein amino acids
a) Primary protein amino acids (Standard amino acids):

Definition: Amino acids that are incorporated into the protein structure during the
stage of translation are called primary protein amino acids (or standard amino acids).
Only primary protein amino acids are represented by genetic code.
Examples : They are 20 in number (Glycine, Alanine, tryptophan etc).
Some primary protein amino acids are modified after their incorporation into
proteins. For example, hydroxylation of proline and lysine to produce hydroxy
proline and hydroxy lysine respectively, y-carboxylation of glutamate to produce
y-carboxy glutamate etc. These post-translationally modified amino acids are called
secondary protein amino acids.
b) Secondary protein amino acids :

Definition: Post-translationally modified amino acids are called secondary protein amino
acids. These amino acids are formed from primary protein amino acids after the stage
of translation by post-translational modification.
These are not represented by genetic code.
Examples: Hydroxy proline, hydroxy lysine, y- carboxy glutamate, cystine etc.
II. Non-protein amino acids:

Amino acids that are not found in the proteins are called non-protein amino acids.
Examples : Ornithine, Citrulline, ~-alanine, y-amino butyric acids (GABA),
Argininosuccinate, Taurine, Homocysteine, Homoserine etc.
Note: Primary protein amino acids (Standard amino acids) are discussed further to
explain the properties of amino acids.

Primary protein amino acids (Standard amino acids):

Definition:
Amino acids, which are incorporated into the proteins during the stage of translation
(protein synthesis), are called primary protein amino acids (or standard amino acids).
Only primary protein amino acids are encoded by genetic code. They are 20 in number.
Structure:
Primary protein amino acids arc all a- amino acids (except proline, which is an
imino acid), as both the amino and carboxyl group are attached to the same a-carbon
atom. The third valency position of a -carbon atom is occupied by a hydrogen atom.
Fourth valency position of a - carbon atom is occupied by a side chain, commonly
called as R group.
By convention, the free amino group is written at the left side and the free carboxylic
group is written at the right side of the structure. R groups are also called side chain
because they project from either side of the protein chain. Hydrogen atom is written
at the opposite end of R group.
Common for all amino acids
1
H a-Carbon
Amino group - - - - HiN -

1/
C - COOH _ ____. Carboxylic group
I
R +-- R group or Side chain
General structure of amino acids
Note: Amino group, carboxyl groups and hydrogen atom are common to all protein
amino acids. Amino acids differ from each other only by their R group, which
determines the identi ty of different amino acids.
For instance, R group is Hin glycine, CH3 in alanine, CH20H in serine, CH2COOH in
aspartic acid, (CHJ 4 N~ in lysine etc.
Names of some of the important R-groups (Side chains):

1) Arginine: Guanidino group
2) Tryptophan: Indole group
3) Histidine: Imidazole group
4) Tyrosine: Hydroxy phenyl group
5) Phenylalanine: Phenyl or benzene group
6) Cysteine: Sulfhydryl group

Structure of amino acids

1) Aliphatic amino aicds
a) Non-branched amino acids b) Branched amino acids
fuN - CH - COOH NH2-CII -COOf l

H2N-fll COOi! H2N-,H -COOII
I
I CH
"
H CH3 CH
Glycine Alanine CH,

/ ~ CHJ /
CH2 Clh
/
Valine CH.1
Leucine Isoleucine
2) Aromatic amino aicds

H2N-CH--COOII H2N-CH-COOII H2N-CII COOII ll2N-CH- COO H 3) Hyroxy amino aicds
I I I
CH2 Cll2 Cll, H2N fll-COOH H2 - Fil -COOH
©
I I
f H2 Oil ~ II
I I
HN NH
"'
H V
OH CH,
Phenylalanine I Threonine Serine

HO
Tryptophan Histidine
Tyrosine
4A) Acidic amino aicds H2N -TH - CO~)H

CH2 SH
H2 -Cl! COOH H2 -CH -COOi!
I I I and Histidine
CH2 r H2
T H2
I
CII, rH2
COOII
I-
CH2
COOH
Aspartic acid G lutami c acid I
N II/
lysine Arginine
5) Amidic amino aicds 6) Sulphux containing amino aicds 7) Imino aicds
112N O I (X)()H H, N - CH - COOH

- I
H2N - Cl!
I
COOi!
HNI 1-COOH
I
Cl-½
I
t2 Cll2
I '----..._/
C=O f H2 1112 Pro line
I
NII, C=O Cysteine s
I I
NH2 C ll i
Aspangine Glutamine Methionine

Classification of amino acids

The 20 primary protein amino acids can be classified on several basis. These are,
I. Based on the Chemical structure:

Am ino acids are classified into 7 structural groups.
1) Aliphatic amino acids:

a) Non-branched cha in a mino acids: Glycine, Alanin e.
b) Branched chain amino acids: Valine, Leuci ne and lsoleucine.
2) Aromatic amino acids: Tryptophan, Phenylalanine, Tyrosine, Histidine
3) Sulphur containing amino acids: Cysteine and Methionine.
4) Hydroxy amino acids: Serine, Threonine and Tyrosine
5) Amino acids with amide group (CON8i): Asparagine, Glutamine
6) Charged amino acids: Include Acidic amino acids, basic amino acids.
a) Acidic amino acids: Aspartic acid, Glutamic acid
b) Basic amino acids: Lysine, Arginine, Histidine.
7) Imino acids (con tain an im ino group): Proline (Pro)
II. Based on the Charge:

Amino acids are classified into 3 groups based on the ch arge on their R - group.
a) Acidic Amino Acids (Monoamino d icarboxylic acids):

These have acidic R groups. E.g.: Glutamic Acid, Aspartic Acid
b) Basic Amino Acids (Diamino monocarboxylic acids):

These h ave basic R groups. E.g.: Lysine, Arginine, and Histidine
c) Neutral Amino Acids (Monoamino monocarboxylic acid s):

They h ave neu tral R groups. E.g.: Glycine, Alanine etc (All other 15 aminoacids).
III. Based on the polarity

1. Polar amino acids: They are hydrophilic in nature
E.g.: Glycine, serine, cysteine, threonine, glutamine, lysine, arginine, glutamic acid e tc.
2. Non-polar amino acids: They arc hydrophobic in n ature.
E.g.: Alanine, Phenylanine, tryptophan, valine, leucine, isoleucine. methionine etc.

IV. Based on the Nutritional requirement:

Based on the nutritional requirement, the amino acids can be classified into two groups-
Essential amino acids and Non-essential amino acids.
Out of 20 primary protein amino acids, our body can synthesize only 10 of them. These
amino acids are called non-essential amino acids or dispensable amino acids. The
remaining 10 amino acids, which cannot be synthesized in the body, are called essential
amino acids or indispensable amino acids. These have to be supplied in the diet.
a) Non - essential amino acids (Dispensable amino acids):

Amino acids, which can be synthesized in the body and hence are not required in the
diet, are called non-essential amino acids. Examples are, Glycine, alanine, serine,
cysteine, tyrosine, aspartic acid, glutamic acid, asparagine, glu tamine, p raline.
b) Essential amino acids (Indispensable amino acids):

Amino acids, which are not synthesized in the body and therefore have to be supplied
through the diet, are called essential amino acids. These are;
1. Methionine
2. Arginine*
3. Threonine Answer hint for Essential amino acids (5 Marks):
4. Tryptophan a. Definition (1 Marks)
5. Valine b. Examples (2 Marks)
c. Importance (2 Marks)
6. Isoleucine
7. Leucine [Mnemonics - MATT VIL PHLy]
8. Phenylalanine Does Mat fly? Yes, Alladdin's Mat.
9. Histidine*
10. Lysine
*Semi-essential amino acids: Among the essential amino acids, arginine and
histidine are called semi-essential amino acids as they are essential in the d iet of children,
pregnant & lactating women (Semi-essential amino acids are not synthesized in sufficient
quantities in these physiological groups) & are not essential in th e diet of normal adults.
Importance of essential amino acids:

Twenty amino acids are required for synthesis of proteins. While the body can produce
non-essential amino acids, the essential amino acids must be provided by the d ietary
proteins. Dietary proteins should provide all essen tial amino acids, as they are the only
sources of essential amino acids to the body. Absence of any one or more essential
amino acids in the diet impairs protein synthesis and lead to negative nitrogen balance.
When the proteins lack one or more essential amino acids, they do not promote proper
growth and maintenance of body tissues.

V. Classification based on metabolic status:

During the catabolism of amino acids, the amino groups are removed from amino
acids to form keto acids (carbon skeletons of amino acids). These carbon chains are
further catabolised. Based on the metabolic fate of these carbon skeletons, the amino
acids are classified into glucogenic, ketogenic and both glucogenic and ketogenic
amino acids.
a) Glucogenic amino acids:

Definition:
After the removal of amino group, if the carbon skeleton of an amino acid can be
converted to glucose in the body, such an amino acid is called glucogenic amino acid.
E.g.: Glycine, Alanine, Serine, Threonine, Glutamic acid, Glutamine, Aspartic acid, Aspragine,
Histidine, Proline, Cysteine, Arginine, Valine, Methionine.
b) Ketogenic amino acids:

Definition:
After the removal of amino group, if the carbon skeleton of an amino acid can be
converted to ketone bodies or acetyl CoA or fat, such amino acid is called ketogenic
amino acid.
E.g.: Only leucine is pure ketogenic amino acid.
c) Both glucogenic & ketogenic amino acids:

Definition:
After the removal of amino group, if the carbon skeleton of amino acid splits into two
parts, one of which can be converted to glucose and the other part can be converted to
ketone bodies or fat, then such an amino acid is called both glucogenic and ketogenic
amino acid.
E.g.: Lysine, lsoleucine, Tyrosine, Phenylalanine, tryptophan.
Purely Glucogenic Both Glucogenic Purely

and Ketogenic Ketogenic
Glycire, Alanine, Serine, Threonine, Lysine, lsoleucine, Leuc ine
Glutamic acid, G lutamine, Aspartic acid, Tyrosine,
Aspragire, Histidine, Pro line, Cysteine, Preny lalanine,
Arginine, Valine, Methionine tryptophan

Answer hint for Amino acid classification question (10 Marks):

b. Classification based on chemical structure (5 Marks)
c. Classification based on Nutritional requirement (2 Marks)
d. Classification based on Metabolic fate, Polarity (1 +1 Mark)
Examples of different amino acids are common questions during viva. These can also
be asked for two marks each in theory examinations also, like name the sulphur
containing amino acids, name the aromatic amino acids etc.
Stereoisomerism in amino acids:

i) Enantiomerism:
Amino acids exhibit D - L stereoisomerism called enantiomerism. Stereoisomerism due
to the position of NH2 around asymmetric carbon atom is referred to as D-L
stereoisomerism (or enantiomerism). If the-NH2 group is on the right side of a- carbon
atom, the amino acid is D-isomer, and if the -NH2 is on the left side, the amino acid is L-
isorner. D and L isomers are mirror images of each other.
H H
I I
H2~ - C - COCH COOH- C - NH.:.
I I
R R
L- Amino acid D-Amino acid

In nature, only L-a-amino acids occur in proteins. Only few D-Amino acids are found.
E.g.: D-alanjne and D-glutamate in the cell walls of gram-positive bacteria and D-
serine and D-aspartate in brain tissue.
ii) Optical isomerism (Optical activity of amino acids):

Optical isomers are stereoisomers, which have the capacity to rotate the plane of
polarized light to right or left. If the light turns right then the amino acid is called
dextrorotatory, denoted as (d) or (+) and if the light turns left then the amino acid is
called levorotatory denoted as (1) or (-).
a-carbon atom of all amino acids (except glycine) is asymmetric. Due to the presence
of asymmetric carbon atom, all amino acids (except glycine) exhibit Optical activity.
H Note: Glycine is the only amino acid that does
not exhibit stereoisomerism and optical
NB2 - C - COOH activity, as it does not have an asymmetric
(chiral) carbon atom and hence does not
H exhibit chirality. This is because two of its
Glycine valencies are occupied by h ydrogen atoms.

Charged properties of amino acids:

Amino acids are charged molecules. Charged properties of amino acids are due to
the presence of both acidic (-COOH) and basic (-NH2) groups. Some amino acids
(charged amino acids) also have additional acidic or basic groups.
Amphoteric nature of amino acids (Ampholytes):

Amino acids are ampholytes, because they behave both as an Acid and as a Base.
a) Behavior as an Acid: In the presence of a base, amino acids can donate a proton
and thus acts as an acid.
H H
I
H JL'< - C - CO O .
I
.. I
H 2N - C - C O 0
I
R R
(AN IONIC l'O RM )
b) Behavior as a Base: In the presence of an acid, amino acid can accept a proton
and thus acts as a base.
H H
I
I
H l -:i,: - C - C O O . .. H 3- N
I
- C - CO OH
I
R R
(CA TI O NIC l'ORM )
Since amino acids can act both as an acid (i.e. proton donor) and a base (i.e. proton
acceptor), they are said to have amphoteric nature (ampholytes).
Isoelectric pH (pl) of amino acids

lsoelectric pH (pl) of an amino acid is defined as the pH at which amino acids exist
as n eutral zwitterions (dipo]ar ion).
E.g.: pl of aspartic acid is 2.9; pl of glycin e is 6.1; pl of his tidine is 7.6
At lsoelectric pH, amino acid s possess equal n umber of positive and negative charges,
hence bear no net charge, d o n ot move in an electric field.
Buffer actions of amino acids (and proteins):

Even; ami110 acid cnn fu11c tion as bllfjer in the ra11ge one pH unit on either side of its pK
value. However, most of the amino acids pK ranges are far from the pH of the blood
(7.4).Thus, in general, amino acids are not useful as buffers in blood, except for one
amino acid, histidine, whose pK value of imidazole ring is 6.1. So, histidine is effective ns
buffer at the physiological pH of blood. The buffering capacity of plasma proteins and
hemoglobin (38 out of 574) is mainly due to histidine content.

Peptide bonds
Definition :
Peptide bonds are anhydride, covalent bonds formed between a carboxyl group of
an amino acid and an amino group of succeeding amino acid.
H R H R
I I I I
ILN-C -CO~ N -C-COOH ~ fuN-C-f OHN~C -COOH
I~ I "' I I
R H H,O RlH
Peptide bond
Peptide bond is represente d by
0
11
- C-N -
I
H
Salient features of peptide bonds:

1) Peptide bond is a strong covalent bond.
2) Peptide bond is an anh ydride bond (i.e. formed by the loss of a water molecule).
3) Peptide bonds are amide linkages.
4) Peptide bond generally exists in -trans configurati on (exception is peptide bond
formed by proline, a imino acid, which has -cis configurati on).
5) Peptide bonds are partial double bond in nature.
0 0 0
II • I
• I
-C=
- C- N - 4111 - C =-=-=- • I
I
H H H
6) Peptide bonds are semi-rigid in nature.
7) All atoms - C, N, 0 and Hare coplanar.
8) During denaturati on, peptide bonds are not affected, because peptide bonds are
strong covalent bonds.
Importance of peptide bonds:

Peptide bonds are responsible for the polymeriza tion of amino acids to form peptides
(oligopepti des and polypeptid es) and proteins.

Amino acid and Protein Chemistr y 91
Physiologically (Biologically) important peptides:

Body contains many importan t peptides (containi ng 2-50 amino acids) that have diverse
physiolog ical functions. These are,
1) Camosin e and Anserine: Both are dipeptide s. Carnosin e is made up of ~-alanine

& histidine; anserine is a derivativ e of carnosine . These peptides are present in muscles.
2) Glutathi one (-y- glutamyl cysteinyl glycine): It is a tripeptid e made up ofy-gluta mic
acid, cysteine and glycine.
Present in erythrocy tes in large amounts. It is a powerful reducing agent and involved
in various reduction reactions in the body.
Glutathio ne functions are discussed in detail in next page.
3) Thyrotropin releasing hormone (TRH): A tripeptid e secreted by hypothal amus,

stimulates the pituitary gland to release thyrotrop in stimulati ng hormone (TSH).
4) Enkepha lins: Pentapep tide neurotran smitters. Enkepha lins inhibit pain sensation .
5) Oxytocin: A nonapep tide hormone secreted by posterior pituitary . It causes uterine

con traction.
6)Vasopre ssin (ADH): A nonapep tide hormone secreted by posterior pituitary . It is

required for smooth muscle contracti on and water reabsorpt ion.
7) Bradykinin: Nonapep tide. It is a vasodilat or.
8) Kallidin: Decapept ide. It is a vasodilat or.
9) Angiote nsins: Angiote nsin II (octapep tide) is derived from Angiote nsin I
(decapep tide). Angioten sin II is a hyperten sive peptides , stimula tes the release of
aldostero ne from adrenal glands.
10) Glucago n (29 amino acids): It is a hypergly cemic h ormone.
11) Gramicidin, Actinom ycin (Antibiot ics) are peptides in nature.
12) Gastrin, Secretin (Gastroin testinal hormones) are also peptides.
13) Insulin (51 amino acids): Secreted from ~-cells of islet of langerha ns. It is a
hypoglyc emic hormone , lowers blood glucose level.
(Note: Some authors include insulin under polypeptides, while others include it under proteins.
By general definition, insulin is a protein.)

Amino acid and Protein Chemistr y 92
Glutat hione (Abbreviated as GSH):

Glutathio ne (or y- glutamyl cysteinyl glycine) is tripeptid e made up of y-glutamic
acid, cysteine and glycine. Glutathio ne is abbrevia ted as GSH, because sulfhydry l
group (SH) of cysteine is the active group of glutathio ne.
( Structure of GSH : y Glutamic acid - Cystein - Glycine )
It exists in 2 forms, reduced glutathio ne (GSH) and oxidized glutathio ne (GS-SG).

GSH GS-SG
(Reduced) (Oxidized)
Reduced form of glutathio ne is biologica lly active and it is a powerfu l reducing

agent, required for many reduction reactions.
Functions:
1. GSH is involved in the anti-oxid ation of toxic oxidants like hydrogen peroxide ,
and superoxi des by its peroxida se activity. This reaction is catalyzed by glutathio ne
peroxidase, a selenium containing enzyme. In this reaction, reduced glutathio ne (GSH)
will be converte d to oxidized glutathio ne (GS-SG).
Glutathione peroxidase
2. GSH required for the intestinal absorption of iron by convering Fe• to Fe• ote that
3 2
(
iron can be absorbed only in Fe • form).

2
3. GSH required for the recoversion of methemoglobin (Fe• ) to Hemoglobin (Fe• ).

3 2
4. Glutathio ne also has a coenzym e role (E.g. Maleyl acetoacetate isomeras e etc.)
5. GSH protects the sulfhydr yl (SH) group of several enzymes I proteins.
6. Meister cycle: Glutathio ne is also involved in the transport of amino acids in the
kidney tubules via Meister cycle or y- glutamyl cycle.
7. GSH is involved in the detoxification of bromobe nzene to mercaptu ric acid.
Importance of glutathione in RBC's: GSH is particular ly rich in RBC's.

• In RBC's, GSH maintains the integrity of RBC cell membrane by detoxifying the toxic hydrogen
peroxide. (which otherwise would disrupt the cell membran e and cause hemolysis ). This
reaction is catalyzed by glutathion e peroxidas e enzyme.
• GSH keeps the hemoglob in in the reduced form (reduced hemoglob in has iron in Fe+2 form).
Wheneve r hemo~lob in (fe+2) is oxidized to methemog lobin (Fe+3), GSH reduces it back to
hemoglob in (Fe+ ). Methemog lobin is unable to carry oxygen.
• GSH protects the sulfhydry l (SH) group of hemoglob in.
Chemistry of proteins :
Proteins are polymers of L-a- amino acids, which are bonded by peptide bonds.
Classification of proteins:
Proteins can be classified on the basis of their Composition, Shape or Functions.
i) Classification of proteins based on chemical composition:

Based on chemical composition, proteins are classified into 3 groups.
a) Simple proteins:
They contain only amino acids. Do not contain any additional groups. Examples are,
• Serum albumin,
• Serum globulins (except immunoglobulins),
• Keratin etc.
b) Conjugated proteins (or Compound proteins):
These have additional Conjugate groups along with amino acids. Examples are,
• Egg albumin (Glycoprotein, containing carbohydrate as conjugate group),
• Hemoglobin (Heme protein containing heme as conjugate group),
• Casein (Phosphoprotein containing phosphate as conjugate group),
• Immunoglobulins (Glycoproteins containing carbohydrate as conjugate group) etc.
c) Derived proteins:
These are the partial hydrolysed products of simple or compound proteins. Examples,
• Gelatin derived from collagen,
• Proteoses & peptones (derived from albumin) etc.
ii) Classification of proteins based on shape (conformation) :

Based on shape, proteins are classified into 2 groups.
1)Globular proteins: These are spherical or oval in shape.
E.g.: Hemoglobin, Album.in and Enzymes.
2) Fibrous proteins: These are elongated and fiber- like structures.
E.g.: Keratin, collagen, elastin etc.
• In globular proteins, the polypeptide chains are folded compactly. The hydrophilic groups of the
constituent amino acids are exposed to the exterior and the hydrophobic groups of the amino acids are
inside to the core of proteins. So, globular proteins are water soluble.
Examples: Hemoglobin, myoglobin, and most of the plasma proteins form globular proteins.
• In fibrous proteins, polypeptide chains are extended. This array of the polypeptide chain gives it tensile
strength & mechanical properties. They are usually components ofstructural proteins.
Examples: Keratin, collagen, and elastin. (These three proteins are also scleroproteins).

iii) Classification of proteins based on the biological function:

Proteins have diverse biological functions, based on which they can be classified as,
1) Catalytic proteins:
All enzymes are protein in nature (exception is ribozymes, which are RNA in nature).
E.g.: Hexokinase, Amylase etc.
2) Defence proteins:
E.g.: lmmunoglobulins as antibodies
3) Structural proteins:
E.g.: Keratin present in hair and nail; Collagen is present in muscles.
4) Hormonal proteins:
Some hormones are protein in nature.
E.g.: Growth hormone, Insulin etc.
5) Contractile proteins:
E.g.: Actin, Myosin and Tropomyosin present in muscle
6) Transport proteins:
E.g.: Serum albumin carries bilirubin, fatty acids etc. Transferrin transports Iron.
7) Storage proteins:
E.g.: Ferritin storage of iron in liver and bone marrow.
8) Visual proteins:
E.g.: Rhodopsin and Iodopsin present in the retina of eye.
9) Membrane proteins:
E.g.: Sodium potassium pump or Sodium potassium ATPase.
10) Haemostatic proteins:
E.g.: Fibrinogen, Prothrombin etc.
11) Buffer proteins:
E.g.: Plasma proteins, Hemoglobin
12) Respiratory proteins:
E.g.: Hemoglobin, Myoglobin
13) Receptor proteins:
E .g.: Insu lin receptors, Glucagon recepto r, s teroid hormone receptors e tc.
14) Genetic proteins:
E.g.: Histones, various tran scrip tion and translation factors.
Answer hint for Protein classification question (5 Marks):
b. Classification based on chemical composition (1.5 Marks)
c. Classification based on biological functions(3 Marks)
F unctional classiification of proteins can alone be asked for 5 Marks.

Protein structures :
• Proteins are made up of one or more polypeptide / protein chains. Proteins with a
single polypeptide chain are called monomeric proteins, while the proteins with more
than one monomeric unit are called oligomeric proteins. Monomeric proteins are
predominant in nature.
• Each polypeptide chain in an oligomeric protein is called subunit or monomer.
Examples for oligomeric proteins are hemoglobin (which has 4 polypeptide chains),
lactate dehydrogenase (which has 4 polypeptide chains).
• Each protein has a unique 3-D structure, which is referred to as its native conformation.
In native form, proteins do not exist in a linear or single dimension form. Instead they
exist as coiled or folded structure or three-dimensional (3-D) conformation. Only such
3-D conformations are biologically active.
• When the proteins are formed on ribosomes, they are synthesized in linear form.
Then they fold and attain native conformation. Formation of active 3-D conformation
from linear form of proteins is considered under structural organization of proteins.
Structural organisation of proteins:

Proteins exhibit four different levels of structural organization.
1) Primary structure
2) Secondary structure
3) Tertiary structure
4) Quaternary structure
Note:
• Secondary, tertiary and quaternary structures are called higher structures.
• Monomeric proteins show only upto tertiary structure. Only the oligomeric proteins
exhibit qua ternary structure. For example, myoglobin, a monomeric protein (having
a single polypeptide chain), shows only first three levels of structure (primary,
secondary and tertiary), whereas hemoglobin, a oligomeric protein (4 polypeptide
chain) shows all the four levels of structure.
Chaperones (Heat shock proteins):

Some proteins can spontaneously undergo folding to attain the active conformation.
But, some proteins require a specialized group of proteins known as chaperones (or heat
shock proteins) that assist in protein folding.
Chaperones reversibly bind with unfolded proteins to cause the proteins to fold and
attain the compact and biologically active conformations.

Primary structure:
Definition :
Primary structure of proteins indicates the number and sequence of amino acid residues
from N - terminal to the C- terminal.
Features:
I) Peptide bonds maintain the primary structure. Peptide bonds form the backbone
of primary structures. Side chains protrude from the sides and in opposite direction.
2) In primary structure, amino acids are arranged in linear chain.
3) The primary structure of each protein has a unique amino acid sequence and numbers,
decided by their genes contained in DNA.
4) Primary structure is not affected during denaturation (as they are made up of strong
covalent peptide bonds).
Importance of Primary Structure :
The primary structure of proteins (i.e. specific sequence and number of amino acid)
determines the higher levels of proteins structure. The biological activities of the
proteins are attributed to their 3- dimensional (higher) structures. Therefore, the
biological activities of proteins are in turn dependent on the primary structure.
Proof: A slight change in the primary structure of proteins may result in the loss of
biological activity of proteins (like, modification of a single amino acid residue as in
mutation). For instance, in sickle cell anemia, change in a single amino acid residue in
the primary structure (change in 6th amino acid in the globin chain), results in loss of
proper function of hemoglobin.
Secondary structures:
Twisting & folding of polypeptide chains results in secondary structures. Secondary
structures refer to the folding of the polypeptide chain, formed by the interaction between
amino acids, which are relatively close in the primary structure of amino acids.
Different secondary shllctures are, a helix,~ sheet, Bends, Loops & Disordered regions.
Among these a helix and psheet structures represent the ordered secondary structures
that are frequently present in proteins. Parts of the polypeptide that are folded in an
ordered fashion often either a helix form or Psheet.
Interspersed between these ordered structures, minor secondary structures like bends
(turns), loops, disordered regions are also present in protein structures.
Normally, a helix and 13 sheet are found in same the protein. For e.g., majority of ordered
secondary structure in hemoglobins is a helix & rest is 13 sheet. Only few proteins like keratin
have exclusive secondary structure, a helix. (So, keratin is also known as a keratin).
Triple helix is a unique structure present in collagen (Structure of triple /1elix and collagen is
explained in detail in Connective tissue chapter.

a Helix:
a Helix is the most common secondary structure found in proteins.
Examples:
a Helix is seen in proteins such as hemoglobin, myoglobin, a Keratin, Collagen etc.
a Keratin has exclusive a-helix structure.
Salient Features :
1) a Helix is a right-handed coiled structure (as proteins are made up of L-amino acids).
2) ex Helix structure is stabilized by extensive hydrogen bonding.

Hydrogen bonds are formed between peptide bonds that are 3 amino acid residues
down the polypeptide chain. (i.e. Hydrogen bond is formed between between CO- & -HN of the
4th residue down the chain). For example, the peptide bond of the 5th amino acid is hydrogen
bonded with the peptide bond of the 9th amino acid, 6th with 10th and so on.
3) The hydrogen bonds are parallel to the a helix.
4) Hydrogen bonds are individually weak, but collectively large number of hydrogen
bonds maintains the helical structure in stable form.
5) Each turn of helix contains 3.6 amino acid residues and each amino acid is separated
by a distance of 1.5 A0 . So the pitch of the helix is 5.4 A0 (3.6 X 1.5 = 5.4 A 0).
6) Proline, hydroxy proline, glycine are considered as helix destabilizing amino acids
(Helix breakers), as they disrupts the formation of a helix, producing a bend. Amino
acids alanine and cysteine promote helix formation.
J-lydrogen bond is formed a-Helix

between 2 peptide bonds Hydrogen bonds are formed between
peptide bonds are 3 amino acid apart
l P1Tc.H
IS S.'iA°
J
------{ M,lrnou£5
1\-t rHGH
COOH

sheet (or J}-pleated sheet) :
1) sheet is an extended structure, where bonding polypeptide chains are arranged in

the form sheets (in a pleated or zigzag pattern, unlike the compact backbone of a helix).
2) sheet is stabilized by hydrogen bonds, formed between the CO & NH of peptide
bonds of adjacent segment of same polypeptide chain. Bonding chains lie side by side.
The hyd rogen bonds are perpendicular to the p olypeptide chains.
3) Parallel / antiparallel sheets: sheet structures can be parallel or antiparallel,
depending on the direction of the bonding chains.
a) Parallel: If the bonding chains run in the same direction N1--~c
(E.g. Flavodoxin) N ------'C 1
b) Antiparallel: If the bonding chains run in opposite direction N1- -~c

(E.g.: Fibroin of silk) C--N
Carbonic anhyd rase shows both parallel and antiparallel sheets.
Parallel Antioarallel
H ...,
® N-C~
"
<?
e~ •
' 0
Minor secondary strnctures:

Interspersed between ordered secondary structures (a-helix and sheet structures), minor
secondary structures like bends, loops, disordered regions are present in protein structures.
• Bends & turns refer to short segment of amino acids that join 2 units of the secondary structures.
For e.g. p bends or p turns refers to the segment that connects ends of adjacent strands of anti-
parallel pleats. Hence the name turns or bends. It involves four amino acid residues, in
which the l51 residue is hydrogen bonded to 4th, resulting in a firm 1800 turn (in reverse direction),
hence it is also called reverse turn or hair pin turn. Praline and glycine often present in P turns.
• Loops: These are irregular regions that contain residues beyond the minimum number necessary
to connect adjacent regions of ordered secondary structures. They play key biological roles. In
some enzymes, the loops that connect the substrate binding domains often contain amino acid
residues that participate in catalysis. For example, in certain DNA binding proteins (like repressors
& transcription factors), loops (li ke helix- loop-helix motifs) provide the portions for binding
with DNA. Though loops lack apparent specific structural regularity, many assume a specific
conformation stabilized through hydrogen bonding, salt bridges & hydrophobic bonds with other
portions of the protein. Super secondary structures refer to the structural motifs such as helix-
loop-helix motifs that are intermediate in scale between secondary and tertiary structures.
• Disordered regions are the structural regions of proteins that are not ordered. In many proteins,
these disordered regions assume ordered conformations upon bindin~ with a ligand.
For queries and sugg!stlons, contact the author at prasad_text@yahoo.com or 9986449575

Tertiary structure:
• Tertiary structure of protein refers to the further folding of secondary structure of
polypeptide chain giving the compact three-dimensional conformation. In monomeric
proteins, tertiary structure results in the formation of final three-dimensional
conformation of the polypeptide chain and formation of the functionally active protein.
• Tertiary structure explains the spatial relationship of amino acids which are far apart
from each other in the linear primary structure, but brought closer as a result of folding.
Tertiary structure results in the orientation of hydrophobic side chains towards the
water free interior and the hydrophilic polar groups towards the surface of the protein.
• Tertiary structure results in the formation of domains. Domains are structurally
connected but functionally independent units of a protein that perform a particular
function, such as binding with substrate (or other ligands) in enzymes etc.
Bonds stabilizing tertiary structure :

• Tertiary structure is stabilized by weak noncovalent bonds like hydrogen bond, van
der Waals forces, ionic bonds and hydrophobic bonds. Even though these bonds
are weak, large number of these bonds gives stability to the structure.
• Some proteins contain strong covalent disulfide (S-S) bonds, which are formed
between two cysteine residues of the same protein chain. Intrachain disulfide bonds
further enhance the stability of the tertiary structure of proteins.
Quaternary structure:
• Proteins having more than one polypeptide chain (oligomeric proteins) show one
more level of higher structure called the quaternary structure. Quaternary structure
refers to the spatial arrangement of the subunits of an oligomeric protein.
• For instance; Hemoglobin has four globin chains. So it shows quaternary structure.
Other examples are Irnmunoglobulins, lactate dehydrogenase etc.
Bonds stabilizing quaternary structure :
• Quaternary structure is stabilized by weak noncovalent bonds like hydrogen bonds,
van der Waals forces, ionic bonds, hydrophobic bonds and also few covalent
disulphide bonds.
• Quaternary structures are interchain interaction between polypeptide chains. (unlike
secondary & tertiary structures, which are intrachain interactions within the same chain).
Note: Some authors include interchain hydrogen bonds (i.e. the hydrogen bonds are formed
between 2 different polypeptide chains) under pleated structure. But it is inaccurate, as
interchain hydrogen bonds are included under quaternary structures. Hydrogen bonding
in pleated structure is intrachain (i.e. hydrogen bond is formed between different
segments of the same polypeptide chain).

Only the oligomeric proteins exhibit quaternary structure:

Monomeric proteins exhibit only 3 levels of structures-primary, secondary and tertiary,
whereas oligomeric proteins exhibit all 4 levels of protein structures including quaternary
structure. In oligomeric proteins, each monomeric unit has its own primary, secondary,
tertiary structure & then these monomeric units interact with each other (quaternary
structure) to form a fully functional oligomeric protein.
Oligomeric proteins lose their biological activity if the subunits are dissociated
Importance of higher stuctures of proteins:

Proteins are biologically active only in their three dimensional conformations. So the
biological activities of proteins are attributed to their higher structures. If a protein
loses its three dimensional form (as in denaturation), it loses its biological activity.
Structural rganization of proteins can be explained by Hemoglobin & Myoglobin structures

• Myoglobin is a monomeric protein, whereas, Hemoglobin is a tetrameric protein having 4
subunits (2a & 2~)- When both Hb and Mb are synthesized by translation, they are formed
in primary stucture. Then they fold by secondary and tertiary structures (and quaternary
structure in case of only hemoglobin) to attain fulJy functional native conformations.
• Myoglobin is a monomeric proteins, it shows only upto tertiary structure.
• Hemoglobin is a oligomeric protein (tetrameric 2a & 2~, shows all 4 levels of structures
including quaternary stucture. Each monomeric unit has its own tertiary structure and then
these monomeric units interact with each other to form fully functional tetrameric Hb.
a a
Primary structure
l
•/ •
Secondary structure
l
Tertiary structure
./
(Myoglobin)
Quaternary structure allP
p a
( Hemoglobin )
Answer hint for Organisation of Protein structure question (10 Marks):

a. Introduction and Definition (1 Mark)
b. Primary structure (3 Marks)
c. Higher structures (secondary, tertiary, quaternary) (5 Marks)
d. Importance (1 Mark)
Individual structures and their bonds can also be asked for 3/5 marks each.

Bonds stabilizing higher stuctures:

Higher structures of proteins are stabilized primarily (and often entirely) by weak non-covalent
bonds like hydrogen bond, van der Waals forces, ionic bonds and hydrophobic bonds. Even
though these bonds are weak, large number of these bonds gives stabilihJ to the structure.
Some proteins contain strong covalent disulfide (5-5) bonds, whicll are formed between two
cysteine residues.
a) Hydrogen bonds:
ln secondary structures, Hydrogen bonds are formed between two peptide bonds. Ill tertian; &
quaternary structures, hydrogen bonds can be formed between two strong electronegative atoms
(like 0, N, 5 etc) by sharing single hydrogen. Hydrogen providing groups are -NH (Histidine,
tryptophan, peptide bond), -OH (hydroxy amino acids like serine, threonine, tyrosine), NH 1
(Arginine, lysine), -SH (Cysteine). Hydrogen accepting groups are carbonyl oxygen (C=O) of
carboxyl groups (aspartate, glutamate) & peptide bonds and disulphide bonds (5-5) etc.
b) Hydrophobic bonds:
Hydrophobic bonds formed between hydrophobic side chains of non polar amino acids like aromatic
amino acids, branched chain amino acids, methionine etc.
These interactions generally take place in the interior of the protein.
c) Ionic bonds (Salt bridges or electrostatic bonds):
Ionic bonds are fom1ed between oppositely charged side chains of charged amino acids. E.g., Ionic
bonds are formed between COO · of side chains of acidic amino acids like aspartate & glutamate
and N H/ of the side chains of basic amino acids like lysine & arginine.
d) van der Waals forces:
van der Waals forces of attractions are formed between the atoms due to oscillating dipoles (neutral
amino acids).
e) D isulfide bonds:
Disulfide (5-5) bonds are strong covalent bonds formed between two cysteine residues to form
cystine residue. lntracliain disulfide bonds stabilize the tertiary structure of proteins and
lnterchain disulfide bonds stabilize the quaternary structure of certain oligomeric proteins.
Peptide
Bond Serine Lysin! Alanine Prenylalanine Cysteine
6
I I I I I
N 0-UOH NH3+ CH, s
I I
H /
/
I
/
0 0 0
11
Q
I I
C C- O C=O CH3 s
Peptide Aspartate Aspartate Alanine Phenylalanine Cysteine

Bond
'-------y--J '-------v---' ----v----
Hydrogen bond Ionic bond Hydrophobic bood Oi;ulfide bond

Insulin
Insulin is a hypoglycemic hormone synthesized from cells of pancreas.
Salient features of human insulin structure (Primary stucture):

• Primary structure of insulin contains 51 amino acids (polypeptide hormone).
• It consists of 2 chains, A chain and B chain. A chain contains 21 amino acids
and B chain contains 30 amino acids.
• Amino terminal of A chain is Glycine and that of B chain is Phenylalanine.
• Carboxy terminal of A chain is Asparagine and that of B chain is threonine.
• The 2 chains are connected b y inter-chain disulfide bridges between cysteine
residues 7th of A chain and 7th of B chain and 20th of A chain and 19th of B chain.
• There is one intra-chain disulfide bridge in A chain connecting 6th with 11th Cys.
Structure of Insulin
S -- -- S
A chain H2N - Gly - Cys - Cys ---- Cys ------- Cys - Asn - COOH
1 6 7 11 20 21
s s
I I
s s
B chain ... - - Cys --------------- Cys ------------- Thr - COOH
HiN - Phe
1 7 19 30
Significance of Insulin structure:

• Functions of insulin is compromised in diabetes mellitus. One of the most important
methods of treating diabetes is the intravenous injection of Insulin.
• Insulins from other animals like cattle, sheep, horse etc differ from human insulin
in having a different sequence of amino acids in the positions 8, 9, 10 in A chain.
Pig insulin differs from human insulin in only one position. 30th amino acid in B
chain is alanine instead of threonine.
• All these insulins have comparable activities when tested in human beings. Thus
this minor altered sequence does not result in appreciable change in biologic
activity. This is a valuable information used in utilizing animal insulin for human
use in the treatment of diabetic patients.
• Nowadays, human insulin is being produced by recombinant DNA technology.

Denaturation of proteins:
Definition: The process of disorganization of native protein structure is called
Denaturation. Denaturation involves the loss of secondary, tertiary and quaternary
structures without breaking the primary structure.
Denaturation causes the unfolding of native 3-dimensional form of proteins.
Reason: Higher structures of proteins (3-D conformation) are maintained by weak
non-covalent bonds, which can be easily disrupted b y a variety of physical and
chemical agents, whereas the primary structure of proteins are not easily broken
because they are maintained by strong covalent peptide bonds.
Denaturation
Native Denatured Form
Agents causing denaturation:

Denaturation can be caused by various physical and chemical agents.
i) Physical agents: Pressure, heat, X-ray, UV radiation, ultrasound etc.
ii) Chemical agents: Acids, alkali, organic solvents (ether, alcohol etc.), high
concentration of urea, salicylates and heavy metals (lead, mercury, silver etc).
Properties: Denaturation involves a change in physical, chemical and biological
properties of protein. Denaturation decreases the solubility of a protein, increases the
precipitability and increase the viscosity of proteins.
Biological relevance of denaturation: Proteins are active only in their 3-D form.
Since 3-D form is lost during denaturation, proteins lose their biological activity.
Denatured proteins are digested easily. This is due to increased exposure of peptide
bonds to proteolytic enzyme action. For instance, cooking denatures dietary proteins.
This is why cooked protein is easily digested.
• So, denatured proteins though biologically inactive, nutritionally advantageous.
• Renaturation: Dena turation is generally irreversible. But, sometimes when the

denaturating agents are removed, the process is reversible forming the original structure
of proteins (as primary structure is intact). This is known as renaturation.
• Coagulation:When heated, irreversible denaturation results in coagulation of proteins.
• Flocculation: Process of protein precipitation at isoelectric pH is called flocculation
and the precipitate is referred to as flocculum . Flocculation is reversible. On application
of heat, flocculum can be converted into an irreversible mass, coagulum.

Structure-function relationship of proteins:

• There's a definite relationship between structure & function of proteins. Proteins are
biologically active only in their 3-0 conformations, called the native conformation, which
is thermodynamically favoured. In 3-D conformation, hydrophobic side chains are
oriented towards the water free interior and the hydrophilic polar groups towards the
outer surface. This is significant as proteins generally exposed to aqueous environment.
• 3-0 conformation results in the formation of domains. Domains are structurally
connected but functionally independent units of a protein that perform a particular
function, such as binding with substrate (or other ligands) in enzymes etc.
• The biological activities of proteins are attributed to their 3-0 structures. If a protein
loses its three dimensional form (as in Denaturalion), it loses its biological activity.
• The three dimensional structural conformation, in turn, is dependent on primary
structure. So, any difference in primary structure may alter the thee-dimensional
structure, which may be functionally inactive or less active. For instance, in sickle cell
anemia, change in a single amino acid residue in the primary structure (change in 6th
amino acid in the globi.n chain), results in loss of proper function of hemoglobin.
• The diversity of relationship between protein structure and functions can be
understood by the study of structure and functions of some important proteins like
hemoglobin, Myoglobin, Immunoglobulins, Collagen, Elastin etc. (Refer Hemoglobin,
Plasma proteins and Connective tissue chapter respectively).
Charged properties of proteins:

Charged properties of protein are attributed to the ionisable group present in side
chains of amino acid resid ues. (Glutamic acid, aspartic acid, lysine, arginine, histidine),
in addition to terminal a-amino and a-carboxylic group.
Amphoteric nature of proteins: Depending on the pl I of solution & isoelectric pH of
proteins, some of the ionisable groups of side chains (of basic & acidic amino acids),
terminal a-amino & o:-carboxylic groups act as proton donors or proton acceptors. So,
proteins are amphoteric in nature (Ampholytes), i.e. they behave both as acids or bases.
Note : The charged state at which a proteins exists at any given time is determined by two
parameters, namely, a) pl (lsoclectric pH) of the proteins a11d b) pH of the medium
• At pH = pl, proteins exist as zwitterions (neutral clrarged molecules).
• At pH < pl, proteins exist as cations (positively charged molewles).
• At pH > pl, proteins exist as anions (negatively charged molecules).
Protein + •- H + + + Protein · _. H + + Protein -
(Cation) (Zwitterion) (Anion)
(pH< pl) (pH= pl) (pH> pl)
When an electric field is applied, both the cationic and anionic forms of proteins move. Anions
moves towards a11ode and cations move toward:. cathode.
This property is utilized in the separation of protein by electrophoresis.

Isoelectric pH (pl) of proteins:

Definition: lsoelectric pH (pl) of a protein is defined as the pH at which the proteins
exist as zwitterions carrying equal number of positive & negative charges.
E.g.: pl of Pepsin - 1.1, Casein - 4.6, Albumin - 4.7, Human Hb - 6.7 etc.
Properties:
1) A t pl, net charge is zero (because, proteins carry equal number of positive and
negative charges) and hence, protein does not move in an electric field.
• At pH< pl, proteins exist as cation forms (positively charged molecules).
• At pH > pl, proteins exist as anion forms (negatively charged molecules).
This property is exploited in electrophoresis of serum proteins. When an electric field
is applied, anions forms moves towards anode & cations move towards cathode.
2) Proteins have least buffering capacity and viscosity at isoelectric pH:
3) Proteins have least solubility and maximum precipitability at isoelectric pH:
At pl, proteins carry no net charge, thus they exert minimum electrostatic repulsion.
So at isoelectric pH proteins tend to aggregate and precipitate.
This property is exploited in isoelectric precipitaion of proteins.
Isoelectric precipitations of proteins:

Definition: Precipitation of proteins at their isoelectric pH is called isoelectric
precipitation.Some proteins are immediately precipitated when adjusted to their
isoelectric pH. The best example is casein, which gets precipitated immediately when
the pH of casein solution is adjusted to 4.6 (lsoelectric pH of casein is 4.6).
Explanation: Proteins are least soluble at their isoelectric pH. At isoelectric pH, proteins
exist as zwitterions, carrying equal number of positive and negative charges and hence
exert minimum electrostatic repulsion. Thus, proteins like casein tend to aggregate
and precipitate readily at isoelectric pH. So, when the pH of casein solution is adjusted
to 4.6 (Isoelectric pH of casein is 4.6).
Biuret test:
This test is used as a general test for proteins, as it is answered by all the proteins.
Procedure: Copper sulphate is added to protein solution in the presence of NaOH.
Observation: A violet color is obtained.
Principle: Cupric ion in alkaline medium forms a violet colored complex with peptide
bond nitrogen of proteins. The minimum requirement for a positive biuret test is the
presence of 2 peptide bonds in the molecule. Biuret test is specific for CO-NH bonds.

Question bank on Amino acid and Protein chemistry .
Essays (10 Marks) :

1. Give an account of structural organization of proteins? Add a note on Denaturation
2. Classification of proteins based on composition, shape and biological functions
Short essays (5 Marks) :

1. Classify proteins based on their functions with examples
2. Describe primary structure of proteins and their significance? Add a note on Peptide bond
3. Essential amino acids
Short notes (2 -3 Marks) :

1. Write the names of three biologically important peptides and their functions
2. a Helix structure and p p leated structure
3. Give brief account on higher structure of proteins and bonds stabilizung them
4. Isoelectric pH of proteins and isoelectric precipitation of proteins

1) Which amino acid can protonate & deprotonate a t neutral pH (AIIMS)
a) Histidine b) Leucine c) Glycine d) Arginine
2) In quaternary structure of proteins, subunits are linked by
a) Peptide bond b) Disulphide bonds c) Covalent bond d) Non-covalent bonds
3) The amino acid that lacks chirality (AIIMS)
a) Lysine b) Leucine c) Histidine d) Glycine
4) All are essential amino acids, except (AI)
a) Phenylalanine b) Lysine c) Leucine d) Glycine
5) Which of the following amino acids is purely ketogenic (COMEDK)
a) Phenylalanine b) Leucine c) ProHne d) Tyrosine
6) The structure that is retained when a protein is denatured
a) Primary b) Secondary c) Tertiary d) Quaternary
7) lmidazole ring is present in
a) Arginine b) Tryptophan c) Histidine d) Proline
8) Example of scleroprotein is
a) Glutenin b) Albumin c) keratin d) Gliadin
9) Which of these amino acids contain indole group?
a) Tryptophan b) Arginine c) Histidine d) Lysine
10) The number of amino acids in insulin is
a)21 b)31 c)41 d)Sl
AnswersforMCQ: J)a 2)d 3) d 4)d 5)6 6)a 7)c 8)c 9)a l 0)d

Nucleotide and Nucleic acid Chemistry 107
Chemistry of Nucleotides and Nucleic Acids
Contents:
• Nucleotide Chemistry
• Definition
• Types
• Biological Importance of free nucleotides
• Nucleic Acid Chemistry

• Definition
• Types
• Difference between DNA and RNA
• DNA: Structure and Functions
• RNA: Types, Structure and Functions
• Genetic codes

Chemistry of Nucleotides
Definition:
Nucleotides are building blocks of nucleic acids (DNA and RNA).
Components:
Each nucleotide is made up of 3 components
i ) Ni trogenous base ii) Pentose sugar iii) Phosphate group
i) Nitrogenous base:
May be either purine bases or pyrimidine bases.
a) Purine bases:
Adenine and guanine are major purine bases
• Adenine (6-aminopurine)
• Guanine (2-amino 6-oxopurine)
b) Pyrimid ine bases:

Cytosine, Thymine and Uracil are pyrimidine bases.
• Cytosine (2-oxy 4-aminopyrimidine)
• Thymine (2,4-dioxy 5-methylpyrimidine)
• Uracil (2,4-dioxypyrimidine)
ii) Pentose sugar:

Pentose sugar can be either ribose or deoxyribose sugar.
DNA has deoxyribose sugar and RNA has ribose sugar.
iii) Phosphate molecules:

Nucleotides can have one, two or three phosphate molecules; consequently Nucleotides
are called mono, di and tri nucleotide phosphates.
E.g.: AMP, ADP, ATP have one, two and three phosphate molecules respectively.
Xanthine and hypoxanthine: Minor purine bases (Not present in DNA and RNA).
• Hypoxai1thine (6-oxopurine)
• Xanthine (2,6-dioxopurine)
• Uric acid (2,6,8-trioxypurine)

Note:
• Nucleoside: Nitrogenous base+ Pentose sugar
• Nucleotide: Nitrogenous base+ Pentose sugar+ Phosphate(s)
[i.e. Nucleotide= Nucleoside + Phosphate(s)]
Similarly, Deoxy nucleotide: Nitrogenous base+ Oeoxy pentose sugar+ Phosphate(s)
Nitrogenous base ucleosides Nudeotides
Adenne Adenosine A..\IIP, ADP, ATP
Guanine Guanosine GMP, CDP,GTP
Cyiosine Cytidne CvlP, CDP, CIP
Thymine Tl~pnidine nfi>, IDP, TIP
"C"raci1 l:ridine L~P, LDP, L7'P
Similarly, Xanthine gives Xanthosine & XMP; Hypoxanthine gives Inosine & IMP.
Functions of free nucleotides:

Nucleotides are the building blocks of nucleic acid. In addition to this, free nucleotides
in the body perform various functions like energy metabolism, protein synthesis,
regulation of enzyme activities, signal transduction and variety of metabolic activities.
I) Functions of adenosine nucleotides:
1) ATP:
• ATP is the energy currency of the cell. It is the universal carrier of energy within the
body. ATP is required for the provision of energy for muscle contraction, transmission
of nerve impulses and transport of nutrients across the membrane.
• ATP is required for the ligase type of enzymatic reactions. Energy is released w hen
ATP is hydrolyzed to ADP and Pi. E.g. Pyruvate carboxylase.
• ATP is also required for energy transfer when ATP is hydrolyzed to AMP and PPi.
E.g. Acyl CoA synthase
• ATP is involved in p hosphate transfer reactions. E.g. Glucokinase reaction.
• ATP is involved in pyrophosphate transfer reactions. E.g. PRPP synthetase.
• ATP is involved in adenosyl transfer reactions. E.g. SAM synthesis.
• Cyclic AMP a secondary messenger is formed from ATP (by adenylate cyclase).

2) Coenzymes:
Few coenzymes have adenosine nucleotides.
E.g. NAO, NADP, FMN, FAD
3) PAPS (Phosphoadenosine phosphosulphate) or Active s ulfate:

PAPS act as a sulfate donor for many sulfation reactions.
E.g.: PAPS is required for the synthesis of sulfated glycosaminoglycans.
PAPS is also required for certain detoxification process.
4) SAM (S-Adenosylmethionine):
SAM functions as a methyl donor in methylation reaction.
E.g.: SAM is required for the synthesis of epinephrine from nor-epinephrine.
II) Functions of guanosine nucleotides:

1) GTP is required for provision of energy during protein synthesis.
2) Cyclic GMP is a secondary messenger formed from GTP by guanylate cyclase.
III) Functi ons of cytidine nucleotides :
1) CDP choline is required for the synthesis of lecithin (Phosphatidyl choline).
2) CDP ethanolamine is required for synthesis of cephalin (Phosphatidyl ethanolamine).
IV) Functions of uridine nucleotides:

1) UDP glucose: (Uracil-ribose-(P)-(P)-Glucose)
UDPG is carrier of glucose in the synthesis of glycogen, glycoproteins and proteoglycans.
2) UDP-glucuronic acid: (Uracil-ribose-(P) - (P) - glucuronic acid)
UDP glucuronic acid is required for detoxification of bilirubin.
Cyclic nucleotides: Cyclic AMP (cAMP) & Cyclic GMP (cGMP) are 2 cyclic nucleotides.
cAMP is formed from ATP by adenylate cyclase enzyme and cGMP is formed from GTP by
guanylate cyclase. Both act as second messengers in hormone actions.
Answer hint for Function of free nucleotides (5 Marks):

a. Functions of Adenosine nucleotides (2 Marks)
b. Functions of Guaosine nucleotides (1 Mark) Exam tip
c. Functions of Cyrosine nucleotides (1 Mark)
d . Functions of Uridine nucleotides (1 Mark)

Nucleotide and Nucleic acid Chemistry 11 1
Nucleic acid Chemistry

Definition: Nucleic acids are polynucleotides.
There are 2 typ es of nucleic acid:
1) Deoxyribonucleic acid (DNA); 2) Ribonucleic acid (RNA)
D ifference between D N A and RNA
' S.ga, • °""'Y

O.N.A.
ri- .
2. itrogenous bases are adenine,
guanine, cyt()!,ine and Thymine
+ R.N.A
Sugar is ribose
Nitrogenous base!, are:

Adenine, guanine, cytosine and u racil.
I
3. Double ~tranded Sing le stranded.
4. A=T ,C=C (Chargaff's rule) A# U;C#G
5. Alkali resistant Easily destroyed by alkali
6. Self replicated Formed from D.N.A.
7. Mii lions of base pairs Us ually only 100-1000 bases

I
8. Present in nucleus Generally present in cytosol
I
DNA (Deoxy-ribonucleic acid):

DNA is the fundamental unit of genetic information.
Structure of D NA:
DNA is a polymer of deoxy-ribonucleotides. Bases present are adenine, guanine, cytosine
& thymine. Sugar present in. DNA is deoxy ribose. The monomeric deoxyribonucleotides
are held together by 31-51-phosphodiester bonds.
There are many different forms of DNA. Among these, B, A and Z forms are important.
• B form: Right handed double helix, has 10 base pairs per turn.
• A form: Right handed double helix, has 11 base pairs per turn.
• Z form: Left handed double helix, has 12 base pa irs per tum.
In physiological conditions, B-form of DNA is predominant.
Watson & Crick proposed the double helix model to explain the structure of B-DNA.

Salient features of double helix model (Watson and Crick model):

1) DNA is composed of 2 polydeoxyribonucleotide chains, which are twisted around
each other spirally. (The pentose sugar & phosphate present on the outer side and
bases are present on the inner side of the chain).
2) DNA forms the right handed double helix structure.
3) Each chain has polarity. The base sequence in the chain is always written from 51 to
3 1 direction. (As the individual monomers are bonded by 31-5 1-phosphodiester bonds).
4) The 2 strands in the DNA molecule run anti-parallel to each other. (i.e. one strand
runs in 5 1 to 3 1 direction while other strand runs in 31' to 5 1 direction).
5) The 2 chains are held together by hydrogen bonds between the bases.
Base pairing rule: The base adenine of one chain is always paired with thymine of
another chain with two hydrogen bonds. Similarly cytosine of one chain is bonded with
guanine of another chain with three hydrogen bonds. This is called base pairing rule.
Thus 2 strands are complementary to each other. This is explained by chargaff's rule.
Chargaff's Rule: It states that the sum of purine nucleotides (A+G) is equal to sum of
pyrimidine nucleotides (C+T). [A+ G = C + T]. This is because of base pairing rule.
Helix Specifications
• Pitch of the helix is 3.4 nm.
• Width (distance between 2 strands) is 2 nm
• Distance between 2 bases in the chain is 0.34 nm
• Each helical turn has 10 base pairs.
Minor
groove
Denaturation of DNA and melting temperature:
The double stranded DNA can be separated by
high temperatures. This is called denaturation. It
Major
groove takes place over a temperature range, the midpoint
is called the melting temperature (Tm). DNA rich
in GC region has higher Tm than AT region, because
GC bond is more stronger than the AT bond, as
there are 3 hydrogen bonds between C & G, while
there are 2 hydrogen bonds between A & T.
Functions of DNA:
DNA is the fundamental unit of genetic information. The genetic information stored
in the DNA serves two functions;
1. DNA is the chemical basis of expression of characters: DNA contains the
information for the synthesis of all the protein molecules of the body.
The information contained in the DNA is first copied into RNA molecules (by
transcription), which then directs the synthesis of proteins (by translation).
2. DNA is the chemical basis of heredity: It provides the template for the transferring
the genetic information from the parent cell to daughter cell (by replication). This
maintains the genotype in offspring.

RNA (Ribonucleic acid):

RNA is the polymer of ribonucleotides. Sugar is ribose. Bases present are Adenine,
Guanine, Cytosine, Uracil. RNA is generally single stranded.
There are 3 major types of RNA
1) Transfer RNA (tRNA)
2) Messenger RNA (mRNA)
3) Ribosomal RNA (rRNA)
Besides these, two minor forms of RNA
1) Heterogeneous nuclear RNA (hnRNA): These are precursors of mRNA
2) Small nuclear RNA (snRNA): These aid in the conversion of hnRNA to mRNA.
tRNA (Transfer RNA):

tRNA is the carrier of amino acid during protein synthesis. Each t-RNA is made up of
75 to 80 nucleotides. tRNA makes up to 20% of total RNA.
Structure: tRNA folds on itself to form a clover leaf like secondary structure.
Salient features:
• 51 end of tRNA start with guanine.
C AcCEPro~ ARM •Darm (DHU arm): 1st arm from 5 end is called
C
Darm (or DHU arm) as it contains the unusual
base, dihydrouracil (DHU).
• Anticodon arm: Anticodon arm contains
anticodons that are complementary to codons of
mR A. This arm contains unusual base
hypoxanthine.
D AltM
• Variable arm (Extra arm): Variable arm (or
extra arm) of tRNA has a length about 3-21 bases.
• T"l'C arm: ext to extra arm is T'PC arm. This
contains unusual bases like thymine and
pseudouracil.
• Acceptor arm (CCA arm): Acceptor arm or 31
end have base sequence of C-C-A.
• Role of diffrent arms of tRNA: DHU arm serves as a recognition site for specific amino acyl
tRJ\JA synthetase enzyme, which adds specific amino acids to tRNA. A11ticodo11 ann i~ required
for binding with codon of 111RNA during translation. It gives specificity to the amino ncyl tRNA.
Variable arm is useful for species identification T,,C arm is involved in binding of the tRNA to
ribosomes. Acceptor amz binds amino acids for the transport to ribosome.
Functions of tRNA:
tRNA carries amino acids to the ribosomes during protein synthesis. Each tRNA is
specific for an amino acid, but some amino acids are carried by more than one tRNA.

rRNA (Ribosomal RNA):

• rRNA constitutes 60 to 70% of total RNA's of the cell.
• Most of the rRNA combines with protein and exist as ribosomes. Thus ribosome is
nucleoprotein particle.
• Ribosomes have 2 sub unit a large subunit and small subunit. The prokaryotic
ribosomes are 70 S ribosomes, made up of larger 50 S and smaller 30 S subunits. The
eukaryotic ribosomes are 80 S ribosomes, made up of larger 60 S and smaller 40 S
subunits. Each subunit (larger and smaller) exists in dissociated form. Association of
these subunits takes place during protein synthesis. The complete ribosome has 2
sites. (A site is amino acyl site and P site is peptidyl site).
Function of rRNA: Ribosomes are factory of protein synthesis. On ribosomes, mRNA,
tRNA interacts to translate the codons present in mRNA to the specific sequence of
amino acids in the polypeptide chain.
mRNA (Messenger RNA):

• mRNA is synthesized from the template strand of DNA. Thus mRNA synthesized
will be complementary to the template strand (or similar to the non-template strand).
• The genetic message encoded in DNA is transcribed to mRNA. But the site of synthesis
of protein is ribosomes present in the cytosol. So mRNA from the nucleus is transported
to the cytoplasm. Since mRNA carries the message from DNA present in the nucleus to
ribosomes present in the cytosol for protein synthesis, it is called the messenger RNA.
mRNA acts as template for protein synthesis.
Structure:
• 51 end of mRNA start with 7 methyl GTP (hence called 7 methyl GTP cap), which
protects mRNA from 5' exonuclease action.
• 3' end of mRNA has a polyadenylate tail of 22 - 250 AMP residues (hence called Poly
A tail), which provides stability and protects from 3' exonuclease action.
• Between the 5 1 and 31 ends, there are number of codons.
sI• nd
7MethylCTP A -A -A -A -A -A -A
r
7 M•th y l C TPcap
r
R lbo n tleo t ides
r
P o ly A ta i l
Function of mRNA:
Each codon is a sequence of 3 bases (triplet codon). Using 4 types of nucleotides (A,
G, C and U) 64 triplet codons are possible. Out of these 64, only 61 codons codes for
amino acids, other 3 codons are called the nonsense codons or chain termination
codons. Since these codons codes for 20 amino acid. Some amino acids are coded by
more than 1 codon. AUG is the chain initiation codon which codes for methionine.
UAA, UGA and UAG are the nonsense codon or chain termination codon, because
the protein synthesis stops or ends whenever these codons occurs on mRNA.

Genetic code:
Definition:
The genetic code is defined as the specific nucleotides sequence present in mRNA.
Genetic code directs the synthesis of proteins with specific amino acid sequences.
Characteristic of genetic code:

a) Triplet codon:
The genetic code is present as codons. Each codon consists of three bases (Triplets) on
rnRNA. Using 4 bases (A, G, C, and U), 64 codons are possible. Out of these 64 codons,
3 codons (UAA, UAG, UGA) are called nonsense codons or chain termination codons
because they do not code for any amino acids and the protein synthesis stops or ends
whenever these codons occurs on mRNA. Other 61 codons code for 20 different amino
acids. In these, AUG (present in 5' end of mRNA), which codes for amino acid
methionine is called Initiator codon. (Some of examples of codons are UCU = Serine,
UUU = Phenylalanine, AGU =Serine, GUU = Valine, GGU = Glycine etc.).
b) Unambiguous (Specific):
A codon always codes for a single specific amino acid.
c) Degenerative:
Most amino acid s are coded by more than one codon.
E.g.: Serine has 6 codons, Glycine has 4 codons.
(Only methionine and tryptophan are coded by single codon)
d) Universal:
Same codon codes for same amino acids in all the organisms.
e) Non overlapping and comma less:
The codons are read from 5' to 3' direction continuously from AUG without a ny
punctuation.
Wobbling and Wobble hypothesis:

The relatively loose pairing between the base at 3/ end of codon and complementary
base at 5/ end of the anticodon is called wobbling.
The first two bases of codon on mRNA always form strong complementary base
pairing with anticodon on tRNA, but the complementa ry pairing of codon and
anticodon can wobble at the third base. This is because the third base (31 base) in
codon sometimes fails to recognize its complementary base in the anticodon (Sf base).
This leads some tRNAs to recognize more than one codon.
E.g., The two codons for arginine, AGA, AGG, can bind to the same anticodon UCU.
Degeneracy of genetic code mostly explained by Wobble hypothesis. The degeneracy
and wobbling phenomenon together reduces the occurrence of mutation.

Question bank on Nucleotide and Nucleic acid chemistry
Short Essays :
1) Explain the function of free nucleotides (Nucleotides of biological importance)
2) Difference between DNA and RNA
3) Structure of DNA (Watson and Crick model)
4) Name the nucleic acids and give their biological functions
5) Structure and functions of tRNA
6) Genetic code

1) What is biological significance of DNA?
2) rRNA
3) Triplet codon
4) Initiato r codon
5) Nonsense codon
6) Wobble hypothesis

1) On complete hydrolysis of DNA, we will get all of the following except (AIIMS)
a) Adenosine b) Purine bases c) Phosphoric acid d} Deoxy pentose sugar
2) Which type of tRNA has the highest percentage of modified base? (AI)
a) mRNA b) tRNA c) rRNA d) snRNA
3) Which base is not found in DNA (AI)
a) Adenine b) Guanine c) Cytosine d) Uracil
4) The initiating codon for protein synthesis is (AIIMS, MAHE)
a) AUG b) UAA c) UUU d) UAG
5) Which form of DNA is seen predominantly (AIIMS)
a) A b) C c) B d) Z
6) Left handed double helix is seen in
a) A form b) B form c) E form d) Z form
7) The unusual base seen in tRNA molecule include all EXCEPT
a) DHU b} Pseudouridine c) Hypoxanthjne d) Xanthine
8) 2-amino 6-oxopurine is
a) Ad enosine b) Guanine c) Hypoxanthine d ) Uric acid
9) Dihydrouracil is p resent in
A) hnRNA B) mRNA C) tRNA D) rRNA
10) Following are the features of Genetic code EXCEPT
A) Universal B) Unambiguous c) Overlapping D) Degenerative
AnswersforMCQ: l)a 2)b 3) d 4)a S) c 6)d 7)d 8)b 9)c lO}c

Enzymes 117
Enzymes and Metabolic Regulations
Contents:
• Definition, chemical nature

• Mechanism of enzyme action: Lock and key model and induced fit theory
• Active site, Substrate binding site, Catalytic site
• Holoenzyme, Apoenzyme, Cofactor, Coenzyme, Activator, Prosthetic groups
• Proenzyme or Zymogen
• Classification with examples
• Enzyme Specificity
• Factors affecting enzyme activity
• Enzyme inhibition and significance
• Control (Regulation) of enzyme activity
• Diagnostic Enzymology (Clinical importance of enzymes)
• Therapeutic Enzymes

Enzymes 118
Definition:
Enzymes can be defined as the "Biocatalysts synthesized by living tissues, which
increase the rate of reaction without getting consumed in the process".
Enzymes are p roteins in nature (exception - ribozymes), thermolabile and colloidal
in character, specific in action .
Note: Enzymes are proteins in nature (exception being ribozymes, which are RNA in nature). So
they follow the physical & chemical properties of proteins. (i.e., like proteins, enzymes are
thermolabile, colloidal in nature & lose their activity during denaturation.
Mechanism of enzyme action :

Enzymes act by binding with the substrate (reactants) and lowering their activation
energy. (Activation energy is the energy required by reactants to undergo reaction).
For a chemical reaction A __. B to occur, the reactant molecule should acquire
sufficient energy to attain an activated condition called the transition state. This energy
is called energy of activation or activation energy (Eact).
(Eact = energy difference between transition state intermediate and reactant molecule.)
Enzymes act by binding with substrate (reactants) and lowering their energy of activation
by providing an alternative pathway, which requires less activation energy. Thus, in
enzymatic reactions more reactant molecules can cross the energy barrier than in
uncatalysed reactions.
Tl
A = Free Energy of the substrate

B = Free Energy of the product
Tl = Free Energy of the Tran,,ition
state intermedlate of the
uncalal)'U'd reaction
T2 = FrL-e Energy of the transition
Note that enzym es do
state intermediate of the not c hange th e free
cataly,ed reaction energies of reactants or
prod u cts, t h erefore
does not alter the final
equilibrium
Course of the Reaction
Importance of enzymes: A reactant can attain this energy of activation by raising the temperature,
which increases the kinetic energy of reaction, and I or altering the pH or adjusting the Ionic
strength. But in living organisms, the temperature, pH and ionic strengths must be maintained
within physiological limits. So, in the absence of enzymes, only a Jew molecules may possess
sufficient energy to cross the energtJ barrier (at a relatively low body temperature of 37° C).

Enzymes 119
So, the prime requisite for the enzymatic reaction is the binding of enzymes with the
substrate to form Enzyme-Substrate complex (ES), which later dissocia tes to form
product and free enzyme.
E + s------ ES E + p
Active site (or Active center) :

Active site of an enzyme can be defined as the dynamic region of enzyme, where the
substrate binds and gets converted to products.
Active site includes both substrate binding site and catalytic site.
a) Substrate binding site:

To initiate an enzymatic reaction the substrate has to bind with the enzyme. Enzyme
has a site where substrate binds. This site is called the substrate-binding site, which is
responsible for substrate specificity.
b) Catalytic site:
After binding with the substra te the enzyme is in ES form. Now substrate is converted
to product. A site called catalytic site of enzyme brings about this transformation.
The catalytic site is responsible for the reaction specificity.
Active site = Substrate binding site + Catalytic site
Additional information on active site:

• The active site is a cleft I crevice in the 3-D conformations of enzymes. It is constituted by
several amino acids that are located at different places in the primary structure ofenzymes, but
brought together as a result offolding of polypeptide chains. Side chains of these amino acids
contribute to the active site. lnside the cleft, side chains ofsome amino acid residues contribute
to substrate binding and some contribute in catalysis.
For example; Lysozyme has 129 Arnino acid residues. Active site is constituted by 35, 52, 62,
63, 101 st amino acids. 35 (Glutamate), 52 (Aspartate), 62 & 63 (Tryptophan), 101 (Aspartate).
Among these, 62, 63, 101 amino acids constitute substrate binding site & 35, 52 constitute
catalytic site.
Invariably substrate binding site and catalytic site overlap each other.
Some times enzymes require cofactors for their activity. In those enzymes where cofactors are
required for the activity, the cofactors are also form a part of catalytic site.
The environment of active site is hydrophobic and water is admitted only if it is one of the
substrate.
Conformation ofenzyme, active site & ES are determined by X-ray crystallography. Now it is
possible to see these with the help of electron microscope.

Enzymes 120
Enzyme substrate complex [ES complex] formation:

Enzyme activity begins with the binding of enzyme with the substrate to form substrate
complex. Michaelis and Menten first proposed the enzyme substrate complex hypothesis
in 1913, which is accepted even today.
Many theories have been put forth to explain the mechanism of formation of ES complex.
Among them Fischer's Template theory (Lock & Key Model) and Koshland's induced
fit theory are the most accepted ones.
1. Fischer's template theory (Lock and key model):

According to this theory, the 3- Dimensional structure of active site of enzymes is
rigid, pre-shaped template and complementary to the shape of the substrate. Thus
the substrate binds to the active site of enzyme, like a key into the proper lock.
This theory have proved to be correct for many enzymes (Enzymes which exhibit
absolute specificity), however it does not explain the flexibility of some enzymes
especially allosteric enzymes.
+
v
Enzyme Substrate ES Complex
2. Koshland's induced fit theory (or Model):

According to this theory, the active site is not a rigid and pre-shaped template and is
not complementary to substrate (i.e. active site is not fully active in the absence of
substrate). The initial interaction of substrates with the enzyme (either the initial
approach of the substrate or the initial superficial binding of the substrate with the
enzyme) will induce a conformational change in the enzyme, resulting in the formation
of the fully functional active site (like strong substrate binding site & proper orientation
of catalytic sites), which then strongly bind with the substrate & carry out the reaction.
-
This theory explains the allosteric regulation of enzymes.
Enzyme Substrate ES Complex
• Substrate Strain Theory:

According to this tl1eon;, when n substrate binds to the active site of enzyme, it brings about the
conformational changes in tlte enzymes, which in turn induces n strain on tlte substrate. The strained
substrate is then co11verted to products.
Tltis theory is n continuation of Induced fit theory & i11 combinntio,1 with it, is co11sidered to be
operative in majority of the enzyme actions.

Enzymes 121
Cofactors (Coenzymes and Activators) :

Most enzymes are proteins. Some enzymes require certain non-protein part in ad dition
to their protein part for their full activity. This non-p rotein part is called the Cofactor.
Protein part + N on-protein part = Active enzyme
(Apoenzyme) (Cofactor) (Holoenzyme)
Cofactors can be organic or Inorganic compounds. If the cofactor is organic compound,

it is called Coenzyme and if cofactor is inorganic compound, it is called Activator.
Examples are,
• Coenzymes: NAO, FAD, TPP etc (Organic cofactors).
• Activators: FeJ+, Cu2+, Mg2+ etc (Inorganic cofactors)
Difference between coenzyme and activator can be shown by the following reaction.
Acetyl CoA carboxylase

Acetyl CoA + C•.? /""" ---......... 11 Malonyl CoA
/ Mn~· ........._
ATP Biotin ADP + Pi
In this reaction, both Biotin and Mn2+ are cofactors of acetyl CoA carboxylase enzyme.
Biotin is the coenzyme and is Mn 2+ the activator.
Prosthetic group:
Definition: If the cofactor is attached tightly (covalently) to the enzyme then it is called
as the Prosthetic group.
E.g.: Binding of biotin to carboxylase enzymes.
Binding of molybdenum to xanthine oxidase enzyme
Metalloenzymes and Metal activated enzymes:

Some enzymes require i11orga11ic (metal) cofactor for their full activity. These inorganic cofactors
are called Activators. The enzymes requiring actimtors are of 2 types,
• Metal/oenzymes
• Metal activated mzymes
a) Metal activated enzymes:

If the metals are loosely fJ01111d with e11zy111es, such enzy111es are called i'vtetnl activated enzymes.
E.g. Mg+2 of enolase enzyme.
b) Metalloenzymes:
If the metals are tightly bound with e11zy111es, such enzymes are called ,Vfetalloenzymes.
£.g. Copper of Phenol oxidase enzyml'; 1110/ybde1111m of Xanthine oxidase en:ymt•

Enzymes 122
Enzyme Nomenclature and Classification:
Enzyme Nomenclature:
Earlier, when very few enzymes were known, they were given whimsical names like
trypsin, pepsin, elastase etc. (some of which are still in use for convenience). These
names do not convey any information about the reaction of enzymes, or the nature of
the substrates they act.
To overcome these difficulties, it was suggested to name the enzymes by adding the
suffix "ase" to the substrate, which is acted upon by these enzymes. E.g. enzyme
acting on lactose is called lactase, enzyme acting on urea is called urease etc. These are
called trivial names. This also led to confusion and ambiguities, as there may be more
than one enzyme acting on the same substrate.
Therefore, International union of Biochemistry and molecular biology (IUBMB)

appointed Enzyme Commission (EC) in 1961, which developed a sys tem of
nomenclature and classification of enzymes in1964. This system is complex and
cumbersome but unambiguous.
According to this system, each enzyme is given a specific name that indicates the
substrate, cofactor (if any) and type of reaction catalyzed them (with suffix 'ase' attached)
followed by a 4 digit enzyme code (EC) number, which are separated by a dot.
• First digit denotes the class,

• Second d igit represents the sub class,
• Third digit stands for sub-sub class and
• Fourth digit is the serial number of enzymes in the sub-sub class list
Examples:
IUBMB name for enzyme alcohol dehydrogenase is Alcohol NAO Oxidoreductase EC 1.1.1.1
IUBMB name for enzyme lactate dehydrogenase is Lactate NAO Oxidoreductase EC 1.1.1.27
Answer hint for Enzyme classification question (5 or 10 Marks):

a. Classification with Basis (1 Mark)
b. Definition of each class{l Marks)
c. Examples (1 Mark)
d. Reactions (1 or 2 based on marks. lif 5 marks, 2 if 10 marks (2 Marks)
• IUBMB system of enzyme classification is given in next page

Enzymes 123
Enzyme classification (IUBMB system):

Enzymes are classified into 6 classes based on the type of reactions catalyzed.
Class I: Oxidoreductases
Oxidoreductase class of enzymes catalyzes the oxidation reduction type of reaction.
( AH2+ B A+ BH2 )
Example:
Glyceraldehyde 3 P
dehydrogenase
Glyceraldehyde 3-phosphatc / 1, 3 Bisphosphoglycerate
NAD+ + Pi NADH+ H+
Succinate dehydrogenase
Su cci nate • Fumerate
7
FAD FADH2
Other examples: Lactate dehydrogenase, Acyl CoA dehydrogenase etc

Class II: Transferases
Transferases catalyze the transfer of a group (other than hydrogen) from one substrate
to another substrate.
( A-x+B A+ B-x )
Example:
Glucokinase
Glucose Glucose 6 -phosphate
/ 4g+~
ATP ADP
Norcpine phnne
Methyl Transrerase
,,,?'"'" .. Epinephrine
SAM SA H
Other examples: Phosphofructokinase (PFK), Transaminase etc
t:Iass III: Hydrolases

Hydrolases ca talyze the splitting of molecules by hydrolysis (using water)
Example:
( A- B + H20 A- H + B -OH]
Lactase
Lactose + H20 .-
Glucose + Galactose
Urease
Urea + HiO r 2NH3 + COi
Other examples: Maltase, Sucrase etc.

Enzymes 124
Class IV: Lyases

Lyases catalyze the splitting of molecules by mechanism other than hydrolysis .
[ A-B + xy A -x + B-y ]
Example,
Glyceraldehyd e 3-phosphate
Fructose 1, 6-bisphosphate
Dihydroxy acetone phosphate
Pynnute decarboxylase
Pyruvatc Acetal dehyde + CO2
Other examples: Aldolase B, Fumarase etc.
Class V: Isomerases
Isomerase class of enzymes catalyze the inter conversion of isomers.
[ A A']
Example,
Phos phohexose isomerase
Glucose 6-phosphate rructose 6-phosphate
Phosphotrios e isomerase
Glyceraldehyd e-3-phosphate . Dihydroxyacetone-3-phosphaie
Other examples: Epimerase, L-methylmalonyl CoA isomerase etc.
Class VI: Ligases

Ligases catalyze the joining of 2 compounds. The energy for ligation is obtained by
ATP hydrolysis. I
+ B
[A 7 A-BJ
ATP ADP+ Pi
Example, Acetyl CoA carboxylase

AcetylCoA +CO2 • Malonyl CoA
/ Mn~
ATP Biotin ADP+ Pi
Aspargine synthase
Aspartate + NH 3 • Asparagine
/ 4+~
ATP ADP+ Pi
Other examples: Pyruvate carboxylase, Glutamine synthetase etc.
Mnemonic s: to remember 6 classes of enzymes is "Oh Thank H eaven, Love Is Life".
For queries and suggestion s, contact the author at prasad_text @yahoo.co m or 9986449575
Enzymes 125
Enzyme Specificity:
Enzymes exhibit various types of specificity towards substrates.
1. Absolute specificity:
Some enzymes are absolutely specific in action. i. e. they act on only one substrate.
E.g.: Glucokinase acts only on glucose.

Glucokinase
Glucose _ _ _,_..._ _ ___,..., Glucose 6-phosphate
/Mg~ ADP
ATP
Glucokinase cannot act on galactose, which is structurally similar to glucose.
Arginase, urease, galactokinase, are other examples.
2. Broad specificity:
Some enzymes show broad specificity i.e. they act on broad group of strucurally
related substrates.
E.g.: Hexokinase acts on all hexoses like Glucose, Fructose and Galactose etc.
Glucose I Hexokinase Glucose 6-phosphate or

Fructose I ... Fructose 6-phosphate or
Galactose Galactose 6-phosphate
ATP ADP
3. Relative specificity:
It refers to the enzymes acting on relative substrates.
Enzymes exhibit two type of relative specificity,

i) Group-dependant
ii) Bond-dependant

Enzymes 126
i) Group-dependant relative specificity:

It refers to the action of enzymes like pepsin, trypsin which preferentially hydrolyze
specific peptide bonds (not every peptide bond).
Example:
Trypsin hydrolyses only peptide bonds involving basic amino acids and pepsin
hydrolyzes only those peptide bonds involving aromatic amino acids and methionine.
ii) Bond-dependant relative specificity:

It refers to the action of enzymes like glycosidases, lipases etc, which are bond specific.
Examples:
Glycosidases acts on glycosidic bonds of carbohydrates, lipases on ester bonds of lipids.
4. Optical specificity or Stereo specificity:

Enzymes w ill act on only one type of stereoisomers (Except for isomerases). This is
called stereospecificity of enzymes.
Examples:
L-amino-oxid ases act only on L-amino acids and

D-amino-oxidases act only on D-amino acids.
L-amino acid oxidase
L- C£ Amino acid ___T\

_______,. ,
L- Amino acid oxidase
a- Keto acid+ NH3
FMN FMNH2
Similarly, D-amino acid oxidase
D - Amino acid oxidase

D-a Amino acid ---7-...,,......,'\----+11 ex-Keto acid +Nlli
FAD FADH2

Enzymes 127
Factors affecting enzyme activity

Velocity or rate of enzyme activity is assessed by the rate of change of substrate to
product per unit time.
Velocity or rate of enzymatic reaction is influenced by various factors,
1. Enzyme concentration
2. Temperature
3. pH
4. Substrate concentration
1. Effect of enzyme concentration on enzyme activity:
Rate of enzymatic reaction is directly proportional to the enzyme concentration i.e.

as the enzyme concentration increases progressively, the rate of enzyme activity also
increases.
When the velocity of enzyme reaction is measured at various enzyme concentrations

(keeping all other factors constant), and the result is plotted on a graph with velocity
on Y axis and enzyme concentration on X axis, a straight line is obtained.
Velodty l
Enzyme Concenlralion ----+
Explanation:
In the beginning of the enzymatic reaction, the number of enzyme molecules is less
and a small number of substrates are converted to products. Provision of more enzymes
enables the conversion of large number of substrate molecules and hence the velocity
increases progressively.

Enzymes 128
2. Effect of temperature on enzyme activity:

When the velocity of enzymatic reactions is measured at various temperatures (Keeping
all other factors constant), and the result is plotted on a graph with velocity on Y-axis
and temperature on X-axis, a bell shaped curve (graph) is obtained.
Vmax
VclOOfy 1
Optimum temp
Temperature
The temperature at which the velocity is maximum is called the Optimum tern perature.
The velocity decreases on either side of the optimum temperature.
Explanation:
As the temperature increases, more and more reactants attain activation energy (energy
required for the reaction to take place), and also kinetic energy of the reactant molecule
increases, which increases the collision (contact) of enzymes and substrate molecules.
So the rate of enzyme activity increases.
At the optimum temperature, the velocity is highest (Vmax). Any further increase in
the temperature will gradually denature the enzymes (Because the enzymes are proteins
in nature). So the rate of enzyme activity decreases, as the temperature increases beyond
the optimum temperature.
Optimum temperature of most of the enzymes in our body is around 40-45°C.
Temperature coefficient or Q10 :

Temperature coefficient is defined as the increase in en7yme activity when the
temperature is increased by l0°C.
For a majority of body enzymes Q10 is 2 for temperature ouc- 40"C.
Rate of enzyme activity increases in fever:

The rate of enzyme nctivihJ incrcnses with i11crensc in temperature. Optimum temperature of
most of the enzymes in 011r body is nro11nd 40-45°C. Body temperature of humans is nbout 37
oc. So, incrense in tile body temperature due to fever increnses the rate of enzyme nctivity.

Enzymes 129
3. Effect of pH on enzyme activity:
When the velocity of enzymatic reactions is measured at various pH s (keeping all

other factors constant), and the result is plotted on a graph with velocity on Y axis
and pH on X axis, a bell shaped curve (graph) is obtained .
Vmax
Velodty r
Optimum pH
pH
The pH at which the velocity is maximum is called the Optimum pH. The velocity
decreases on either side of the optimum pH.
Explanation:
The enzyme activity is d ue to a particular charged state of enzymes (particularly
active site) and substrates. At optimum pH, enzymes and substrates are in appropriate
charged state to carry out the reaction and the velocity is maximum.
Any decrease or increase in pH would disturb the specific charged state of the enzyme
and substrates required for the reaction; hence velocity decreases on both sides of
optimum pH. ·
Optimum pH of Pepsin is around 1-2

Acid phosphatase is around 4-5
Alkaline phosphatase is around 9-10
Optimum pH of most of the enzymes in our bod y is around 6-8.
Pepsin loses its activity iu the intestine:

Pepsin is a proteolytic enzyme acting on protei11s in stomach. Optimum pH of Pepsin is
aro11 nd 1-2. So, acidic environment of stomach suits the activity of pepsin. pH of intestinal
juice is alkaline (pH 7-8). Hence, when pepsin reaches intestine, it loses its activity.

Enzymes 130
4. Effect of substrate concentration on enzyme activity:

When the velocity of enzymatic reactions is measured at various substrate
concentrations (Keeping all other factors constant), and the result is plotted on a
graph with velocity on Y-axis and substrate on X-axis, a rectangular hyperbolic curve
(graph) is obtained.
Vmax
Vclocity 1
Substrate Concentration
Explanation:
At low substrate concentrations much of the enzymes are free. As the substrate
concentration increases, the rate of enzyme activity increases proportionally till all
the enzyme molecules exist as ES. At this point velocity is maximum (Vmax). Any
further increase in the substrate concentration from this point will not increase the
velocity, because all the enzymes are saturated and there are no free enzymes to
carry out the reaction.
Answer hint for factors influencing enzyme activity (10 Marks):

a) Definition and factors (2 mark)
b) Role of enzyme concentration (2 mark)
c) Role of temerature (2 mark) Exam tip
d) Role of pH (2 mark)
e) Role of substrate concentration (2 mark)
Michaelis Menten equation (MM equation):

Michaelis Menten equation links velocity of enzyme reaction and substrate concentration.
Where, V = Velocity of enzyme reaction

V = Vmax [S] Vmax = Maximum velocity
s = Substrate concentration
Km+ [S]
Km = Michaelis constant

Enzymes 131
Km or Michaelis constant :
Definition:
Km is defined as the substrate concentration at half maximum velocity (½ Vmax).
Km is expressed in moles/ L.
Significance of Km:
1) Km is the characteristic feature of an enzyme for its substrate: It is a constant for
an enzyme for its substrate. Km is termed as the signature of the enzymes.
2) Km is the measure of affinity of enzyme for its substrate:
Lower the Km value higher w ill be the substrate affinity of enzymes and vice versa.
E.g.: Glucose is phosphorylated by glucokinase (liver enzyme) & hexokinase (present in all tissues).
Both hnve different Km vnlue for glucose. Km of glucokinase is 10 mmol/L & Km of hexokinnse is
0.05 11111101/L. This i11dicntes hexokinase has more affinity thnn glucokinase for glucose.
3) Enzymes have 50 % efficiency at Km:At Km, enzymes have half the maximum
velocity i.e. only 50% of enzymes are active (other 50% are free).
4) Km value is helpful in understanding the natural substrate of enzymes that act on
more than one substrates. For instance, hexokinase can phosphorylate glucose, fructose, galactose,
mannose etc. But this enzyme has the lowest Km (maximum affinity) for glucose than other substrate. So,
it can be concluded that the glucose is the natural substrate of hexokinase enzyme.
5) pH, temperature and inhibitors affect Km values.
6) lsoenzymes have different Km values for the same substrates.
Calculation of Km: Lineweaver-Burk Plot or Double

Measure the velocity of enzymatic reactions at reciprocal plot for determination of Km:
various substrate concentrations (keeping all Sometimes, it is not possible to achieve high
other factors constant) and plot a graph with substrate concen tration to reach the Vmax
velocity on y-axis and substrate concentration conditions. So, Vmax, ½ Vmax or Km values are
in x-axis. Then measure the Vmax. Then calculate not possible to determine. In such conditions, a
the ½ Vmax. Draw a parallel at½ Vmax to the reciprocal ofMichaelis-Men ten equation is taken,
x-axis, till it touches the curve. From this point which gives a straight line graphical representation,
of intersection drop a perpendicular to the x-axis. which is then extrapolated to get the reciprocal of
This intersection gives the Km value. Km. This is called Double Reciprocal Plot or
Lineweaver-Burk Plot.
Ve!Ocity
'
Slope • Km
Vm.ax
½Vmax
- I -1._
Sul>lttate Concentration
Km [SJ

Enzymes 132
Enzyme inhibition
A wide variety of compounds (organic or inorganic) can reduce the enzyme activities.
These compounds are called enzyme inhibitors. Enzyme inhibitors bind to the enzyme
and reduce their activity. This process is called enzyme inhibition.
There are 3 types of enzyme inhibitions,
1) Competitive enzyme inhibition
2) Non competitive enzyme inhibition
3) Un competitive enzyme inhibition
I. Competitive enzyme inhibition :

In competitive enzyme inhibition, the inhibitor is structurally similar the substrate
(substrate analogue). Hence inhibitor competes with the substrates for binding with
the active site of the enzyme. ·
The binding of the inhibitor to the active site of the enzymes prevents the substrate
from binding with the enzyme, which results in decreased ES formation and reduced
rate of enzyme reaction.
+ s ES E + p
E
+ El No products
+
v-
Substrate
1:vl-
ES Complex
or
Product
Enzyme
•-
Inhibitor
c:!J- No Product
EI Complex
The formation of ES complex or EI complex (or degree of inhibition) depends on the

relative concentration of substrates and inhibitors.
Competitive enzyme inhibition is reversible. It can be overcome by a hjgh substrate
concen tration. Thus competitive enzyme inhibition does not alter the Vmax, but
increase the Km value.
Example:
Malonate is the competitive inhibitor of succinate dehydrogenase enzyme.
Succinate dehydrogenase enzyme converts succinate to fumarate. Malonate has a
structure similar to succinate, hence competes with succinate to bind with succinate
dehydrogenase. Hence malonate competitively inhibits succinate dehydrogenase enzyme.

Enzymes 133
CH2COOH HOOCCH
I II
CH2COOH Succinate dehydrogenase CHCOOH
Succinate Fumarate
<
COOH Note that the structure of malonate is similar to succin ate.
CH2
COOH
So, malonate competes with succinate to bind with the active
s ite of s u ccinate deh ydrogenase enzyme, hence
Malonate competetively inhibits the enzyme.
Biomedical importance of competitive enzyme inhibition:

The mechanism of competitive enzyme inhibition is used in chemotherapy (treatment
with the help of chemical compounds) of certain diseases. These are synthetic chemical
compounds, which are designed to have structures similar to desired substrates whose
reaction needs to be competitively inhibited. These compounds, when ingested,
competitively inhibit specific enzymes blocking the unwanted reactions. (Such
compounds are referred to as ' Antimetabolites').
Antimetabolites:
Antimetabolite is chernical compound that inhibits the use ofa metabolite (a normal compound
that is a part of normal metabolism). Antimetabolites often have similar structure to that of
structures of metabolite that they interfere with.
Examples are,
1) Treatment of gout (Inhibition of Xanthine oxidase by allopurinol drug):

Gou t is a clinical condition caused due to the increased levels of uric acid in the blood
and subsequent accumulation of uric acid in the joints causing severe pain (arthritis).
The enzyme xanthine oxidase produces uric acid from hypoxanthine (which in turn is
obtained from catabolism of purine nucleotides).
..______
Hypoxanthi ne - - - - - Xanthine Uric acid
Allopurinol _...,0..__•~ Xanthineo~dase
Allopurinol is a structurally similar to hypoxanthine, so when ingested, it competitively

inhibits xanthine oxidase enzyme, resulting in decreased uric acid production.

Enzymes 134
2) Treatment of cancer by antifolate compounds (anticancer agents):

(Inhibition of Folate reductase by arninopterin and amethopterin):
Cancer is a disease of uncontrolled cell division. FH4 (Tetrahydrofolate), the coenzyme
of vitamin B9 (folic acid) is required for DNA synthesis, cell division and cell
multiplication. It is formed from folate by folate reductase enzyme.
Aminopterin and amethopterin (a methotrexate) are structural analogues of folic acid.
So when ingested competitively inhibits folate reductase enzyme, thus reduces FH4
production and hence reduces cell division and multiplication. Thus these com-
pounds are used as anti cancer drugs (agents), especially in Leukemia.
Note: However, these compounds cannot be taken for a long time therapy because
folate is required for growth and multiplication of normal cells of the body.
Folate ' Dihydrofolate (FH2) / Tetrahydrofolate (FIL)

Aminopte.rin & 0 ""- /
Amethopterin ----• Folate reductase
3) Treatment of certain bacterial infections by sulfa drugs (like sulfonamide):

Folic acid is required for the growth and multiplication of cells in human beings, bacteria
etc. Human beings cannot synthesize folic acid, whereas bacteria can synthesize folic
acid from a compound called PABA (Para-aminobenzoic acid) by their enzyme
machinery. Human beings obtain folic acid only through diet;
Sulfonamide (a sulfa drug) is a structural analogue of P ABA, so when ingested it
competitively inhibits the synthesis of folic acids from PABA in bacteria. Since this is
the only source of folic acids for bacteria, bacteria cannot grow and multiply. Thus
bacterial infections can be controlled.
Note: These sulfa drugs do not affect human beings because human beings do not
synthesize folic acids from PABA and obtain the vitamin only through diet.
4) Treatment of alcohol toxicity (Inhibition of alcohol dehydrogenase by ethanol):

Alcohol toxicity of methanol is due to the production of formaldehyde (a toxic
compound} from methanol, by action of alcohol dehydrogenase.
In methanol poisoning, ethanol is ingested as a treatment. Ethanol is structurally
similar to methanol and also is a substrate of alcohol dehydrogenase. Hence, ethanol
competitively inhibits the oxidation of methanol and production of formaldehyde.
Methanol is then excreted from body through urine.
Other examples that use competitive inhibition mechansim are,

5) Treatment of Tuberculosis by Isoniazids like INH (Pyridoxine analogue)
6) Treatment of Thrombosis or intravascular clotting by Dicumarol (Vit K analogue)
6) Treatment of Hypercholesrolemia by Lovastatin (HMG Coa analogue)

Enzymes 135
II. Non competitive enzyme inhibition:

In non-competitive enzyme inhibition, the inhibitor is not structurally similar to the
substrate, hence does not compete with the substrates for the active site.
Inhibitor
•
+ S - - - ES +I - -
v- 2Yl~
+ Subslnte
ES Complex
E +I + S ____. ES I - No product
-El
c____J ~ - No Product
En~yme
• - ~ ~ E Sl complu
Inhibitor
El Complex
V
Substrate
Types: Non-competitive enzyme inhibitions can be reversible or irreversible. But both

show similar kinetics (I.e. Km value is unchanged, but Vmax is lowered).
a) Reversible non competitive enzyme inhibition : In Non-competitive enzyme
inhibition, the inhibitor is not structurally similar to the substrate, so, does not compete
with the substrates for the active site of the enzyme. Inhibitor may or may not bind with
active site. Instead it may (mostly) bind to enzyme at a site other than active site. Since
Sand I may combine at different sites, formations of both EI and ESI complexes are
possible. Since ESI may breakdown to form products at a slower rate than does ES, the
reaction may be slowed but not stopped. So Inhibitor does not interfere with the binding
of substrate with the enzyme, but it lowers the maximum velocity attainable.
Examples:
1) Anti-trypsin, non-competitively inhibits trypsin in reversible manner
2) Trypsin inhibitor present in ascaris worm. (So, ascaris is not digested by intestinal juice).
3) Antibodies prepared against enzyme proteins.
b) Irreversible non competitive enzyme inhibition (Enzyme poisons): A variety
of enzyme 'poisons' reduces the enzyme activity non-competitively. Here the inhibitor
is not structurally similar to the substrate, but these inhibitors generally bind to the
active site of enzymes (binds covalently) and inactivates them, which is irreversible.
This process is not readily reversible by increasing the substrate concentration; however
the presence of substrates exerts a protective role by blocking or slowing the access of
inhibitor to the active site. Usually these enzyme poisons are not biological compounds.
Examples:
1) Inhibition of enzymes by heavy metals like Hg•2 Ag•, Pb•2 etc.
2) Inhibition of enolase (A glycolytic enzyme) by fluoride
3) Inhibition of cytochrome oxidase enzyme by cyanide.
4) Inhibition of SH enzymes by Iodoacetate (SH enzymes are enzymes with SH groups
at acive site, e.g. Glyceraldehyde 3-P dehydrogenase, papain etc).
5) Inhibition of acetylcholine esterase by DIFP (Diisopropylfluorophosphate), a nerve gas.

Enzymes 136
Clinical significance of Non-competitive enzyme inhibition:

1) Fluoride is added to blood durirzg blood glucose estimation: During blood glucose estima tion,
the RBC consumes blood glucose by glycolysis, thereby reducing blood glucose level (about 10%
l{Jer hour). This is more prominent, if there's a long time gap between blood collection & glucose
~stimation. Fluoride is an inhibitor of glycolytic enzyme enolase, when added to blood prevents
glycolysis and hence prevents lowering the blood glucose level.
12) BAL (British Anti-Lewisite) is used as an antidote for heavy metal poisoning. (Heavy metals
~ct as enzyme poisons by reacting with -SH group of certain enzymes, called -SH enzymes). BAL has
15everal SH groups, which can combine with heavy metals and remove them.
III. Uncompetitive enzyme inhibition

In Uncompetitive enzyme inhibition, the inhibitors do not bind to free enzymes, but can
bind only to ES Complex and decreases the velocity of enzymatic reactions.
Uncompetitive enzyme inhibitors decrease both Vmax and Km of enzymes.
ubstrale lnh.ibitor
Examples:
1) Inhibition of Placental alkaline phosphatase enzyme by phenylalanine
2) Inhibition of S-adenosyl methyl transferase by ATP ( In yeast)
Lineweaver-Burk Plot (Double reciprocal plot for different Inhibitions

Competitive
+I ...L
V
inhibitor o inhibitor
·...!._ ...L
Km IS! I
i;; .."" N
Lineweaver-Burk Plot or D"ouble reciprocal plot ( Competitve inhibition)
Noncompetitive
Uncompetitive
inhibitor
inhibitor o inhibitor
I
v
\'m,, '"'"
/ _L
--------
I 1
It.JD ir' "'i
Noncompetitve inhibition Uncom etitve inhibition

Enzymes 137
Suicide Inhibition (or Mechanism based inactivation):

It is a special type of enzyme inhibition, where the inhibitor makes use of the enzyme's
own catalytic mechanism to inactivate it (Hence, called mechanism based inactivation).
In suicide inhibition, the structural analog suicide inhibitor is initiaJly inactive. On binding
with the active site of the enzyme, the inhibitor is converted to a more effective inhibitor
(by enzyme itself), which irreversibly binds to the enzyme and inhibits further reactions.
Here, enzyme literally commits suicide by itself producing the effective inhibitor, which
inactivates itslef. Examples are,
1) Inhibition of Ornithine decarboxylase (ODC) by Difluromethylornithine (D FMO):
Oifluromethylornithine is a suicide inhibitor of enzyme ornithine decarboxylase (ODC)
enzyme. DFMO is initially inactive, but on binding with the enzyme, it forms irreversible
covalent complex with the pyridoxal phosphate (coenzyme of the reaction) and the
amino acid residues of the enzyme and inactivates the enzyme ODC.
Significance:
Oifluromethylornithine (DFMO) have been used in the treatment of trypanosomiasis
(African sleeping sickness), a disease caused by trypanosoma).
Ornithine decarboxylase (ODC) enzyme catalyses the conversion of ornithine to
putrescine, which is necessary for trypanosoma survival. Difluromethylomithine (DFMO)
is a suicide inhibitor of enzyme ornithine decarboxylase (ODC) enzyme. When the
ODC in trypanosoma is inhibited, multiplication of the parasite is arrested. In mammalian
cells, the turnover rate of ODC is very high, and so the inhibition by DFMO is only
temporary. So, DFMO kills the parasites, with no side effects to the patient. Besides,
DFMO does not inhibit any other body enzymes.
2) Inhibition of Xanthine oxidase by Allopurinol:
Allopurinol (structural analogue of hypoxanthine) is the suicide inhibitor of xanthine
oxidase. When allopurinol binds with xanthine oxidase enzyme, it is converted to
alloxanthine, which is a strongly binds with xanthine oxidase inhibiting them.
3) Inhibition of thymidylate synthase by 5-fluorouracil :
5-fluorouracil (structural analogue of pyrimidine) is the suicide inhibitor of thymidylate
synthase. When 5-fluorouracil binds with thymidylate synthase enzyme, it is converted
to fluorodeoxyuridylate (FdUMP), which is a strongly binds with thymidylate synthase
inhibiting them.
4) The bactericidal action of Pencillin is based on suicide inhibition of transpeptidase.
5) The anti in flamatory action of asp irin is also based on su icide in hibition .
Answer hint for enzyme Inhibition question (10 Marks):

a) Definition and types (1 mark)
b) Competitive inhibition (Definition, examples, significance) (5 mark)
c) on-competitive inhibition (Definition, types, e.g.) (2 mark)
d) Uncompetitive inhibition (Definition, examples) (1 mark)
e) Suicide lnhibition (Definition, example) (1 mark)

Enzymes 138
Control (Regulation) of enzyme activities:

Enzymes mediate v irtually all biochemical reactions in the body. So the regulation of
enzymes activities is the major mechanisms by which metabolic process are controlled.
Enzyme activity is regulated by different mechanisms,
I) Induction (lncreased syn thesis of enzymes) (Coarse regulation)

& Altering the concentration
}
Repression (decreased synthesis of enzymes) of enzymes at gene tic level
a) Allosteric activation
II) Allosteric regulation
b) Allosteric inhibition
(Fine regulation)
Altering the activity
a) Irreversible of exis ting en zymes
III) Covalent modification
b) Reversible
IV) Miscellaneous:
• Multi enzym e complex
• Compartmentalization
• Isoenzymes
Key enzymes (Regulatory or rate limiting enzymes):

Rate limiting (Regulatory or key) en zym e is one which catalyze the committed s tep
of a pathway, which d ecide the fate and speed of the entire pathway. (Committed
step is one whose reaction product is unique to that pathway).
The entire metabolic pathway can be controlled by targeting this en zyme. Genera lly,
these key enzymes are inducible / repressible enzymes or a llosteric enzymes or bo th
(or even covalent modifications). These enzymes are used as regula tory tool in
regulation of metabolic pathways.

Enzymes 139
I) Induction and repression of enzyme synthesis:

The rates of synthesis of certain enzymes are regulated by induction and repression .
These work at the gene level (at the leveJ of transcription) and alter (increase or
decrease) the concentration of enzymes.
• Induction refers to the increased synthesis of enzymes and inducers are compounds
which cause induction.
• Repression refers to the decreased synthesis of enzyme and repressors are compounds
which cause repression (decrease the synthesis) of enzymes.
Examples:
1) Insulin is a hypoglycemic hormone. Insulin (inducer) increases the synthesis of
glycolytic enzymes like glucokinase, PFK (by induction). Insulin also represses the
synthesis of gluconeogenic enzymes like pyruvate carboxylase.
2) Glucagon is the inducer of pyruvate carboxylase and repressor of PFK.
Enzyme Pathway Inducer Repressor
Glycolysis Insulin Glucagon

Phosohofructokinase
Pyruvate carboxylase Gluconeogenesis Glucagon Insulin
Significance: Usually ind ucible/ repressible enzymes are regulatory enzymes.
II) Allosteric regulation:

Some of the enzyme called the allosteric enzyme possess an allosteric site (Alla =other;
Steric = site), which is different from active site. Certain substances referred to as
allosteric modifiers (allosteric effectors or modulators) bind at the allosteric site and
either increase or decrease enzyme activity. Allosteric enzymes have different sites
for different allosteric modifiers.
• If the allosteric modifiers increase the enzyme activity, then they are called allosteric
activators or positive modifiers and the process is ca lled allosteric activation.
• If the allosteric modifiers decrease the enzyme activity, then they are called allosteric
inhibitors or negative modifiers and the process is called allosteric inhibition.
Enzyme Pathway Allosteric activators Allosteric inh1bitors
(or Positive (or Negative
allosteric mod ifier) allosteric modifier)
Phosphofructokmase Glycolysis AMP ATP
lsocitrate dehydrogenaSE TCAcycle ADP ATP
Acetyl CoA carboxylase Fatty acid synthesis Citrate Palmitate
Significance: Generally, most of the regulatory enzymes are allosteric enzymes.

Enzymes 140
Classification of allosteric enzymes:

Allosteric enzymes are classified into 2 classes based on effect of al losteric modifiers
onKrn&Vmax:
a) K-Class allosteric enzymes (K enzymes): The allosteric modifier changes the Km
(increased) but Vmax is unchanged. So, these exhibit substrate saturation kinetics
similar to competitive inhibition. E.g. Phosphofructokinase.
b) V-Class allosteric enzymes (V enzymes): The allosteric modifier changes the Vmax
(decreased), but the Km is unchanged. So, these exhibit substrate saturation kinetics
similar to non competitive inhibition. E.g. Acetyl CoA carboxylase.
1. Almost all the allosteric e11zymes are oligomeric. So Jar, only 2 monomeric al/osteric e11zy111es
have bee11 identified; ribonucleotide dipltosphate reductase 1111d pymvate UDP-N-acelyl glucosamine
trans/erase. O/igomeric al/osteric e11zymes have more thall one substrate binding & al/osteric sites.
So, allosteric enzymes produce a sigmoidal curve (instead of usual hyperbola), when the velocity
is plotted agai11st the substrate conce11 tratio11. (Figure A). This is due to cooperativity.
2. Homotropic and Heterotropic allosteric regulation :
• If the 111odulator is the substrate itself, then the process is called homotropic allosteric
regulation. Such effect is always positive and e11zy111e is always an oligomeric enzyme.
• If the modulator is not the substrate, then the process is called heterotropic allosteric reg11latio11.
This can be positive or negative & can occur in monomeric or oligomeric allosteric enzymes.
Cooperativity of allosteric enzymes:

Allosteric enzymes demonstrate the phenomenon of cooperativity due to the oligomeric
nature of the allosteric enzymes. They produce a sigmoid substrate saturation curve.
Cooperativity is defined as a change in the activity of one subunit due to the binding of
modulato r to another subunit of alloste ric enzyme. (Figure A).
Binding of modulator (homotropic or heterotropic) to one subunit brings about the
conformational changes in the other subunH, either activating (positive cooperativity)
or inhibiting (negative cooperativity) the active site of the enzyme. (Figure B).
Po~itive modifiers (activators) show positive cooperativity and negative modifiers
(inhibitors) show negative cooperativity.
0 Hyperboilc cur ve of
non allosteric enzymes 0
l Sigmoid cu rve of
allosteric e nzym es 1
1
2
Positive
modulator
o modulator
3 Negative
modulator

Enzymes 141
III) Covalent modification:

A) Irreversible Covalent modification (Proenzymes or Zymogens, Zymases):
Some enzymes are synthesized and secreted as inactive proenzymes or zymogens.
These precursors are converted to active form "Zymases" in the site of their activity.
This involves irreversible breakjng of specific covalent peptide bonds. For Example;
1) Proteases of GI tract such as trypsin, chymotrypsin, pepsin etc are synthesized as
zymogens. For instance, Trypsin is secreted by pancreas as inactive trypsinogen
(zymogen form). Trypsinogen is transported to intestine, where they are converted to
trypsin by breaking of specific covalent peptide bonds by enterokinase, which is
irreversible. Pepsin & chymotrypsin are also activated by a similar process.
2) Blood coagulation factors are also synthesized in inactive form.
Significance:
• The synthesis of proteases as catalytically inactive form protects the tissue of origin
(e.g. pancreas) from auto digestion.
• Proenzymes facilitate rapid activity in response to physiological demand, when denovo
synthesis of the required enzymes are slow (e.g. Blood clotting factors).
B) Reversible Covalent modification:

Some enzymes are regulated by reversible covalent modification by phosphate groups.
Phosphorylation and dephosphorylation of these enzymes are catalyzed by a variety of
protein kinases and phosphatases respectively. Some enzymes are active in their
phosphorylated form and inactive in their dephosphorylated form & vice versa.
Examples: Glycogen phosphorylase is active in their phosphorylated form (a) and inactive
in their dephosphorylated form (b), but Glycogen synthase enzyme is active in their
dephosphorylated form (a) and inactive in their phosphorylated form (b).
ote that a is active form & bis inactive form.
Reversible covalent modification of glycogen phosphorylase enzyme:

ATP~na~ADP
Glycogen phosphorylase b 11 Glycoge·n phosphorylase a
(Dephosphorylated form) (Phosphorylated form)
4~ -----
(Inactive form) Pi Phosphatase H20 (Active form)
Gl ycog en sy nthase a
ATP ~nase::::
Reversible covalent modification of glycogen synthase enzyme:
_ _ _...;;:::,_,..~~---+-
_
A., DP
Glycoge n sy nthase b
(Dephosphoryla te d form ) ---,,,,,--..::::---- (Ph os ph o rylated fo rm )
:.:------=--
Pi -----H 20
Phos phatase
(Active form) (Inactive form)

Enzymes 142
Feedback regulation:
In many metabolic pathways, when end products are produced in sufficient amounts,
they block their own excess synthesis by regulating the activity of the key enzymes,
which catalyses an earliest irreversible step (committed step) of the pathway. This is
called feedback regulation. There are 2 types of feedback regulation,
1) Feedback allosteric inhibition:

If the end product of a pathway inhibits the activity of the key enzyme that catalyzes
the committed step of the pathway, then such feedback regulation is called feedback
(allosteric) inhibition or end product inhibition.
Example: Regulation of ALA synthase (Key enzyme of heme synthesis). Excess
production of heme inhibits ALA synthase enzyme by feedback inhibition.
S u cci n y l CoA + G lyci n e
A LA synthase
ALA - - - - Heme
2) Feedback repression:
lf the end product of a pathway represses the synthesis of the key enzyme that catalyzes
the committed step of the pathway, then such feedback regulation is called feedback
repression. Feedback repression operates in the genetic level.
Example: Regulation of HMG CoA reductase (key enzyme of cholesterol synthesis).
Excess of cholesterol represses HMG CoA reductase enzyme by feedback repression.
( -)
(HMG CoA reductase gene) )()0000(_/
(-) !
HMG CoA reductase
HMG CoA - -- - - - - Mevalonate - - -- - C holeste rol
Feedback Regulation = Feedback repression+ Feedback inhibition
Feedback in h ibition
l
El
( E ffec t direc tly o n e n zy me ~)
E2 E3
A B ----• c

Enzymes 143
IV) Miscellaneous factors:
a) Multienzyme complex:
Localization of several enzymes catalyzing a sequence of consecutive reactions of a
metabolic pathway into a macro molecular complex is referred to as Multienzyme
1
complex or multienzyme.
Examples : Fatty acid synthase complex, cx-keto glutarate dehydrogenase complex,

Pyruvate dehydrogenase complex etc.
The multienzyme complex is active only in complex form i.e. individual enzyme
activities cannot be separated by fractionation of multienzyme complex.
Significances:
• Multienzyme complex increases the efficiency (speed) of the overall pathway by
directly transferring the intermediates from one enzyme to the next one, and thereby
avoiding their dilution in the medium. Thus, this system ensures the uninterrupted
sequence of reactions right up to the completion of the pathway once started.
• The intermediates remaining bound to the m ultienzyme complex are protected from
diversion into other metabolic pathways.
b) Enzyme compartmentalization:
• Enzymes present in cells are situated in different intracellular compartments like
cytosol, mitochondria, ribosome, lysosome etc. For example, enzymes of TCA Cycle,
~-oxidation of fatty acids etc. are present in mitochondria, where as enzymes of
glycolysis, glycogenesis, glycogenolysis, and fatty acid synthesis etc. are present in
cytosol. This provides a point for finer regulation of enzyme activities as these reactions
can be independently regulated.
• There are certain substances that are synthesized and degraded in different
compartments. E.g.: Fatty acids, which are synthesized in cytosol and degraded in
mitochondria. This facilitates their separate reciprocal regulation, so that both the
pathways do not occur simultaneously. This facilitates to prevent futile cycle.
• Sometimes some reactions of the pathway take p lace in one compartment and the
succeeding reactions of the pathway in another compartment. For example, heme
synthesis and urea synthesis etc. The intermediates have to be shuttled (transported)
across the membranes (compartment barriers). This is carried out via the shuttle
mechanisms. This also provides a point for finer regulation of enzyme activities.

Enzymes 144
c) Isoenzymes (Isozymes):
Definition :
Multiple forms of an enzyme catalyzing the same reactions are called l soenzymes.
An enzyme may exist in several molecular forms in the same species. These multiple
forms of the enzymes are called Isoenzymes or Isozymes.
Examples:
1) Lactate dehydrogenase enzyme exists in 5 different isoenzyme forms.
Lactate dehydrogenase
Pyruva tc - -- - - - - - - -- - - - - - Lactate
LDH1 / LDH2 / LOH3 / LDHi / LDHs
2) Creatine kinase (CK) has 3 isoenzymes.
Characteristics of isoenzymes:
1) lsoenzymes have different structures, different physical and chemical properties,
but all the isoenzyme forms of an enzyme catalyze the same reaction (i.e. act on the
same substrate to produce same products).
2) Isoenzymes have different amino acid compositions, electrophoretic mobility and

immunological properties. Isoenzymes can be separated by electrophoresis.
3) Isoenzymes have different Km values (and Vmax) for the same substrates.
4) Isoenzymes generally have more than one polypeptide chains (Oligomeric units)
E.g.: LDH has 4 polypeptide chains, CK has 2 polypeptide chains.
Lactate dehydrogenase isoenzymes:
Isoenzymes S u bunit make up Tissue of origin Percentage
LD H-1 HHHH ((H1) H ca rt 30%
LD H-2 11 IJ II M ( H 3M ) RB C 35~•o
LD H-3 HHMM ( ll 2M 2) Bra in 2 0°·0
LD H-4 HMMM ( HM ,) Live r 10%
ILD H-5 MM MM (M 4) Mu sc le 5%
For queries and suggestions, contact t he author at prasad_text@yahoo.com or 9986449575

Enzymes 145
Creatine kinase isoenzymes:
Isoenzym es S ubunit make up T issu e of origin Pe rcen tag e
CK-3 (CK-MM) MM Muscle 90%
CK-2 (CK-MB) MB Heart 9'l·o
I CK-] (CK-BB) BB Brain 1%
Regulatory role of isoenzymes:

Different isoenzymes of an enzyme are present in different tissues. Their activity is
con trolled by different regulators (inducers, repressors and allosteric mod ifiers)
according to the tissue / body needs. Thus, isoenzymes provide a point for the
regulation of enzyme activity.
Clinical significances of isoenzymes:

Generally different isoenzymes are rich in different tissues. These isoenzymes are
released into the blood during normal wear & tear of tissues. Blood contains these
isoenzyrnes in certain levels. When these tissues get damaged, corresponding isoenzymes
are released into the blood and their levels in the blood increases. Thus, by measuring
the isoenzyme levels in blood, we can d iagnose certain diseases. For Example,
• LDH isoenzymes: LOH 1 isoenzyme is rich in heart muscles and LOH5 1soenzyme is
rich in m uscles. So during myocardial infarctions, LOH 1 isoenzymes increases in blood.
Similarly during m uscle dystrophies, LDH5 isocnzymes increases in blood.
• CK isoenzymes: CK-MM isoenzyme is rich in skeletal muscles and CK-MB isoenzyme
is rich in heart muscles. So, in diseases of skeletal muscles (like muscle dystrophies)
the level of CK-MM isoenzyme increases in the blood and in myocardial infarctions the
level of CK-MB isoenzyme increases in the blood
Answer hint for isoenzyme question (5 Marks):

a) Definition (1 mark)
b) Examples (1 mark)
c) Properties (1 mark)
d) Regulatory Role {l mark)
e) Clinical significance (1 mark)
Functional, N on-functional enzymes:

Enzymes, which have specific functions i11 blood are hence called f unctional enzymes (like lipoprotein
lipase, cernloplas111in etc) and enzymes, which have no specific fun ctions in the blood are en/led
non-functional enzymes (E.g. AST, ALT, LDH, CK etc). Non-functionnl enzymes nre derived from
different organs nnd released f rom into the blood during nonnnl wear and tear of these tissues.

Enzymes 146
Diagnostic enzymes (Clinical significance of enzymes):

Diagnostic enzymes a re intracellular enzymes and have no specific role in the blood
(non func tionally enzymes). These are present in specific tissues and released into the
blood during the normal wear and tear of these corresponding tissues. In healthy
individuals, their blood concentration is in very low and within certain normal levels.
When these tissues are damaged, the corresponding enzymes are released into the
blood in more amounts and their level in the blood markedly increases. The presence
of increased levels of these enzymes in blood, reflects the possible damage of
corresponding tissue, which is rich in that particular enzyme. Thus, by measuring
the levels of these enzymes in the blood, we can relate the increased blood enzyme
level conditions to the disease of corresponding tissues, i.e. diagnosis of diseases.
These enzymes are called diagnostic enzymes (or Clinical enzymes).
The degree of elevation of these diagnostic enzymes often correlates with the extent
of tissue damage. They are also useful during prognosis of the disease.
Examples:
1) ALT (Alanine transaminase) is rich in liver. Normal serum level is 3 - 35 IU/L.
During liver diseases, ALT is released into the blood and their level increases in the
blood, which reflects a possible liver damage.
2) AST (Aspartate transaminase) is rich in heart and in liver. Normal serum level is
4 - 40 IU / L of serum. Elevated levels of AST in serum indicates a possible heart
attack or liver disease.
Both ALT and AST levels are increased in liver diseases, but ALT> AST.
3) LDH, and CK-MB isoenzyrnes are rich in hea rt. So, during myocardial infarctions
LDH1 and CK-MB enzyme level increases in the blood (Refer isoenzyme section).
4. Aldolase enzyme is rich in muscle tissues. It increase in muscle diseases.
5. GGT (y-glutamyl transferase) is a enzyme marker for the detection of alcoholism.

t
Other examples:
Diagnostic enzymes Principle sources Norma l serum Conce ntration
Concentration increases in
All..aline phosphatase BONE, and 3-13 KA unit>/ di of serum Rickets & other bone diseases;
(ALP) Biliary canahculi Obmuctive jaundice.
Acid phosphatase PROSTATE I - 4 KA units/ di ml of seru m Pro,rntic cancer
Amylase Pancreas. 80-180 Somogyi units/di Pancreatiti s

Salivary glands Paroutis
Lipa~e Pancreas 0.2- 1.5 llJ / L PancreatiLis

Enzymes 147
Answer hint for diagnostic enzymes question (5 Marks):

a) Definition and explanation - 2 marks
b) Exam p les - 1 mark
c) Table of diagnostic enzym es (More examp les) - 2 marks
Cardiac markers (Enzyme profile in myocardial infarction):

Cardiac markers are the group of enzymes or proteins that are measured d uring the
diagnosis of m yocard ial infarction. These are,
1) Creatine Phosphokinase (CPK or CK): ormal serum value is 20-80 IU/1.
During myocardial infarction, CPK (particularly CK-MB isoenzyme) is the first enzym e
to be released into the circulation. CPK val ues starts to increase w ithin 3 hours of
infarction . So CPK values are very useful in early detection of MI w here ECG values
are ambiguous. CPK values reach the peak in 36 hours and come back to reference
level within 3 days. CPK values (precisely CK-MM values) also increases in muscle dystrophy.
2) Aspartate Transaminase (AST): Normal serum value is 4-20 IU / 1.
AST va lues rises sharply with in 6-12 hours of MI, reaches the peak within 48 hours
& comes back to reference level w ithin 5 days AST values also increase in Liver diseases.
3) Lactate dehydrogenase (LDH): Normal serum value is 100 -200 IU / I.
In MI, the LDH (precisely LDH1 isoenzyme) values increases. It starts to increase in
12 to 24 hours after the MI, reaches the peak in 3 days and comes back to the reference
range in 8 to 10 days.
LDH is the last enzyme to r ise during MI and last enzyme to return to referen ce
value. So LDH values are u seful in patients admitted more than 48 hours after
Myocardial infarction (a time period where serum CPK values almost comes back to
reference values).
4) Cardiac Troponin (CT):
Cardiac troponins are proteins that are useful in early detection of MI.
Car diac troponins, p a rticularly Cardiac Troponin I (CTI) are released into the
circula tion within 4 hours after the onset of MI, reach a peak value by 12 - 24 hours,
and remain elevated for about a week. So, it is a useful cardiac marker at any time
interval after the heart a ttack.
Enzyme marker Onset Peak Duration
CK- MB 3 hou rs 36 rours 3days
AST 6-12 hours 48 hours 5 days
LOH 12-24 hours 3days 8-10 days
Tropo nin 4 hours 12-24 hours 1 week

Enzymes 148
• Hepato-biliary markers (Enzyme markers in hepatic and biliary diseases):

i) Enzymes Indicating liver damage (Hepatic jaundice);
• ALT (SGPT): Normal level is 3 - 35 U / liter of serum
• AST (SCOT): Normal level is 4 - 40 U / liter of serum
• LOH (Particularly LOH isoenzyme 4): Normal level is 60 - 200 U / L.
These levels increase in liver damages (i.e. Hepatic jaundice).
ii) Enzymes indicating biliary diseases (Obstructive jaundice):
Alkaline phosphatase (ALP) : Normal serum level is 3 - 13 KA units/ dl).
GGT (y-glutamyl transferase): Normal serum level is 7 -50 U/ L
5'-Nucleotidase: Normal serum level is 2-17 U/ L
These enzyme levels increase during posthepatic jaundice (or obstrnctive jaundice).
5'-Nucleotidase is a better indicator of biliary diseases, as it is biliary specific, whereas, ALP also
increases in bone diseases and GGT also increases in alcoholic liver diseases.
• Pancreatic markers (Enzyme markers in Pancreatic diseases):

Amyalse : Normal serum level is 10 - 90 U / L.
Lipase: Normal serum level is 0.2 - 1.5 U/ L
These enzyme levels increase during pancreatitis.
• Choline esterase (ChE):

ChE is present in nerve endings & also in RBCs. Reference level in serum is 2-10 U/L.
Organophosphorous pesticides like parathione irreversibly inhibi t ChE in RBCs.
Measurement of ChE in RBCs is particularly useful in persons working with these
pesticides to determine the extent of exposure and damage.
Therapeutic enzymes:
Enzymes that are used in the treatment of certain diseases are called therapeutic enzymes
• Streptokinase obtained from streptococcus and urokinase obtained from urine of
human beings are used in the lysis of the intra vascular clots as they convert plasminogen
to plasmin which lyses the clot.
• Asparginase is used in the treatment of leukemia. Tumour cells have a high
requirement for asparagine. Administration of intravenous asparginase enzyme
decreases the plasma level of asparagine and availability of asparagine to the tumor
cells is decreased. This depresses the feasibility of tumor cells.
• Pa pain is used in the treatment of inflamation.
• Antitrypsin is used in the treatment of emphysema.
• Collagenase is used in the treatment of burns and ulcers.

Enzymes 149
Question Bank on Enzymes
Essay questions (10 Marks):

1. Classify enzymes according to IUB system? Give examples and reactions catalyzed by them?
2. Explain the factors affecting enzyme activity
3. Write an detiled account of enzyme inhibition
Short answers (5 Marks):

1. Give an account on competitive enzyme inhibition
2. Give an account of isoenzymes
3. Give an account on diagnostic enzymology
Short Answers (2 to 3 Marks):

1. Fischer's Lock and Key model / Koshland's Induced fit theory)
2. Cofactors/ Coenzymes /Activators / Prosthetic groups / Proenzyme (Zymogen)
3. Serum enzyme marker in myocardial infarction
4. Allosteric enzymes Induction and repression
11

1) All of the following enzymes are involved in oxidation-reduction reactions, except (AI)
a) Dehydrogenases b) Hydrolases c) Oxygenases d) Peroxidases
2) One of the following enzymes is not a protein (AIIMS)
a) DNAase b) Abzyme c) Eco RI d) Ribozyme
3) Hexokinase is a (AIIMS)
a) Transferase b) Reductase c) Oxidoreductase d) Oxidase
4) Enzyme+ Coenzyme constitutes (AIIMS)
a) Apoenzyme b) Proenzyme c) Protoenzyme d) Holoenzyme
5) Kinases require (AIIMS)
a) Mn•2 b) Cu-2 c) Mg• 2
d) Inorganic phosphate
6) AST (SGOT) enzyme is most abw1dant
a) Brain b) heart c) Spleen d) Retina
7) All of the following are marker enzymes of Liver disease EXCEPT
a) SGOT b) ACP c) ALP d) SGPT
8) Which of the following enzyme is used to lyse intravascular clot?
a) Collagenase b) Hyaluronidase c) Asparginase d) Streptokinase
9) Which of the following enzymes belongs to the class lyase?
a) Glucokinase b) Aconitase c) Malate dehydrogenase d) Aldolase
10) An example for ligase is
a) Aldolase b) Acetyl CoA carboxylase _c) Fumarate d) Hexokinase
Answers for MCQ: 1) b 2) d 3) a 4) d 5) c 6) d 7) d 8) b 9) c 10) c

Overview of Metabolism 150
Overview of Metabolism
Contents:
• Definition
• General characteristics of Metabolism
• Categories (Anabolism and Catabolism)
• Stages of Metabolism
• Summary of Metabolism

Basic Aspects and Overview of Metabolism
Metabolism:
Definition: The term metabolism can be defined as the entire biochemical reactions
tha t are taking place in the body.
The compounds that take part in these reactions are called metabolites.
Metabolites include substrates (starting compounds), products and intermediates.
In a reaction pathway,
A - ••
- ---1 B C-- - -~ • D E
A is the starting compound
E is the end product
B, C and Dare the intermediates
Metabolism consists of 2 categories:
1) Anabolism
The chemical reaction pathways leading to the synthesis of a compound is termed as
anabolism. Anabolism generally requires energy.
2) Catabolism
The chemical reaction pathways leading to the degradation of compound is called
catabolism. Catabolism generally releases energy.
Metabolism = Anabolism + Catabolism

There are some pathways, wruch have both synthetic (anabolic) and degradative
(catabolic) roles. These are termed as amphibolic pathways.
Production of Energy :
Major function of food is to provide energy. Carbohydrates, fats and proteins (Amino
acids) are principle energy yielding compounds of the food.
The extraction of energy from these compounds can be studied in 4 stages,

Stage 1: Digestion and absorption: Fuel molecules are present in complex forms in the
food. In GIT, these compounds are converted into simpler monomeric units by digestion and
then absorbed. For examples, starch and glycogen are digested to glucose, fats to fatty acids
and glycerol and proteins to individual amino acids. These are then absorbed.
Stage 2: Formation of acetly CoA: Compounds formed in stage l (glucose, fatty acids,
glycerol, amino acids) are converted to acetyl CoA. In these pathways, energy is released
which are trapped as NADH++ H+ & FADH2• Some energy is directly trapped as ATP (substrate
level phosphorylation).
Stage 3: Citric acid cycle: Acetyl CoA formed from all these compounds undergoes Citric
acid cycle (TCA cycle) to give CO2 & Hp. So, TCA cycle is the final metabolic pathway for
the oxidation of acetyl CoA obtained from carbohydrates, fats & amino acids (proteins). Energy
released are trapped as NADH• + H. Some energy is directly trapped as GTP.
Stage 4: Oxidative phosphorylation: NADH+ + H• & FAD8z formed in stage 2 & 3 enter
electron transport chain present in mitochondria in the presence of oxygen to form ATP by
oxidative phosphorylation. Note: According to current concept, NADH gives 2.5 ATP &
FADH2 gives 1.5 ATP in ETC. Explanation is given ETC chapter.
CARBOHYDRATE PROTEIN FAT
l
Monosaccharides (Mainly glucose) Amino acids Glycerol and fatty acid
Stage 2 I1Fonnation of acetyl CoA)
ACETYLCoA
ATP
..--- FA0H, ........
ATP
CO, +H,O
Stage 4
(Oxidative phosphorylation)
• Sources and fate of Acetyl CoA:

Acetyl CoA is a central molecule in all 3 metabolisms (Carbohydrate, lipid & protein).
a) Sources: Acetyl CoA is formed from carbohydrates (from pyruvate by pyruvate
dehydrogenase), lipids (by ~-oxidation of fatty acids, ketolysis), catabolism of ketogenic
amino acids, oxidation of ethanol, cleavage of citrate (by citrate lyase) and HMG CoA.
b) Fate: Main fate of acetyl CoA is TCA cycle. It is also used for synthesis of fatty acids,
cholesterol, ketone body, acetylcholine, melatonin & detoxification of sulfa drugs.

Note to the students:

Invariably, cycles or metabolic pathways are asked in essay type of question, carrying
8 to 10 marks. Mere writing the reactions of pathway is not enou gh . Here's a guideline
to write answers to cycle or metabolic pa thways questions.
M a rk Dis t r i b u tion for Essa y Quest i ons on Cycles / Meta boli c pathways :
a) Defin i ti o n 0.5 mark
b ) Sites
i) T iss u e s ite - 0.5 m ar k
ii) In tr ace ll u la r s ite - 0.5 ma rk
c) S tartin g a nd En d prod u cts - 0 .5 m ark
d ) P a th way - - - - -- 4 ma r k- 5marks
e) E n ergetics (if any) - - - - - - 1 m ark
f) Regu la tion _ _ __ __ .._ 0 - 1 mark
g) Sign ificance - - - -- - 1 - 2 ma r ks
Total 8 -10 mark s ote that this is only a guideline to answer the cycles /
metabolism questions. Based on the type of questions, this
format may vary (For e.g., gluconeogenesis does not have
energetics, but sign ificance carr ies more weightage).
Exam tips for energetics
in energy pathways
ATP, GTP = 1 ATP
FADH2 = 2ATP
NADH = 3ATP
Exam tips:
According to new concept,
FADH 2 gives 1.5 ATP Kinase enzymes requires ATP, Mg•2
& NADH gives 2.5 ATP Dehydrogenase: NAD/NADP / [AD
Reactions in cytosol Partly in cytosol, partly Reactions in

in mitoch ondria Mi tochon clria
Qycolysis, Glycogenesis, Heme synthesis TCA cycle, ~-ox idation,
Qycogenolysis, HMP shunt, Urea cycle Pyruvate dehydrogenase
De novo syn thesis of fatty acids, GI ucone ogeneifa EfC and Oxidati,·e
Transaminatim, Purine synthesis Pyrimidine syn thesis phosphorylation
Ketogenesis, Ketolysis

Digestion and Absorption 154
Digestion and Absorption
Contents:
• Definition
• General characteristic of digestion and absorption,
• Digestion and absorption of

• Carbohydrates
• Proteins
• Lipids
• Malabsorption Syndrome, Lactose intolerance

Digest ion and Absorption

General Aspects
Most of the foodstuffs taken in the diet are in the complex form, which cannot be
absorbed from GIT unless they are broken down into simpler molecules.
• Digestion is the process of hydrolyzin g large and complex food materials into the
smaller and simpler form in the GIT.
• Absorption is the transport of digested products from the intestinal lumen into
blood or lymph across the intestinal mucosa! cells.
Digestion - General aspects:

• Digestion is the process of hydrolyzin g large and complex food materials into the
smaller and simpler form, which can be absorbed in the GIT.
• The major foodstuffs are carbohydra tes, proteins and fats (triacylglycerols). Digestion
is brought about by action of enzymes that are present in different digestive juices
like saliva, gastric juice, pancreatic juice, intestinal juice and bile.
• During the process of digestion, polysaccharides and disaccharid es are converted
to monosacch arides, proteins into amino acids and triacyl glycerols (fats and oils)
into glycerol and fatty acids. These simple forms are ready for absorption.
Note: Cooking of the food and mastication (in the mouth) considerab ly improves the
digestibili ty of food. Cooking causes the hydration of polysacch arides and
denaturati on of proteins, which helps in digestion. Masticatio n helps in breaking
down of food particles, thus increases solubility and surface area for enzyme action.
Absorption - General aspects:

• Absorption is the transport of digested products from the intestinal lwnen into
blood or lymph across the intestinal mucosal cells.
• The small intestine is the main absorptive organ. About 90% of the ingested
foodstuffs are absorbed in the course of passage through the small intestine. Note
that water is mainly absorbed in the large intestine.
Absorption of substances into the intestinal cells involves the passage of substances
across the cell membrane and it takes place by 2 processes.
1) Simple diffusion: Does not require carrier molecules and energy.
2) Facilitated transport: Requires a carrier protein, but does not require energy.
3) Active transport: Requires both carrier protein molecule and energy.
Answer hint for Digestion and Absorptio n (10 Marks):

a) Digestion - 6 mark
b) Absorption - 3 Mark
c) Si · ·cance (an Additional oints) - 1 mark

Digest ion and Absorption of Carbohydrates

I. Digesti on of carbohydrates:
• Major dietary carbohyd rates are polysacc harides (starch) and disacchar ides (sucrose,
lactose). Small amounts of monosac charides (fructose, glucose and pentoses) and
dextrins are also present in the food. Diet may also contain trehalose (a disaccha ride
present in mushroo ms) and maltose (disaccha ride present in malt and beer).
• Generally starch forms more than 50% of carbohydrates in human food.
• Digestion of carbohyd rates occurs briefly in mouth and largely in intestine.
• Glycogen digestion: There is practically no dietary source of glycogen; any glycogen
stored in animal organ is quickly converte d to lactic acid at the time of slaughter.
However , if synthetic glycogen is given, it is digested similarly as starch.
a) D igestion of starch:
• Digestion of starch begins in mouth and continue s in intestine.
• Salivary amylase and pancreat ic amylase are the important enzymes of starch
digestion. CI· is an activator of these enzymes.
i) Digestio n in the Mouth
• Salivary amylase (ptyalin) of saliva starts the digestion of cooked starch in the
mouth. Very little digestion takes place in the mouth as the food remains in the
mouth for a very short period of time.
• Salivary amylase randomly hydrolyz es starch at internal a.-1, 4 glycosidi c bonds to
give short oligosacc harides (branche d or unbranch ed) and maltose.
Salivary amylase Maltose+ Maltotriose
Starch Unbranch ed oligosaccharides
(also Glycogen) CJ- Branched Oligosaccharides (a -Limit dextrins)
ii) D igestion in the Stomach

• When the food gets mixed with gastric juice, the action of amylase stops due to
acidity. (pH of gastric contents is less than 3 due to presence of HCl, which denature s
and inactivate s salivary amylase).
• However some sa livary amylase action continue s for some time a t the centre of the
food bolus in the stomach.
iii) D igestion in the Small Intestin e
Pancreatic amylase continue s the digestion of starch in the intestine. Action is similar
to salivary amylase.
Pancreatic amylase Maltose + Maltotriose
Starch Unbranch ed oliga;accharides
(also Glycogen) O· Branched OLiga;accharides (a -Limit dextrins)
For queries and suggestio ns, contact the author at prasad_te xt@yahoo.com or 9986449575
Additional points on lipase action:

• Both salivary and pancreatic amylases are endoglucosidases, which act on internal ex -1, 4
glycosidic bonds. They cannot act on terminal ex -1, 4 glycosidic bonds and any a -1, 6 glycosidic
bonds. They also do not act on a -1, 4 bonds adjacent to a -1, 6 glycosidic bonds.
• Starch has two components, amylase and amylopectin. Amylase of starch is linear, straight chain
(having only a -1, 4 bonds) and amylopectin of starch are branched chain structures (having both
a -1, 4 and a -1, 6 bonds). Action of amylases on amylase give maltotriose, maltose, unbranched
oligosaccharides, where as action of amylases on amylopectin give maltose and maltotriose, branched
oligosacclzarides (i.e. a -limit dextrins) and unbranched oligosacclwrides. a -limit dextrins
generally have 6 - 8 glucose molecules with one a -1, 6 branch point.
• As amylases do not net 011 terminal bonds, glucose is not normally produced by amylase action.
Since amylase cannot act 011 ex -1, 4 bonds adjacent to the branch points (a -1, 6), isomaltose are
also not produced by amylase action.
• Amylases cannot hydrolyze linkages. So, amylases cannot digest cellulose.
iagrammatic representation of action of amylase (Salivary/Pancreatic) on stare
(Amylose of starch)
Salivary amylase I
Pancreatic amyalse
l (Amylopectin of starch)
0-0
00 0-0-0
Maltose Maltotriose
0-0-00-0-0 0 0 Branched oligosaccharide

Unbranched oligosaccharide (a-Limit dextrin)
Further digestion of these disaccharides and oligosaccharides:

The final digestion of these disaccharides and oligosaccharides is carried out b y
disaccharidases (Maltase and Isomaltase enzym es), which are present in the brush
border of small intestine. These enzymes are also referred to as b rush bord er enzymes.
l)Maltase digests maltose, maltotriose, limit dextrins and unbranched (linear)
oligosaccharides. Maltase removes single glucose residues from the non-reducing ends.
Maltase enzymes do not act on branch points (a-1, 6 bonds).
2) Isomaltase digests isomaltose as it breaks a-1, 6 bonds. Isomaltase also removes the
branch points (1 • 6) from a-Limit dextrins (branched oligosaccharides).

• Maltose Maltase 2 Glucose molecules
• Maltotriose Maltase 3 Glucose molecules
• Unbranched Maltase n Glucose molecules

ol igosaccharides
• a- Limit dextrins Isomaltase Small Unbranched

oligosaccha rides
• a- Limit dextrins Maltase lsomaltose, Glucose molecuJes
• lsomaltose lsomaltase 2 Glucose molecules
Thus the complete digestion of starch yields glucose residues.
Note that Maltase breaks a -1,4 bonds and lsomaltase breaks a -1,6 bonds.
• Maltose Maltase 2 Glucose molecules

0-0 0 0
• Ma Itotriose Ma ltase 3 Glucose molecules

0-00 0 0 0
• Unbranched oligosaccharides Maltase n Glucose molecules
0-0-0-0-0-0 000000
• o:- Limit dextrins Isomaltase Unbranched oligosaccharides
0-0--0-0
0-0-0-0
• a- Limit dextrins Maltase lsomaltose, Glucose molecules

000
000
• lsomaltose Isomaltase 2 Glucose molecules
l
0
0

b) Digestion of sucrose:
i) Brush border enzyme sucrase (Invertase) hydrolyses sucrose into a molecule of

glucose and a molecule of fructose.
Sucrase
Sucrose - - - - • Glucose + Fructose
ii) Some sucrose is digested in the stomach by HCl
HCl
Sucrose Glucose+ Fructose
c) Digestion of lactose:
Brush border enzyme lactase, hydrolyses lactose into a molecule of glucose and a
molecule of galactose.
Lactase
Lactose _ _ __., Glucose + Galactose
d) Digestion of trehalose:
Brush border enzyme trehalase, hydrolyses trehalose into 2 molecules of glucose.
Trehalase
Trehalose 2 Glucose molecules
Additional points:
Brush border enzymes:

Outer surface of small intestinal epithelial cells (brush border cells) contain many
disaccharidases and oligosaccharidases, which hydrolyze dietary and digestion
derived disaccharides and oligosaccharides. These enzymes are also referred to
as brush border enzymes. These enzymes are firmly attached to the cell surfaces
with their catalytic domains protruding into intestinal lumen.
Examples: Lactase, Maltase, Sucrase, Isomaltase, Trehalase
Sucrase - Isomaltase enzymes are initially synthesized as single polypeptide

chain which is later cleaved into two subunits, each having a distinct enzyme
activity. Both these enzymes are embedded in brush border of small intestine.

II. Absorption of carbohydrates:

The products of carbohydrate digestion are mainly glucose and small amounts of
fructose and galactose molecules.
Absorption of monosaccharides is a carrier mediated process.
Monosaccharides are absorbed from intestinal lumen to the intestinal cells by 2 carrier
mediated mechanisms, namely, 1) Secondary active transport 2) Facilitated diffusion
1) Glucose and galactose are absorbed by secondary active transport.

• Active transport requires both carrier protein and energy.
• A carrier protein called sodium dependent glucose transporter (SGLT 1) binds both
glucose and Na• at separate sites and transports them from intestinal lumen to intestinal
mucosal cells. This is symport (a type of co-transport) transport mechanism.
2) Fructose (for a small extent glucose & galactose) is absorbed by facilitated diffusion.
• Facilitated diffusion requires a carrrier protein, but does not require energy.
• A carrier protein called sodium independent transporter (GLUT 5) transports fructose
& also glucose & galactose (for a small extent) from intestinal lumen to intestinal mucosa.
Transport of Monosaccharides from intestinal cells to the Capillaries:

Glucose and other monosaccharides are transported from the intestinal cells into the
capillaries by GLUT-2 (sodium independent glucose transporter).
It is a uniport facilitated diffusion system.
Glurosc / Fn11:1ow / Galar loS<· Glurnw / Galat·to~l' Lumen of Intestine
(Na' -indc11cntlc111
lrnn,-porll·rt ,'/a• -d~pl'nclcnl 1rnn,,por1cr
GI.UT-5 - - - - , •• Brus h hordcr of Intestina l

Epithelial Cell
GI.UT-1
To capillark~
Galat·1n,l' / Fru,·10,t· I Gh1<·0,t· J Na* 1 I\. '
0 l'\11•-indc11c11dcnt
tran:..1wrtcr
N:. • -dcpcntlcnt
tr:111:..porlcr ' N.1•-1<• ATJ>asc

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

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

Digestion and Absorption of Lipids

More than 90% of the dietary lipids are fats and oil (triacylglycerols). The rest is
mainly made up of cholesterol and phospholipids.
I. Digestion of lipids:
Lipids digestion poses a problem as lipids are insoluble in water and lipid digesting
enzymes are in water medium. This problem is overcome by emulsification of fats by
bile salts in the intestine (and peristaltic movement in stomach).
Very little digestion takes place in mouth because lipids are not emulsified in the mouth
and hence lingual lipase cannot act on them.
Emulsification of lipids:
Definition: Combination of bile salts with lipids to form micelles is termed as emulsification.
• Lipids digestion poses a problem as lipids are insoluble in water and lipid digesting enzymes
cannot act on them. This problem is overcome by emulsification of lipids by bile salts in the
intestine (and peristaltic movement in stomach).
• Bile salts have the property of reducing the surface tension. This property enables them to
emulsify lipids and form micelles. Micelles are amphipathic. In micelles, lip id molecules are
dispersed as fine emulsions that increase their surface area, enabling enzymes to act on them.
• Emulsification and micelle formation also helps in the absorption of lipids.
A) Digestion of triacylglycerols (fat):
i) Digestion in the Stomach

Lingual and gastric lipases initiate lipid digestion by hydrolyzing triacylglycerols to
form mainly 1, 2 diacylglycerols and free fatty acids. Peristaltic contractions of stom ach
emulsify lipids for some extent. Owing to the retention time of 2-4 hours in s tomach,
up to 30 % of fats can be digested in stomach. The enzymes are soon destroyed at low
p H of the stomach.
ii) Digestion in the Intestine

Pancreatic lipase enzyme hydrolyses triglycerides in the intestine. When acid chyme
from stomach comes to intestine, the alkaline content of the pancreatic and biliary
secretion neutralizes it and changes the pH of this matter to the alkaline sid e. This is
necessary for the action of pancreatic lipase. Bile also contains bile salts, which emu lsify
fats in th e intestine.

Action of pancreatic lipase:

• This enzyme hydrolyzes triglycerides in the intestine. Colipase, a protein secreted by
pancreas, acts as cofactor for pancreatic lipase.
• Pancreatic lipase attacks the primary ester linkage i.e. C-1 and C-3 of triacylglycerols,
hydrolyzing ester linkage a t C-3 first and then at C-1 producing 1,2-diacylglycerol
first and then 2-monoacylglycerol. A fatty acid molecule is released at each step.
• An isomerase then shifts the ester bond from position C-2 to C -1 to convert
2-monoacylglycerol to 1-monoacylglycerol, which is then hydrolyzed by the lipase
to form glycerol and fatty acid.
Triacylglycerol
Lipase
(Colipase)
l Fatty acid
1, 2- d iacylglycerol
Lipase
(Colipase) pl Fatty acid
2- Monoacylglycerol
Isome,~•!
1- Monoacylglycerol
Lipase
(Colipase) tL
"a. Fatty acid
Glycerol
Note: Isomerisation is a relatively slow process. As a result, the major end p roducts of
the digestion of Triglyceride are 2-monoacylglycerol (72%), 1-monoacyl glycerol (6%),
glycerol (22%) and fatty acids.
B) Digestion of Cholesterol ester and Phospholipids:

Pancreatic secre tion also contains cholesterol esterase and phospholipase A2.
i) Cholesterol esterase hydrolyzes cholesterol ester to cholesterol and fatty acid.
ii) Phospholipase A 2 hydrolyzes phospholipids to lysophospholipid and fatty acid.
Products of lipid digestion:

The products of lipid digestion are fatty acids (short chain, medium chain & long chain),
2-monoacylglycerol, 1-monoacylglycero l, glycerol, cholesterol and lysoph ospholipid.

II. Absorp tion of lipids:

The lipid digestion prducts that are to be abosrbed are fatty acids (short chain, medium
chain & long chain), 2-monoa cylglycer ol, 1-monoa cylglycer ol, glycerol, cholester ol
and lysophos pholipid . There are two ways by which these are absorbed ,
• Direct: The water-sol uble products of lipid digestion like glycerol as well as s mall
and medium chain fatty acids are directly absorbed from the intestinal lumen in to the
portal vein and taken to the liver and immedia tely utilized for energy.
• As bile salt micelles: The water-ins oluble products like 2-monoacylglycerols, long
chain fatty acids and small amounts of 1-monoacylglycerols, cholesterol, phosphol ipids
and lysophos pholipids are incorpor ated into bile salt micelles and absorbed into the
intestinal cells.
In the intestinal mucosa! cells, triacylglycerols are reconstit uted. Then triacylglycerols
and other lipids are incorpora ted into chylomic rons and transport ed from intestine to
periphera l tissues.
Summary of Digesti on and Absorption of triacylglycerols
Intestinal lumen Intestinal epithelial cells Lacteals

(Lymphatic vessels)
Trlacylglyce rol
FA1~;;;:; 0 = Monoacylglycerol pathway
1,2-Diacylgly cerol
0 = Phosphatidic acid pathway
FA
1 Pa11creati
lipase
2-Monoacylglycerol -+--
(~72 %)
2-Monoacylglycerol
_B
i Triacylglycerol Triacylglyce rol
Fatty Acids _ _ __ __ _ _T_ru_·o_ki_nas_e..
Isom erase
Fattv acvl CoA
Thiokinase
A
l
Chylomicrons
1-Monoacylg lycerol 1-Monoacy lglycero._t _. Glycero.-. Glycerol

(-6 %) Intestinal G/yu rol 3-P
+--- - - -
i
.~ Pancreatic lipase kinase
FA II> lipase Portal vein
(-22 %)
Glycerol --t-- ---- ----- - - - - -- -+-1• Glycerol
For queries and suggestio ns, contact the author at prasad_te xt@yahoo .com or 9986449575
Bile salts and role of micelles in digestion and absorption of lipids :

Bile salts are essential for the digestion and absorption of lipids. They combine with fats &
other lipids and reduce their surface tension to form micelles. This process is called
emulsification. This process facilita te the action of lipolytic enzymes.
Micelles are spherical particles with a hydrophilic exterior & hydrophobic interior core. Bile
salt and lecithin that are present in bile are amphipathic and hence form micellar aggregation.
Products of lipid digestion are incorporated into micelles to form mixed micelles.
These mixed micelles fuse with the cell membrane of intestinal mucosa! cells whereby the
products of lipid digestion are internalized into the mucosal cells.
The bile salts are left behind which are mostly reabsorbed from the ileum and returned to
the liver. They are excreted back to the bile (enterohepatic circulation).
Normally Over 98% of the dietary lipid is absorbed.
Re-esterification inside Mucosal Cell:

Inside the intestinal mucosa! cells, the long chain fa tty acids are re-estenfied to form
triglycerides by two pathways.
• Monoacylglycerol pathway (MAG pathway): In this pathway, the fatty acids are first
activated to fatty acyl CoA by the acyl CoA synthetase. Two such activated fatty acids interact
with 2-monoacylglycerol to form TAG. Majority of molecules take up this MAG pathway.
• Phosphatidic pathway: In this pathway, free glycerol absorbed from the intestinal lumen
directly enters into the blood stream. So free glycerol is not available for the re-esterification.
But the cells can convert glucose to glycerol-3-phosphate and then add 3 molecules of fatty
acyl CoA to synthesis TAG (Phosph atidic pathway).
The absorbed lysophospholipids and much of the absorbed cholesterol are also re-acylated
with acyl CoA to regenerate phospholipids and cholesterol esters.
• Chylomicron Formation:
Triacylglycerol, cholesterol ester, cholesterol, phospholipids and fat-soluble vitamins along
with apoproteins are incorporated into chylomicrons.
The chyle (milky fluid) from the intestinal mucosa! cells loaded with chylomicrons are
transported through the lymphatic lacteals into the thoracic duct and then emptied into
systemic circulation. The serum may appear milky after a high fat meal (post-prandial
lipemia) due to the presence of chylomicron in circulation. Normally, the lipemia is cleared
within few hours by the uptake of chylomicrons by tissues.
Short and medium chain fatty acid s do not need re-esterification. They can directly enter
into blood vessels. They are better absorbed than long chain fatty acids.
Cholelithiasis- cholesterol gall stone disease: Bile salts & phospholipids are responsible
for keeping cholesterol in soluble form in bile. When cholesterol concentration in bile
increases or in the absence of bile salts, cholesterol may get precipitated in gall bladder
producing cholesterol s tones, a condition known as cholelithiasis (Gall stone).
Cholelithiasis may block the flow of bile to intestine and lead to malabsorption syndrome
symptoms like steatorrhea due to defective digestion and absorption of lipids (as bile
salts are required for the digestion & absorption of lipids) and obstructive jaundice.
For queries and S!Jggestions, contact the author at prasad_text@yahoo.com or 9986449575

Malabsorption syndrome :
Malabsorption syndrome is comprised of a group of clinical disorders, which are due
to defective digestion or defective intestinal absorption of nutrients.
Although absorption of all nutrients (fat, carbohydrate, protein, vitamins and minerals)
may be adversely affected, malabsorption of fat and fat soluble vitamins is generally
the most important manifestations of general malabsorption syndromes.
Findings:
a) Steatorrhea (Fatty s tool): Characterized by foul smelling fatty stool due to excessive
excretion of fats / lipids(> 6g/ day). This is due to the defective digestion or absorption
(or both) of lipids. Steatorrhea is the most characteristic symptom of malabsorption.
b) Vitamin and mineral deficiencies: Some of the clinical manifestations (mainly anemia,
rickets and osteomalacia) are due to the inadequate absorption of vitamins (Both fat
soluble and water soluble) and minerals like calcium and iron.
c) Protein deficiency: Marked protein deficiency is also seen.
Symptoms: Abdominal distention, diarrhea, foul smelling stool, loss of weight, anemia
and manifestations of vitamin and mineral deficiencies are the major symptoms.
Causes of malabsorption:
1) Due to acquired disorders:
a) Due to defective digestion:
• Pancreatitic: Panreatitis, Pancreatic tumor, Zollinger-Ellison syndrome etc
• Biliary: Obstruction, cirrhosis, hepatitis etc.
• Gastric: Partial or total gastrectomy, gastro jejunostomy
b) Due to defective absorption:
• Disease of the gut: Jejuno-ilietis, tuberculosis, amydoilosis
• Decreased surface are of intestinal mucosa: Castro-colic fistu la, extensive intestinal resection
• Bacterial or parasitic infection of small intestine; e.g.: Blind loop syndrome
• Primary intestinal malabsorption: Tropical sprue, gluten-sensitive enteropathy.
2) Due to genetic disorders:
• Inborn errors of mono and disaccharide digestion and absorption
• Inborn errors of amino acid absorption
• Ma/absorption associated with abetalipoproteinemia
• Ma/absorption is associated with hypogammaglobulinemin, agammaglobulinemia
Chyluria: Urine appears milky due to the presence of lipid droplets. There is abnormal
connection between the urinary tract and lymphatic drainage system of the intestine.
Chylothorax: Chyle may leak into the pleural cavity from the thoracic duct or its
tributaries due to trauma (stab wounds, crush injuries, surgical injury) or obstruction
by tumor. It is common ly mistaken for pus. Chyle contains fat globules.

Digestion and Absorption of Proteins

Dietary proteins are denatured on cooking and therefore more easily digested. In
denatura tion unfolding of protein molecule takes place and thus peptide bonds
become more accessible for the enzyme action.
I. Digestion of Proteins :
A group of enzymes called the proteases or peptidases in the gastrointestinal tract
digests dietary proteins. These enzymes hydrolyze specific peptide bonds in proteins.
Peptidases are mainly two types ;
• Exopeptidases: Such as carboxypeptidases and aminopeptidases hydrolyze only
terminal peptide bonds connected to the last amino acid residues of the peptide chain
and release that terminal amino acid.
• Endopeptidases: Such as pepsin, trypsin, chymotrypsin and elastases hydrolyze
specific peptide bonds in the middle of the peptide chain to cleave the polypeptide
into smaller protein chains or peptides.
Proteolytic enzymes are secreted as inactive zymogens. They are converted to active
zymase form in the intestine. This activation involves the cleavage of small peptides.
This prevents the auto-digestion of the secretory acini.
• Digestion of proteins also require di peptidases and tripeptidases, which are present
in brush border of intestine.
Digestion of proteins begins in stomach and then continues in intestine.
i. Digestion in the Stomach:

Protein digestion starts in the stomach by HCl, pepsin and rennin of the gastric juice.
a) HCI
HCl is secreted by parietal cells of gastric gland . It makes the pH optimum for the
action of pepsin, activates pepsin, denatures the proteins and also kills
microorganisms. Theoretically HCl can also hydrolyze proteins, but this process
requires high temperature. So, HCl cannot break peptide bonds at room temperature.
b) Rennin (Chymosin)
Rennin is a protease. It is active in infants and is involved in the curdling of milk.
Rennin denatures casein of milk to paracasein, which coagulates in the presence of
calcium ions to form insoluble calcium-paracaseina te, which then can easily be digested
by pepsin. Rennin is absent in adults.

c) Pepsin
Pepsin is secreted by the chief cells of stomach as pepsinogen, which is activated to
pepsin by HCl. Later pepsin itself converts pepsinogen to pepsin (Autocatalysis).
HCl
Pepsinogen C-
____::.--- Pepsin
Pepsin is an endopeptidase hydrolyzing specific peptide bonds. Proteins are broken

into polypeptides of lower molecular weight proteases and peptones by Pepsin.
Pepsin
Dietary proteins - - -- - - - Proteoscs and Peptones
ii. Digestion in the small intestine :

Most of protein digestion occurs in the duodenum & jejunum of small Intestine. Pancreatic
juice contains proteolytic enzymes like endopeptidases such as trypsin, chymotrypsin
& elastase; exopeptidases like carboxypeptidases, which are secreted as zymogens and
converted into active 'Zymase' in the intestine. Enterokinase, present on the intestinal
microvillus membranes activates trypsinogen to trypsin. Trypsin then autocatalyses its
own activation & also catalyses the activation of chymotrypsinogen, proelastase &
procarboxypeptidases to chymotrypsin, elastase & carboxypeptidases, respectively.
Enterokinase
Trypsinogen trypsin
Trypsin
Trypsin ogen trypsin
( _)
Trypsin
Chymotrypsinogen Chymotrypsin
Try psi n
Proelastase Elastase
Trypsin
Procarboxypeptidase - - - - --+ Ca rb oxypeptidase
Action of trypsin, chymotrypsin and elastase enzymes in the small intestine:

Trypsin, chymotrypsin & elastase are endopeptidases, which hydrolyzing specific
peptide bonds. They acts on proteases and peptones (produced by action of pepsin on
proteins in stomach) and also proteins to form small polypeptides & oligopeptides.
Trypsin, Bastase
Proteoses and Peptones - - -- -- + Small polypeptides + Oligopeptides
Chymotrypsin

Complete digestion of proteins to amino acids require aminopeptidases, di peptidases

and tripeptidases, which are present in intestinal juice.
• Carboxypeptidases hydrolyze one amino acid at a time from the carboxy-terrninal
peptide bonds of polypeptides and oligopeptides
• Aminopeptidases hydrolyses one amino acid at a time by attacking the peptide bonds
from amino-terminal amino acids of polypeptides and oligopeptides.
• Di peptidases and Triipeptidases hydrolyses dipeptides and tripeptides respectively
(which are produced during the course of reaction) to am ino acids.
Aminopeptidase, Carboxypeptidase
Small polypeptides+ Oligopeptides Amino acids
Dipeptidase, Tripeptidase
( Summary of digestion of proteins )
Pepsin
Proteins Proteoses,
(Stomach) Peptones
Trypsin
(Intestine) Chymotrypsin
l Elastase
Small Polypeptides + Oligopeptid es
Carboxypeptidases
(Intestine) Aminopeptidases
Tripeptidases
Dipeptidases
Amino acids
Specificity of endopeptidases:
Pepsin, trypsin, chymotrypsin and elastase are endopeptidases, which hydrolyze specific
peptide bonds, contibuted by specific amino acids.
Chymotrypsin Phe, Tyr, Trp, Val, Leu (Aromatic, uncharged amino

Acids)
Elastase Ala, GI , Ser small amino acid residues)

Absorption of amino acids:

Under normal circumstances the dietary proteins are almost completely digested to
their constituent amino acids and w ith a small amount of di- and tripeptides and
oligopeptides, all of which absorbed. In infan ts, entire IgA of mothers milk is absorbed.
Free amino acids are absorbed by tw o types of transport systems:
1) Sodium dependent secondary active transport systems:

Free amino acids are absorbed by various sodium dependent secondary active transport
systems. A sodium dependent amino acid transporter binds both amino acid and Na+
at separate sites and transports them both from intestinal lumen to intestinal mucosa!
cells. This is a symport (a type of co-transport) transport mechanism. There are at least
6 different amino acid transporters specific for various amino acids have been identified,
• Small neutral amino acid (Alanine, serine, threonine)
• Large neutral amino acids (Isoleucine, Leucine, Valine, phenylalanine, tyrosine, tryptophan)
• Acidic amino acids (Aspartate and glutamate)
• Imino acids (proline and hydroxy proline) and glycine
• Cysteine and Dibasic amino acids (Ornithine, Arginine, Lysine), termed as 'COAL'.
• For f3-amino acids (f3-alanine)
2) y-glutamyl cycle (Meister cycle):

In intestine, kidney and brain, another transport system called the y-glutamyl cycle or
Meister cycle operates to absorb amino acids. Meister cycle is not a common mechanism
for amino acid absorption. This cycle requires 3 ATP'S for the absorption of a single
amino acid. However, it takes place whenever a rapid absorption of certain neutral
amino acids like cysteine & glutamate is required.
(Sodium dependent active transport) ( -y-glutamy l cycle (Meister cycle) )
Amin o acid
Amin o acids
Na·
Na•
3 ~a ·
r--Tuns porler
2K
l
Intestinal lumen
membrane
G lutathione
(y-GI u--cys-gl y)
Amino acid
Cys- gly
y-glutamyl-amino acid
Glu
Intestinal lumen
membrane
of intestin.il
ATP
A DP + P,
Na- K
pu mp
of intestinal
mucosa F
Cys - Qy-Glu
mucosa
Amino acid 3Na , 2 K, Intestinal mucosat cell A mi no acid Intestinal mucosa I cell
Di- and tripeptides are absorbed by a proton linked transport system into the brush
border of intestinal mucosa! cells, where they are hydrolyzed to free amino acids,
which are then transported into the portal vein. Therefore, only free amino acids are
found in the portal vein after a protein meal.
Oligopeptid es also rarely get absorbed in intestine by endocytosis. Sometimes, many
such peptides are large enough to stimulate antibody formation - this is the basis of
food allergy. E.g.: Wheat allergy in some people due to partially digested wheat proteins.

Qustion bank on Digestion and Absorption
Essay questions :
1. Give an account of digestion and absorption of carbohydrates
2. Give an account of digestion and absorption of lipids?
3. Give an account of digestion and absorption of protein
Short Essays :
1. Malabsorption syndrome
2. Lactose intolerance
Short Answers :
1. Role of bile salts in digestion and absorption of fat
2. Amylases and Pepsin
3. Digestive enzymes of Pancreatic juice and Intestinal juice
4. Write briefly on intestinal absorption of a) Glucose b) Amino acids c) Lipids

1. Which of the following enzymes is stable at acidic pH (AI)
a) Pepsin b) Trypsin c) Chymotrypsin d) Carboxypeptidase
2. Alpha amylase acts on which bond (AI)
a) Alpha 1-4 bond b) Alpha 1-6 c) Beta 1-4 d) Beta 1-6
3. Detergent action of bile salts is due to (MAHE)
a) Hydropathic b) Acts as a zwitter ion c) Arnphipathic d) All
4. The enzyme responsible for protein d egradation in stomach is (AI)
a) Trypsin b) Pepsin c) Ch ymotrypsin d) Aminopeptidase
5. Which one is not an endopeptidase (JIPMER)
a) Trypsin b) Pepsin c) Chymotrypsin d) Arninopeptidase
6) Steatorrhoea is characterized by daily excretion of fat more than
a) 2gm b) 4 gm c) 6 gm d) 8 gm
7) Characteristic feature of Micelle is
a) Nonpolar b) Charged c) Amphipathic d) Polar
8) Which of the following is a carboxyproteases?
a) Pepsin b) Trypsin c) Carboxypeptidase d) Renin
9) Bile acids are required for the digestion and absorption
a) Lipids b) Protein c) Minerals d) Carbohydrate
10) Renin changes the casein of milk to paracasein in the presence of
a) Mg++ b) Mn++ c) Ca++ d) Ba++
Answers for MCQ: 1) a 2) a 3) c 4) b 5) d 6) c 7) c 8) c 9) a 10) c

Carbohydrate Metabolism 172
Carbohydrate Metabolism
Contents:
• Introduction
• Glycolysis
• TCA cycle
• Substrate level phosphorylation
• Glycogen metabolism
i) Glycogenesis
ii) Glycogenolysis
• HMP shunt pathway
• Gluconeogenesis
• Cori cycle
• Galactose metabolism
• Fructose metabolism
• Hormonal regulation of glucose
• Glycosuria
• Diabetes mellitus
• Glucose tolerance test (GIT)
• Glycated hemoglobin, Fructosamine, Microalbuminuria
Use of Italics :
additional information and may not be needed for the basic understanding of the ch apter.
So, students who want to omit these segments in first reading, can do so withou t
of the subject. However, these italic segments may be asked in MCQ, Viva. Short

Introduction to carbohydrate metabolism:

• All forms of life require energy for their growth and maintenance. Among the energy
yielding compounds, carbohydrates occupy a central role in energy metabolism.
• Carbohydrates are easily available and is a cheap source of food for man. Eventhough
fats and proteins can also give energy, carbohydrates should be present in minimum
amounts in the food. This is because some of the tissues in the body (like brain,
erythrocytes) have an obligatory requirement for glucose and glucose is the preferred
source of energy for most of the body tissues.
• All the carbohydrate metabolic pathways are connected with glucose. Dietary starch
gives glucose during digestion. Fructose (obtained from sucrose) and galactose (obtained
from lactose of milk) are converted to glucose in the body. So, glucose occupies a
central stage in carbohydrate metabolism.
Entry of glucose into cells:

Glucose enters into the cells by 2 mechanisms;
1) Insulin dependent transport system: This mechanism requires insulin for their
entry into the cells. This occurs in muscle and adipose tissues.
2) Insulin independent transport system: This mechanism does not require insulin for
their entry into the cells. This occurs in liver, RBC's and brain tissues.
Overview of glucose metabolism:

Glucose is principal energy source in body. It is metabolized to pyruvate aerobic
conditions (or lactate in anaerobic conditions) by glycolysis to give energy. In aerobic
conditions, pyruvate further oxidized to acetyl CoA, which then enters TCA cycle for
complete oxidation to CO2 and H 20 releasing energy. In fed conditions, excess of glucose
is converted to glycogen by glycogenesis, and still excess of glucose is converted to fat
by lipogenesis. When blood glucose is lowered (i.e. when glucose is exhausted),
glycogen is broken down to glucose by glycogenolysis to maintain the glucose level.
When the glycogen is, then body gets energy from alternative sources like fats and
proteins. But, tissues like brain has an obligatory requirement for glucose, it is produced
from non-carbohydrate sources like lactate, glycerol and glucogenic amino acids by
gluconeogenesis. Small amount of glucose also can enter minor metabolic routes like
HMP shunt pathway, uronic acid pathway, lactose synthesis, sorbitol pathway etc.
Glycolysis: Breakdown of glucose for energy is called glycolysis.
TCA cycle: Oxidation of acetyl CoA to CO2 & H 20 releasing energy is called TCA cycle
Glycogenesis: Formation of glycogen from glucose is called glycogenesis.
Glycogenolysis: Breakdown of glycogen is called glycogenolysis.
Gluconeogenesis: Formation of new glucose from non-carbohydrate sources like lactate,
glycerol and glucogenic amino acids is called gluconeogenesis.

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

a) In tissue under hypoxic conditions
b) In vigorously contracting skeletaJ muscles
c) In RBC's, Lens etc., which lack mitochondria

Glucose
IReaction PathwayI ATP
Mg·' Hexokinase in all tissues. Glucokinase in liver
A DP
Glucose 6•Phosphate
t Phosphohexose isomerase
Fructose 6·phosphate
ATP
Phosphofructokinase (PFK)
ADP ~ i
Fructose 1,6-bisphosphate
Glyceraldehyde 3·phosphate Dihydroxyacetone phosphate

p~ Phosphotrobe isomerase
NAD+ Glyceraldehyde 3-Phosphate dehydrogenase
NADH+H+
1, 3-bisphosphoglycerate
A DP ~
Mg•2 Phosphoglycerate kinase
ATP
t
3-pbosphoglycerate
Phosphoglycerate mutase
2-phosphoglycerate
Mg·2 j Enolase
H20 Y!
Phosphoenolpyruvate
ADP:i
Mg·2 Pyruvate kinase
ATP
Pyruvate
In aerobic conditions, pyruvate is the end product of glycolysis, but in anaerobic
condition, pyruvate is further converted to lactate by lacta te dehydrogenase (LDH).
ADH• + tt• NAD•
Pyru vate •1-----'-----L,._____ •~Lactate
4 -i
Lactate dehydrogenase
LDH utilizes NADH formed in glyceraldehyde

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

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

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

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

Regulation of glycolysis :
The energy requirement of the cell regulates the rate of glycolysis. When the cell
requires energy, the glycolysis operates in a faster rate and when the cell has sufficient
energy, glycolysis runs slowly. Hexokinase I Glucokinase, Phosphofructokinase
(most important enzyme), Pyruvate kinase are the regulatory enzymes of glycolysis.
Indu cer Re pressor Activator Inhibitor
Hexol<lnase Glucose 6-phosphate

- -
Glucokinase Insulin Glucagon
- --
Phosphofructokinase Insulin Glucagon AMP,
Fructose 6-phosphate ATP, Citrate
Fructose 2,6-bis phosphate
Pyruvate kinase Insulin Glucagon Fructose 1,6-bis phosphate ATP
Pasteur Effect: Under aerobic conditions, glycolysis is inhibited. The inhibitory effect
of oxygen on glycolysis is known as Pasteur effect.
Difference between Hexokinase and Glucokinase

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

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

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

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

The bypass around phosphoglycerate kinase step is known as Rapaport leubering
cycle or 2, 3-BPG shunt. In this shunt, no ATP is generated.
Glucose
( Rapaport leubering cy cle )
l
I, 3-Bisphosphoglycerate
ADP Phosphoglycerate mutase
2, 3-Bisphosphoglycerate
ATP ~ H 20
J phosphatase
3-phosphoglycerate Pi
l
Pyruvate
Importance of 2,3 -BPG:
• 2, 3 - BPG is a regulator of oxygen transport in erythrocytes. It combines with Hb and

reduces the affinity of hemoglobin to 0 2. So, in presence of 2, 3 - BPG, oxyhemoglobin
will unload 0 2 more easily in tissues.
• Rapaport leubering cycle and the concentration of 2, 3-BPG in erythrocytes increases

in hypoxic conditions. This favors the release of 0 2 to tissues even when p0 2 is low.
• The energy requirement in erythrocytes is minimal. In 2, 3 -BPG shunt, the energy

yielding phosphoglycerate kinase step is bypassed and hence no energy is formed.
This allows the glycolysis to proceed without the synthesis of extra energy.

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

Pyruvate is oxidatively decarboxylated to acetyl CoA by pyruvate dehydrogenase (PDH)
enzyme complex. Acetyl CoA then enters TCA cycle. PDH is a muJtienzyme complex.
Pyruvate dehydrogenase
Pyruvate Acetyl CoA
CoASH FAD, TPP CO,
Lipoic acid
NAO• ADH+H•
Pyruvate dehydrogenase enzyme is a multie11zyme complex. ft contains 3 enzymes & 5 coenzymes.

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

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

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

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

Functions of TCA Cycle :

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

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

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

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

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

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

transaminases (ALT and AST).
Oxidative decarboxylation reactions:

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

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

Production of ATP (Phosphorylation):

Production of ATP from ADP and Pi is called phosphorylation. There are 2 types,
a) Substrate level phosphorylation:

Production of ATP, where the en ergy is trapped directly from the substrate without
the involvement of electron transfer chain is called substrate level phosphoryla tion.
E.g.: 2 reactions from glycolysis, 1 reaction from TCA cycle & creatine kinase reaction.
Phosphoglycera te kinase
1) 1, 3-Bisphosphoglycerate 4 • 3-phosphoglycerate
ADP
/Ms:K.. ATP
Pyruvate kinase
2) Phosphoenolpyruvate Pyruvate
ADP ATP
Succinate thiokinase
3) Succinyl CoA ••- --~~---•
/4g~ Succinate
GDP+ Pi GTP
Creatine kinase (Refer connective tissue

4) Creatine phosphate
7 '-; Crea tine
chapter for creatine
ADP ATP kinase reaction)
b) Oxidative phosphorylation:
Production of ATP, where the energy is trapped from the oxidation of reducin g
equivalents s u ch as NADH- and FADH 2 in the ETC is termed as oxidative
phosphorylation . (Refer Oxidative phosphorylation chapetr).
NAOH+H+give 3 ATP in ETC and FAOH2 gives 2 ATP in ETC. According to current
concept, NADH+H+give 2.5 ATP in ETC and FADH2 gives 1.5 ATP in ETC.
Energy released in complete oxidation of glucose:

Glucose Aerobic condi tions Anaerobic conditions
l Glycolysis
2 Pyruvate
SATP 2 ATP
l Pyru vale dehydrogenase

2 Acetyl CoA
= 3X 2= 6AlP OATP
l TCA cycle
COz+ HiO
= 12 X 2 = 24 ATP OATP
To tal 38ATP 2 ATP

Carbohydra te Metabolism 185
Glycog en metabo lism (Glycogenesis & Glycog enolysi s)

Glycogen is an animal storage polysaccha ride. It is synthesize d (Glycogene sis) during
fed (energy rich) conditions and broken down (Glycogenolysis) during fasting (energy
deficient) conditions.
Glycoge nesis (Glycogen synthesis)

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

ATP ADP UTP PPi
~g+2Jf \. 1'
Glucose Glucose 6-phosphate • ., Glucose lpho p a h t ~ UDPGlc
Hexokinase/ Phosphogluc o UDPGlc
Glucokinase mutase pyophosphor}'lase
UDP glucose is the donor of glucose in glycogen synthesis.
b) Lengthen ing of chain & formation of glycogen:

It requires two enzymes i) Glycogen synthase ii) Branching enzyme.
i) Glycogen synthase: By the action of glycogen synthase enzyme, UDP glucose adds
its glucose to a pre-existing glycogen primer & lengthens it by one glucose molecule
to form glycogen amylase (l iberating free UDP). The new glucose is added in a(l • 4)
direction. (Glycogen primer is synthesize d by glycogeni n, a glycoprotei n).
UDP gluco~e UDP

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

When the chain has been lengthen ed to a minimu m of 11 glucose residues, a second
enzyme, the branchi ng enzyme transfers a part of a(l • 4) chain (minim um length
of 6 glucose residues) to a neighbo ring chain, produci ng a new a(1• 6) linkage, thus
forming a new branch point.
Then the branche s grow by further additio n of glucose in a(1 • 4) directio n by
glycogen synthas e enzyme and then further branchi ng by branchi ng enzyme.
VD P
;1')
Diagramatic representation of glycog enesis
Signifi cance:
Glycogen is an animal storage polysaccharide. It is stored in liver and muscle. When
the blood glucose level increases, excess glucose is convert ed to glycoge n.
Regula tion of glycog en synthe sis:

Glycogen synthas e is the regulato ry enzyme of glycogenesis. It is regulate d by covalen t
modification. (Refer enzyme chapter).The phospho rylated form of glycogen synthas e
is inactive, whereas dephosp horylate d form of g lycogen synthas e is active.
Glycoge n synthas e activity is decreas ed by adrenal ine and enhanc ed by insulin.
ATP ~ n as:.----- -::: : DP

Glycogen synthase a Glycogen synthase b
(De phospho ry la ted fo rm) (Phospho ry la ted fo rm )
(Active form)
:::---------
Pi Ph osphatase H 20
(Inactive form)
Reversi ble covalen t modific ation of glycoge n synthas e enzyme
9986449575
For queries and suggest ions, contact the author at prasad_te:xt@yahoo.com or
Glyco genol ysis (Glyco gen break down)

Definit ion:
GlycogenoJysis means the breakdo wn of glycogen.
Site:
a) Tissue site: Liver and Muscle
b) Intracellular site: Cytosol
Staiting material:
Glycogen
End product:
i) Glucose in liver and
ii) Glucose -6-phos phate in muscles (as muscle lacks glucose-6-phosphatase)
.
Reaction pathway:
Glycoge n is broken down by combin ed action of 3 enzyme s, namely,
a) glycoge n phosph orylase b) glucan transferase c) debranc hing enzyme.
a) Glycog en phosph orylase :

It acts on glycogen & breaks the termina l a(1 • 4) glycosidic bonds of glycoge n and
releases the termina l glucose molecule as glucose ]-phosp hate.
Pi
Glycoge n
(Glucos e) n
----= ----- ---+ Glycogen
Glycogen
+ Glucose 1-phosp hate
phosphorylase (Glucos e) n-1
Action of glycogen phospho rylase continu es till about 4 glucose residues remain on
either sides of the branch point.
Muscle glycogen phospho rylase is a PLP depend ent enzyme .
b) Glucan transferase: It transfer s 3 glucose residue s from one chain to another

chain
and thus exposin g the a (1 • 6} glycos~dic bond (branch point).
c) Glycoge n debranc hing enzyme: It splits the a(1 • 6) bond at the branch point and
releases free glucose. After the remova l of branch, action of glycogen phospho rylase
continues.
Thus the action of these 3 enzyme s leads to the complet e breakd own of glycoge
n
to give mainly glucose 1-phosp hate (90%) and few free glucose molecu les (10%).
For queries and suggest ions, contact the author at prasad_ text@ya hoo.com or
9986449575
Carbohy drate Metabol ism 188
Diagra matic repres entatio n of glycog enolys is
Fate of glucos e 1-phos phate:
a) In liver: Glucose 1-phosp hate is convert ed to glucose in liver.

Mg•2 H20 Pi
Glucose 1-phosphate - - - -+ Glucose 6-phosphate ':::- 2! •
Glucose
Phosphogl uco mutase Glucose-6-phosphatas e
b) In muscle : Muscle lacks glucose 6-phosp hatase enzyme . So, in muscle

glycogenolysis, the end p roduct is glucose 6-phosp hate.
Signifi cance of glycog enolys is:

a) Hepatic glycogenolysis takes place in order to maintai n blood glucose level.
b) Muscle glycogenolysis takes place for the provision of energy.
Regulation of glycog enolysi s :

Glycogen phosphorylase is the regulatory enzyme of glycogenolysis. It is regulate d by
covalen t modification. (Refer enzyme chapter ). The phosphorylated form of glycogen
phosphorylase is active, wh ereas dephos phoryla ted form is inactive. Glycog en
phosph orylase activity is activated by glucago n and adrenalin; inhibite d by insulin.
ATP ADP
Glycogen phosphor ylase b • Glycogen phosphor ylase a
(Dephosp horylated form) (Phospho rylated form)
4~ -----
(Inactive form) Pi Phosphat ase HiO (Active form)
Reversi ble covalen t modific ation of glycoge n phosph orylase enzyme
or 9986449575
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Clinical significance of glycogen metabolism
Glycogenosis (Glycogen storage diseases)

The inherited genetic defects related to the glycogen metabolism (Synthesis and
degradation) are called glycogenosis or glycogen s torage diseases.
These genetic diseases are characterized by deposition of abnormal type or abnormal
quantity of glycogen in the tissues.
Glycogenosis are the group of genetic diseases that result due to enzyme defects of
glycogen metabolism. There are several types of glycogen storage diseases. In each
type there is deficiency or low activity of a particular enzyme.
Types: There are 6 major types of glycogen storage diseases.
Type Name Enzyme defect Characteristics
Hepatomegaly (Due to
Von-Gierke's disease GI ucose-6-phospha tase liver cells loaded with
I in liver glycogen, hypoglycemia,
Lactic acidosis, Ketosis.
Fatal, accumulation of
II Lysosomal acid maltase
Pompe's disease glycogen in lysosomes,
(a-1 • 4 glucosidase)
heart failure.
Limit dextrinosis/ Accumulation of a

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

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

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

von Gierke's disease results from absence of glucose-6-phosphatase in liver.
Features include hypoglycemia, hepatomegaly, lactic acidosis, ketosis, hyperuricemia.
• Hypoglycemia: Blood glucose is maintained by hepatic glycogenolysis in the
interdigestive periods. In von Gierke's disease, there's no con version of glucose 6
phosphate to glucose, leading to hypoglycemia.
• Hepatomegaly: There is an abnormal accumulation of glycogen in liver due to inability
of breakdown of glycogen, leading to hepatomegaly. Thus, this accumulated glycogen
in liver is not useful.
• Lactic acidosis and ketosis: Glucose 6-phosphatase is also required for gluconeogenesis
for the conversion of lactate to glucose in liver. Failure of lactate to glucose leads to
accumulation of lactate, causing lactic acidosis and subsequent ke tosis.
• Hyperuricemia and Gout:von Gierke's disease is associated w ith other secondary
manifestations like hyperuricemia and gout. Glucose 6-phosphatase deficiency blocks
the conversion of glucose-6-phosphate to glucose. Accumulated glucose-6-phosphate
is then diverted to HMP shunt pathway resulting in elevated levels of ribose-5-phosphate
and hence purine n ucleotides and nucleic acids synthesis. Breakdown of excess purine
nucleotides & nucleic acids causes hyperuricemia (Refer nucleotide metabolism chapter).
Type II or Pompe's disease:

Lysosomal a. 1, 4-glucosidase (also called acid maltase) defect. Normally this enzyme
is involved in the degradation of glycogen in lysosomes. In its absence glycogen
accumulates in lysosomes, maninly in heart, which lead to cardiac failure & early death.
Type III or Cori's disease (limit dextrinosis):

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

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

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

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

HMP Shunt pathway
Synonyms:
Hexose Mono Phosphate pathway, Pentose Phosphate Pathway (PPP) and direct
oxidative pathway of glucose
Definition:
HMP shunt pathway is an alternative pathway of the oxidation of glucose. About
1% of glucose enters this pathway.
Site:
a) Tissue Site:
Mainly liver, RBC, adrenal cortex, testis, mammary glands.
b) Intracellular site:
Cytosol.
Starting compound:
Glucose 6-phosphate
Reaction pathway:
HMP shunt pathway has 2 phases
a) Oxidative phase.
b) Non-oxidative phase.
a) Oxidative phase:
In the oxidative phase, glucose 6-phosphate is oxidatively decarboxylated to ribulose
5-phosphate with the production of two molecules of NADPH.
Glucose 6 -P 6 -P gluconate
Glucose 6 -P 7 '\ •
dehydrogenase
6 -phosphogluconatE: 7 ~~
dehydrogenase
Ribulose 5 -P
02
NADP NADPH + H • NADP NADPH + H •

Carbohydra te Metabolism 192
b) Non-Oxid ative phase:

Non-oxida tive phase, three molecules of ribulose 5-phospha te are rearranged to from
two molecules of fructose 6-phospha te and a molecule of glyceraldeh yde 3-p hospha te.
These molecules then enter glycolysis.
Ribulose 5 P
!Ep;me,as e I
Ribulose 5 P
lsomerase I
Ribulose 5 P
Ep;merase
Xylulose 5 P
Xylulose 5 P Ribose 5 P
Sedoheptu lose 7 P Glycerald ehyd e 3 P
Fructose 6 P Erythrose 4 P
Fructose 6 P Glycerald ehyd e 3 P
Fructose 6-phospha te and glyceralde hyde 3-phospha te, then enter glycolysis.
Summary of non-oxida tive pathway:
3 Ribulose 5-P 2 Fructo e 6-P + Glyceraldehyde 3-P

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

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

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

Clinical significance of HMP shunt pathway:

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

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

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

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

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

Reaction pathway: GLUCOSE

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

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

G OH ~
Fumarate MITOCHONDRIA a -Ketoglutarate .-- ~
Succinyl CoA ------- ~
Answer hint for gluconeogensis from diffrent individual sources
(like alanine, lactate, glycerol or propinyl CoA) questions:
This is a all-in-one representation . Start from the individual source that
is asked (for e.g. lactate), draw gluconeogenesis from that point.

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

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

Pyruvate carboxylase Adrenalin Insulin CoA
Phosphoenolpyruvate
carboxy kinase Same Same
-- --
Fructose 1,6- AMP, Fructose 2,6-
bis phosphatase Same Same Citrate bisphosphate
Glucose 6-phosphatase Same Same -- --

Cori cycle (Lactic acid cycle)

Cori cycle is a process in which the lactate formed from glucose/ glycogen by glycolysis
in skeletal muscle & erythrocytes, is trasported to the liver & kidney, where it is converted
back to glucose, which again become avalible via blood for oxidation in the tissues.
MUSCLE / RBC BLOOD LIVER
c,,,.... l
Glucose
Anaerobic
Glycolysis
Glucose ....,1--- G lucose
Jc,.,....,,.~..
Lactate Lactate Lactate
Explanation: Lactate is mainly formed from anaerobic glycolysis of glucose in skeletal

muscles and RBC's. This lactate diffuses into the blood, carried to the liver. In liver,
lactate is converted to glucose by gluconeogenesis & transported to the tissues (Including
muscle tissues) via blood. This recycling of lactate is called Cori cycle.
Significance: Cori cycle provides an indirect way of utilizing muscle glycogen to maintain
blood glucose level in fas ting conditions.
Glucose-alanine cycle:
In fasting conditions, alanine is released from muscle. This alanine is mainly formed by
transamination of pyruvate produced from glucose (or glycogen) by glycolysis (and
metablosim ofsuccinyl CoA generated by the catabolism of branched chain amino acids isoleucine
& valine). Alanine is then taken up by liver and converted back to pyruvate by
transamination, which is then converted to glucose (by gluconeogenesis), which enter
the blood and used by muscle. This process is called glucose-alanine cycle.
MUSCLE LIVER
Glucose Glucose
l
G lycogen Glucose
~ colysi{ Gluconeogenesis
l
Pyruvate Pyruvate
TranS,,1mination l Tra.nsa.mination
AJanjne Alanine AJarune
Significance: Glucose-alanine cycle provides an indirect way of utilizing muscle glycogen

to maintain blood glucose level in fasting conditions.
Alanine (like glutamine), is a non-toxic transport form of NH3 from muscle to liver.
ln muscle, NH3 combines with a-ketoglutarate to form glutamate, which then transfers the amino
group to pyruvate to form alanine by ALT. Alanine is transported to liver & transfers back the
amino group to glutamate by ALT. Glutamate releases N H3 by GOH, which is detoxified to urea.

Uronic acid pathway:

This is an alternative pathway for the oxidation of glucose (like the HMP shunt
pathway) and this pathway is also called glucuronic acid pathway.
Formation of Uronic acids (UDP -Glucuronate):
Glucose
1Glu,okinm / Hexokinase
Glucose 6-phosphate
1 Phosphoglucomutase
Glucose 1- phosphate
UTP
PPi 1 UDP glucose pyrophosphorylase
UDP- Glucose
2NAD+
UDP-glucose dehydrogenase
2NADH+H+
UDP -Glucuronate
H20 Glucuronidase
UDP
D -Glucuronate
Significance:
Glucuronic acid is used for detoxification processes and synthesis of
glycosarninoglycans.
• UDP-glucuronic acid is the active form of glucuronic acid which is used for the
conjugation and detoxification of bilirubin, steroids and certain drugs (morphine,
salicylate etc). Conjugation makes these compounds water soluble and allows them
to be excreted in urine and bile.
• UDP-glucuronic acid is also required in the synthesis of glycosaminoglycans and
proteoglycans.

Fate of UDP-Glucuronate :
UDP -Glucuronate
NADPH+H•
Glucuronate reductase
NADP•
L-Gulonate
NA~ H,0 L-gluconolactone oxidase

NADH..-H· an enzyme required to
L-Gluconolactone
convert L-gluconolactone to
1
(3-keto L-gulonate]
(Further reactions do not occur in humans > v itamin C, is absent in
CO, spontaneous humans_ So, Vitamin C is
L-Xylulose not fanned in humans.
NADPH +H- ~ Xylitol de.hydrogenase - - -- - Absent in Pentosuri•
NADP- 1
Xylitol
NAO, D-Xylulose reductase

NADH+H·
D-Xylulose
AT P ~ Kinase
Mg•'
ADP
D-Xylulosc 5-phosphate HMP shunt pathway
Clinical significance
Essential Pentosuria:
• It is a rare inherited disease due to the absence of xylitol dehydrogenase enzyme.
• Xylulose cannot be further converted to xylitol, resulting in the accumulation and
excretion of large amounts of L-xylulose in urine.
• It is asymptomatic and does not ca use any harm. Urine Benedict's test w ill be tested
positive. So, the result should be carefully analyzed & differentiated from diabetes.
Polyol pathway or sorbitol pathway:

Sorbitol pathway converts glucose to sorbitol (by aldose reductase) and then sorbitol to
fructose (by sorbitol dehydrogenase). This pathway is absent in liver.
Glucose
__
7
___'---,..
ADPH + H •
___• Sorbitol __
aldose reductase
I\JADPH•
sorbitol dehydrogenase
7
___
NA[}
• ,---+
"1ADPH+ H•
Fructose
This pathway is increased in high glucose concentrations (as in diabetes) in lens, peripheral
nerves, glomeruli (but not in liver). Complications like diabetic cataract, peripheral
neuropathy and nephropathy are attributed to the accumulation of sorbitol in these tissues.

Fructose metabolism:
Fructose is obtained from fruits, honey and digestion of dietary sucrose. Fructose is
absorbed by facilitated diffusion. It is then carried to liver by portal blood. It is either
oxidized to pyruvate by glycolytic pathway via fructokinase or forms glucose.
Fructose
ATP Fructose is converted to

ADP -.;::{ •ro•""'"-'"•-~• fructose-1-phosphate, by
fructokinase. Fructose-1-
Fructose I-phosphate
phospha te, then gives
I Aklolase B (specific to fructose-1-P) glyceraldehyde, which
forms glyceraldehyde 3-
1 phosphate and enters
Dihydroxy acetone prosphate Glyceraldehyde
glycolysis or forms glucose.
ATP
Tdokinase
ADP
GIye era ldehyde 3-phasphate

Glycolytic pathway •
_ Pyruvate
Aldolase
Fructose 1, t -bisphosphate
i Gluconeogenesis
Gluco,;e
Hereditary Fructose intolerance :
• It is an inherited metabolic defect, caused due to the deficiency of Aldolase B. It is
characterized by hypoglycemia & vomiting after consumption of fructose (or sucrose).
• Elevated fructose-1-phosphate inhibits glycogen phosphorylase leading to decreased
hepatic glycogenolysis & hypoglycemia. Glycogen accumulates in liver causing
hepatomegaly, hepatic failure & jaundice. Depletion of inorganic phosphate lowers
ATP synthesis. So, less inhibition of de novo purine synthesis by ATP & hyperuricemia.
• Diets free from (or low in) fructose (fruits, honey) and sucrose is beneficial.
Essential Fructosuria:
• It is caused due to fructokinase deficiency. This condition is benign and asymptomatic.
There is no abnormality other than accumulation & excretion of fructose in urine.
Fructose is better utilized in patients with diabetes mellitus. Fructose is more lipogenic than glucose.
Intestinal absorption of fructose is energy independent facilitated diffusion. Entry of fructose into
the cell is passive diffusion & it does not require insulin. Fructokinase,Jirst step offructose metabolism
is insulin independent. Fructose-1-phosphate, formed from fructokinase step, bypasses the regulatory
PF K step to enter glycolysis. So,Jructose is better utilized in diabetics. For the same reason, fructose
is more lipogenic than glucose. Since, fructose metabolism is not regulated by insulin; there is
increased production of pyruvate by glycolysis, subsequent acetyl CoA formation & lipogenesis.

Metabolism of Galactose:
Galactose is need ed for the synthesis of glycolipids, glycoproteins, proteoglycans &
lactose. It is obtained from digestion of lactose of milk. It is absorbed by active transport
& then carried to liver by portal blood. While the portal blood that enters liver contains
glucose, galactose (& fructose), systemic blood that leaves liver contains only glucose.
Galactose
ATPM~ Galactokinase
ADP ,~
Epimerase
Glucose-1-phosphate
I Used for the syn thesis of glycolipids,
+ Glycoproteins, Proteoglycans & lactose.
! •In mammary glands, UDP-galactose is required

for lactose synthesis. But, this UDP-galactose
Glucose
is obtained from glucose (not galac/ose).
Galactosemia:
• Galactosemia is an inborn error of metabolism of galactose metabolism, mainly due
to the absence of galactose-1-P uridy l transferase enzyme. Babies born with this
defect cannot utilize galactose obtained from lactose of milk, the only food of babies.
• Normally, systemic blood does not contain any galactose. But, in this condition, there
is a high level of galactose in blood (galactosemia) and increased excretion of galactose
in urine (galactosuria). The galactose 1-phosphate inhibits glycogen phosphorylase
enzyme decreasing the conversion of hepa tic glycogen to glucose and consequent
hypoglycemia. Glycogen accumulates in liver causing hepatomegaly, hepatic failure
and jaundice. Part of galactose is reduced to gaJactitol or dulcitol, accumulation of
these in lens causes cataract and blindness.
• Soon after the babies are born, they must be screened for any inborn errors. If any
baby is diagnosed to be galactosemic, it should be fed with lactose-free milk foods.
• For normal growth and development UDP galactose is needed. In the affected children,
this can be obtained from UDP glucose by reverse reaction of epimerase.
• Galactokinase deficiency, though rare, will also causegalactosemia. Generally, these individuals
do not develop hepatic complications. However, cataract is manifested.

Glycosuria
Definition: Glycosuria refers to excretion of sugars (mainly glucose) in urine.
1. The term glycosuria refers to the excretion of all sugars in urine. However since glucose is
the most common sugar excreted in urine, the term glycosuria is often (though incorrectly)
used to denote the excretion ofglucose in urine. Glycosuria is detected by Benedict's test.
2. Glucose is reabsorbed almost completely in renal tubules & hence urine contains almost no
glucose. When blood glucose level exceeds 180 mg/dl, glucose cannot be completely reabsorbed
& will be excreeted in urine. This level is called the 'Renal threshold for glucose'.
Conditions: Glucosuria (excretion of glucose) occurs in diabetes mellitus and renal
diabetes. Other conditions of glycosuria are lactosuria (during pregnancy and lactation),
galactosuria (in galactosemia), fructosuria, pentosuria etc.
Types:
a) Hyperglycemic glycosuria: When the blood glucose level exceeds the renal threshold
(160 - 180 mg/ dl), glucose is excreted in urine.
E.g.: Diabetes mellitus, hyper secretions of thyroid hormones, cortisol etc.
b) Renal glycosuria: In some people, renal threshold i.e. reabsorptive capacity of the
renal tubules for glucose is reduced, resulting in the excretion of glucose in urine even
when the blood glucose level is normal. This condition is known as renal glycosuria.
c) Alimentary glycosuria: This occurs, following a rich carbohydrate meal due to
augmented absorption from intestine. This results in hyperglycemia above the renal
threshold and leads to glycosuria. This is also seen in Hyperthyroidism.
d) Glycosuria of pregnancy: Due to decreased renal threshold in pregnancy, the glucose
can be excreted in urine of pregnant woman.
e) Transient glycosuria: Transient glycosuria occurs in some people during emotional
stress. Excessive production of catecholamines causes hyperglycemia and resultant
glycosuria. Once stress is removed, glycosuria disappears.
Factors adding glucose to circulation (Hyperglycemic factors):

a) Diet: Glucose absorbed from the small intestine
b) Hepatic glycogenolysis
c) Gluconeogenesis from non-carbohydrate sources
d) Lipolysis (More energy from fat reduces glucose utilization).
Factors removing glucose from circulation (Hypoglycemic factors):
a) Uptake of glucose into the cell
b) Catabolism for energy i.e. Glycolysis
c) Glycogenesis: (Glycogen synthesis from glucose)
d) Lipogenesis: Formation of fat (from glucose) in adipose tissues and storage
e) Other minor pathways like HMP shunt pathway, uronic acid pathway etc.

Hormonal regulation of glucose

Normal fasting blood glucose level is 60 to 100 mg/ dl. The level above this range is
termed as hyperglycemia and level below this range is called hypoglycemia.
Blood glucose level is under the influence of various hormones. These hormones are of
two types, hyperglycemic hormones and hypoglycemic hormones.
Hypoglycemic hormones:
Hormones, which decrease the blood glucose level, are called hypoglycemic hormones.
E.g.: Insulin
Hyperglycemic hormones:
Hormones, which increase the blood glucose level, are called hyperglycemic hormones.
E.g.: Glucagon, Adrenalin, Glucocorticoids, Growth hormones and Thyroid Hormones.
Individual action of these hormones are given below,

1) Insulin:
Insulin is a hypoglycemic Hormone. It is secreted by cells of islets of langerhans of
pancreas. Hyperglycemia is the stimulation for insulin secretion.
Insulin lowers the blood glucose level by following means,
a) Insulin increases the uptake of glucose by muscle and adipose tissues (Insulin is
not required for uptake of glucose by liver).
b) Insulin stimula tes glycolysis.
c) It stimulates glycogenesis.
d) It stimulates HMP shunt pathway.
e) It inhibits gluconeogenesis.
t) It inhibits glycogenolysis.
g) Insulin stimulates lipogenesis (i.e. synthesis of fat from glucose).
h) Insulin inhibits lipolysis. (so, energy from fat is restricted & glucose is utilized).
2) Glucagon:
Glucagon is a major hyperglycemic hormone (so glucagon is an anti-insulin hormone).
Glucagon is secreted by a cells of islets of langerhans of pancreas. Hypoglycemia is
the stimulant for glucagon secretion.
Glucagon increases the blood glucose level by following means,
a) Glucagon stimulates hepatic glycogenolysis.
b) It stimulates gluconeogenesis.
c) It decreases glycogenesis.reduces gluose utilization).
d) It inhibits glycolysis, TCA cycle, HMP shunt pathway etc.
e) It stimulates lipolysis. (More energy from fat reduces glucose utilization).

3) Epinephrine (Adrenalin):
Adrenalin (Epinephrine) is a hyperglycemic hormone, secreted by adrenal medulla.
Adrenalin increases the blood glucose level by following means,
a) It stimulates glycogenolysis.
b) It stimulates gluconeogenesis.
c) It inhibits glycolysis.
d) It stimulates lipolysis. (More energy from fat reduces glucose utilization).
e) It inhibits insulin secretion.
4) Glucocorticoids (Cortisol):
Cortisol is a hyperglycemic hormone. It is secreted by adrenal Cortex.
Cortisol increases the blood glucose level by following means,
a) It stimulates gluconeogenesis
b) It stimulates glycogenolysis
c) It decreases glycolysis
d) It stimulates lipolysis. (More energy from fat reduces glucose utilization).
e) It inhibits insulin secretion.
5) Growth hormones:
Growth hormone is a hyperglycemic hormone. It is secreted by anterior pituitary.
Growth hormones increases the blood glucose level by following means,
a) It decreases the uptake of glucose by muscle tissues.
b) It decreases glycolysis.
c) It mobilizes fatty acids from adipose tissues into blood, which inhibits glucose
utilization.
6) Thyroid hormones:
Thyroid hormones are secreted from thyroid glands.
Thyroid hormones increases the blood glucose level by following means,
a) It increases the absorption of glucose from intestine.
b) It stimulates glycogenolysis.
c) It stimulates gluconeogenesis
d) It stimulates lipolysis
~nswer hint for Hormonal regulation of glucose question (10 Marks):

1'1) Introduction and types of hormones - 2 mark
D) Role of Insulin - 3 Mark
") Role of Glucagon, Epinephrine, Cortisol,
Growth hormone, Thyroid hormone -1 Mark each
Liver plays an important role in the regulation of blood glucose,
1. When there is hyperglycemia, liver helps in restoring the normal level by increasing utilization
of glucose and accelerating glycoge11esis
2. When blood glucose falls, as in fasting conditions or decreased intake, liver helps to maintain the
blood glucose by accelerating glycogenolysis, gluconeogenesis & by depressing tl1e glucose utilization.

Diabetes mellitus
Definition:
Diabetes mellitus is a metabolic disease caused either due to insulin deficiency or failure
in insulin action.
Classification:
The diabetes mellitus can be classified into 2 groups.
a) Ty pe 1 diabetes mellitus (Insulin dependent diabetes mellitus; IDDM):

Type 1 diabetes starts early in life (So, it is also called as juvenile onset diabetes). This
results from total lack or decreased production of insulin from -cells of pancreas.
b) Type 2 diabetes mellitus (Non-insulin dependent diabetes mellitus; N IDDM):
Type 2 d iabetes is more commonly seen in persons above 40 years (So, it is also called
as maturity onset diabetes). In this type, the defect is lack of insulin action and not
prodcution of insulin. Blood often contains normal levels of insulin.
The defect is du e to decrease in the number of target cell insulin receptors and lack of
sensitivity to insulin. Type 2 d iabetic patients are normally obese.
Metabolic changes and clinical complications of diabetes mellitus:

1) Hyperglycemia:
Function of insu lin is to reduce the blood glu cose level. So, in diabetes mellitus (wh ere
there is absolute or relative insulin deficiency), hyperglycemia is main symp tom.
2) Glucosuria:
When the blood glucose level exceeds th e ren al threshold level (180 mg/ dl), the glucose
is excreted in urine (Glucosuria).
3) Polyuria:
Glucosuria is accompanied by water due to osmotic effect of glucose. So, the person
passes more urine (Polyuria, Excessive excretion of urine).
4) Dehydration and Electrolyte depletion:
Due to polyurea, body water level comes down leading to dehydration. Electrolytes are
also lost in urine leading to electrolyte depletion, ma inly sodium & potassium.
5) Polydypsia:
To compensate for the water loss, thirst centre is activated and more water is taken
(Polydypsia).
6) Muscle wasting:
Since carbohydrates do not provide energy, the body proteins are broken down to give
energy leading to muscle wasting.

7) Delayed wound healing:

In diabetes, since carbohydrates do not give energy, the body gets energy from proteins
and proteins are not available for growth, maintenance and tissue repair (i.e. wound
healing). So, wounds heal very slowly.
8) Loss of body weight:
Loss of body fat and muscle wasting lead to loss of body weight.
9) Polyphagia:
To compensate the loss of weight due muscle wasting, diabetics eat more (Polyphagia).
10) Cataract:
Hyperglycemia also causes the sorbitol formation from glucose (by polyol pathway),
which is accumulated in lens causing cataract.
11) Retinopathy:
Glycosylation of retinal proteins and sorbitol formation causes retinopathy and then
total blindness.
12) Neuropathy:
Glycosylation of neural proteins causes neuropathy. Formation of advanced glycation
end products (AGE) and deposition of sorbitol in Schwann cells causes peripheral
neuropathy, which may lead to foot ulcers and gangrene.
13) Nephropathy:
The hyperglycemia also causes glycosylation of basement membrane proteins of nephron
causing nephropathy.
14) Chronic recurrent infections:
When glucose level in blood increased, bacteria get good nutrition for multiplication.
So, the diabetics may get chronic recurrent infections such as boils, abscesses etc.
15) Ketosis and Diabetic ketoacidosis, Kussmaul's respiartion:
• Ketosis: In the absence of Insulin, lipolysis is increased & more acetyl CoA is produced.
Since oxalocecetate is deficient (because it is used up in gluconeogenesis), acetyl CoA
cannot enter TCA cycle. So Acetyl CoA is directed to the synthesis of ketone bodies.
This leads to ketonemia, ketonuria, acetone breath (sweet smell) and consequent ketosis.
• Kussmaul's respiartion: DKA patients exhibit a breathing pattern known as kussmaul's
respiartion (deep breathing due to a compensatory hyperventilation).
• DKA: The ketone bodies are strong acids. So, increased level of keto acid formation
in diabetes leads to acidosis. The acidosis due to ketone bodies in diabetes is called
diabetic ketoacidosis (DKA). If not treated, diabetic pateints enter coma and die.
Death in diabetics is mainly due to ketoacidosis, dehydration and electolyte imbalance.
Hyperosmolar nonketonic hyperglecemia: Seen manily in type II diabetics.

In this condition, plasma glucose level is very high (upto 900 mg / dl), but without ketosis.
High blood glucose level elevates osmolality of plasma, leading to loss of water and
electrolytes. Coma results from dehydration of brain cells due to hypertonicity of ECF

Diagnosis of diabetes mellitus:

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

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

Glucose tolerance test (GTT)

Definition: Glucose tolerance test or GTT is a test to measure the capacity of the body
to dispose off an additional load of glucose entering into the body.
Oral GTT refers to measureent of tolerance after a oral dose of glucose.
GTT is usually performed in diagnosis of doubtful cases of diabetes mellitus.
Procedure:
• The patient is instructed to have a good carbohydrate diet for 3 days prior to the
test and the patient is starved overnight (12 hours fasting condition).
• In the morning a sample of blood and urine is collected in fasting condition.
• Then the patient is given glucose load (75 g of glucose in a glass of water).
• Then the blood and urine samples are collected at an interval of half an hour for the
next 2½ hours (30, 60, 90, 120, 150 minutes).
• Plasma glucose level is estimated quantitatively in all the blood samples. A graph is
plotted with plasma glucose va lues (in mg/ dl) on y-axis and time (in minutes) on
x-axis. Urine samples are tested for sugar by benedict's qualitative test.
Response: Three types of responses are seen.

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

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

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

250
Blood glucose 200

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

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

Question bank on carbohydrate metabolism

1. Describe glycolysis
2. Describe citric acid cycle
3. Describe HMP shunt pathway
4. Describe glycogen metabolism in liver in the fed and fasting state. Add a note on glycogenosis
5. Describe Gluconeogenesis
6. Hormonals regulation of blood glucose level (Glucose homeostasis)

3. Glucose 6-phosphate dehydrogenase deficiency
4. Fructose metabolism / Galactose metabolism
5. Diabetes mellitus
6. Describe GIT and Glycosuria
7. Formation of Uronic acid and its significance
8. 2, 3 BPG shunt (Rapaport leubering cycle)

1) Oxaloacetate+ Acetyl CoA - > Citrate+ CoASH. This reaction is
a) Reversible b) Irreversible c) Can be reversed by Catalase d) Competitive
2) NADPH is used in
a) Fatty acid synthesis b) Ketogenesis c) Glycogenesis d) Glycolysis
3) Total number of dehydrogenases in Krebs cycle
a)3 b)2 c)4 d)5
4) Acetyl CoA can be converted into all of the following, except
a) Glucose b) Fatty acids c) Cholesterol d) Ketone bodies
5) Which of the following metabolic pathways does not generate ATP
a) Glycolysis b) TCA Cycle c) Fatty Acid Oxidation d)HMP Pathway
6) McArdle's is due to deficiency of
a) Acid Maltase b) branching enzymes c) Liver phosphorylase d) Muscle phosphorylase
7) PEPCK is a key enzyme in
a) Gluconeogenesis b) Glycolysis c) Polyol pathway d) None of these
8) UDP galactose is essential for the synthesis of
a) Lactose b) Maltose c) Sucrose d) s tarch
9) Hereditary fructose intolerance is due to the absence of
a) Aldolase B b) Aldolase A c) Phosphofructokinase d) Fructokinase
10) TCA cycle takes place in the
a) Cytosol b) Mitochondria c) Lysosomes d) ucleus
Answers for MCQ: 1) b 2) a 3) c 4) a 5) d 6) d 7) a 8) a 9) a 10) b

Lipid Metabolism 212
Lipid Metabolism
Contents:
• Introduction to lipid metabolism
• Lipolysis
• Oxidation of fatty acids

a) p -oxidation of fatty acids, Carnitine shuttle
b) a -oxidation of fatty acids, Refsum's disease
c) c.o -oxidation of fatty acids
• Lipogenesis
a) Denovo synthesis of fatty acids
b) Triacylglycerol synthesis
• Ketone body metabolism:

a) Ketogenesis
b) Ketolysis
c) Ketosis
• Lipoproteins (Chylomicrons, VLDL, LDL and HDL)

• Cholesterol metabolism: Synthesis, degradation, cholesterol transport
• Hypercholesterolemia & its effects (Atherosclerosis, Coronary heart diseases)
• Approaches to treatment of atherosclerosis
• Fatty liver and lipotropic factors

Introduction:
Major lipids in the body are triacylglycerols, fatty acids, cholesterol, phospholipids
etc. Triacylglycerols constitute the majority of lipids in the body.
Triacylglycerols are the storage form of energy ( fatty acids), which are stored mainly
in adipose tissues. During the conditions of restricted diet (like starvation and diabetes
mellitus), the stored triglycerides are broken down to glycerol and fatty acids by
successive action of three different lipases. This process is called lipolysis.
Lipolysis:
Definition: Complete degradation of triacylglycerol by lipases to give glycerol and
three fatty acids is called lipolysis. Lipases are the lipolytic enzymes.
Lipolysis Oipases)
Triacylglycerol + 3H20 Glycerol + 3 Fatty acids
In adipose tissues, TAG Lipase (also callled hormone sensitive lipase), DAG lipase,
MAG lipase, hydrolyze triacylglycerol to glycerol and 3 fatty acids.
(HSL)
TAG lipase
Triacylglycero l / ~ Diacylglycero~
D AG lipase
"=
MAG lipase
Monoacylglycero7 Glycerol
H1 O fatty acid H 1O fatty acid H1 O fatty acid
Hormone sensitive lipase, HSL (TAG lipase of adipose tissues):

HSL removes the fatty acids from TAG to form DAG and free fatty acid.
HSL is the regulatory step in lipolysis. It is stimulated by epinephrine, nor-epinephrine,
glucagon, growth hormone, ACTH, thyroid hormones etc and inhibited by insulin.
Epinephrine, nor-epinephrine, glucagon (hormones that cause rapid lipolysis), do so
by stimulating the activity of adenylate cyclase enzyme, which increases cAMP
production. cAMP activates protein kinase C, which activates Hormone sensitive lipase.
Other important lipases:

• Lipoprotein lipase is a lipolytic enzyme present in capillary walls. It hydrolyzes TAG
present in chylomicrons and VLDL to liberatefree fatty acids and glycerol.
• Pancreatic lipase and lingual lipases nre required during digestion of lipids.
• Phospholipases are lipases which hydrolyze phospholipids.
N o te: The fatty acid released from adipose tissue are transported in the plasma as free
fatty acid-albumin complex and taken up by tissues, which can utilize fatty acid as a
source of energy. E.g. skeletal tissue, heart muscle, renal cortex etc.

~-oxidation of fatty acids

Introduction:
Fatty acids are rich sources of energy. This energy is released when fa tty acids
undergo ~-oxidation. The ~-oxidation of fatty acids occurs by a sequential removal of
two carbon atoms from the carboxylic end of fatty acids.
This pathway is called ~-oxidation because the oxidation occurs at the carbon atom
of fatty acids.
Site:
a) Tissue site: Liver, muscle, renal cortex, adrenal medulla, heart etc.
b) Intracellular site: Mitochondria
Starting compound:
Palmitic acid is the commonly occurring fatty acid in the food & the body.
Reaction pathway:
Fatty acids are present in cytosol and the ~-oxidation of fatty acids takes place in the
mitochondria. So fatty acids are activated and then transported into mitochondria.
~-oxidation occurs in 3 stages:

i) Activation of fatty acids
ii) Transport of fatty acyl CoA into mitochondrial matrix (Carnitine shuttle)
iii) ~-oxidation reactions
i) Activation of fatty acids:

Fatty acid s are con verted to fatty acyl-CoA b y thiokinase en zyme (or acyl-CoA
synthe tase). 2 high-energy bonds are utilized in this activation step.
Thiokinase (Acyl-CoA synthetase)
11
Fatty acid + CoASH Fatty acyl-Co
.,,,,.,,-- Mg+2
ATP AMP +PPi
ii) Transport of fatty acyl CoA into mitochondria (Camitine shuttle):

Fatty acyl-CoA cannot enter mitochondria as such. A specialized carrier molecule
called carnitine transports fatty acyl-CoA across the mitochondrial membrane. This
process is called carnitine shuttle.

iii) Reactions of P-oxidation :

It is cyclical process. Each cycle consists of a sequence of four reactions -
1. Oxidation 2. Hydration 3. Oxidation 4. Thiolytic cleavage
Each cycle results in the removal of 2 carbon compound (as Acetyl CoA) from fatty
acyl CoA, resulting in the formation of a new fatty acyl CoA, which has two carbon
atoms less than the original fatty acyl-CoA. These 4 reactions are repeated in a cyclical
manner till the fatty acyl CoA is completely broken down to acetyl CoA molecules.
Fatty acyl CoA
F
FAD
Acyl CoA J 1-0x:idatioo step
Deh}drogenase FADH,
Reactions of a, P unsaturated acyl CoA
rk
(also called A1- trans-Enoyl-CoA)
~-oxidation H,O
Enoyl CoA
hydratase Iu - Hydration step l
P•h}droxy acyl CoA
Undergoes
repeated
cycles P-Hydroxy NAD'
acJI CoA 1m Oxidation step ]
Dehydrogenase
NADH+H'
Thiola: k e t o ~ . \ CoASII
I•
l.____ Acetyl CoA
Fatty acyl CoA _ _ __
V Cleavage step
This ratty acyl CoA has 2 carbons

le.ss than original rauy acyl CoA
End products of ~-oxidation:

a) P-oxidation of even chain fatty acids gives only acetyl CoA molecules.
b) P-oxidation of odd chain fatty acid give both acetyl CoA and propionyl CoA. After
the removal of successive acetyl CoA molecules, one molecule of propionyl CoA is
obtained in the terminal P-oxidation step or cycle. This propionyl CoA is converted to
succinyl CoA, which is an intermediate of TCA cycle.
Function of Ji-oxidation:
1. Provision of energy (especially in conditions of limited carbohydrate availability).
2. Ketone body formation (from acetyl CoA).
Regulation of P-oxidation:
P-oxidation is regulated by availability of free fatty acids, which in tum depends upon
adipose tissue lipolysis by hormone sensitive lipase.
HSL is stimulated by glucagon, epinephrine, norepinephrine etc & inactivated by Insulin.

Energetics of 13- Oxidation:

Palmitic acid is the commonly occurring fatty acid in the food & the body. So the
energy derived from ~-oxidation of Palmitic acid is calculated.
• Each cycle of ~-oxidation produces one NADH + H+ and one FADHr These enter ETC
& give 3 and 2 ATP respectively. So one cycle of ~-oxidation produces 5 ATP.
Therefore 7 cycles produces 35 ATP.
• Totally 8 acetyl CoA are produced. Each acetyl CoA enters TCA cycle and gives 12
ATP. So 8 acetyl CoA gives 96 ATP. Therefore 35 + 96 = 131 ATP.
Total 131 ATP are formed. But the initial activation step of fatty acid utilizes 2 high
energy bonds. So the net production of ATP from palmitic acid is 131- 2 = 129 ATP.
According to the current concept, NAOH gives 2.5 ATP, FAD8i gives 1.5 ATP in ETC. So,
each cycle of P-oxidation gives 4 ATP, So, 7 Cycles give 28 ATP. Each TCA cycle gives 10 ATP
& 8 acetyl CoA gives 80 ATP. So, total energy gained in P-oxidation is 108 (28 + 80 ATP).
Net yield of ATP is 106 ATP (Total 108 - 2 ATP's used in initial activation step).
Carnitine shuttle:
The P-oxidation of fatty acids takes place in mitochondria but the fatty acids received from
blood is in cytosol. Mitochondria is impermiable to fatty acyl CoA. Carnitine, a carrier molecule
transports fatty acyl CoA from cytosol to mitochondria, hence called camitine shuttle.
Jnner mitochondrial Mitochondrial matrix
Membrane
Translocase (Carrier protein)
Fatty acyl CoA Carnitine .,__...__,.__ Carnitine Fatty acyl CoA
Carnitine ¥A
transferase
Carnitine
transferas~
CoASH Acylcarnitine -,-.-,...,,,--.,.... Acylcarnitine CoASH
Fatty acyl CoA combines with carnitine to form fatty acyl carnitine by enzyme CAT I (carnitine
acyl transferase I). Acy) carnitine is transported into mitochondrial matrix by translocase in
exchange with carnitine, which is transported to cytosol. In mitochondrial matrix, acyl carnitine
combines with CoASH to form fatty acyl CoA back & carnitine by CAT ll. This transport of
fatty acyl CoA inlo mitochondria with the help of carnitine is called Camitine shuttle.
Clinical Significance:
• Carnitine deficiency, CAT I deficiency and Jamaican vomiting sickness (caused by eating
unripe fruit of akee tree, which contain toxin hypoglycin, inhibitor of medium & short chain acyl
CoA dehydrogenase), all result in reduced fatty acid oxidation and ketogenesis with hypoketotic
hypoglycemia, skeletal muscle weakness, cramps, vomiting conv11lsions etc.
• Sudden infant deatl1 syndrome [SIDS] is unexpected death of infants associated with deficiency
of medium cltain acyl CoA dehydrogenase, mainly during carbohydrate deprivation. Prevalence is 1
in 10,000 births (More than PKU).

a-oxidati on of fatty acids:

Removal of one carbon (in the form of CO ) at a time from the carboxyl end (a carbon)
2
of fatty acids is termed a-oxidatio n. This pathway does not produce ATP.
The enzyme required for a-oxidation are a-hydroxy lase and a-oxidase.
Site:
Tissue site: Brain.
Intracellular site: Microsomal endoplasmic reticulum.
Importance of a oxidation: Phytanic acid (obtained from milk and phytol of chlorophyll)
has a methyl group at C3 that prevents ~-oxidation. This CH group is removed by a-
3
oxidation to form pristanic acid, which then undergoes ~- oxidation.
a-hydroxylase a-oxidase
Phytanic acid <X--OH phytanic add Pristanic acid - - . ~xidation
Refsum's disease:
It is caused due to absence of enzyme a -hydroxylase that leads to the accumulati on of
phytanic acid in brain.
Symptoms include neurological disorders and retinitis pigmentosa.
co -Oxidation of fatty acids :

This is a minor pathway involving oxidation of w-carbon atom by the enzyme
hydroxylase. The co-CH3 (methyl) group is converted CH 0H, then to COOH to produce
2
a dicarboxylic acid. This reaction takes place in liver microsomes.
CH3 CH2 CH2 ............... CH~ COOH
ro -oxidation 1
COOH-Cl '2 CH2 ............... CH2 COOH
Then ~-oxidation takes place from both the ends, usually up to adipic acid (C") or
suberic acid (C8), which are excreted in urine.
Peroxisomal fatty acid oxidation:

This system facilitates the oxidation of very long chain fatty acids (Cw C:?1). This is a
modified form ~-oxidation and leads to the formation of acetyl CoA and 8i0 (from
2
FAD-linke d dehydrog enase). This dehydroge nation is not linked directly to
phosphory lation & ATP generation. This system doesn't attack short chain fatty acids;
~-oxidation ends at octanoyl-CoA (8 carbon atoms), which along with acetyl CoA is
transferred to mitochond ria and further oxidized.
This system shortens the side chain of cholesterol in bile acid formation.
It also takes part in the synthesis of ether glycerolipids, cholesterol and dolichol.

Lipogenesis:
When excess of glucose (more than the energy need of the body) are consumed in the
diet, the surplus is generally converted to fa tty acids and then stored in the form of
triglycerides.
De novo synthesis of fatty acid:

(Or Extra-mitochondrial or Cytoplasmic synthesis of fatty acid)
Fatty acids are synthesized mainly by de novo synthesis, which occurs in cytosol.
(Hence the name Extra mitochondrial or Cytosolic synthesis of fatty acids). Palmitic
acid is the major fatty acid synthesized.
Tissue site: Primarily in Liver and Mammary glands, also to a smaller extent in
adipose tissues, brain and kidney.
Intracellular site: Cytosol
Starting material: Acetyl CoA.
End product: Palmitic acid
Transport of mitochondrila acetyl CoA to cytosol.:

De novo synthesis of fatty acid takes place in the cytosol. But, acetyl CoA is formed
from pyruvate in the mitochondria. So, it should be transported to cytosol. But, acetyl
CoA cannot enter cytosol, as mitochondrion is impermeable to acetyl CoA.
Acetyl CoA first combines with oxaloacetate to form Citrate and then citrate is transported
to cytosol. In cytosol, citrate cleaves back to acetyl CoA and oxaloacetate by cytosolic
ATP-citrate lyase.
(Mitochondria)
Acetyl CoA + Oxaloacetate Citrate

Citrate synthase
(Cytosol)
ADP+ Pi ATP CoASH
Acetyl CoA + Oxaloacetate .V /

ATP - Citrate lyase
Citrate
This acetyl CoA is then used for the de novo sythesis of fatty acids.

Lipid Metabolis m 219
Reaction s: Cytosolic fatty acid synthesis requires two enzymes systems.

1) Acetyl CoA carboxylase
2) Fatty acid synthase
1) Acetyl CoA carboxylase system:

Eight Acetyl CoA are required for the synthesis of one molecule of palmitic acid. Out
of these 8 acetyl CoA, only one molecule of acetyl CoA takes part in the synthesis as
such. The remainin g 7 -acetyl CoA molecules take part in the reaction after they are
converte d to malonyl CoA by the enzyme acetyl CoA carboxylase.
ATP Biotin ADP+ Pi
Acetyl CoA + COi

~Mn+:c:::: • MalonylC oA
(2C)
Acetyl CoA carboxylase (3C)
2) Fatty acid synthase complex:

Fatty acyl synthase complex (present in cytosol) unites one molecule of acetyl CoA
with seven molecules of malonyl CoA by a series of reactions to form palmitic acid.
Acetyl CoA - - - - - - - - . - • Palmitic acid

Fatty acid synthase
8 Acetyl CoA- [
7 Acetyl CoA - - - - + 7 Malonyl CoA
Acetyl CoA
Carboxylase
Fatty acid synthase is a multifunctional enzyme. It is a dimer of two identical subunits

(proteins), each monome r containin g the activities of seven enzymes and an acyl
carrier protein (ACP). Seven enzymes are,
i) Acetyl transacylase
ii) Malonyl transacylase
iii) Keto acyl synthase
iv) Keto acyl reductase
v) Enoyl reductase
vi) Enoyl hydratas e
vii) Deacylase
Note:
l. Dissociation of dimer results in loss of fatty acid synthase activity.
2. In prokaryotes, the acyl carrier protein (ACP) is a separate protein, but in eukaryot es
ACP is a part of fatty acid synthase complex.

Lipid Metabolis m 220
Active sites of fatty acid synthase system:

ACP has an SH group in the 4'- Phospho -panateth eine moiety, called the Pan-SH.
Another SH group present in Cysteine moiety of the enzyme ~-keto acyl synthase
and referred as Cys-SH.
The Pan-SH and Cys-SH bind & hold the reactant molecules of fatty acid synthesis.
The Pan-SH of one monome ric unit is in close proximity with Cys-SH of other monome r
and vice versa. This is possible because the two monome rs lie in 'head-to- tail'
(antiparallel) orientatio n.
Fatty acid synthase complex
1) Acetyl transacylase
2) Malonyl transacylase
3) Keto acyl synthase
4) Keto acyl reductase
5) Enoyl reductase
6) Enoyl hydratas e
7) Deacylase
Note that the functional division of fatty acid synthase enzyme is different from subunit
division. The actual functional unit consists of one half of one monome r interactin g
with the complem entary half of another monome r. The two functiona l subunits
operate independ ently and synthesiz e two fatty acids simultaneously. (So, only dimer
is functionally active).
Reaction pathway:
I) Binding of Acetyl and malonyl units to Fatty acid synthase complex:

The initial reactions involve the binding of acetyl CoA and malonyl CoA to ACP of
fatty acid synthase complex.
a) First, a molecule of acetyl CoA is attached to ACP of fatty acid synthase complex.
Acetyl transacylase
Acetyl CoA + ACP Acetyl-ACP + CoASH
b) Next, this two-carbon acetyl unit is tran sferred to -SH of cysteine residue of the
-keto acyl ACP synthase of fatty acid synthase complex.
Acetyl-ACP + Enzyme-SH Acetyl-S-enzyme + ACP
c) The Vacant ACP now accepts m alonyl group from malonyl CoA
Malonyl transacylase
Malonyl CoA + ACP Malonyl - ACP + CoASH
Now, since both malonyl and acetyl group are attached to the fatty acid synthase
enzyme, the enzyme is called acetyl-malonyl enzyme
II) Elongation process:
Involves the sequence of 4 reactions, w hich adds 2 carbon units to the growing chain.
All the reactions take place when the reactant molecules are still attach ed with ACP.
Step 1: Condensation:
Acetyl and Ma lonyl units condense to give a 4-carbon compound, 13 -keto acyl ACP
with a release of a molecule of CO2 •
13-ketoacyl
ACP synthase
Acetyl-enzyme + Malonyl ACP .. 13-ketoacyl ACP

Step 2: Reduction:
13,-keto acyl reductdse
P-ketoacyl ACP / ~ . ., P- hydroxyacyl - ACP
NADPH + H• NADP+
Step 3: Dehyd ration
Hydratase
P- Hydroxy acyl-ACP a,punsaturated acyl-ACP
(also called 2,3-UnsaturaLed acyl ACP)
Step 4: Reduction:
Enoyl reductase
a, p Unsaturated acyl ACP _______ ~..----+11 Acyl (Butyryl) -ACP
7
NADPH+H• NADP•
(2,3-UnsaturaLed acyl ACP)
Thus, after the sequence of these 4 reactions, a four-carbon compound (butyrate) is

obtained, which is still a ttached to ACP.
III. Continuation of binding of malonyl CoA and elongation process:

Next this four-carbon butyrate is transferred from the ACP to the cysteine residue of P-
keto acyl ACP synthase of fatty acid synthase complex. Now the ACP is vacant.
Then next molecule of malonyl unit binds to ACP.
Then the four reactions (Condensation, Reduction, Dehydration and Reduction) are
repeated so that a 6-carbon fatty acid is synthesized.
This process repeated till a 16-carbon fatty acid (pal.mi tic acid) is formed which is still
attached to ACP (as Palmitoyl - ACP).
IV. Release of Palmitic acid:

Next a palmitoyl thioesterase (deacylase) enzyme releases palrnitic acid from ACP of
fatty acid synthase complex.
Deacylase
Palmitoyl - ACP + H20 Palmitic acid + ACP

Summary: Fa tty acid synthase Fatty acid synthase

De novo synthesis I I
Malonyl CoA + ACP A cetyl CoA +Enzyme - SH
of fatty acids
1
Malonyl
I ra nsa cylas c
M alony l AC P + Acetyl enzyme

l A ce t y l
Ira nsa cyla se
jA. Conden sation! l Ketoacyl synthase
jl-ketoacyl A CP
N ADPH· + H -~
j B. Redu ctionf Ketoacyl redu ctase
NA DP•
jl-hydroxyacyl ACP
j C . Dehydra tion! H y d ra ta se
a, II uns.iturated acyl AC P
r
NA DPH •+ H - ~
j D . Reduction! Enoy l reductase
NA D P ·
A (b o <y ,yO A C,
Steps A , B, C, D is
rep ea led 6 mor e times
Palm itoyl AC P
l Thioesterase
Palmitic acid+ Fatty acid synthase
Provision of NADPH for fatty acid synthesis:

NADPH required for fatty acid synthesis is mainly provided by HMP shunt pathway.
Malic enzyme (NADP malate dehydrogenase) and cytosolic isocitrate dehydrogenase
enzymes also provide NADPH+H+.
Regulation:
Acetyl CoA carboxylase is the regulatory enzyme of fatty acid synthesis. It is allosterically
activated by citrate and inhibited by long-chain acyl-CoA like palmitoyl CoA.
Insulin induces acetyl CoA carboxylase enzyme and stimulate fatty acid synthesis.
Glucagon, epinephrine, norepinephrine and thyroxine inactivate the enzyme by
promoting phosphorylation (covalent modification) and inhibiting fatty acid synthesis
Significance:
Fatty acids are synthesized when there is a surplus of energy. The excess carbohydrates
& amino acids are converted to fatty acids and then stored as fat.

Fate of Palmitic acid:

Palmitate is the end product of de novo synthesis fatty acid that occurs in cytosol. It
may be used for chain elongation, desaturation or esterification to triglycerides.
a) Chain elongation:
There are two systems for the synthesis of long chain fatty acids from palmitate.
1) Microsomal elongation system: This is brought about by specific enzymes called

elongases. This is similar to fatty acid synthase system and uses malonyl CoA and
NADPH. In brain 22C and 24C fatty acids are synthesized by this system.
2) Mitochondrial elongation system: This system uses Acetyl CoA as 2C donor and
utilizes NADH. This is less active.
b) Desaturation:
Fatty acyl desaturase, a microsomal enzyme have ability to introduce double bonds
and form unsaturated fatty acids.
This enzyme requires NADH and molecular Or
Oleic acid can be synthesized from stearic acid and palmitoleic acid can be produced
from palmitic acid.
Fatty acyl desaturase
Palmitic acid Palmitoleic acid
(NADH, molecular 02)
Fatty acyl desaturase

Stearic acid Oleic acid
(NADH, molecular 02)
Note: Mammals lack the capacity to introduce double bonds beyond carbon
atom 9. So, linoleic acid and linolenic acid cannot be synthesized in the body.
(Hence these are called essential fatty acids).
However, arachidonic acid can be synthesized from linoleic acid by desaturation
and chain elongation.
c) Esterification:
The palmitic acids formed are also esterified with glycerol to form triacylglycerols.

lipid Metabolism 225
Triacylglycerol (TAG) synthesis

Triacylglycerol (TAG) synthesis occurs mainly in liver and adipose tissues.
Pathway:
TAG is synthesized from glycerol 3 -phosphate and fatty acyl CoA.
This glycerol 3-phosphate is obtained from 2 pathways.
a) From DHAP obtained from glucose.
b) From glycerol (by Glycerokinase enzyme). Adipose tissue lacks this enzyme. So, adipose
tissue can produce glycerol 3-phosphate only from glucose. Thus TAG is synthesized
in adipose tissues only when glycolysis is activated i.e. fed condition.
i
G lu cose
( TAG synthesis) G lycoly s is
G lyce rokina s e
N AO
NAD H + H •
=l
Dihyd roxyace co n e ph osp h ate (D H A P )
G ly ccrol 3-phos phate

Dehydrogenase
G lycero I --~--,_--------• I G lycero I 3-phospha ce i

ATP A DP Fatt y a cy l C o A
A c y l tran s fera sc
C oA S H
F att y acy l C oA
CoAS H
:::i
Lysophospha1ida1e
A cy l tran s fera s c
Phospha1ida1e
H iO
Pi
---d
_,,,,-! Ph os ph a ta se
==i
Diacy lglycerol
Fatty acy l CoA

A cy l trans f e r ase
CoASH
Tr iacy lg lycero l
Fate of triacylglycerols synthesized in adipose tissues and liver:

i) In adipose tissues, the triacylglycerols stored in the cells, which serves as "depot
fat". This stored fat is ready for mobilization whenever the body requires fuel.
ii) In liver, the newly synthesized triacylglycerols are exported out of liver as VLDL.
In liver very little triacylglycerols are stored.

Phospholipid metabolism
Phospholipid synthesis:
Glycerophospholipids and sphingophospholipids are two phospholipids.
1) Glycerophospholipid synthesis:
Lecithin, Cephalin and Phosphatidyl serine three important glycerophospholipids.
They can be formed by transferring phosphory-base to diacylglycerol. CDP acts as
carrier of these phosphoryl-base.
a) Lecithin (Phosphatidyl Choline) synthesis:

Diacylglycerol - - - ~ - - : : : o - , , - - - - - - + i , Phosphatidyl choline
/ "-. (Lecithin)
CDP Choline CMP
b) Cephalin (Phosphatidyl ethanolamine) synthesis:
Diacylglycerol =--------+i, Phosphatidyl ethanolamine

---~---:::::-.............
/ 'A (Cephalin)
CDP Ethanolamine CMP
c) Phosphatidyl serine synthesis:
Phosphatidyl ethanolamine + Serine - - - - - + Phosphatidyl serine+ Ethanolamine
2) Sphingophospholipid synthesis:
Sphingomyelin is the sphingophospholipids.
Sphingosine Fatty acyl sphingosine ., Sphingomyelin

/ 'il (Ceramide) /
Fatty acyl CoA CoASH CDP Choline CMP

Lipid Metabolism 22:J
Catabolis m of phosphol ipids:
1) Degradati on of glyceroph ospholipid s:

Glycerophospholipid s are degraded by phospholip ase enzym es, which attack the
phosphod iester bonds. The action of different phospholip ases is given below.
Phospholipase A1 (and Phospholipase B)
CH20 fFatty acid

I Phospholipase A2 (and Phospholipase B)
CH O i Fatty acid
I Phospholipase C
CH, 0 j Pf Nitrogenous base

Phospholipase D
Phospholipase A 1
i) Phospholipid - - - - - - - - Monoacylg lycero phosphoryl base + Fatty acid
Phospholipase A2
ii) Phospholip id Lysophospholipid + Fatty acid
iii) Phospholipase B can hydrolyze both acyl groups (Cl & C2)
Phospholipase C
iv) Phospholipid Diacylglycerol + Phosphoryl base
Phospholipase D
v) Phospholipid Phosphatid ate + itrogenous base
Significan ce: Product of phospholipase A enzyme is lysolecithin is detergent has

2
hemolytic effect. The enzyme is present in the viper snake venom. This explains th e
hemolysis and the consequen t renal failure in viper bite.
2) Degradat ion of sphingop hospholip ids:

Enzyme sphingomy elinase degrade Sphingornyelin.
Niemann Pick disease is a disease caused d ue to s phingomy elinase deficiency.

Lipid storage diseases (Sphingolipidoses):

The term Sphingolipidoses is used to collectively represent a group of genetic disorders
that result in the accumulation of any of the sphingolipids (Sphingomyelin or glycolipids).
The result can be seen dramatically in nervous tissue in which neurological deterioration
occurs that lead to early death. (This is because; nervous system has a very high turnover
of sphingolipids. Sphingolipidoses lead to accumulation of these sphingolipids).
Disease Accumulated sphingolipid Enzyme absent Symptoms
Niemann-pick Sphingomyelin Sphingomyelinase Enlarged liver & spleen, mental

retardation, fatal in early life
Gaucher's Glucocerebroside ~-glucosidase Enlarged liver & spleen, mental

retardation, fata l in early life
Krabbe's Galactocerebroside ~- ga!actosidase Severe mental retardation,

Total absence of myelin
Fabry's Ceramide trihexoside a. -galactosidase Reddish purple skin rash,

Renal failure
Metachromatic Sulfated Aryl sulfatase A Mental retardation, demylination
leukodystrophy galactocerebroside Progressive paralysis & dementia
Tay-sachs Ganglioside GM2 ~-hexosaminidase A Mental retardation, blindness,

Seizures, death by 2-3 years
Children born with sphingolipidoses will have serious mental defects. So, it is very
important to diagnose these diseases prenatally by amniocentesis. Pregnancy may be
terminated if sphingolipidoses are confirmed.
Sphingolipidoses form a group of lysosomal storage diseases. Sphingolipids are normally
degraded by a number of Iysosomal hydrolases. Defect in any one of these enzyme result in the
accumulation and abnormal storage of the corresponding sphingolipids in lysosomes.
Propionyl CoA metabolism (or Propionate metabolism):

Propionyl CoA is produced from ~-oxidation of odd chain fatty acids. Propionyl CoA
is also produced during the metabolism of valine, methionine etc. Propionyl CoA is
converted to methylmalonyl CoA, whuch is then converted to succinyl CoA (this reaction
required Vitamin B12• Succinyl CoA then enters TCA cycle.
Deficiency of Vitamin B12 results in methylmalonyl aciduria (Details in Vitamin B12 section).
Propionyl CoA melhybnalonyl Co A
ca rboxylase Racemase mutase
Propiony!Co/"'f. K D-methylmalonylCoA L- melhybnalonyl CoA - - - - - • Sucd nyl CoA
+ Mn+2 deox)'ldenosylcobabmin I
CO, Biotin (vitamin 8 ,.) 't
ATP ADP+Pi TCA cycle

Ketone body metabolism :

Acetoacetate, ~- hydroxybutyrate, acetone are collectively known as ketone bodies.
Ketone bodies are alternative fuel molecules formed from acetyl CoA in liver. Generally,
acetyl CoA obtained from fa tty acids oxidation enters TCA cycle, but a small amount
of acetyl CoA is used for the synthesis of ketone bodies in liver. These ketone bodies
are then transported to extrahepa tic tissues (mainly brain & muscle) and used as fuel.
Ketone body metabolism incl udes Ketogenesis and Ketolysis.
Ketogenesis (Synthesis of ketone bodies)

Definition:
Synthesis of ketone bodies from acetyl CoA is ketogenesis.
Site:
a) Tissue site: Exclusively liver
Starting compound:
Acetyl CoA
Reaction pathway:
Acetoacetate is p roduced first and P-hydroxybutyrate and acetone are formed from
acetoacetate. So, acetoacetate is called the primary ketone bod y; P-hydroxybutyrate
and acetone are called the secondar y ketone bodies.
Acetyl CoA CoASH
Acetyl CoA Thiolase \.,_ /
+ Acetoacetyl CoA HMG- CoA
'Acetyl CoA 1- HMG CoA synthase
CoASH
HMG CoA lyase
(Ketogenesis ) Acetyl CoA
Dehydrogenase
P-hydroxy butyrate Acetone I

Note: Ketone bodies formed in the liver are transported to extrahepatic tissues like
heart muscles, renal cortex, brain, where they can be utilized for energy.
Liver cannot utilize ketone bodies as it lacks the ketolytic enzyme thiophorase.
Ketogenesis increases when there is a high ra te of fatty acid oxidation in th e liver.

Ketolysis (Ketone body utilization):

Definition:
Breakdown of ketone bodies for energy is called ketolysis.
Site:
a) Tissue site: Heart muscle and renal cortex prefer ketone bodies to glucose for energy.
Skeletal muscle and brain use ketone bodies when glucose is not ava ilable.
Reactions:
P-Hydroxyb utyrate
NAD+
P-Hydroxybutyrate dehydrogenase
ADH+HT
Acetoacetate
Succiny l CoA
Thiophorase Liver lacks the enzyme thiophorase.
Succinate So, liver cannot utilize ketone bodies.
CoASH i
Acetoacetyl CoA
Thiolm
2Acetyl CoA TCA cycle
Significance:
Formation of ketone bodies during starvation (especially initial stages) is beneficial.
Ketone bodies serve as a fuel for Extrahepatic tissues (mainly for brain and muscle),
especially when the availability of carbohydrate is reduced (such as starvation).
Normally, the rate of ketone body synthesis in the liver is such that they can be easily
metabolized by extrahepatic tissu e and there is a fine balance.
Normal levels of ketone bodies:
a) Norma I serum level of Ketone body is 1 mg/ dl
b) Normal amount excreted in urine is< 100 mg/day.
But, when there is a high rate of fa tty acid oxidation in the liver (as in or uncontrolled
diabetes and prolonged starvation), ketogenesis increases and exceed the limit of their
utilization by extrahepa tic tissues resulting in accumulation of ketone bodies in blood,
finally leading to clinical condition, ketosis.

Ketosis:
Ketosis constitu tes 3 clinical conditions, namely ketonemia, ketonuria, acetone breath.
i) Ketonemia: Presence of higher level of ketone bodies in the blood (> 1 mg/ dl) is
called Ketonemia.
ii) Ketonuria: Excretion of large amount of ketone bodies in the urine (> 100mg /
day) is termed as Ketonuria.
iii) Acetone breath: Acetone can also be eliminated through the lungs giving acetone
smell to the breath, this condition is called the acetone breath.
Condition in which ketosis occurs:

Conditions where ketone body synthesis is increased & development of ketosis are,
1) Uncontrolled diabetes mellitus 2) Prolonged Starvation
Explanation for development of ketosis in uncontrolled diabetes & Prolonged starvation:

In diabetes and starvation, synthesis of ketone bodies is more than normal.
a) Diabetes mellitus: In DM, since glucose cannot provide energy, lipolysis increases in
adipose tissues to release fatty acids into the blood (So, free fatty acid level is elevated in
OM). This leads to increased P-oxidation to produce more acetyl CoA. Since oxaloacetate is
deficient (as they are used for the synthesis of glucose by gluconeogenesis}, the acetyl CoA
cannot enter TCA cycle. Excess of acetyl CoA is diverted to the formation of ketone bodies.
b) Starvation: Similarly in starvation, since glucose is deficient, lipolysis and consequent
P-oxidation increases to give acetyl CoA. Since oxaloacetate is deficient (as it is used in the
synthesis of glucose by gluconeogenesis & lack of glucose to produce pyruvate, which in
tum gives oxaloacetate), acetyl CoA cannot enter TCA cycle & diverted to ketogenesis.
Thus, in uncontrolled diabetes and prolonged starvation, the rate of synthesis of ketone body
increases, which exceeds the capacity of extrahepatic tissue to utilize them. This leads to the
accumulation of ketone bodies in the blood, leading to ketonuria, excretion of ketone bodies
in the urine (ketonuria) and smell of acetone in the breath constituting the condition ketosis.
Ketoacidosis (Diabetetic ketoacidosis and starvation ketoacidosis):
Acetoacetic acid and P-hydroxy butyric acid are moderately strong acids and are buffered
by buffer system (mainly by bicarbonate buffer) of the body. However, their continuous
production results in bicarbonate deficit & decrease in blood pH resulting in acidosis, termed
as ketoacidosis. This may be fatal in uncontrolled diabetes and prolonged starvation.
Diagnosis:
The presence of ketone bodies in urine is detected by Rothera's test & Gerhardt's test.
Rothera's test: Saturate 5 ml of urine w ith solid ammonium sulphate. Add a few drops
of freshly prepared sodium nitroprusside followed by 2 ml of liquor ammonia along
sides of test tubes. Presence of a purple ring at the junction of 2 layers indicates the
presence of ketone bodies in urine.
Gerhardt's test: FeC12 solution is added drop by drop to 3 ml of urine. A port-wine
color indicates the presence of acetoaceta te.

Cholesterol synthesis
Body requires around 800mg of cholesterol per day. About 500 mg of cholesterol is
synthesized in a day and other 300mg is provided by diet.
Site:
i) Tissue site : Liver, intestine, skin, testes, ovaries, etc.
ii) Intracellular site : Cytosol & microsomal fraction of cells.
Starting compound:
Acetyl CoA
Reaction pathway:
The cholesterol synthesis can be studied in 5 steps.
Step 1: Synthesis of mevalonate

Step 2: Synthesis of isopentenyl pyrophosphate from mevalonate
Step 3: Synthesis of squalene from isopentenyl pyrophosphate
Step 4: Synthesis of lanosterol from squalene
Step 5: Synthesis of cholesterol from lanosterol
Step 1: Synthesis of mevalonate:
Thiolase HMG CoA

synthase
Acetyl CoA --~..----11 Acetoacety l CoA + Acetyl CoA / ' ( " HMG CoA
+ CoASH "-
H20 CoASH
Acety1 CoA
2 (NADPH+H)+ HMGCoA
reductase
Mevalonate

Complete reaction sequence of cholesterol synthesis
Step 1) Synthesis of mevalonate from acetyl CoA: (Already discussed before).
Step 2: synthesis of isopentenyl pyrophosphate:
Mevalonate ',<;'
ATP Mg"' ADP
Mevalonate
• Mevalonate S P
ATP Mg"'
'\.0
ADP
/f • Me,•alonate pyrophosphate
Mevalonate
kinase phosphokinase
Mevalonate
pyrophosphate
ADP+ Pi decarboxylase
CO2
Isopentcnyl pyrophosphate
Step 3: synthesis of sgualene:
lsopentenyl pyr ophosphate (S C) • lsomerase • Dimethylallyl pyrophosphate (S C)
Geranyl pyrophosphate synthase
Farnesyl pyrophosphate synthase
Farnesyl pyrophosphate (lSC) Farnesyl pyrophosphate (1S C)
Squalene (30 C)
Step 4) Synthesis of lanosterol: Squalene is cyclised to form lanosterol.

Lanosterol is the first steroid compound produced.
Epoxidase Cyclase
Squalene _ _ _ __ __ Squalene epoxide - -- -- -- Lanosterol
Step 5) Synthesis of cholesterol: Finally lanosterol undergoes several steps

(like oxidation, desaturation, removal of methyl group etc.) to form cholesterol.
Lanosterol ----,•• Zymosterol ___. Desmosterol ___. CH OLESTEROL

Regulation of cholesterol synthesis:

HMG CoA reductase is the regulatory enzyme of cholesterol synthesis. This enzyme
is regulated by feed back regulation & reversible covalent regulation.
1) Feed back repression:

Excess of Cholesterol (the product) will repress the regulatory enzyme by feedback
(or end product) repression.
2) Reversible covalent regulation (Hormonal regulation):

HMG CoA red uctase is activated in dephosphorylated form and inactive in
phosphorylated form. Insulin and thyroid hormones favors the dephosphorylated
form of HMG CoA reductase enzyme.
Insulin and thyroid hormones stimu late cholesterol synthesis by favor ing
dephosphorylated form of HMG CoA reductase. Glucagon and glucocorticoids favors
the phosphorylated form of HMG CoA reductase and inhibits cholesterol synthesis.
Thus, in starvation, where glucagon and glucocorticoids are elevated, the HMG CoA
reductase enzyme is in inactive form. This is the reason why HMG CoA does not
lead to cholesterol synthesis in starvation.
Fate (Catabolism) of Cholesterol:
1) Conversion to bile acids / bile salts:

80% of cholesterol in liver is converted to bile acids / bile salts.
2) Conversion to 4 classes of steroid hormones

i) Glucocorticoids e.g.: Cortisol
ii) Mineralocorticoids e.g.: Aldosterone
iii) Male sex hormones e.g.: Testosterone
iv) Female sex hormones e.g.: Progesterone, Estradiol etc
3) Conversion to 7-dehydro cholesterol for the synthesis of Vitamin D

7 -dehydrocholesterol, a derivative of cholesterol can be converted to cholecalciferol
(vitamin 0 3) by ultraviolet rays of sunlight in the skin.
(Skin)
7- dehydrocholesterol • • • • • • • • Cholecalciferol (vitamin DJ
Ultraviolet rays of sunlight
4) Conversion to cholestanol and coprastanol and excretion through feces:

ln large intestine, the cholesterol is converted to cholestanol and coprastanol by
intestinal bacterial flora, which is excreted in feces.

Bile acids / Bile salt:

The term bile acids and bile sa lts are often used interchangeably. At physiological
pH, bile acids present as anions and combine with sodium or potassium ions and
exist as sodium or potassium salts of bile acids (or bile salts).
Synthesis:
Bile acids are synthesized in the liver from cholesterol.
!
Cholesterol
(Uve,) 7-a-hydroxylase (Vffamin C, NADPH, O,)
7 - hydroxycholesterol
/ ........
!
Primary bile acids Cholic acid Chenodeoxycholic acid
1· Conjug,tion wilh glydne o, taurine
i1/
Conjugated Glycocholic acid or Glycochenodeoxycholic or
Bile acids Taurocholic acid Taurochenodeoxycholic
~ dium I polassium
Bile salts Sodium or potassium salts of bile acids

(or Bile sa Its)
Steps:
• First step in the synthesis of bile acids is the formation of 7 - hydroxycholesterol
from cholesterol by the enzyme 7-a-hydroxylase.
• 7 -hydroxycholesterol then undergoes series of reactions to form primary bile acids
like cholic acid and chenodeoxycholic acids.
• Primary bile acids undergo conjugation reaction with g lycine or taurine to form
conjugated bile acids like Glycocholic acid , taurocholic acids;
Glycochenodeoxycholic acids and Taurochenodeoxycholic etc.
• In the bile, these conjugated bile acids exist as Na or K salts of bile acids (or bile
salts).

Fate:
• Conjugated bile salts synthesized in the liver accumulate in the gall bladder and
then secreted into the duodenum (of intestine) by bile duct.
• In the intestine, bile salts act as emulsifying agents and help in the digestion and
absorption of lipids and lipid soluble vitamins.
• After their function in the intestine (mainly duodenum) almost all bile acids are then
absorbed from the ileum of intestine and return to liver via the portal circulation. •
From liver, bile salts are agin secreted ino bile. This cycling of bile salt between intestine
and liver is known as 'enterohepatic circulation'. Thus, almost all (up to 99%) of bile
salts are recycled and reused. Only a small portion of bile salts (about 0.5 g / day) is lost
in the feces. ·
• A portion of primary bile acids in the intestine undergoes deconjugation and
dehydroxylation to form secondary bile acids like deoxycholic acid & lithocholic acid.
• Primary bile acids: Cholic acid and chenodeoxycholic acids.

• Conjugated bile acids: Glycocholic acid, Glycochenodeoxycholic acid,
Taurocholic acids and Taurochenodeoxycholic.
• Bile salts: Sodium or potassium salts of conjugated bile acids
• Secondary bile acids: Deoxycholic_acid and lithocholic acid.
Functions:
• Bile salts are required for the digestion and absorption of lipids and lipid soluble
vitamins in the intestine. They combine with fats and other lipids to form micelles.
This process is called emulsification.
• Bile salts serve as emulsifying agents and reduce the surface tension of lipids and
thus help in the digestion and absorption.
Clinical significance of bile salts:

• Urinary bile salt is of clinical importance. It is used as a diagnostic in the differential
diagnosis of jaundice.
• Normally bile salts are not found in the urine. They are detected in the urine during
hepatic (+) and obstructive (post-hepatic) jaundice (+++).
• Urinary bile salts can be detected by Hay's test.
Note:
Bilirubin follows the same route of enterohepatic circulation with bile salts, so
bilirubin is a lso excreted along with bile salts in hepatic and obstructive
(post-hepatic) jaundice.

Lipoproteins :
Introduction: Lipids are water insoluble compounds. So, they can't be transported
in blood. Lipids are made water-soluble by combining with protein to form lipoproteins,
which are water-soluble and can be transported in blood. Thus, lipoproteins are the
transport form of lipids in the blood.
The protein part of lipoproteins is called apolipoproteins. Lipoprotein contains variable
amount of triglycerides, cholesterol, cholesterol ester, phospholipids and fatty acids .
• "1 'Ml•,,
\ ' IIJl///l!lf•,.. ~~---- Phospholipid monolayer
Peripheral apoprotein
Yi 1 ~---·...,__
' - - - - Triacylglycerol
.I. I i
Free cholesterol ) \.\p\ : Cholesterol ester
~--. . . . ~<!) <

--~
/ : Integral apoprotein
( Generalized structure of a lipoprotein )
Lipoprotein classification:
1) Based on the density :
Lipoproteins are divided into four groups based on their densities.
a) Chylomicrons - Density is < 0.96
b) VLDL (Very low density lipoprotein) - Density is 0.96-1.006
c) LDL (Low density lipoprotein) - Density is 1.006-1.063
d ) HDL (High density lipoprotein) - Density is 1.063-1.21
2) Based on electrophoretic mobility :
Lipoproteins can also be classified based on their electrophoretic mobility.
a) Chylornicrons stay at the region of application of serum.
b) VLDL moves to pre-~ region, hence VLDL is called as pre lipoprotein.
c) LDL moves to region, so LDL is also called lipoprotein.
d) HDL moves to a region, hence called a lipoprotein.
[ Summaryl
Class of Density Electrophoretic %Composition Major lipid
Lipoprotein Mobility Lipid Protein Present
Chylomicrons < 0.96 Origin 98 % 2% Mainly Triglycerides
VLDL 0.96-1.006 Pre-13 reITTon 91 9 Mainly Triglycerides
LDL 1.006-1.062 13 region 79 21 Mainly Cholesterol
HDL 1.063-1.21 ex region 50 50 Mainly Cholesterol
& Phospholipids

lipid Metabolism 238
Functions of lipoproteins:
Lipoproteins are the transport form of lipids. They transport different lipids in blood.
1) Chylomicrons: Chylomicrons transport exogenous triglycerides from intestine to
peripheral tissues like adipose tissue and skeletal muscle.
2) V LDL: VLDL transports endogenous triglycerides from liver to extrahepatic tissue
like heart, adipose tissue, muscle, blood vessels etc.
3) LDL: LDL transports cholesterol from liver to extrahepatic tissues.
4) HOL: HDL transports cholesterol and cholesterol esters from extrahepatic tissue
back to the liver.
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Diagramatic representation of lipid transport
Answer hint for lipoprotein question (5 Marks):

a) Explanation - 1 mark
b) Classification (2 ways) - 2 Marks
c) Functions with diagram - 2 marks

Cholesterol transport:
Total serum cholesterol level is 150-220 mg/di. 30% of which is in esterified form and 70% is
free form. Cholesterol and cholesterol esters are transported in the blood by HDL and LDL.
LDL transports cholesterol from liver to extra hepatic tissues. So, LDL cholesterol is some time
referred to as bad cholesterol . HDL transports cholesterol & cholesterol esters from extra hepatic
tissue to the liver. Thus HDL has a protective function and is referred to as good cholesterol
and the transport of cholesterol through HDL is termed as reverse cholesterol transport.
Apolipoproteins : Protein part of lipoprotein is called apolipoprotein.

Lipoprotein class Apolipoprotein present
Chylomicrons Apo A, Apo B-48, Apo C, Apo E
VLDL Apo B-100, Apo C, Apo E
LDL Apo B-100
HDL Apo A, Apo C, Apo E
LCAT (Lecithin Cholesterol Acyl Tra11sferase): This enzyme is synthesized by liver & is found in plasma.
LCAT converts surface phosplwlipids and free cholesterol ofHDL into cho/esteryl ester and lysolecithin.
LCAT
Cholesterol+ lecithin - - - ---+ O,olesterol ester+ Lysolecithin
A fatty acid residue is transferred from lecithin to cholesterolforming lysolecithin and cholesteryl ester. The
non-polar clwlesteryl ester then moves into the hydrophobic interior of HDL, whereas lysolecithin is
transferred to the plasma albumin. This helps in the forma tion of mature HDL.
LDL receptors: Function of LDL is to transport cholesterol from liver to extrahepatic tissues. The LDL
particles bind to specific LDL receptors, (which are clustered in theform ofpits). Tire intracellular side of
LDL receptors is coated with specific protein called clathrin, which stabilizes the shape of the pits.
Apo B 100 is responsible for the recognition of LDL receptor. LDL binds with the receptor and whole
receptor-LDL complex is internalized by endocytosis.
Deficiency of LDL receptors causes type-Ila hyperlipoproteinemia (or Familial hyperclzolesterolenzia),
resulting in increased plasma LDL level (serum cholesterol level).
Oxidized LDL: Nowadays, oxidized LDL is considered as one of the most important risk factor in the
development ofatherosclerosis. The free radicals and other oxidants convert normal LDL to oxidized LDL.
Oxidized LDL is taken up by macrophages. Excessive uptake of Oxidized LDL causes the transformation
of macrophages i11to foam cells, which participate in formation of atherosclerotic plaques. Conversion of
LDL to oxidized LDL depends both 011 concentration ofoxidants and the concentration ofantioxidants.
Lipoprotei11 lipase (LPL) or tile clearing/actor: Lipoprotein lipase enzyme is present in capillary walls
ofadipose tissues and muscles. it hydrolyzes triacylglycerols present in clzylomicrons and VLDL to liberate
free fatty acids and glycerol. Tissues take up thefatty acids and glycerol goes to the liver. So, LPL is also
called clearingfactor. Deficiency of LPL causes type-1 hyperlipoprotei11emia, resulti11g i11 accumulation of
city/omicrons and VLDL (serum triglyceride) level.
Lipoprotein (a) or LP(a): LP(a) is structurally similar to LDL, but only distinguishing factor is the
presence of additional apolipoprotein (a), which is covalently attached to Apo B100. LP(a) is seen
only in some individuals. When present in large quantities in plasma, it is an indicator of increased
the risk of coronary heart diseases. LP(a) inhibits fibrinolysis .

Lipoprotein turnover:
1. Chylomicrons
Initially nascent chylomicrons are assembled in the intestine from triacylglycerols,
phospholipids, cholesterol, apo A and apo B-48. They are transported to blood via lymphatics.
In the blood, apo C and apo E are added to these chylomicrons from HDL to form complete
chylomicrons.
In extrahepatic tissue capillaries, lipoprotein lipase hydrolyzes TAG to glycerol and free fatty
acids. The free JathJ acids are esterified back to triacylglycerols and stored by adipose tissue.
Glycerol is taken up by the liver.
Chy/omicrons, after loosing triacylglycerols, lose apo A and apo C also to form chylomicron
remnants. Apo A and apo C return to HDL. Apo E is retained. Chy/omicron remnants are
taken up by liver by receptor mediated endocytosis, mediated by apo E.
2. VLDL
Liver synthesizes nascent VLDL consisting of triacylglycerols, phospholipids, cholesterol esters,
cholesterol, apo B-100, apo C and apo E. It becomes complete VLDL in the blood after addition
of extra apo C and apo Efrom HDL.
Lipoprotein lipase hydrolyzed triacylglycerols of VLDL to release fatty acids. Then it loses its
apo C (which is returned to HDL) and becomes VLDL remnant (also called as the intermediate
density lipoprotein [IDL]). IDL is either taken up by liver or loses its apo E to become LDL.
3.LDL
It is obtained form VLDL. It consists of triacylglycerols, phospholipids, cholesterol and its
esters and apo B-100. About half of the circulating LDL is taken up by extrahepatic tissues
(which have Apo B-100 specific LDL receptor) and degraded in these tissues to release cholesterol.
Thus, LDL transports the cholesterol to the extrahepatic tissues. The remaining LDL is degraded
in liver.
4.HDL
It is synthesized and secreted by liver and intestine. The nascent HDL is discoid in shape and
its core is almost empty. LCAT (Lecithin cholesterol acyl trans/erase) enzyme bound with
HDL converts phospholipid and free cholesterol to form cholesterol ester and lysolecithin. The
cholesterol ester is then transferred to the core of nascent HD L. As more and more cholesterol
ester enters to the core of nascent HDL, it becomes spherical in shape to form mature HDL.
HDL consists of triacylglycerols, phospholipids, cholesterol and its esters, apo A, apo C and
apo E. It serves as reservoir of apo C and apo Efor formation of chylomicrons and VLDL.
HDL is finally taken by liver and releases the cholesterol. HDL transports the cholesterol from
extrahepatic tissues to liver.

Lipid Metaoolism 241
Clinical significance of lipid fractionation:

Disorder of plasma lipoprotein is called dyslipoprotenemia.
Dyslipoprotenemia include hyperlipoproteinemin and hypolipoprotenemia.
I) Hyperlipoproteinemia (also called hyper lipidemia):

The condition of elevation of one or more lipoprotein fraction in the plasma is known as
hyperlipoprotenemia. According to Frederickson 's classification there are 5 types of
hyperlipoproteinemia
a) Type-1 hyperlipoproteinemia:
Metabolic defect: Lipoprotein lipase enzyme deficiency.
Plasma chylomicron and VLDL (Plasma TG level) level are increased) increases.
b) Type-II a hyperlipoproteinemia (or Familial hypercholesterolemia):

Metabolic defect: LDL receptor deficiency. Plasma LDL cholesterol is increased.
c) Type II b hyperlipoproteinemia:
Defect: Overproduction of apo B. Both LDL and VLDL increases.
Both plasma TG and cholesterol level increases.
d) Type III hyperlipoproteinemia: Increase in IDL
e) Type IV hyperlipoproteinemia: Increase in VLDL
f) Type V hyperlipoproteinemia: Increase in VLDL & chylomicron
II) Hypolipoproteinemia:
Condition ofdecreased lipoprotein fraction is termed as hypo!ipoproteinemia.
a) Familial hypo-~-lipoprotenemia:
Defect: Failure in the synthesis of apo B lipoproteins.
LDL level increases in the blood.
b) Abetalipoprotenemia:
Defect: Absence of Apo B100. LDL fraction is campletely absent.
c) Familial a-lipoprotein deficiency (Tangier disease):

Defect: HDL deficiency, due to reduction in Apo A synthesis.

Serum cholesterol level is associated with atherosclerosis and CHD.

Normal level of ch olesterol in serum is 150-220 mg/ di.
Elevated serum ch olesterol level is the major risk factor in promoting atherosclerosis.
Hypercholesterolemia and development of atherosclerosis and CHO:

Hypercholesterolemia is mostly associated w ith increased LDL cholesterol levels.
Increased cholesterol level (m ainly LDL fraction) leads to the deposition of cholesterol
in the intimal side (inner side) of the arteries, resulting in the formation of fibrous
plaques and consequent thickening and hardening of arterial wall causing the condition
Atherosclerosis. Coronary arteries, aorta and cerebral vessels are predominantly
affected. The atherosclerotic plaques lead to narrowing of blood vessels. So the blood
flow through them becomes turbulent and there is increased tendency for clot formation.
The plaques and clot occlude the artery (Occlusion) and finally stop the blood flow
through them (Thrombosis). Thrombosis leads to decreased oxygen supply (Ischemia)
to the tissues. Finally, ischemic death (Infarction) of tissues occurs. Since coronary
artery is one of the major arteries affected, the myocardial infarction is the main
consequences of atherosclerosis and hypercholesterolemia.
Causes of Hypercholesterolemia (and atherosclerosis and CHO):

• Diabetes mellitus: Due to increased cholesterol synthesis since tlzc availability of aceh;l CoA is
increased.
• Obstructive jaundice: Cholesterol is mainly excreted through bile. Tn obstructive jaundice,
there is an obstruction in the cholesterol excretion through bile, causing hyperclzolesterolemia.
• Hypothyroidism: Thyroid hormones play a role in reducing serum cholesterol level. So,
cholesterol level increases in hypothyroidism.
• N e phrotic syndrome: T11 nephrotic syndrome, lipoprotei11 lipase (which is required to clear
lipids fro111 blood) may be lost in the urine.
• Familial Hypercholesterolemia (Familial type II a hyperlipoproteinemia): due to the
defect in LDL receptors (required for hepatic cholesterol uptake), cholesterol level increases in blood.
• Other risk factors tha t alter the serum cholesterol level are heridity, high BP, smoking,
obesity, lack of exercise, emotional stress, excess coffee drinking, su crose consumption .
• H ereditary factors play an important role in influencing serum cholesterol levels.
Besides these, dietary factors also play an important role in determining serum
cholesterol level. Unsatura ted fatty acids decrease the serum cholesterol level, while
saturated fatty acids increase the serum cholesterol levels. Cholesteryl esters formed
with the unsaturated fatty acids are believed to be rapidly removed from the circulation.
But, exact mechanism is not known yet.
• Moderate alcohol consumption is shown to have a beneficial effect on a therosclerosis.
• Premenopausal women have lower incidence of atherosclerosis and CHO, it is probably
due to the beneficial effects of estrogen.

Biochemical tests for the detection of atherosclerosis and CHD:

1. Serum total cholesterol, LDL and HDL cholesterol fractions:
Normal levels:
Normal level of choles.terol in serum is 150-220 mg/ dl.
Normal LDL cholesterol level is < 130 mg/ dl.
Normal HDL cholesterol level is > 60 mg/ di.
Normal LDL: HDL ratio is below 2.5.
Cholesterol is mainly transported by HDL and LDL.
LDL transports cholesterol from liver to extrahepatic tissues. So, LDL cholesterol is
some time referred to as bad cholesterol. HDL transports cholesterol and cholesterol
esters from extra hepatic tissue back to the liver. So HDL cholesterol level has a
protective role on development of atherosclerosis, it is referred to as good cholesterol.
Increase in LDL cholesterol level and / or decrease in HDL cholesterol level increases
the risk of atherosclerosis. So in hypercholesterolemia, it is essential to know which
fraction (HDL or LDL) increases in the blood.
Interpretation of values:
• Serum total cholesterol levels below 180 mg/ dl is desirable; level between 180 and
220 are borderline; levels above 220 mg/ dl considered a moderate risk; levels above
240 mg/ di is considered a definite and considerable risk factor in atherosclerosis.
• Serum LDL levels below 130 mg/ dl is desirable; levels between 130 and 160 are
borderline; and levels above 160 mg/ di considered a definite risk factor in
development and progression of atherosclerosis.
• Serum HDL levels above 60 mg/dl is desirable; levels between 60 and 40 are
borderline; levels below 40 mg/ d l considered a definite risk factor in atherosclerosis.
• LDL: HDL ratio more than 2.5 are considered dangerous.
2. Other indicators of atherogenesis and cardiovascular diseases:

• Plasma homocysteine:
Normal level is <15 micromole/L. Elevated plasma homocysteine level has been implicated
in the development of atherosclerosis. Homocysteine thiolates LDL particles. These particles
aggregate and endocytosed by macrophages and increase the tendency of atherogenesis.
• Pl asm a C-Reactive Protein (CRP):

An elevated blood level of CRP, particularly high sensitivity CRP (hs-CRP) is an indicator
of chronic inflammation that appears in atherosclerosis.
• Apoli poprotein estimation:

Apo A-I is a measure of HDL-cholesterol and apo Bis a measure of LDL-cholesterol. ormal
ratio of Apo B: Apo A-I is 0.4. Increased ratio is considered as a risk factor in atherosclerosis.

Approaches to treatment of atherosclerosis:

Major risk factor of atherosclerosis and coronary heart diseases (CHD) is
hypercholesterolemia. So, the most important approach of treatment of atherosclerosis
is to bring the serum cholesterol level back to normal level.
A. Hypocholesterolemic agents
The compounds, which decrease the serum cholesterol level in the blood are called
hypocholesterolemic agent. These are,
a) Statins: like Lovastatin, atorvastin, simvastatin, fluvastatin etc inhibit HMG CoA
reductase and decrease cholesterol synthesis, hence decreases blood cholesterol level.
b) Sitosterol: Sitosterol is a plant sterol. It decreases the intestinal absorption of
cholesterol by competition.
c) Cholestipol and cholestyramine (Bile acid binding resins): They bind to bile acids
and prevent their reabsorption from intestine and increase their faecal loss.
d) Ezetimbe: Reduces serum cholesterol by blocking intestinal absorption of cholesterol.
e) Clofibrate, Gemfibrozil and nicotinic acid: Lowers serum cholesterol & triacylglycerol
by decreasing the secretion of triacylglycerol and cholesterol containing VLDL by liver.
f) O-thyroxine: D-thyroxine reduces the LDL level. A dose of 4-8 g per day is given.
B. Lifestyle modifications:
1. Reduce dietary cholesterol
Restriction of diets rich in cholesterol like egg and meat is beneficial.
2. Reduce dietary saturated fatty acids
Fat or oils containing saturated fatty acids tend to increase the serum cholesterol level.
So, the diet containing saturated fatty acids should be restricted. These are butter,
ghee, palm oil etc
3. Increase dietary unsaturated fatty acid (both mono and poly)
Fat/ oils containing unsaturated fatty acids decrease the serum cholesterol level.
Cholesteryl esters formed with the unsaturated fatty acids are believed to be rapidly
removed from the circulation. So, the diet containing unsaturated fatty acids is
recommended. These are vegetable oil (like sunflower oil, olive oil etc) & fish liver oil.
4. Increase green leafy vegetables
Green leafy vegetables contain high dietary fiber content, which increase the bowel
motility and reduce the reabsorption of bile salt. Thus enterohepatic circulation of bile
acids is disrupted and less bile acids return to the liver from intestine. This results in
more cholesterol directed to bile acid synthesis and thus reducing the cholesterol level.
5. Regular exercise is beneficial
Regular exercise lowers the serum LDL level and increases the serum HDL level.
Exercise also reduces obesity, which is one of the major risk factors in atherosclerosis.
6. Reduce sucrose intake

Fatty liver
Definition: Normally liver cells contain only 5 % of fats. If the fat level increases above
5 % in the liver cells, then the condition is known as fatty liver.
Liver has the ability to take up fatty acids from the blood and esterify it to fat. This fat
(Triglycerides) along with the endogenously synthesized triglycerides are incorporated
into VLDL & transported out of the liver to the extra-hepatic tissues. This is a very
finely balanced process. Imbalance in the rate of triacylglycerol synthesis and export
causes fatty liver. Excessive accumulation of fat in the liver is pathogenic. It leads to
fibrotic changes in the liver causing fibrosis, then cirrhosis and finally hepatic failure.
Fatty liver is seen in two conditions:
1) Increased synthesis of triacylglycerol in liver
2) In the metabolic block of VLDL formation
1. Increased synthesis of triacylglycerol in liver:
Causes: diabetes mellitus, starvation, high fat diet, high calorie intake, alcoholism etc.
• Diabetes mellitus & starvation: Increased mobilization of fat from adipose tissues
by increased lipolysis during diabetes mellitus and starvation (due to unavailability or
underutilization of carbohydrates), leading to increased levels of free fatty acids in the
blood and subsequent increased uptake of fatty acids by liver and synthesis of excess
amount of fat, which exceeds the rate of VLDL formation and capacity of fat disposal
from liver. This results in the accumulation of fat in the liver leading to fatty liver.
• High fat diet: Directly elevates free fatty acids in blood, excess uptake and excess
production fat in liver leading to fatty liver.
• High calorie intake: In high calorie intake, surplus sugar is converted to fat in liver.
• Alcoholism also leads to fatty liver: Oxidation of ethanol by alcohol dehydrogenase
leads to excess production of energy (NADH), which leads to inhibition of fatty acid
oxidation and causes increased esterification of fatty acids to form triacylglycerol (fat),
resulting in the fatty liver. Oxidation of ethanol produces acetaldehyde, which is oxidized
by aldehyde dehydrogenase, to form acetate, which is converted to acetyl CoA. Excess
of acetyl CoA is drawn to the synthesis of fatty acid and then fat leading to fatty liver.
2. In the metabolic block of VLDL formation:
Endogenous triacylglycerols are transported out of liver by VLDL. Any metabolic block
in VLDL formation, fat is not properly transported out of the liver, leading to the
accumulation of fat in the liver causing fatty liver.
• VLDL is a lipoprotein consisting of triglycerides (fat), phospholipid, cholesterol and
proteins. If any one of this is absent, VLDL is not synthesized properly.
• Choline, PUFA are required for the synthesis of phospholipid. So in the deficiency of
PUFA (essential fatty acids) or choline, the phospholipids are not synthesized properly
& without phospholipids, VLDL are not formed.
• Puromycin, chloroform, CC14, lead, arsenic inhibit the protein synthesis in the liver.
Protein energy malnutrition (PEM) also reduces the protein synthesis due to unavailability
of amino acids. Since protein is required for VLDL synthesis, VLDL formation is blocked.

Lipotropic factors:
Definition:
Lipotropic factors are the factors that are required for normal mobilization of fat from
the liver and thus preventing the risk of fatty liver. Examples are,
1. Choline: It is required for the synthesis of phospholipids, which are required for the
synthesis of VLDL. So choline is an important lipotropic factor.
2. Methionine and betaine: These are required for the formation choline, which is
required for phospholipid synthesis, hence VLDL formation. So, methionine and
betaine are also lipotropic factors.
3. PUFA: PUFA are required for synthesis of phospholipids. So, PUFA also are lipotropic
factors.
4. Inositol is required for synthesis of phospholipids. So, it is also a lipotropic factor.
5. Vitamin E and Selenium are also considred lipotropic factors due to their antioxidant
activities.
Answer hint for Fatty liver and lipotropic factors question (5 Marks):
a) Definition - 1 mark
b) Explanation - 1 Mark
c) Types - 1 mark
d) Lipotropic factors - 1 mark
Role of liver in lipid metabolism:

Liver has a central role in lipid metabolism.
1. Bile synthesis: Liver facilitates the digestion and absorption of lipids by the production
of bile, which contains cholesterol and bile salts. Bile salt is synthesized from cholesterol
in the liver. It is then transported to gall bladder and then to intestine, where they are
requ ired for digestion and absorption of lipids and fat soluble vitamins.
2. Metabolism of fatty acids: Liver is actively involved in the synthesis and oxidization
of fatty acids.
3. Triacylglycerol and phospholipid synthesis:
Liver synthesizes triacylglycerols and phospholipids.
4. Ketogenesis: Liver converts fatty acids to ketone bodies (Ketogenesis). Note that
ketogenesis takes place exclusively in liver.
5. Lipoprotein metabolism: Liver plays an integral role in the synthesis and metabolism
of plasma lipoproteins.

Question bank on Lipid metabolism

1. Describe beta (~) oxidation of fatty acids? Write its energetics?
2. Explain extramitochondrial synthesis (Denovo synthesis) of fatty acids
3. Explain ketone body metabolism. Add a note on ketosis

1. Carnitine shuttle
2. Fatty acid synthase enzyme
3. Hypercholesterolemia and associated disorders. Add a note on hypercholesterolemic agents.
4. Write the formation of mevalonate from acetyl CoA.
5. Discuss the regulatory enzyme of cholesterol synthesis? (Write the reaction and regulation)
6. What is the fate of cholesterol?
7. Lipoprotein and their significance
8. What is fatty liver? Explain the causes of it. Add a note on lipotropic factors.

1) Apoproteins present in LDL (MAHE)
a) B-48 b) B-100 c) C-1 d) C-llI
2) Which cholesterol is designated as "Good Cholesterol" (COMEDK)
a) VLDL b) LDL c) HDL d) IDL
3) Which organ does not utilize ketone bodies (AIIMS)
a) Liver b) Adipose tissue c) Skeletal muscle d) Heart muscle
4) Rothera's test used for detection of (AI)
a) Proteins b) Glucose c) Fatty acid d) Ke tones
5) Sphingomyelinase deficiency is seen in (AI)
a) Niemann pick disease b) Farber's disease c) Tay-Sachs disease d) Krabbe's disease
6) The term beta-lipoprotein refers to
a) Chylomicrons b) VDL c) LDL c) HDL
7) Primary Ketone body or the first ketone body that is formed is
a) Acetone b) Acetoacetate c) ~-Hydroxy butyrate d) Propionate
8) Dietary triglycerides is transported to the extrahepatic tissue through
a) VLDL b) LDL c) HDL d) Chylomicrons
9) Refsum's is disease is due to accumulation of
a) Phytanic acid b) Glucocerebrosides c) Sphingomyelin d) Gangliosides
10) Regulatory enzyme for cholesterol biosynthesis is
a) HMG CoA Lyase b) HMG CoA Reductase c) Squalene synthase d) HMG CoA synthase
Answers for MCQ: 1) b 2) c 3) a 4) d 5) a 6) c 7) b 8) d 9) a 10) b

Amino acid and Protein Metabolism 248
Metabolism of Amino acids and Proteins
Contents
• Nitrogen Balance
• Catabolism of Amino acids
• Urea Cycle
• Metabolism of individual amino acids (Glycine, Phenylalanine, Tyrosine, Tryptophan,
Histidine, Methionine, Cysteine, Arginine)
• Specialized products formed from amino acids

Protein turnover and Amino acid pool:

Body proteins are in a dynamic state and being constantly degradaded & resynthesized,
which is called protein turnover. Average protein turnover is about 400 g per day.
Amino acid pool represents the amount of free amino acids prsent at any given time in
the body. It is about 100g. Major sources of amino acids in the pool are, 1) amino acids
formed from degradation of body proteins; 2) amino acids obtained from dietary proteins;
3) Non-essentajl amino acids produced in the body. Major fates of amino acids in the
pool are; 1) synthesis of body proteins, 2) formation of specialized products, 3) catabolism
to keto acids and NJ\ (NJ\ is then detoxified to u rea)
Nitrogen balance:
The difference between nitrogen intake (mainly in the form of dietary proteins) and
nitrogen output (in the form of urea, uric acid, creatinine etc) is called nitrogen balance.
3 nitrogen balance conditions can be defined.
1) Nitrogen equilibrium: Nitrogen equilibrium is a condition when the nitrogen intake
(is equal to total nitrogen output). (Nitrogen intake= Nitrogen output).
Condition: In a normal healthy adult, nitrogen balance is in nitrogen equilibrium state.
2) Negative nitrogen balance: Negative nitrogen balance is a condition, where the
nitrogen intake is less than the nitrogen output. ( itrogen intake < Nitrogen output).
There is a net loss of nitrogen from the body.
Conditions are Diabetes mellitus, Starvation, Wasting diseases like tuberculosis &
in acute illness (such as trauma, bums, infection, injury), Protein energy malnutrition,
Poor quality protein intake, Old age, Glucocorticoids, stress hormones etc.
3) Positive nitrogen balance: Positive nitrogen balance is a condition, where the nitrogen
intake is m ore than the nitrogen output. ( itrogen intake> N itrogen output). There is
a ne t retention of nitrogen in the body.
Conditions are Children, Pregnancy, lactation, Convalescence (Recovery) after illness,
surgery, Growth hormone, insulin, testosterone etc.
Causes and reasons for negative nitrogen balance: Diabetes mellitus & prolonged
sta rvation (due to increased protein catnbolism to give energy as glucose is unable to provide
energy in diabetes & starvation), Wa sting diseases like tuberculos is & acute illness such as
trauma, bums, infection, injury (due to increased tissue destruction & increased depletion of protein
stores), P rotein energy m aln11tritio11, Poor qualitiJ prot ein intake, S tarvation (due to defective
protein intake. Deficiency of even a single amino acid can affect new protei11 synthesis), Old age
(due to depletion of body proteins), cortisol, s tress hormones (which promote prote111 breakdown).
Causes a nd reasons fo r p ositive n itrogen bala n ce: Childre11 (nitrogen is retained for body
growth during the period of nctivr growth), Pregnancy and lactation (more nitrogen is retained
due to increased requirr111ent), Convalescence (Recotll'ry) after illness, surgery (Tissue repair
require protein). Growth hormone, ills11lin, testosterone show positive 11itroge11 balance.
Note that a high intake of proteins does not lend to positive nitrogen balance. It increases both
anabolism and catnbolism of proteins, so that the nitrogen eq11ilibriu111 is maintained.

Amino acid and Protein Metabolis m 250
Catabolism of Amino acids:

Each day, around 400 g (1-2%) of body proteins are degraded to amino acids. Around
75-80 % of these amino acids are reutilized for the synthesis of new proteins. The other
20-25% of amino acids are cataboliz ed to a-keto acids and ammonia .
a Amino acid -----.':::- -• a- keto acid (carbon skeleton of amino acids)
H3
~reacy cle
Urea
• During the catabolis m amino acids, the amino group is removed from amino acids (as
ammonia ) to form cx-keto acids (which are called the carbon skeletons of amino acids).
• Ammoni a, thus removed is then d etoxified to urea and excreted in urine; whereas,
carbon skele tons can be further converte d to intermed iates of energy producin g
metabolic pathways (glycolysis, TCA cycle) and metaboliz ed to CO2 & Hp giving
energy or converter ted to glucose, fatty acids or ketone bodies.
Amino acid are catabolized by mainly two processe s,

1) Transdea mination - Major process.
2) Deamina tion (Oxidativ e deaminat ion and Non-oxid ative) -Minor processes .
I. Transdeamination:
The amino group of the most of the amino acids is removed by a coupled reaction
called transdeam ination i.e. transami nation followed by oxidative deamina tion of
glutamate. In transami nation, amino groups from different amino acids are funneled
to cx-ketoglu tarate to form glutamate. This glutamate is then transport ed to liver and
oxidative ly deaminat ed to a-ketoglu tarate & ammonia .
Transdeamination = Transamination + Oxidative deamination of glutamate
Transamination :
Definiti on:
Transam ination is the process of transfer of amino group from an amino acid to a keto
acid, convertin g the original keto acid to a new amino acid and the original amino acid
to a new keto acid, without the liberation of ammonia .
Transam ination is a reversibl e process. lt is catalyzed by enzyme transami nases
(Aminotr ansferase s). PLP (Pyridoxa l phosphate) is the coenzym e of transamin ases.
Transaminase
Amino acid 1 + Keto acid 2 Keto acid 1 + Amino acid 2
PLP
75
For queries and suggestions, contact the author at prasad_te xt@yahoo .com or 99864495
Examples:
1) AST (Aspartate transami nase) or SCOT (Serum Glutama te Oxaloacctate Transaminase)
Aspartate + o.-ketoglutarate • AST (PLP) • Oxaloacetate + Qutama te
2) ALT (Alanine transaminase) or SGPT (Serum glutama te pyruvate transaminase)

:
Alanine+ a,.. ALT (PLP)
ketoglutarate Pyruvate +Glutamate
Features: All amino acids, except lysine, threonine and proline, undergo transamination.
Differen t transam inases are specific for their amino acids. Keto acid, which receives the
amino group from these amino acids, is a-ketoglutarate . As a result of transam ination,
amino acids are convert ed to correspo nding a-keto acids by transfer ring the amino
group to a -ketoglu tarate, which forms glutama te. Conseq uently, amino groups of all
amino acids that undergo transam inated are concentrated in glutamate. So, glutama te
serves as a "collect ion center" for amino groups from differen t amino acids.
Importance of transamination:
1) Degradation of amino acids: Transam ination helps in the degrada tion of amino
acids by convert ing amino acids to corresp onding a-keto acids (by transfer ring the
amino group to a-ketog lutarate to form glutama te). Conseq uently, amino groups of all
amino acids that undergo transam ination are concent rated in glutama te. Glutam ate
then transpo rted to liver & releases the ammon ia by glutama te d ehydrogenase.
2) Synthes is of non-essential amino acids: Transam ination reaction is freely reversible.
So transam ination used in the synthes is of differen t n on-esse ntial amino acids by
redistrib ution of amino groups among amino acids and keto acids.
3) Provision of energy: Transam ination produce s compou nds like oxaloacetate, pyruvat e,
cx-ketoglutarate etc, which can enetr glycolysis or TCA cycle and provide energy.
4) Role in glucone ogenesi s: GJucogenic amino acids like aspartate, alanine etc undergo
tra nsamin ation to form oxaloac etate, pyruva te, which can fo rm glucose by
gluconeogenesis.
5) Clinical signific ance of transaminases: AST and ALT are used as diagno tic enzymes
.
• ALT: ALT is rich in liver. Normal serum level is 3-35 TU/1. During liver diseases,
ALT level
increases in the blood.
• AST: AST is rich in heart & liver. ormal serum level is 4-40 IU/L. Elevated levels of AST
is an indicatore of possible heart attack or liver disease.
Answer hint for Transaminase question (5 Marks):

a) Definition (1 mark)
b) Example s (2 marks)
c) Functions, Clinical significance (1 + 1 marks)
• Glutamate is then transported to liver and releases this ammonia by glutamate dehydrogenase.
For queries and suggestions, contact the author at prasad_ text@ya hoo.com
or 9986449 575
Amino acid and Protein Metabol ism 252
Oxidat ive deami nation of glutam ate (by glutama te dehydro genase enzyme):
The remova l of an amino group from glutama te to release NH3 and a-ketog lutarate is
catalyzed by glutama te dehydrogenase (GOH) enzyme. It takes place in liver exclusively.
Glutamate d ehydrogen ase
Glutamate ---~- ,-,--- -• a-ketoglutarate + Nl-1 3
NAD(P) • NAD(P)H• + H•
Significance of GDH:
• GOH plays a signific ant role in catabolism of amino acids: In transamination, amino
groups of amino acids are transferred to a -ketoglu tarate to produce glutama te. Thus,
glutama te serves as a "collection center" for amino groups from differen t amino acids.
This amino group is released from glutama te as ammon ia by the action of GOH in liver.
This ammon ia is then further catabolized to urea.
• GOH plays a role in synthes is of non-essential amino acids: GOH is freely reversible,
It can provide glutama te for glutama te related synthesis of non-essential amino acids.
• GOH plays a role energy production: GOH produce s a-ketog lutarate , which can
enter TCA cycle and gives energy.
Summary of transdeamination:
e
Transde aminatio n constitu tes two processe s, transam ination followed by oxidativ
deamina tion of glutama te.
1) Transamination: Takes place in all the tissues, where the amino group from amino
acids
that are transaminated is funneled to a-ketogl utarate to form glutamate.
This glutama te is then transported to liver.
2) Oxidative deamina tion of glutamate: In liver, glutama te is oxidatively deamina ted
to a-
ketoglutarate releasing amino group as ammonia .
all
Thus, these two processes take place in physically different places (i.e. transamination in
cells and oxidative deamina tion of glutamat e in liver). But, these are physiolo gically coupled
reactions. Hence, the name transdeamination.
Transdeamination = transamination + oxidative deamin ation of glutamate
Amino acids Y,_ a-Keto glutarate
Transa minati x all tissues)
a- Keto acids glutamat e
Transport ed to liver _ _ _ . )
Glutam ate--- -- - - - - - a-Ketogl utarate + N H ••

Glutamate dehydrogenase
acids
So, effective ly, in the process of transdea mination , amino group from different amino
are removed as a mmonia to form ketoacids of corresponding amino acids.
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9986449 575
II. Deamination (Minor pathways of amino acid catabolism to release N~):

Deamination is the direct removal of amino group as NH3• (whereas, transamination
involves only the transfer of NH3). Some amino acids are degraded by deamination.
Deamination can be oxidative or non-oxidative.
1. Oxidative deamination:
a) L-amino oxidase: It can act on all amino acids (except hydroxy amino acids and
dicarboxylk amino acids) to form a-keto acid and ammonia. It uses FMN as coenzyme.
L - a Amino acid ___
/______
'\: _._ a- Keto acid+ NH:i
b) D-amino oxidase: It can act on glycine and any D-amino acids (that may be formed
by bacterial metabolism) to form a-keto acid & ammonia. It uses FAD as coenzyme.
D - Amino add oxidase
D-a Amino acid - - - / - ~ ' \ ~ - -..
- a-Keto acid +NHJ
FAD FADH2
Glutamate dehydrogenase (GOH): GOH is also an oxidative deamination reaction.
c)
GOH is mainly associated with transdeamination reactions.
2. Non-oxidative deaminations:
Serine, threonine, cysteine and histidine are deaminated by non-oxidative deamination
reactions to release ammonia.
Histidase
a) Histidine Urocanate + NH3
PLP
Cysteine desulphydratase
b) Cysteine Pyruvate + NH3 + H 2S
PLP
Serine dehydratase
c) Serine Pyruvate + NH3
PLP
Threonine dehydratase
d) Threonine a-ketobutyrate + NH,
PLP

Disposal of ammonia:
Ammonia is continuously formed in the t'ody, mainly by catabolism of amino acids. A
small amount of ammonia is also produced from catabolism of purine & pyrimidine
and bacterial action in intestine. Ammonia is highly toxic, even in mi.nu te amounts. So,
it should be immediately detoxified by conversion to urea (by urea cycle that takes
place exclusively in liver) and excreted in urine.
In transdeamination process (major catabolic route of amino acids), final release of
ammonia by GDH is in liver itself, so, ammonia can be immediately detoxified to urea.
But, ammonia produced in extra-hepatic tissues (by oxidative & non-oxidative
deamination processes or catabolism of purine & pyrimidine catabolism etc) should be
transported to liver for the detoxification to urea.
Ammonia transport (Glutamine formation) (Mainly in brain, m uscle):
Although N~ is constantly formed in all tissues, its concentration in very low in blood
(10-20 µg/ dl). This is due to the rapid removal of blood NH3 by liver & conversion to
urea and by formation to glutamine, a non-toxic transport form of N H 3.
Glutamine synthetase combines N~ with glutamate to form glutamine. Glutamine
formation is very significant in brain as even minute amow1ts of N~ is toxic to brain.
G lutamine synthetase
Glutamate + Nrk • Qutamine + HzO
ATP
/M~
ADP+ Pi
Glutamine is then released into the blood and transported liver (also to kidney for acid
base balance). So, glutamine is present in highest concentration in blood (8 mg/ dl) among
amino acids. In liver, glutamine is cleaved by glutaminase enzyme to form glutamate
and free ammonia. (Glutaminase activity is also present in kidney, which is required for acid
base balance). Glutamate then releases ammonia by glutamate dehydrogenase reaction.
GIutaminase Glutamate d ehydrogenase
Glutamine • Glutamate / ';.,_ '---; • a.-ketoglutarate
NA D(P)• N AD(P) H + H• N}-iJ
Ammonia is then detoxified to urea by urea cycle and then excreted th rough urine.
• From muscle, NH3 can also be transported in the form of alanine (Refer gluocose-a/anine cycle).
Ammonia toxicity:
NH3 is highly toxic. Even minute amounts of NH3 is toxic to CNS and can cause ammonia
intoxication. Its mainly caused due to hyperammonemia and accumulation of N~ in brain.
Reason for ammonia intoxication:
Accumulated ammonia in brain reacts with a-ketoglutarate to form glutamate, resulting in
depletion of a-ketoglutarate, impairing TCA cycle function in brain.
Symptoms of am monia intoxication:
Slurred of speech, blurred vision, tremors, vomiting, lethargy, aversion to high protein food,
disorientation, irritability, mental retardation. In severe cases coma and death.

Urea cycle or Krebs-Hanseleit cycle (Detoxification of ammonia):

Definition:
Process of conversion of toxic ammonia to nontoxic urea is called urea cycle.
Site:
a) Tissue site: Liver
b) Intracellular site: Partly in mitochondria partly in cytosol.
Starting compound:
Ammonia, CO2
End product:
Urea
Reaction pathway:
k
Carbamoyl phosphate
2ATP
Carbamoyl phosphate Synthetase I
2 ADP+ Pi
Ornithlne transcarbamoylase
~ trulline Aspartate
Steps 1 & 2 occcur
Ornithlne in mitoch ondria;
Arginosuccinate steps 3 to 5 occur
Synth~e in cytosol
ATP
AMP+ Pi
Arginine .
~ nosucclnate
Arginosoccinate
Fumarate
Lyase
Energetics of Urea cycle:

• Urea cycle consumes 4 high energy bonds, 2 in CPS-I & 2 in arginosuccinate synthase.
• Urea synthesis generates 3 ATPs, as fumarate formed in urea cycle enters TCA cycle.
Net utilization (4-3) = 1 ATP. So, urea cycle requires 1 ATP to operate & is endergonic.

Significan ce :
Urea cycle convert toxic amonia to non-toxic urea that is then excreted through urine.
Regulatio n:
Carbamoyl phosphate synthetase-1 (CPS-I) is the regula tory enzyme of urea syntheses.
CPS-I is allosterically activated by N-Acetyl-glutamate ( AG).
NAG is syn thesized from acetyl-CoA and glutamate and degraded by a specific
hydrolase. NAG synthesis is stimulated by arginine.
The consumpti on of high protein diet increases the level of NAG.
When the amino acid catabolism increases (starvation, diabetes etc), the concentrati on of
glutamate increases, which in turn increases the formation of NAG.
Inborn errors of Urea cycle (Urea cycle disorders):

Deficiency of any of the urea cycle enzymes lead to defective conversion of H1 to urea
and consequen t elevation of NH, level in the blood resulting in hyperammonemia.
Name of the disorder Enzyme defect Accumulated product
Hyperammo nemia type I Carbamoyl phosphate synthetase I Very high H 3 increase

Hyperammonemia type II Ornithine transcarbam oylase High H 3 increase, 0rnithine
Ci trullinemia Arginosuccinatc synthetase Nrf;, Citrulline
Arginosuccinic aciduria Arginosuccinate lyase Arginosucci nate, NH3

Hyperargin enemia Arginase Arginine, Low increase
Severity of tire co11ditio11 depends 011 tire site of the block. If tire block is at earlier steps (step 1 or 2),
the co11ditio11 is more severe, since NH3 itself accumulates (am111011ia i11toxicatio11), whereas block
at later enzymes (Step 3, 4, mainly 5) is less harmful, as some of the NH 3 has already been removed.
Clinical symptom s of urea cycle disordere s:

• Vomiting, lethargy, aversion to high protein food, intermittan t ataxia, disorientat ion,
irritability, mental retardation. In severe cases (untreated cases), coma and death.
Treatmen t:
Significant improveme nt is noted on a low protein diet.
Urea cycle and TCA cycle are interdependent (Referred to as Krebs bicycle).
• Fumarate formed in Urea cycle enters TCA cycle and oxa loacetatc of TCA cycle undergoes
transaminat ion to produce a~partate, vvhich ente~ urea cycle.
• ATP genera ted in TCA cycle is required for urea cycle.
• CO, released in TCA cycle is utili.ted (detoxified) in urea cycle.
Therefore, TCA cycle & un•a cycle arc interdepend ent, together referred to as Krebs bicycle.

Hyperammonemia (Increase in blood ammonia level):

Normal ammonia concentration is in blood is 10-20 µg / di. Increase ammonia in blood
is termed as hyperammonernia. There are two types of hyperammonernia,
• Congenetal hyperammonemia:
Hyperammonemia due to urea cycle d isorders is termed as congenetal
hyperammonernia. (Refer urea cycle disorders).
• Acquired hyperammonemia (severe liver disease increases Blood NH3):
NH3 is detoxified to urea in the liver. In patients with severe liver diseases (like vira l
hepatitis or hepatotoxins like alcohol etc), the conversion of NH3 to urea is decreased,
resulting in increase in blood NH3 level (and decrease in blood urea level).
Symptoms: Hyperammonernia cause the symptoms of ammonia toxicity like tremors,
slurred speech, blurred vision, vomiting, disorientation, lethargy, finally coma & death.
Blood Urea level and it's clinical significance:

Normal blood urea level is 12-36 mg/ dl. Increased urea in blood is called uraemia.
Blood urea levels are in the upper range in people whose protein intake is high.
Clinical increase of blood urea is classified into 3 categories:
1) Pre renal causes:
In severe vomiting, diarrhoea and severe burns, the blood volume is decreased, hence
glomerular filtration decreases and consequently blood urea concentration is elevated.
Any disease which increases protein catabolism, like trauma, surgery, pyrexia, leukemia,
diabetes melllitus etc. also increases blood urea levels.
2) Renal causes:
All forms of kidney diseases like acute and chronic glomerulonephritis, later stages of
nephrosis, polycystic kidney and hydronephrosis increases blood urea levels.
3) Post renal causes:
Any type of obstruction in the lower urinary tract (Stricture urethra, tumours,
Enlargement of the prostate, stones in bladder) cau ses elevated blood urea levels.
Decreased blood urea:

In liver diseases and in conditions of low protein diet, blood urea levels are decreased.
Answer hint for Individual amino acid essay question no Marks):

Metabolism of amino acids like glycine, phenyalaninc, tryptophan etc can be asked for essay
a. Introduction (Chemical nature, Essential/non-essential, glucogenic/ketogenic) (01 Marks)
b. Synthesis (if its a non-essential amino acids, Degradation ( 1+2 Marks)
d . Clinical significance and Disorders (02 Marks)

Metabolism of Glycine:
Glycine is the simplest amino acid. Glycine is a non-essential, glucogenic amino acid.
Glycine is optically inactive.
Synthesis of glycine:
1) By glycine synthase enzyme (From carbon dioxide and ammonia):
Glyci n e syn thase
11-----------• Glycine+ NA D•
•
~ PL~
N S N lO FH.
methylene FH 4
2) From serine (by Serine hydroxy methyl transferase enzyme) :

Se rin e hy dr oxy
me th y l tra nsferase
Serin e • • Glycine
FH , N S N 10
met h y l e n e F H .
3) From glyoxylic acid (by glycine transaminase)

Glycin e transaminase
Glyoxylate •11--~--~P-L~~~----• G~c~e
Glutamate a -Ketoglutarate
4) From Threonine (By threonine aldolase):

Threonine aldolase
Threonine + - - - - - - - - - + Glycine+ Acetaldehyde
5) Glycine can also be synthesized from choline

Choline • Betaine • Dirnethylglycine _ _.._• Gly cine
Degradation of glycine:
1) Glycine cleavage:
Glycine is mainly degraded by reversible reaction of glycine synthase enzyme.
Glycin e cleavage
Glycine+ NAO • " ~ ~ CO2+ NH,• + NADH + H·
PLP
FH, NS N lU
methylene FH,

2) Serine formation:
Glycine can also be cataboliz ed through serine. First glycine is converte d to serine (by
reversible reaction of serine hydroxy methyl transferase enzyme). Serine then degraded
to pyruvate by serine dehydrat ase enzyme. So, glycine is glucogen ic amino acid.
Serine hydroxy Serine
methy l transferase
Glycine •• --7---=-~,,..----•• Serine de hydratase
(PLP)
Pyruvate + NH3
N 5 N1° methylen e fH4 fH4
3) Transamination reaction.
Glycine can also give glyoxyla te by transamin ation reaction.
Glycine transaminase
Qycine + a-ketoglutarate Qyoxylate + Qutamate
PLP
4) Oxidativ e deamination:
Glyci ne oxidase
Glycine + H:zO • 7" ~ yoxylate Oxalate
FAD FADH2 l
Excreted in urine
Functions of Glycine:
Besides being incorpora ted into proteins, glycine forms various specializ ed products .
It is also required for various conjugati on reactions. These are,
l ) Formatio n of proteins
2) Formatio n of glutathio ne
3) Formatio n of creatine
4) Synthesis of heme
5) Formatio n of purine nucleotid es
6) Formatio n of serine
7) Formatio n of conjugat ed bile acids (by conjugati on process)
8) Detoxification of benzoic acid (by conjugati on process)
9) Function s as neurotran smitter
1) Formation of proteins :
Glycine is required for the formation of proteins (like all other protein amino acids).
Being small molecule, glycine can be accommo dated where the protein chains bends or
turns sharply. Being non polar, it is generally accommo dated in the interior of proteins.
In collagen, every 3rd amino acid is glycine.
2) Formation of purine nucleotid es:
Entire glycine molecule is incorpora ted into purine nucleotid es during their synthesis.
C-4, C-5 and N-7 of purine ring comes from glycine.

3)Formation of glutathion e (y-Glutamyl cysteinyl glycine):

Glutathion e is a tripeptide containing glutamic acid, cysteine and glycine.
Refer Amino acid/ protein chemistry for glutathione function.
Glutamate Cysteine
~ l u t a m yl cy,t,ine syntha,e
y-Glutamyl cysteine
Glycine ,
lI Glutathione synthase
Glutathione (y-Glutamyl cysteinyl glycine)
4) Formation of creatine:
Creatine present in muscle as creatine phosphate is formed from glycine and arginine.
Am.ido transferase Methyl transferase
Arginine 7'\. • Guanido acetate / '\. • Crea tine
Glycine Onuthme SAM SAH
Creatine is then phosphory lated to form creatine phosphate by creatine kinase.

Creatinc C reatine kinase C rcahnc phosphate
4
'
ATP ADP
Importa nce: Creatine phosphate serves as an immediate store of energy in muscle
cells. During muscle contraction, energy is derived from ATP hydrolysis. Then the ATP
can be regenerate d form Creatine phosphate (Lohmann's reaction).
5) Heme:
Heme (p rosthetic group present in heme proteins) is synthesize d from glycine.
ALA synthase
Qycine + Succinyl CoA - - - - - + ALA • • • • Heme
Importance: Heme is a non protein part present in hemoglobin and other heme proteins.
6) Formation of serine:
Glycine can form serine by serine hydroxy methyl transferase enzyme.
Glycine •• --------
----/----=--~
Serine hydroxyl m eth y l tra n sfe rase
• Seri ne
N 5 N 10 m e th y I e n e F H ,
7) Functions as neurotransmitter:
Glycine is also functions as inhibitory neurotrans mitter in the brain stem & spinal cord.
For queries and suggestions, contact the author at prasad_tex t@yahoo.com or 9986449575
8) Synthesis of conjugated bile acids:

Glycine is required for the formation of conjugated bile acids (Glycocholic acid and
glycochenodeoxycholic acid).
Cholic acid + Glycine Glycocholic acid
Chenodeoxycholic acid + Glycine Glycochenodeoxycholic acid
9) Detoxification of benzoic acid (by conjugation process):

Glycine is required for detoxification of benzoic acid by conjugating process.
Benzoic acid + Glycine Benzoyl glycine (Hippuric acid)
G
Proteins+- Purines
L
Serine Glutathione
y
Glucose Heme
C
Formate Conjugation
(Bile acids, Detoxification)
Creatine
N
Disorders of glycine metabolism:
1) Glycinuria:
Cause: Defect in the renal reabsorption of glycine (transporter defect).
Characteristics: Excretion of large amount of glycine in urine. Blood level of glycine
will be normal.
2) Hyperglycinemia:
Cause: Defect in the glycine cleavage enzyme system.
Characteristics: Increased glycine level in blood, urine and CSF leading to decreased
neurotransmission (Since glycine acts as a inhibitory neurotransmitter).
3) Primary hyperoxaluria:
Cause: Defect in the enzyme glycine transaminase.
Characteristics: Increased excretion of oxalates in urine. It is due to failure to convert
glyoxylate to glycine by glycine transaminase. Glyoxylate then converted to oxalate,
which can deposit in renal tissues as calcium oxalate and causing renal stone.

Metabolism of Serine:
Serine is a hydroxy amino acid. Serine is a non-essential, glucogenic amino acid.
I. Synthesis of Serine:
1) From Glycine by serine hydroxy methyl transferase (Refer glycine)
2) From Phosphoglycerate
Dehydrogenase Transaminase Phosphatase
3-Phospho • 3-Phosphohydroxy Phoophoserine • Serine
glycerate pyruvate / PLP "-
NAO. NADH•+ H Glutamate a-KG H20 Ii.JPO•
3) From Hydroxypyruvate (by transaminase)

Transaminase
Hydroxypyruvate 4/ • Serine
/ PLP 16.
Pyruvate Alanine
II. Catabolism of Serine:

1) Deamination to pyruvate: Serine gives to pyruvate by serine dehydratase. Pyruvate
can be convereted to glucose. So, serine is a glucogenic amino acid.
Serine dehydratase
Serine Pyruvate + Nl-b
PLP
2) Transamination to hydroxypyruvate
Transaminase
Serine •• --7--P- -~---
1
• Hydroxypyruvate
Pyruvate Alanine
III. Functions of Serine:

1) Synthesis of Proteins: Serine is required for the formation of proteins (like all other
protein amino acids). Hydroxy group of serine of proteins serves as site for attachment
of phosphate group during the phosphorylation of proteins. Hydroxy group of serine
also serves as site for attachment of carbohydrates during the formation of glycoproteins.
2) Formation of pyruvate: Serine can give pyruvate, which can be converted to glucose.
3) One carbon metabolism: Serine donates 1-carbon group to 1-carbon pool (to from
N 5, N 10 methylene FH4 from FH4 ) to form glycine by serine hydroxy methyl transferase.
4) Formation of Azaserine: Serine is used for the synthesis of anti-cancer drug Azaserine.
5) Formation of glycine:

Metabolism of Sulphur containing amino acids

Methionine and cysteine are sulfur containing amino acids.
Metabolism of Methionine:
Methionine is a sulfur containing, essential and glucogenic amino acid.
Metabolism of methionine is routed through the formation of SAM (S -Adenosyl

methionine). SAM is called active methionine. SAM acts as a methyl donor and
participates in many transmethylation reactions (Explained later). During the reaction,
SAM is converted to SAH (S-Adenosyl Homocysteine).
SAH then gives Homocysteine by releasing adenosine. Homocysteine has 2 fates,
a) Homocysteine can form back methionine by methionine synthase enzyme.
b) Homocysteine can combine with serine to form cystathionine, which is converted to
a-ketobutyrate and cysteine. a-ketobutyrate then converted to succinyl CoA. Since
succinyl CoA is formed, methionine is a glycogenic amino acid.
Methionine can give cysteine, making cysteine a non-essential amino acid.
Methionine
Methionine ATP
adenosyl
transferase PPi+Pi
SAM
Methyl acceptor.; Methionine

Transmethylase synthase
Methylated products
SAH
Hydrolase l,- H i()
+.
Adenosine NSmethyl FH,
Serine Homocysteine
c,........... ,.,•.
Cyst~thionine
Cystathioninase !
a -ketobutyrate + cysteine
!
Succinyl CoA

Functions of Methionine:
Methionine is a sulfur containing, essential and glycogenic amino acid.
1. Formation of proteins:
Methionine is required for the formation of proteins (like all other protein amino acids).
Methionine is the first amino acid to be incorporated during protein synthesis.
2. Methionine can give cysteine.
3. Methionine is required for the synthesis of SAM (S-Adenosyl Methionine), an
important methyl donor in many methyla tion reactions.
SAM (S-Adenosyl methionine) is called active methionine:

Formation: SAM is formed from methionine by methionine adenosyl transferase.
Methionine Adenosyl transferase
Methionine+ ATP - - - - - - - - - - - • SAM + PPi + Pi
Functions of SAM (Transrnethylation or Methyl transferase reactions):

SAM acts as a methyl donor & d onates the methyl group to methyl acceptors to form
methylated prodcts; and SAM itself gets converted to SAH (S-Adenosyl Homocysteine).
This p rocess is called transmethylation catalyzed by methyl transferase enzyme.
Examples of transmethylation are,
M e th y l tra n sf e ra se
1) Guanidoacetate ----~-------• Crea tine
SA M / ~ S A H
Me th y l t ra n sfe ra se
2) Norepinephrine ----~--~--------• E pin cp h rin e
SAM ' ...._S AH
Meth yl lran sferase

3) N-acet yl sero tonin • Melatonin
SAM ~ SAH
M eth y I transfe rase

4) Ethanolamine ----~-------• Choline
SAM / ~ SAH
5) Cephalin Methyl tra n sferase Leci thin

(Phosphatidyl ethanolamine)
SAM / " SA H
• (Phosphatidyl choline)
M eth y I transferase
6) Carnosine - - ---,,,......,.- - - - -~• Anserine
SAM / ~SAH

Metabolism of Cysteine
• Cysteine is a sulfur containing, non essential and glucogenic amino acid.
• Cysteine can be formed from methionine. So, cysteine is a non-essential amino acid.
Catabolism of Cysteine:
1) Cysteine undergoes catabolism to form pyruvate and ~S.
Cyste in e desulphydratase
Cysteine Pyru,·ate + NH , + H , S
PLP
• Pyruvate can form glucose. Thus, cysteine is glucogenic.
• H 2S may be oxidized to sulfites & then finally to sulfates, which can produce PAPS.
2) Cysteine on decarboxylation gives ~-mercapto ethanolamine, whcih can form CoASH.
Functions of Cysteine:
1) Formation of proteins: Cysteine is required for the formation of proteins. -SH group
of cysteine forms the active site of some enzymes. Cysteine can form cystine by
condensation of 2 cysteine residues by disulphide bond (& stabilizes tertiary &
quaternary structures of proteins).
2) Formation of glutathione: (Refer glycine for synthesis, protein chemistry for functions)
Glutathione is a tripeptide containing glutamic acid, cysteine and glycine.
3) Synthesis of CoASH (Coenzyme A): Cysteine is required for the formation of CoASH,
a coenzyme of pantothenic acid. Active-SH group of CoASH is derived from cysteine.
Decarboxylation
Cysteine •• ---~~--• • ~-Mercapto ethanolaminc /- - •
• CoASH
PLP 1' Pantothcnic acid
c~
4) Synthesis of taurine: Cysteine is required for the formation of taurine. Taurine is
required for the production of conjugated bile acids.
Cysteine Cysteic acid Taurine
+0,
" co,
•
5)Synthesis of PAPS: ~S produced from cysteine can form sulfate, which can react
with ATP to give PAPS.
Cysteine --+ H 2S ----. Sulfite ----. Sulfate / • PAPS
2ATP ADP+ PPi
PAPS (Phospho Adenosine Phospho Sulfate) or active sulfate is involved in various
sulfation reactions like synthesis of glycosaminoglycans, sulfatides, detoxification etc.
6. Role in iron absorption: Cysteine (along with glutathione, Vitamin C) facilitate iron
absorption by reducing Fe•3 form to Fe+2 form. Iron can only be absorbed in Fe•2 form.
7. Role in regeneration of Hb: Cysteine has a role in converson of Met Hb to Hb.

Disorders of Sulphur containing amino acid metabolism:
1) Homocystinuria:
Cause: Mainly due to deficiency of cystathionine ~-synthase (Homocystinuria type I).
2 forms of Type I are known, vitamin 86 responsive form & vitamin 86 unresponsive form.
Characteristics: Increased blood homocysteine & excretion of homocystine in urine.
Clinical findings include thrombosis, osteoporosis, and mental retardation.
Treatment: Diet low in methionine and rich in cysteine. Dietary supplementation of
vitamin B6 is also useful in vitamin B6 responsive form.
Homocystinurias:
Homocysteinurias are group of disorders characterized by increased urinary excretion
of homocystine. There are 3 different types,
Type I: Cystathionine ~-synthase enzyme deficiency
Type II: N5, N 10 methylene FH4 reductase deficieny
Type III: Methionine synthase (N5 methyl FH4 homocysteine methyl transferse
deficiency), due to defect in synthesis of active form of cobalarnine (methyl cobalarnine),
2) Cystathioninuria:
Cause: Decreased activity of cystathioninase enzyme.
Characteristics: Increased urinary excretion of cystathionine. Mental retardation, anemia.
3) Cystinuria:
Cause: Defect in the renal reabsorption of cysteine and also lysine, ornithine, arginine
(as they use the same carrier system).
Characteristics: Increased urinary excretion of cysteine & also lysine, ornithine, arginine.
Patients also may develop cysteine stones in the renal tract. This is because cysteine is
sparingly soluble in water and in acidic pH, cysteine crystals are formed, which are
deposited in the renal tubules causing renal stones, hematuria.
4) Cystinosis (Cysteine storage disease):

Cause: Carrier mediated transport defect of cysteine in lysosomes.
Characteristics: Cysteine crystals get deposited in lysosomes of tissues like liver, spleen,
lymph nodes, bone marrow and kidney. Deposition of cysteine in kidney causes renal
stones. Patients usua lly die of young age due to acute renal failure.
Defect Name of Disorder

Cystathionine ~-synthase Homocystinuria type I
Cystathioninase Cystathioninuria
Impairment in the renal reabsorption of cysteine Cystinuria
Defective carrier mediated transport of cysteine Cystinosis

Metabolism of Phenylalanine and Tyrosine:

Phenylalanine and tyrosine are two structurally related aromatic amino acids.
a) Phenylalanine: Aromatic, Essential, partly glucogenic, partly ketogenic amino acid.
b) Tyrosine: Aromatic, Non-essential, partly glucogenic, partly ketogenic amino acid.
Note: Phenylalanine is an essential amino acid, but tyrosine is a non-essential amino

acid. (Because tyrosine can be synthesized from phenylalanine in the body)
Conversion of phenylalanine to tyrosine:

Tyrosine is formed from phenylalanine by the enzyme phenylalanine hydroxylase,
which is an irreversible reaction.
Phenylalanine hydroxylase
Phenylalanine Tyrosine
Phenylalanine hydroxylase:
Tyrosine is formed from phenylalanine by the enzyme phenylalanine hydroxylase,
which is an irreversible reaction. (So, tyrosine cannot replace the nutritional
requirement of phenylalanine).
Pheny lalanine hydroxylase

[ Phenylalanine J ---77"------~-=--------+• [ Tyrosine J
Tetrahydro HiO Dihydro
biopterin biopterin
dihydrobiopterin
NADP+ reductase N ADPH + H+
Phenylalanine hydroxylase is present exclusively in liver. This enzyme requires

te trahydrobiopterin as a coenzyme. Phenylalanine hydroxylase is a mixed function
oxidase (monooxygenase), which requires a molecule of oxygen. One oxygen atom
(0) is added to the para position of phenylalanine as OH group & other oxygen
atom is reduced to H 20 .

Catabolism of phenylalanine and tyrosine:

Note: A single pathway is responsible for the catabolism of both phenylalanine and
tyrosine. First, phenylalanine is converted to tyrosine by phenylalanine hydroxylase.
The rest of the reactions are same for .both the amino acids.
Phenylalanine hydroxylase
Phenylalanin--------- Tyrosine
....- +"-a.
,--F_u_m
_ a-ra_t_e~] [,. A-ce_t_o-ac_e_t_a_te---,]
.
Fumarate is glucogenic and acetoacetate is ketogenic. So, Tyrosine (and phenylalanine)

is partly glucogenic and partly ketogenic.
Phenylalanine
hydroxylase
Phenylalanine - - - - - - + Tyrosine
a-Kg ~
Tyrosine transaminase
Glutamate
P- h ydroxy phenylpyruvate
Complete reaction sequence]

p-hydroxy phenylpyruvate hydroxylase OR
Cu ' 2, Vitamin C p-hydroxy phenylpyruvate dioxygenase
CO2
Homogentisate
02~
Fe2• l Homogentisate oxidase
Maleylacetoacetate
GSH 1 Maleylacetoacetate isomerase
Fumarylacetoacetate
H20 i;,,.,.,,.
~rylacetoacetate hydrolase
Fu.marate Acetoacetate

Functions of Phenylalanine and Tyrosine:

Besides its incorporation into proteins, the only other function of phenylalanine is to
get converted to tyrosine. All other functions of phenylalanine is through tyrosine.
So, the functions of tyrosine and phenylalanine are,
l. Formation of proteins
2. Formation of Catecholamines (Dopamine, Epinephrine and Norepinephrine)
3. Formation of Melanin
4. Formation of thyroid hormones (T3 and TJ
1. Synthesis of catecholamines (Dopamine, Epinephrine and Norepinephrine):

• Catecholamines are amine derivatives of catechol (Di hydroxy phenyl).
• Catecholamines include Dopamine, Norepinephrine and Epinephrine.
• Catecholamines are formed from tyrosine in adrenal medulla.
DOPA
Tyrosine hydroxylillU! decarboxylase
Tyrosin/e • DOPA --.:;. • [ Dopamine )
(Dihydroxy PLP COi
CJl. H20 phenylalanine)
Oi
Dopamine
Tetrahydro Dihydro
biopterin biopterin ~-hydroxylase
[ Norepinephrine ]
Note: The term 'nor' in norepinephrine
means that the compound does not contain SAM
Methyl
"R" or methyl group (No R group = Nor). transferase
SAH
Epinephrine
Functions of catecholamines:
Epinephrine and norepinephrine are hormones. They play an important role in
carbohydrate and lipid metabolism
Dopamine and norepinephrine are neurotransmitters.
Degradation of Epinephrine (catecho lamines):

Epinephrine is degraded by COMT & MAO enzymes to form VMA, which is excreted in urine
(COMT) (MAO)
Ca techo 1-0-meth yl transfe rase Monoamine oxidase (VMA)
Epinephrine Metanephrine Vanillyl mandelic acid
Pheochromacytoma: These are tumors of adrenal medulla.

So, there is increased production & breakdown of catecholamines. It is diagnosed by the presence
of high amounts of VMA (normal <6 mg/day) in urine.

L-DOPA is used in the treatment of Parkinsonism:

In Parkinsonism, there's a marked reduction in the dopamine content in brain. Dopamine can't be
used in the treatment ofParkinsonism, because it cannot enter brain cells. But, L-DOPA, a precursor
of dopamine, can enter the brain and then get converted to dopamine.
2. Synthesis of thyroid hormones:

Triiodothyronine (T3} & tetraiodothyronine (T4 or Thyroxine) are two thyroid hormones.
They are synthesized from tyrosine and iodine in the thyroid gland.
Thyroid hormone synthesis is explained in detail in iodine metabolism under minerals.
3. Synthesis of melanin:
Melanin is a black pigment present in skin, hair, eye, substantia nigra etc.
It is synthesized from tyrosine in melanocytes (the pigment forming cells).
/b •
Tyrosinase Tyrosinase
Tyrosine Dopa ----,.....,---•• Dopaquinone
Cu•2 J,
02 H20 Qi H20 J, Non enzymatic
J, (Spontaneous)
J,
*
[ Melanin ]
Melanin synthesis requires only one enzyme, tyrosinase, which catalyzes the first two
reactions. The remaining reactions are non enzymatic and take place spontaneously.
Function of melanin:
Melanin protects the underlying cells from the harmful effects of sunlight.
Phenyalanine & tyrosine are required for the formation of proteins.
Summary of phenylalanine and tyrosine functions

p Dopamine
H T
E y Norepinephrine
N Protein R
y 0 Adrenaline
L Glucose s
A I Thyroid hormone
L Fat N
A E Melanin
N
]
N
E

Inborn errors of phenylalanine and tyrosine metabolism
1) Phenylketonuria (PKU):
PKU is one of the most common metabolic disorders. The frequency is 1 in 10000 births.
Inability to convert phenylalanine to tyrosine leads to PKU.
Metabolic defect:
Absence or deficiency of Phenylalanine hydroxylase.
There are 5 types of PKU
Type I: Due to deficiency of phenylalanine hydroxylase - Classic PKU

Type II: Due to deficiency of dihydrobiopterin reductase
Type III: Due to deficiency of dihydrobiopterin reductase
Type IV: Defect in dihydrobiopterin biosynthesis
Type V: Defect in dihydrobiopterin biosynthesis
Biochemical abnormalities in PKU :

In PKU, phenylalanine cannot get converted to tyrosine. So phenylalanine accumulates
in body and then metabolized by other minor routes.
Block
Phenylalanine I -t:::==:t-----. Tyrosin,
o.-KG
Transamination
Glutamate
Phenyl pyru vate
Oxidation and
Decarboxylation
I Phenyllactate Phenylaceta te
Conjugation Glutamine
with glutamine l
I Phenylacetylglutamine I
Net outcome of PKU is that the phenylalanine is not converted to tyrosine, leading to
accumulation of phenylalanine in the tissues and blood. This leads to the excretion of
large amounts of phenylalanine in the urine.
Besides phenylalanine, urine will also contain phenylpyruvate, phenyllactate and
phenylacetate in the form of phenylacetyl glutamine.
Diagnosis:
i) Estimation of blood phenylalanine:
Normal level: 1 to 2 mg/dl. This may be demonstrated by chromatography.
PKU: > 20 mg/ dl.
Guthrie test: It is a rapid screening test. Certain strains of bacillus subtilis need phenylalanine
for growth, without them bacteria cant grow. Bacterial growth is proportional to the phenylalanine
content in the patients blood. This test is done after baby is fed with breast milk for 2 days.
ii) Urine ferric chloride test:

When ferric chloride is added to the urine of PKU patients, the urine turns green.
This test is non-specific, as certain other compounds may also give a false positive test.
ii) DNA probes:
DNA probes facilitate prenatal diagnosis of defects in ezymes causing PKU.
Prenatal diagnosis of PKU by DNA probe is preferred (as blood phenylalanine may not be detectable
until 2-3 days. Early diagnosis is important as mental retardation caused by PKU is irreversible.
Manifestation and symptoms:

i) Mental retardation, low IQ, seizure, irritation, fai lure to walk, agitation etc. Exact
reason for mental retardation is not well understood. It could be due to,
• Due to lack of synthesis of neurotransmitters from tyrosine.
• Low serotonin levels (High levels of phenylalanine inhibit the formation of serotonin
from tryptophan)
• Defect in myelin formation
ii) Hypopigmentation (light skin, hair blue eyes) due to lack of melanin.
iii) Eczematous dermatitis due to lack of melanin.
iv) Mousey odor (Lactic odor) of urine due to phenyllactate.
Management of PKU:
The principle treatment for PKU is provision of diet containing low phenylalanine and
rich tyrosine. (As tyrosine becomes an essential amino acid).
This special diet is continued till 5 years of age; then normal diet can be given. But it is
advisable to have the restricted d iet for many years in patient's life.

2) Alkaptonuria:
Metabolic defect: lack of homogentisate oxidase enzyme.

Frequency: One in 2.5 lakh births.
[ Block
Homogentisate - - - - - - - - - - - + Maleylacetoacetate
Homogentisate oxidase
Manifestations:
1) Homogentisate accumulates in body, blood and is excreted in urine (So, this disease
is also calJed homogentisic aciduria) .
2) Urine becomes black on exposure to air. This is due to oxidation of homogentisate in
urine by the 0 2 in air to a brownish black pigment.
3) Homogentisate is oxidized by the enzyme polyphenol oxidase to form benzoquinone
acetate which polymerizes to form a pigment alkapton. Late in the disease, there is
generalized pigmentation of connective tissues (Ochronosis). Ochronosis results from
binding of alkapton to connective tissue macromolecules.
4) This is a form of arthritis may be seen in middle age; this is due to the accumulation
of the alkapton bodies in the joints.
Block
Homogentisate Maleylacetoacetate
l Polyphenol oxidase
Homogentisate oxi~ase
Benzoquinone acetate
1 Polymerization
[ Alkapton bodies - - -- -• Binds to connective tissues
D iagnosis:
1) Urine becomes black on standing. This is a simple traditional test.
This is due to oxidation of urinary homogentisic acid on exposure to air.
2) Ferric chloride test: When FeCl3 is added to urine, it turns green. This is a non
specific test, because even phenylketonuirics also give a positive FeC13 test.
Treatment:
Alkaptonuria is a benign condition. Patient leads a normal Life except that the urine
becomes black. However, consumption of low phenylalanine in diet is recommended.

3) Albinism:
Defect: Defect in the synthesis of the pigment melanin.
Biochemical basis (Causes):

• Defective melanin synthesis in albinism can be due to various fac-tors.
The most common cause of albinism is tyrosinase defect, the enzyme responsible for
melanin synthesis.
• Decrease in melanosomes in melanocytes.
• Failure in melanin polymerization
• Limitation of tyrosine molecules.
• Failure in uptake of tyrosine by melanosomes.
Symptoms:
Skin and hair becomes white (albino in Greek means white).
The most important function of melanin is the protection of the body from sunlight. So
lack of melanin in albinism makes the albinos sensitive to sunlight, susceptible to skin
cancer and photophobic (Intolerance to light due to lack of melanin in the eyes). No
impairment in the eye sight is seen in albinos.
Tyrosinemia:
1) Tyrosinemia type I (or Tyrosinosis):

Also caLied congenital tyrosinosis or hereditary tyrosinosis.
Metabolic defect: Defect in fumarylacetoacetate hydrolase enzyme.
It is 2 types: Acute tyrosinosis and chronic tyrosinemia.
Symptoms: Both the types are rare but very dangerous disorders.
In acute tyrosinosis, infants exhibit diarrhea, vomiting and "cabbage like" odor. Without
treatment death occurs within 6 o 8 months from acute liver failure.
In chron ic tyrosinemia, symptoms are similar, but milder. Death generally ensues by
age of 10.
Treatment: Diet low in tyrosine and phenylalanine.

2) Tyrosinemia type II (Richner-Hanhart Syndrome):

Metabolic defect: Defect in enzyme tyrosine transaminase
Clinical findings:
This disease is characterized by elevated plasma levels of tyrosine (4-5 mg/ dL) skin
lesion (dermatitis) eye lesion and moderate mental retardation.
There is urinary excretion of tyrosine and abnormal metabolites of tyrosine like p-OH
phenylpyruvate, p-OH phenyllactate, p-OH phenylacetate, N-acetyl tyrosine and
tyramine.
3) Neonatal tyrosinemia:
Metabolic defect: Relative lack of p-hydroxy pheny lpyruva te hydroxy lase
(p-hyd roxy phenylpyruvate dioxygenase).
Risk factor: Premature birth, dietary vitamin C deficiency, high protein diet.
Clinical findings: Blood levels of tyrosine & phenylalanine are elevated. Urinary levels
of tyrosine, p- hydroxyphenyllactate, N-acetyl tyrosine and tyramine.
Treatment: Low protein diet. Infants respond to vitamin C usually.
Metabolism of Arginine:
Arginine is a basic, essential / semi-essential, glycogenic amino acid.
Arginine is cleaved by arginase to produce ornithine and urea. Arginase enzyme defect
lead to a urea cycle disorder known as hyperargininemia.
Omithine can give glutamate by degradation. (So, arginine is a glucogenic amino acid).
Functions of arginine:
1) Nitric Oxide (NO) formation:

Arginine is a substrate for the production of nitric oxide. The enzyme nitric oxid e
synthase cleaves the nitrogen from arginine to form NO.
Importance: NO is called as a wonder molecule having variety of biological functions.
• NO is a vasodilator. It plays an important role in regulation of normal blood pressure.
• NO acts as a neurotransmitter, mainly in pre-synaptic terminals.
• NO has bactericidal activity of macrophages.
• NO causes the relaxation of smooth muscles.
• NO inhibits platelet aggregation, hence protects against coronary heart diseases.
• NO is involved in penile erection. (Action of NO is through cGMP. Sidenafil (Viagra),
inhibits the degradation of cGMP & hence prolong the action of NO, so, it's used in
treating erectile d ysfunction.
2) Synthesis of creatine: Refer glycine for synthesis and function.

Metabolism of Tryptophan
Tryptophan is an Aromatic, Essential, partly glucogenic & partly ketogenic amjno acid.
Catabolism of tryptophan:
1. Major catabolic pathway (Kynurenine pathway):
Catabolism of tryptophan gives alanine (glucogenic) and acetoacetyl CoA (Ketogenic).
So tryptophan is both glucogenic and ketogenic amino acid.
Tryptophan
i
~!---
~ -- ---, - ---~
[ Alanine J [Acetoacetate ]
Tryptophan
Complete reaction sequence 02 --{ Tryptophan pyrrolase (Tryptophan oxygenase)

of kynurenine pathway
N - formylkynurenine
H~ Kynureni ne fonnylase
Formate ~
Kynureninc
NADPH ' H~i> Kynureni"• hydro,yla,e
A DP• ~
--.-'-;,~ =:..
Al anine. ~
~h:~n:::::,"~raru,,t, on~,
1
2- Amino 3- carboxy muconaldehyde
C•l Oecarboxylase
I Acetoacetate I +- +- 2- aminomuconaldehyde
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2. Minor catabolic pathways:

These normally take place in minute amounts. Products of minor catabolic pathways
are xanthurenate, kynurenate, anthranilate, indolacetate, which are excreted in urine in
small amounts. Excretion of these compounds is increased (maninly xanthurenate),
when the major pathway is hampered.
Putrefaction of tryptophan:
Bacterial putrefaction of unabsorbed tryptophan in large intestine produces indole and
ska tole. A part of indole and skatole are absorbed and oxidized to indoxyl and skatoxyl, and
then detoxified in liver by PAPS to form indoxyl sulfate etc, which is excreted in urine as
indican (K· salt of indoxyl). The remaining indole & skatole are excreted in stools. The
obnoxious smell of feces is due to these compounds.
Tryptophan Indolepyruva te - - - Indoleacetate
Indoxyl Indole +---

!
Skatole ----+ Skatoxyl
PAPS --.,j
PAP ~
Indoxyl sulfate
!
Indican (Potassium salt of indoxyl)
Functions of Tryptophan:
It is an Aromatic, Essential, partly glucogenic and partly ketogenic amino acid.
Besides being incorporated into protein, tryptophan is also used for synthesis of niacin
coenzymes (NAO+I NADP+), serotonin and melatonin.
1) Synthesis of NAD+/NADP+ (Nicotinic acid pathway)

3-hydroxyanthranilate formed from tryptophan can be diverted to NAO+ formation.
Tryptophan
-1,
l
l
3-Hydroxyan thra nilate
l
NMN NAO+ and NADP+
NAO+/ NAOP+ (coenzymes of vitamin niacin) can be synthesized from tryptophan.

The amount of tryptophan directed to NAO+ synthesis is very small (only 3%). From 60
mg of dietary tryptophan only 1 mg equivalent of nicotinamide is formed. (60:1 ratio).
Note: Synthesis of niacin coenzymes from tryptophan requires PLP. So, pellagra like
symptoms is seen in deficiency of vitamin B6 deficiency.

2. Synthesis o f serotonin (5-hydroxy tryptamine):

Serotonin is a neurotransmitter synthesized from tryptophan.
Normally 1% of Tryptophan is diverted for the synthesis serotonin.
Tissue site:
Mast cells, CNS and argentaffin cells of GIT mucosa.
Tryptophan (5-hydroxy tryptamine)
hydroxylase Decarboxylase
Tryptophan ;;J"' 5- hydroxytryptophan PLP ~ [ Serotonin
02
/ Oi . H20 Dihydrobiopterin
Tetrahydrobiopterin
'\
Function:
• Serotonin is a excitatory neurotransmitter in the brain. It is considered to be a mood
elevator and antidepressant. Deficiency of serotonin may result in depressive psychosis.
• Serotonin is a powerful vasoconstrictor and stimulator of smooth muscle contraction.
• Serotonin also increases the motility of GIT (Peristalsis).
• Serotonin induces sleep.
Degradation of Serotonin:
Serotonin is degraded by MAO (monoamine oxidase) to form 5-hydroxyindoleacetate, which is
excreted in urine.
Monoamine oxidase (MAO)
Serotonin 5-hydroxy indoleacetate (5-HIAA)
• Drug iproniazid inhibits MAO and elevates serotonin levels, therefore, iproniazid is a
psychic stimulant (it causes mood elevation). ·
3. Melatonin:
Melatonin (a hormone) can be synthesized from tryptophan in pineal gland of the brain.
Serotonin Methyl
N-acetylase transferase
Serotonin / ' \ " N-acctyl serotonin 7"'\ "IMelatonin I
Acetyl CoA CoASH SAM SAH
Functions:
Melatonin is a hormone involved in circadian rhyth ms or diurnal variation. It p lays an
important role in sleep wake cycle. Melatonin inhibits the secretion of MSH and ACTH.
Melatonin also acts as a neurotransmitter.
Tryptophan is required for the formation of proteins.

Summary of Tryptophan functions

T
Proteins R NAD•, ADJ>+ (Niacin coenzymes)
y
Glucose p Serotonin
Fat
T
0
p
!
Melatonin
H
A
N
Inborn errors of Tryptophan metabolism:

1. Hartnup disease:
Defect: Impairment in the intestinal absorption and renal reabsorption of neutral amino
acids, including tryptophan.
Symptoms and manifestations:
1) General neutral amino aciduria: Neutral amino acids like tryptophan, phenylalanine,
tyrosine, methfonine, valine and their abnormal metabolites are excreted in urine.
2) Putrefaction: Dietary un-absorbed tryptophan undergoes bacterial putrefaction in
the intestine to produce indican, which is excreted in large amounts in urine and feces.
3) Pellagra like symptoms (i.e. Diarrhea, Dermatitis and Dementia) due to reduced
availability of tryptophan for the synthesis of NAD• and NADP•.
2. Malignant carcinoid (Argentaffinoma)

Serotonin is produced by argentaffin cells of GIT and is necessary for GIT motility. In
carcinoid tumor of argentaffin cells (called the malignant carcinoid or argentaffinoma),
serotonin is produced in large amount. ormally only 1% of Tryptophan is channeled
to serotonin formation. But in carcinoid syndrome, upto 60% of tryptophan is diverted
to serotonin formation. This metabolic diversion reduces the production of NAD• and
NADp+ from tryptophan. So pellagra like symptoms appears in malignant carcinoid.
Other symptoms include cutaneous vasomotor flushing, sweating and diarrhea. These
symptoms are due to effect of serotonin on smooth muscles and GIT.
Diagnosis:
Normally urine contains less then 5 mg/ day of 5-!,ydroxyindoleacetate (5-HIAA), a
degradation product serotonin. During malignant carcinoid syndrome, the level of 5-
HJA goes up to 500 mg/ day. Serotonin level of blood also elevated. During the course,
the patient shouJd abstain from certain foods like banana, tomato etc.

Metabolism of Histidine
Histidine is aromatic, semi essential and glucogenic amino acid.
Histidine has an Imidazole ring. Histidine is also a basic amino acid.
Catabolism
Catabolism of histidine gives glutamate, which is glucogenic.
Histidine
NH3
PL1H· 1s ti"d ase
'i
Urocanate
H,O Urocanase
lmidazole propionate
H,O "{Hydmlase
N- formimino glutamate (FIGLU)
One carbon
FH
Formimino F~ /
4
---dl Glutamate formimino transferase
Metabolism
Glutamate --+ Glucogenic
Note:
• Glutamic acid is transaminated to form a-ketoglu tarate, which can be converted to

glucose. So Histidine is a glucogenic amino acid.
• Formimino FH4 is a form of one carbon compound. Through the formimino group,
histidine contributes to the one carbon pool.
• In vitamin B9 (folk acid) deficiency, the FH4 (coenzyme of vitamin B9) is not available.
So, the conversion of FIGLU to N- formimino FH4 and glutamate is blocked. This
results in the elevated excretion of FIGLU in urine.
• This forms the basis of histidine loading test (FIGLU excretion test), which is
commonly employed to assess folate deficiency.

Functions of histidine:
1) Formation of proteins:
H istidine is required for the formation of proteins (like all other protein amino acids).
Histidine is vital for the function of hemoglobin as 0 2 is a ttached to histidine of Hb.
2) Formation of histamine:
His tidine gives the histamine on decarboxyla tion by en zyme histidine d ecarboxylase.
Histidine decarboxylase
Histidine • Histamine
PLP CO2
• Major function of histamine is to reduce the blood pressure by vasod ialation.

• Histamine regula tes HCl secretion by gastric mucosa.
• H istamine participates in allergic and inflammatory reactions.
3) Histidine is constituent of Camosine and Anserine:
Carnosine and anserine are dipeptides found in muscle contain histidine (Functions
unknown, believed to be functioning as buffers in muscles).
Carnosine: Histidine + P-alanine Anserine: Methyl cam osine.
4) Buffering action:
pKa value of histidine is 6.8 and it is very close to the p hysiological pH. So, histidine is
responsible for the buffering action of proteins.
Inborn errors of histidine metabolism:

1) Histidinemia:
Defect: Absence of deficiency in the enzyme histidase.
Characteristics: Elevated levels of plasma histidine and increased excre tion of Irnidazole
pyruvate and his tidine in urine, menta l retardation and defect in speech.
~lock 1 ..
Histidine --:_:-_-
I _ -:_-:_-:_-:_-:_~+-:•• Urocanate
l Transamination
lmidazole acetate - - - Imidazole pyruvatc _ _ _ _.,. Imidazole lactate
FIGLU excretion test:

FIGLU excretion test is conducted to differentiate the megaloblastic anemia caused
due to vitamin B9 (folic acid deficiency) from tha t of vitamin B12 deficiency.
In the catabolism of histidine, FH4 (coenzyme folic acid) is required in the glutamate
formi.mino transferase step (Converts FIGLU to glutamate). In folic acid deficiency,
there is a block in this step resulting in accumulation and excretion of FIGLU in urine.
Test involves giving a large dose of histidine to the anemic patient under investigation.
i) In folate deficiency, high dose of histidine results in the excretion of FIG LU in urine.
ii) In vitamin B12 deficiency, there is no excretion of FIGLU.

Maple syrup urine disease (branched chain ketonuria):
Defect:
Absence or deficiency of a-ketocacid decarboxylase (of a-ketocacid dehydrogenase
complex) of branched chain amino acids (Valine, Leucine, Isoleucine).
Biochemical and clinical manifestations:

• Most characteristic feature of this disease is smell of urine, which resembles that of
maple syrup or burnt sugar (hence the name, maple syrup urine disease).
• Elevation of plasma and urine levels of branched chain amino acids like valine, leucine,
isoleucine and their ketoacids.
• Extensive brain damage occurs.
Treatment:
Replacing dietary protein by a mixture of amino acids that excludes valin e, leucine
and isoleucine. When plasma levels of branched chain amino acids come back to normaJ,
infants are restored milk & other foods in amounts that do not exceed metabolic demand.
MSUD is a lethal disease. Without treatment, death occurs by the end of 1st year. The
disease is evident in the very first week of life. The child is difficult to feed, may vomit
and may be lethargic.
Treatment should be initiated within the first week of life to avert dire consequences.
Amino aciduria:
Increased urinary excretion of amino acids more than the normal amounts is termed
as amino acid uria. It generally results due to some defect in the metabolism of certain
amino acids leading to increased excretion of that particular amino acid and/ or its
products.
Examples:
Phenyl ketonuria, Alkaptonuria, Glycinuria, Hartnup disease, MSUD, Homocystinuria,
Cystinuria etc. These aminoacidurias are discussed in the individual amino acid
metabolism sections. (Explain few of them if aminoaciduria question is asked).
Detection:
Chromatography is one of the most technique employed to detect different types of
aminoaciduria.
Significance:
Most of the amino aciduria is associated with mental retardation. So, it is very critical
that these are detected early so that the mental retardation could be prevented. Delay
in diagnosis will appreciably reduce the intelligent quotient.

Functions of glutamic acid:

1. Synthesis of glutathion e:
Glutathio ne is a tripeptide containing glutamic acid, cysteine and glycine.
(For functions of glutathion e refer amino acid chemistry chapter).
2. Synthesis of GABA (Gamma Amino butyric acid):

Glutamate decarboxylase
Glutamate - - - - - - . . , , - - - - - - ~ GABA
PLP '-:;: CO2
GABA is an inhibitory neurotran smitter.
3. Role in urea synthesis :

Glu tamic acid takes part in urea synthesis as -acetyl glutamate in the carbamoy l phosphate
synthetase step. N-acetyl glutamate is needed for the proper conformation of enzyme.
4. In transamin ation reactions:

Both glutamic acid and aspartic acid play a very important role in transamin ation reactions.
Amino groups of all amino acids are funneled into glutamate.
5) Synthesis of glutamine :
G Iutami ne synthetase
Glutamate + NH.•
ATP
fag~AD Glutamine + Hi)
P+ Pi
Glutamin e formation is very important for the transport of NH from brain to liver.
3
Functio ns of glutami ne:

1. Synthesis of purines: Amide nitrogen (N) of glutamine becomes 3rd and 9 th N of purine
structure. Besides, the ~group of the 2nd carbon atom of guanine is also from glutamine .
2. Synthesis of pyrimidin es: Amide N of Glutamin e becomes the 3rd N of pyrimidin e structure.
Besides, the~ group attached to 4th carbon atom of cytosine is also from glutamine .
3. Synthesis of NAO: Amide of glutamine becomes amide N of nicotinam ide part of NAO.
4. Synthesis of glucosam ine: Amide N of glutamine becomes the N~ group of glucosamine.
Glucosam ine and its derivativ e N- acetyl glucosam ine a re constitue nts of different
glycosamin oglycans.
5. Transport of ammonia: NH3 formed in the tissues (mainly brain & muscle) is trapped in
the form of non-toxic transport form glutamine and transporte d to liver.
6. As a source of urinary NH : 60% of NH required for excretion of H• by kidney as NH4-+ is
3 3
formed from glutamine by glutamine by hydrolysis by glutamine (remainin g 40 % of NH4• is
formed by oxidative deaminati on).

Decarboxylation reactions (Formation of bioge nic amines):

When amino acids are decarbo xylated corresp onding (biogenic) amines are formed.
This reaction is catalyze d by carboxylase enzyme s, which require PLP as coenzym e.
R
Otcarbox ylast I
N H . - CH ,
PLP ----.::::: •
co ,
Amino acids Amines
Biogenic amines are product s of decarboxylation of amino acids or their derivatives.

Examples: Histami ne, GABA, melaton in, serotoni n, tyramin e, tryptam ine etc. These
biogeni c amines have diverse physiological functions.
1) Histam ine - by decarboxylation of histidine:

Histidine gives histami ne on decarbo xylation by enzyme histidin e decarboxylase.
Histidine decarboxylase
Histidine ""'-. i- Histamin e.
PLP COi
His tamine is a powerfu l vasodila tor. It reduces the blood pressur e. Histami ne regulates
HCl secretio n by gastric mucosa. It also participates in allergic & inflamm atory reactions.
2) GABA (Gamma Amino Butyric Acid) - by decarboxylation of glutamate:

Glutamate decarbox ylase
Glutama te - - - - - - - - - - + i - GABA.
PLP -<..:.....;: C 0 2
tamate
GABA is an inhibito ry neurotra nsmitte r. It is produce d by the action of glu
decarbox ylase on Glutama te. GABA is cataboliz ed by GABA oxidase.
Sodium valproa te is u sed in the treatme nt of epilepsy : Low levels of

inhibi tory
,
neurotra nsmitter GABA results in epilepsy . Sodium valproat e is a GABA oxidase inhibitor
so, it reduces GABA catabolism, thereby increase s the concentr ation of GABA.
3) Dopam ine (by decarbo xylation of DOPA) :

DOPA Decarboxy lase DOPAMJNE
"<..;!
PLP CO 2
4) Seroton in (by decarboxylation of 5-hydroxytryptophan):

O ecarboxy lase
5- hydroxy tryptoph an [ Serotoni n ) or 5-hydrox y tryptamin e
PLP CO2
• Tyramine and Tryptamine are two other importa nt biogenic amines. They formed
from decarbo xylation of tyrosine and tryptop han respectively.
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Polyamines:
Polyamines are compounds having many amine groups. They are aliphatic amines.
Example:
Putrescine, Spermidine, Spermine.
Synthesis:
Polyamines are synthesised from Putrescine and SAM (which is obtained from
methionine).
Ornithine
SAM
CC>i 0rnithincdecarboxylase (ODO
PLP SAM decarooxylase
CQ PulTescine
De::arboxylatro SAM ._.J Sperrridine synthase
Methylthioadenosine ..-------1
Spermidine
Decarboxylated SAM ---J Spermine synthase
Methylthioadenosine ...---------i Spermine

Functions:
• Polyamines play an important role in stabilization of structures like DNA, ribosomes,
tRNA, certain subcellular organelles. Since polyamines are cations, they bind with
anionic structures like DNA, ribosomes, tRNA etc.
• Polyamines are involved in synthesis of DNA, RNA and proteins.
• Polyamines required for cell division, proliferation and growth.
Excretion of polyamines is found in all types caner. E.g. leukemias, cancer of lungs,
kidney, gall bladder etc. Monitoring the levels of polyamines in serum and urine is
useful in detection of severity of cancer.
• DFMO, an inhibitor of ODC (required for polya mine synthesis, which are required for cell
division) is used as an anticancer drug. (Refer suicide inhibition in enzyme chapter).
• DFMO is also used in the treatment of Indian Kala-azar and African sleeping sickness
(caused by trypanosoma). ODC has a long halflife in these parasites. DFMO inhibits ODC &
polya1nine synthesis in them, and inhibiting their multiplication.
• Although, DFMO inhibits ODC in humans, it does not affect humans, because the half life of
ODC is only five minutes and is continuously produced.

Question bank on Amino acid metabolism

1. Describe urea cycle. What is its importance? Add a nore on its disorders
2. Expain Glycine / Phenylalanine / Sulphur containing amino acids / tryptophan metabolism

1. Discuss the formation and disposal of ammonia.
2. Outline the transamination with suitable examples. Give its clinical significance
3. Functions of glycine / Tyrosine and phenylalanine / Tryptophan /
4. Biogenic amines / SAM / Transmethylation reactions
Short Answers (2-3 Marks):

1. Ammonia transport or Glutamine formation and its importance
2. Ammonia intoxication / PKU / AlkaptonuriaAlbinism / Glycinuria / Cystinosis / Cystinuria
Cystathionuria / Homocystinuria / Maple syrup urine disease / Hartnups disease

1) Ammonia in the brain is converted into (AIIMS)
a) Urea b) Glutamine c) Glutamic acid d) Creatinine
2) Melatonin is derived from (AIIMS, AI)
a) Tryptophan b) Lysine c) Histidine d) Phenylalanine
3) Branched chain ketoacid decarboxylation is defective in (AI)
a) Maple Syrup urine disease b) Hartnups disease c) Alkaptonuria d) Gangliosidosis
4) Decarboxylation yields a vasodilator from (AIIMS)
a) Aspartate b) Arginine c) Histidine d) Serine
5) Melanin is synthesized from (AIIMS)
a) Glycine b) Leucine c) Tyrosine d) Phenylalanine
6) The coenzyme required for the production of biogenic amine is
a) FAD· b) NADP+ c) Pyridoxal phosphate d) Thiamine pyrophosphate
7) The calming chemical in the brain is
a) Serotonin b) Melatonin c) Histamine d) Nicotinic acid
8) Hartnups disease involves the defect in the absorption of
a) Histid ine b) Tryptophan c) Praline d) Lysine
9) N itric oxid e is synthesized from
a) Alanin e b) Arginine c) Histidine d) Glutarnic acid
10) All of the following can be formed from tryptophan except
a) Niacin b) serotonin c) Melatonin d) Melanin
Answers for MCQ: 1) b 2) a 3) a 4) c 5) c 6) c 7) a 8) b 9) b 10) d

Intermediary of Metabolism 287
Intermediary Metabolism
Contents:
• Metabolism in starvation condition
• Metabolism in fed (absorptive) condition
• Integration of Metabolism
• Methods of study of Intermediary Metabolism

Integration of metabolism:
The co-ordination between different metabolisms is called integration of metabolism .
Biochemistry of Starvation:
Definition:
• Starvation refers to a state of prolonged food deprivation (whereas fasting refers to
voluntary restrain from food). Strictly speaking, starvation starts immediately after
the absorption of the meal is completed.
• Body has energy stores of about 1,600,000 Kilocalories, which can last for one to
three months.
Metabolic changes during starvation:

• Glucose is the prime energy source of the body. When the carbohydrates are not
available, body gets the energy from alternative fuel sources like proteins (amino
acids) and fats (fatty acids). Some tissues like brain have an absolute requirement
for glucose.
• During starvation, at first, glucose level is maintained by hepatic glycogenolysis.

Glycogen reserve is so low that, it cannot meet the energy requirement beyond
one day. As the level of glycogen begins to deplete, gluconeogenesis is accelerated,
which ensures the continuous supply of glucose to brain and other tissues. The
amino acids (mainly alanine) released from muscle forms the major source for
gluconeogenesis. So, muscle wasting and negative nitrogen balance are seen in
prolonged starvation.
• In the mean ti.me, lipolysis increases to provide fatty acids, consequently, fatty
acid oxidation and ketone body synthesis is increased.
• In prolonged starvation, gluconeogenesis from protein is diminished owing to

the reduced release of amino acids from muscle. Meanwhile, brain adapts to
ketone bodies replacing half of the glucose oxidized. In severe cases, glucose
contributes less than 5 % of the total substrate oxidized in the body.
• Finally, increased ketone body production lead to ketosis and acidosis. The acidosis
condition due to starvation is called starvation ketoacidosis. Death from starvation
usually occurs due to ketoacidosis.

Fed or absorptive state

1. Comse of carbohydrates: Dietary carbohydrates are digested to monosaccharide
(mainly glucose), which then absorbed into the blood. This glucose is taken up most
tissues (insulin dependant or independent uptake) and oxidized by various tissues for
energy. The excess glucose is then converted and stored as glycogen in the liver and in
muscle. (When blood glucose is high, glucokinase is induced in liver, which
phosphorylates glucose, which is then converted to glycogen).
In the liver, excess glucose may be converted to triacylglycerols, which are packaged in
very low density lipoproteins (VLDL) and released into the blood. The fatty acids of
VLDL are stored in adipose tissue as fat.
2. Dietary fats (triacylglycerols) are digested to fatty acids and 2-monoglycerides (and
also 1 monoacylglycerols). These digestive products are absorbed in the intestine and
re-esterified to triacylglycerols in intestinal epithelial cells, packaged into chylomicrons
and secreted via lymph into the blood circulation. The fatty acids of chylomicrons may
be stored in adipose tissue or oxidized by various tissues for energy purposes.
3. Dietary proteins are digested to amino adds and absorbed into the blood to contribute
to amino acid pool. The amino acids may be used by various tissues to synthesis
proteins and various nitrogen containing compounds (such as purine, pyrimidine, heme,
creatinine and catecholamine) or they may undergo oxidation to produce energy (ATP).
• Fat burns in the flame of carbohydrates.

Acetyl CoA formed by fatty acid oxidation is then further catabolized through the
TCA cycle by combining with oxaloacetate. Oxaloacetate required for this is synthesized
from pyruvate formed by the glycolytic pathway. So, for complete oxidation of fatty
acids, carbohydrates are required.
• Glucose cannot be synthesized from fats (lipids).
The fatty acids undergo ~-oxidation to give rise to Acetyl CoA which is completely
oxidized to CO2 andf1iO. Acetyl CoA formed cannot be converted back to Pyruvate as
PDH reaction is irreversible. Hence lipids ca nnot be converted to glucose
(Carbohydrate).
• Sustained high dietary intake of glucose does not lead to sustained increased
glycogen level in liver or muscle. After a meal, blood glucose levels rise and body
tissues take up this glucose. In liver and muscle tissues, some of the excess glucose is
converted to glycogen. The muscles store two thirds of the body's total glycogen and
the liver stores the other one third. Sustained high dietary intake of glucose does not
lead to sustained increased glycogen level in liver or muscle. This is because liver
and muscle has a limited capacity to store glycogen. If glucose intake continues after
muscle and liver glycogen stores are saturated, the glucose is not excreted or wasted.
It is converted to fat and stored in adipose tissue.

Methods of study of intermediary metabolism

Various techniques have been employed to study the chemical reactions that take place
in the body. Many of these methods employ animal experiments to study metabolism,
as the fundamental metabolic pathways of most organisms are essentially identical.
These methods may be broadly divided into 4 groups,
1. Use of whole organisms or its components.

a) Whole organisms: The ultimate aim of a biochemist is to know the metabolism in
the organism as a whole. Many of the symptoms of nutritional disorders are studied
by altering the nutritional doses in experimental animals & monitoring their response.
In humans, the Glucose tolerance test (GTT), employed to measure the response towards
glucose metabolism is a good example of the use of whole organism.
Surgical extirpation of an organ is one of the oldest and commonly employed methods
to study the metabolism. For example, the role of pancreas in the etiology of diabetes
mellitus was discovered in studies of the surgically depancreatized dog.
b) Isolated organs, tissue slices, whole cells, and subcellular organelles, are frequently
used to explain biochemical reactions and metabolic pathways. Liver, brain, kidney,
pancreas and other tissues, cut into slices approximately 50 im thick and studied.
2. Studying the inborn errors of metabolisms:

There are hundreds of inborn errors of metabolism that are caused due to mutations.
These conditions provide an experimental approach to study metabolic pathways.
3. Use of metabolic probes:

Metabolic inhibitors and mutagens are two commonly used metabolic probes to study
metabolic pathways. These metabolic probes block a specific reaction in a metabolic
pathway and help to study the pa thway. For example, Inhibitors of electron transport
chain have been largely responsible to understand and trace out the sequence of electron
carriers. There are hundreds of inborn errors of metabolism that are caused as a result
of genetic mutations. These conditions provide an experimental approach to study
metabolic pathways.
4. Application of isotopic tracers:

Isotopes are the atoms with the same number of protons but different neutrons. The
administered isotopes are indistinguishable chemically in the body, but they can be
traced and thus, isotopes allow the labeling of molecules. So, when specific isotopes
are used, the required molecules of the living systems are labeled and traced without
altering their chemical properties.
Application of isotopes in biochemistry has revolutionized the study of metabolisms.
Most of the metabolic pathways are studied using this method.

Question bank on Intermediary Metabolism:

1. Explain the metabolic changes that take in starvation
2. Explain the metabolic changes that take in fed condition (Absorptive state)
3. Explain the various methods of study of intermedia ry metabolism
Short Answers (2-3 Marks):

1. Use of tracers in the study of intermediar y metabolism
2. Starvation
1) First reaction that takes place during starvation

a) Lipolysis b) Glycogenolysis c) Glycogenesis d) Gluconeoge nesis
2) During starvation, which amino acid is the major source of gluconeoge nesis
a) Phenylalani ne b) Alanine c) Leucine d) Glycine
3) The function of glucokinase is to provide glucose-6-p hosphate

a) At constant rate glycolysis b) Under fasting state
c) After a meal for glycogen synthesis d) For ATP generation
4) The major pathway that utilizes NADPH is

a) Gluconeoge nesis b) Ketogenesis c) fatty acid synthesis d) Glycolysis
5) Substrate level phosphoryl ation occurs in
a) Citric acid cycle b) Heme biosynthesis c) Uronic acid pathway d) ~-oxidation
6) Insulin facilitates all the process EXCEPT
a) Glycolysis b) Breakdown of fat in adipocytes c) Membrane transport d) Lipogenesis
7) Acetyl CoA for the fatty synthesis is generated in the cytosol by
a) Acetyl CoA synthase b) Glycerokinase c) ATP citrate lyase d) Pyruvate dehydrogen ase
8) The TCA Cycle intermediat ed from which glutamate may be formed is

a) Succinate b) Malate c) Oxalosuccin ate d) ~-ketogluta rate
9) Which of the following Hormones causes negative nitrogen balance?
a) Corticostero ids B) Growth hormone c) Insulin d) Androgens
10) Which of the following amino acid is purely ketogcnic in nature
a) Serine b) Leucine c) Valine d) Methionine
Answers for MCQ: 1) b 2) b 3) b 4) c 5) a 6) b 7) c 8) d 9) a 10) b
Electron Transport Chain and Oxidative Phosphorylation 292
Electron Transport Chain (ETC) & Oxidative phosphorylation
Contents:
• Overview
• Electron T ransport Chain (ETC)

• Oganization
• Individual Complexes and Mobile carriers
• Inhibitors
• Oxidative phosphorylation
• Mechanism
• Chemiosmotic hypothesis
• Inhibitors
• Uncouplers

Electron Transport Chain (ETC) & Oxidative phosphorylation

Overview:
• Living organisms require continuous supply of energy, which is provided by energy rich
molecules like glucose, fatty acids and amino acids. Energy is released when these fuel
molecules are oxidized by a series of reactions finally yielding CO2 and 8i0.
• In these fuel molecules, energy exists as reducing equivalents (electron, hydrogen or hydride).
During oxidation of these fuel molecules (i.e. in glycolysis, P-oxidation of fatty acids, TCA
cycle etc), these reducing equivalents are transferred to specialized coenzymes like NAD+
and /or FAD to form NADH or FADH2 respectively.
• The electrons from these reduced coenzymes (NADH or FADH2) are transferred through a
specialized set of electron carriers collectively called as 'Electron trans port chain' or 'ETC'
that are present in inner mitochondrial membrane. Molecular oxygen acts as the final electron
acceptor, which is eventually converted to water. (Since ETC requires oxygen, it is also called
as Respiratory chain). This process is also termed as mitochondrial respiration as this process
takes place in mitochondria.
• As electrons are passed down the ETC finally to oxygen, they lose much of their free energy.
Part of this free energy is utilized to produce ATP from ADP and Pi (inorganic phosphate) by
ATP synthase complex located in inner mitochondrial membrane. Thus, the phosphorylation
of ADP to form ATP is coupled with the energy released by oxidation of NADH/FAD8i in
Electron transport chain. So, this process is termed as Oxidative phosph orylation.
B asic infonnation about Oxidation, R edu ction, R edox pair, EOI, JiEO, G, ll.G and ll.G 0•
• Oxidation I Reduction: Loss of electrons is oxidation and gain of electrons is reduction.
• Redox pair: Wizen a substance can exist both in reduced form and oxidized state, the pair is
called as redox pair. For example, fe+2 I Fe-3 are redox pair, where Fe~2 is the electron donor and Fe•3
is electron acceptor.
• £ 01 or Standard redox potential (redox potential) of a redox pair: Redox pair has a tendency to
gain or lose electrons (electron affinity), measured in terms of standard redox potential (E0 volts).
• Flow ofelectrons: Lower E0 indicates a lower electron affinity, so higher tendency to loss electrons
& Higher E0 indicates a higher electron affinity, so higher tendency to gain electrons. Consequently,
electrons are transferred from lower redox potential to higher redox potential.
For example £0 of NAD+/NADH is -0.32 Volts & of FMN/FMNH2 is -0.12 Volts. That means
electrons are transferred from NAO+/NADH to FMN/FMNH2•
• tiE0 or Standard redox potential change is the difference in redox potential of 2 redox pairs.
• G or free energy of a compound is defined as the amount of energy available for doing work.
• AG or Free energy change is difference between free energy of the reactants and the product.
• AG0 or Standard Free energy change is difference between free energy of the reactants & the
product under standard conditions. (Reactant concentration at 1.0 M, pH at 7.0, temperature 25°C) .
.cic0 is related to tiE0 by formula, (where, n is the number of electrons transfrerred and
AG0 = -n F AE:1 F is Faraday constant (23.06 Kcal)
• For e.g., E° of NAD+(NA DH is -0.32 volts; £Do/ 1/2 0 / Hp is +0.82 volts. So, tiE0=1.14 volts.
(n =2; F = 23.06 Kcal). So, tiG0 = -2 X 23.06 X 1.14 = -52.6 Kcal.

The process of mitochondrial respiration can be discussed under two headings;

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

Electrons from NADH and FADH2 are transferred to 0 2 through a series of electron
carriers known as Electron transport chain (ETC). ETC is loca ted in inner
mitochondrial membrane
Components and organization of ETC:

Electron carriers in ETC are organized in to 4 complexes and 2 mobile carriers.
These complexes are arranged i n increasing o rde r of redox potentials. This
arrangement ensures the continuous flow of electrons from NADH / FAD8i to 0 2•
(As electrons flow from lower to higher redox systems.) Each electron carrier in ETC
can receive electrons from an electron donor and subsequently dona te electrons to
the next carrier in the chain. Molecular oxygen (02) acts as the final electron acceptor,
which is eventually converted to water.
Components of electron transport ch ain (ETC)

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

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

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

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

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

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

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

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

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

Barbiturates such as amobarbitol, amytal (sedative)
Piericidin A (antibiotic)
Complex II Malonate
Complex III Antimycin A (antibiotic)

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

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

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

Chemiosmotic hypothesis (also known as Mitchell hypothesis):

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

Inhibitors of Oxidative phosphorylation:

There are a number of inhibitors that inhibit oxidative phosphorylation.
Protein inhibited Inhibitors

ATP synthetase Oligomycin (antibiotic)
ATP-ADP transporter Atracty loside
Uncouplers of oxidative phosphorylation:

Uncouplers separate the oxidation process in the ETC and phosphorylation processes,
hence the name 'Un co up lers'. Uncouplers act as proton channels in inner
mitochondrial membrane and cause the leakage of protons back to the mitochondrial
matrix preventing the development of proton gradient that is required for ATP
synthesis. Thus, the uncouplers allow the oxidation to proceed, but not the
phosphorylation. Electrons are allowed to flow in ETC and the energy produced by
electron transport in ETC is released as heat.
Ionophores: The term 'lonophores' refers to the lipophilic substances that promote
the transport of ions across the biological membranes. All the Uncouplers are Ionophores.
Examples:
• 2,4-dinitrophenol (DNP), 2,4-dinitrocresol, Dicumarol (an anticoagulant), Thyroxine
(a physiological uncoupler), antibiotics like Gramicidin A, valinomycin & nigercin,
which act as ionophores that allow passage of cations through the mitochondrial
membrane dissipating the proton gradient that is required for phosphorylation.
• Thermogenin, a protein present in the mitochondria of brown adipose tissue in
newborns and hibernating animals (which maintains their body temperature).
Brown adipose tissue:

Brown adipose tissue is a different type of adipose tissue (than the usual white adipose
tissues). It is generally present in newborns and also in hibernating animals. They are
called brown adipose tissues due to the presence of high content of mitochondria.
These tissues have a protein called 'thennogenin' (also called uncoupling protein, UCP),
which allows the oxidation in ETC to proceed, but not the phosphorylation. The energy
produced by electron transport is released as heat. So, brown adipose tissue is involved
in keeping the baby warm and also the hibernating animals.
The brown adipose tissues also have a possible protective role against obesity.
Answer hint for ETC and Oxidative phosphorylation question (10 Marks):
a) Definition, Overview - 1 mark
b)ETC - 4Mark
c) Chemiosmotic theory - 3 mark
d) Inhibitors - 1 mark
e) Uncouplers - 1 mark

1. Proton gradient is an electrochemical gradient:

Proton gradient generated due to pumping of electrons by £TC is an electrochemical gradient
i.e. both electrical gradient (more positive charges on the outside of the in11er membrane tlum on
the inside) & chemical (pH) gradient (outside of the membrane is at a lower pH than the inside).
2. ATP sites:
Complex l, Ill and TV act as proton pumps and s11pport the synthesis of ATP. So they are referred
to as ATP site 1, 2 and 3 respectively. Complex Tl does not act as proton pump, so it does not favor
ATP formation.
3. Number of ATP molecules produced f rom NA DH and FADH2:
Complex I, Ill and IV act as proton pumps and support the synthesis of ATP. So tlzey are referred
to as ATP site 1, 2 and 3 respectively. Complex IT does not act as proton pump, so it does not favor
ATP formation. For each pair of electrons transferred through each of the 3 ATP sites in ETC,
approximately one ATP molecule can be produced.
• NADH: Electrons from NADH pass through all the 3 ATP sites. So, one molecule of NADH can
produce about 3 ATP in ETC.
• FADH 2: Electrons from FADH2 bypass the ATP site 1 and transferred through only 2 ATP sites.
So, one molecule of FADH2 can produce only about 2 ATP in ETC.
4. P : 0 Ratio:
It is defined as the number of molecules of ATP produced per atom of oxygen consumed.
P:0 ratio of NADH is 3 and FADH2 is 2.
5. More than 90% of ATP in the body is formed by oxidative phosphorylation:

Oxidative phosphorylation ensures a constant supply of ATP. So, about 10 grams of ATP in tlze
body at any given time meets all the energy needs.
6. Cytochromes
Cytochromes are heme enzymes helping in cellular oxidation. All the et;tochromes except cytochrome
oxidase are anaerobic dehydrogenases.
Cytochromes contain an iron atom which can alternate between Fe3• (ferric) & Fe2• (ferro11s) state
during oxidation & reduction. There are many types of cytochromes, cytochrome aaJ' b, c and c1,
cytochrome b5 and P450 etc.
• Cytochrome aa3, b, c and c1 present in inner mitochondrial membrane as members of an electric
transport chain. They facilitate the transfer of electrons from flavoproteins to cytochrome oxidase
and then to water.
• Cytochrome b5 and P450 occur in membrane of smooth endoplasmic reticulum.
Current concept in energetics of ETC:

Recently, Peter Henkel proved that the actual energy production from NADH & FADH2
in ETC is lesser than earlier calculated figure of 3 and 2 respectively. This is due the
leakage of protons. So, according to the current concept, NADH gives 2.5 ATP and
FADH2 gives 1.5 ATP in ETC. So, P:O ratio of NADH is 2.5 and FADH2 is 1.5.
Consequently, energetics from aerobic glycolysis is adjusted to 7 ATP, energetics from
TCA cycle is adjusted to 10 ATP and energetics from P-oxidation of palm.itic acid is
adjusted to 106 ATP.

Transport of reducing equivalents from cytosol to mitochondrial matrix:

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

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

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

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

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

High energy compounds:

Definition: The term high energy compounds are referred to those compounds which
possess more than 7.0 Kcal / mol of free energy (.1G0 ).
Lipmann proposed the concept ofhigh energi; compounds containing high-energtJ acid anhydride
bonds. Since most ofthese bonds are phosphoanhydride bonds, most ofthe high-energtJ compounds
are high-energy phosphates. The high energy bond is represented by a symbol ~ (Swiggle).
High energy compounds (.1G 0 (Kcal/mol)

1. Nucleotides like ATP, GTP, UTP, UDP-glucose etc - 7.3
2. Thi olesters (CoA derivatives) like Acetyl CoA, Succinyl CoA etc - 7.7
3. Sulfonium compounds like S-Adenosyl Methionine or SAM -10.0
4. Phosphoguanidines or Phsophagens like Crea tine phosphate -10.3
5. Acyl Phosphates like 1,3 -Bisphosphoglycerate - 11.8
6. Cyclic AMP -12.0
7. Carboxylic phosphorus anhydrides like Carbarnoyl phosphate -12.3
8. Eno) phosphates like Phosphoenol Pyruvate -14.8
Classification of high energy compounds:

High energy compounds are broadly classifed into 5 groups,
1) Pyrophosphates: ATP, GTP, CTP
2) Enol phosphates: Phosphoenolpyruvate
3) Acylphosphates: 1,3 bisphosphoglycerate, carbamoylphosphate
4) Acylthioesters: Acetyl CoA, succinyl CoA, S-Adenosylrnethionine
5) Guanidium phosphates: Crea tine phosphate
Importance:
• ATP: ATP is the most important high energy compound in biological system. It consists
of adenine, a ribose & triphosphate moiety. ATP is writtenAMP~P-P. The two terminal
phosphoanhydride bonds in the triphosphate unit of ATP (shown by~) are two high
energy phosphate bonds. ATP is the energy currency of the cell and the free energy
released from ATP hydrolysis (to ADP & phosphate) drives all the endergonic reactions
in the cell. ATP captures the chemical energy released by oxidation of nutrients (by
oxidative phosphorylation & substrate level phosphorylation) and transfers it to synthetic
reactions that require energy. Thus, ATP is continuously being hydrolysed and
regenerated. This is called ATP-ADP cycle.
(Function of ATP is given in nucleotide and nucleic acid chemistry chapter).
• Crea tine phosphate is as a storage form of high energy phosphate in muscle & brain.
Jt can rapidly provide high energy phosphate to regenerate ATP in contracting muscle
by 'Lohmanns reaction (Refer connective tissue chapter).

Question Bank on ETC and Oxidative phosphorylation:

1) Explain electron transport chain (ETC) and oxidative phosphorylation

1) Describe the components of electron transport chain. Name inhibitors of ETC.
3) What is oxidative phosphorylation? Explain the chemiosmotic theory.

1) Name the inhibitors of each components of Electron Transport Chain
2) Uncouplers
3) Brown adipose tissues

1) Dinitrophenol causes (AI)
a) Inhibition of ATP synthase b) Inhibition of electron transport c) Uncoupling of oxidation
and phosphorylation d) Accumulation of ATP
2) Tissue in which fuel oxidation serves not to produce ATP but to generate heat is (AI)
a) Adrenal gland b) Skeletal muscle c) Brown adipose tissue d) Heart
3) In gluconeogenesis reducing equivalents from mitochondria to cytosol shuttled by (ABMS)

a) Malate b) Aspartate c) Glutamate d) Oxaloacetate
4) Cytochromes are (MAHE)

a) Pyridine nucleotides b) Metal flavoproteins c) Peroxidase d) Iron containing proteins
5) All the following are inhibitors of cytochrome oxidase except (AIIMS)

a) Carbon monoxide b) Amytal c) Cyanide d) Azide
6) Mitochondrial membrane is impermeable to

a) FAD b) NADPH c) ATP d) NADH
7) One molecule of FADH2 produces the following number of ATPs

a) Zero b) One c) Two d) Three
8) Which of the following in NOT an uncoupler of oxidative phosphorylation

a) Dinitrophenol b) Thyroxine b) Dinitrocresol d) Tyrosine
9) Brown adipose tissue is brown because of the increased presence of

a) Endoplasmic reticulum b) Lysosomes c) Mitochondria d) Peroxisomes
10) Inhibitors of ATP synthetasc is

a) Streptomycin b) Gentamycin c) Puromycin d) Oligomycin
Answers for MCQ: 1) c 2) c 3) a 4) d 5) b 6) c 7) c 8) d 9) c 10) d

Nutrition 304
Nutrition
Contents:
• Introduction
• Importance of nutrition
• Calorimetry - Calorific va lues, calorimeter
• Resp iratory quotien t
• Energy requirement of a person
• Basal metabolic rate
• Special d ynamic action
• Physical activities
• Calculation of energy requirement of a person
• Balanced d iet
• Recommended dietary allowances
• Role of carboh ydrates in diet
• Role of lipids in diet
• Role of proteins in diet
• Nitrogen balance
• N utritional disorders (Marasmus, Kwashiorkor etc.)

Nutrition 305
1. Terminologies:
• Food:
Food is one of the basic necessities of life just as air and water. Basic function of food is
to provide energy. Food also provides the raw materials for growth, maintenance,
tissue repair, regulation and protection of the body.
Food can be defined as Any material consumed by a living organism to provide
II
energy, growth, maintenance, tissue repair of the body".
• N utrients:
Nutrien ts are defined as the chemical components of food which are required to
maintain optimum health. There are six major groups of nutrients; n amely,
carbohydrates, lipids, proteins, vitamins, minerals and water.
• Nutrition:
Nutrition may be defined as the science concerned with foods / nutrients and their
relationship to health. Nutrition deals with dynamic processes of food utilization
(Ingestion, digestion, absorption, transport, storage, metabolism, disposal of end
products) in the body. In simple words, nutrition is "food at work in the body".
Difference between food, nutrients and nutrition:

"Food is what we eat and nutrients are what we get from these foods".
Food depends on culture. The food habits of one culture may appear weird and unacceptable to the
people of another culture. For example, a person eating chicken might feel disgusted to see another
person eating frog legs. But, that same person eating frog legs might be horrified to see someone
eating dog meat. The person eating dog meat might possibly be shocked to see people eating snakes.
So, food is different for different individuals based on their food culture. But what we get (i.e.
nutrients) from these different foods is same. Whether we eat chicken, frog legs or dog meat; all
these foods provide the same nutrients (But in different proportions). So, " Food is what we eat,
nutrient is what we get from food; nutrition is the process of utilization of foods/ nutrients
in the body".
•Energy :
Energy is defined as the "capacity to do work".
Prime purpose of food is to provide energy.
• Unit of energy is calorie (c). This is too small for measuring the energy values of
foods. So, energy content of foods is expressed in Kilo-calories (Kcal or Cal or C).
1 Kilocalorie = 1000 calories
• SI unit of energy is joule (J). 1 calorie = 4.184 joule (or 1 Kcal= 4.184 Mega joules).
Energy yielding nutrients in the food are carbohydrate, lipids and protein.

Nutrition 306
• Calorific value (energy content) of food:

Definition:
Calorific value of foods is defined as the amount of energy released when one gram of
the nutrients (Carbohydrate, lipids or protein) is oxidized. It is expressed in Kcal/g.
Calorific values are expressed in two types;
i) Actual calorific values:
Actual calorific va lue is defined as the amount of energy obtained when one gram of
food is completely oxidized in bomb calorimeter. Values are,
• Actual calorific value of carbohydrate is 4.1 Kcal/ g
• Actual calorific value of fat is 9.45 Kcal/g
• Actual calorific value of protein is 5.65 Kcal/ g
ii) Physiological Calorific value of food:

Physiological calorific value is defined as the amount of energy {in Kcalories) obtained
when one gram of food is oxidized in the body. Physiological calorific value gives the
measure of energy actually available to the body from these nutrients. Values are,
• Physiological calorific value for carbohydrate is 4 Kcal/ g _ _ _ _ _ _ _ _ _
• Physiological calorific value for fat is 9 Kcal / g. These values are called
• Physiological calorific value for protein is 4 Kcal/ g. Atwater - Br ant values
Physiological calorific values are lesser than actual calorific v alues: This is due to loss
of energtJ when the food is oxidized in the body owing to " loss in digestion and incomplete
oxidation". This difference is more in proteins as they are not completely oxidized in the body.
Nutrient Physiological calorific values Actual calorific values
Carbohydrate 4 Kcal / g 4.1 Kcal/ g
Fat 9 Kcal / g 9.4 Kcal/ g
Proteins 4 Kcal/ g 5.4 Kcal/ g
Significance:
Calorific values help in calculating the amount of n utrients required in the preparation
of balanced diets.
Answer hint for Calorific values question- 5 Marks

a) Definition - 1 Mark
b) Actual calorific va lues - 1 Mark
c) Physiological calorific values - 1 Mark Exam Tip
d) Comparison and reason -1 Mark
d) Significance - 1 Mark
If calorific vaJue question is asked for 2 marks, write only definition (0.5 Mark) and
Physiological calorific values (1 Mark), significance (0.5 Mark).

Nutrition 307
• Respiratory quotient (RQ):

Definition:
Respiratory quotient is defined as the ratio of the volume of CO2 produced to the volume
of 0 2 consumed.
RQ = Volum e of CO2 produced
Volume of 02 consumed
Values:
a) RQ of carbohyd rates:
The carbohydrates are completely oxidized in the body.
C6H1206 (Glucose) + 6 02
So, RQ for carbohydrate= 6CO2 I 6 02 = 6 I 6 = 1
b) RQ for fat and fatty acids:

Fats have relatively low RQ, because they have low oxygen content, and hence require
more for 02 for oxidation.
RQ for fat is calculated to be 0.7
RQ for fatty acid is calculated to be 0.7
c) RQ for proteins:
RQ cannot be measured for proteins, as proteins have variable chemical nature and
also proteins are not completely oxidized. By indirect method RQ of proteins is found
to be around 0.8
RQ of a mixed diet:
Generally we consume mixed diet, which contains variable proportion of carbohydrates,
fats and proteins. RQ for a mixed d iet is around 0.8.
Significance of RQ:
RQ is an indicator of metabolic status. RQ is helpful in understanding the type of food
consumed at any given time. RQ in diabetics may be decreased up to 0.7 indicating
that diabetics derive majority of energy from fats/ proteins and not from carbohydrates).
Answer hint for Respiratory Q u o tient q uestio n - 5 Marks

b) Values - 3 Marks
c) Significance - 1 Mark
lf RQ question is asked for 2 marks, write only definition (0.5 Mark) and values (1 Mark),
significance (0.5 Mark).
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Nutrition 308
Energy requirement of a person

Energy requirement of a person depends on various factors. It mainly depends on the
energy expended by a person. There should be a balance between energy expenditure
and energy intake to maintain normal health. That means energy requirement of a
person fairly depends on his / her energy expenditure.
The energy is expended / required) by an individual mainly for 3 processes. These are,
a) Basal metabolic rate (BMR)
b) Thennogenic effect (SDA or Specific Dynamic Action) of food
c)Physical activity
Note: Additional energy requirement during physiological conditions such as
pregnancy and lactation should also be considered suitably.
a) Basal metabolic rate (BMR):

Definition:
Basal metabolic rate is defined as the minimum energy required by a person at complete
physical, mental & digestive rest (i.e. in the post-absorptive state).
Requirement of BMR:
BMR is the minimum amount of energy required to carry out basal vital body functions
like respiration, heart rate, circulation, renal functions, brain functions, conduction of
nerve impulse and transport across membrane etc.
Calculation of BMR:
BMR is measure using Benedict's-Roth apparatus (Closed circuit indirect calorimetry).
Procedure: The subject breathes in oxygen for 6 minutes. Let the volume be Y liters.
Calorific value of 0 2 is 4.8 Kcal/ liter. So, Heat produced in 6 minutes= 4.8 x Y Kcal
Heat produced in a hour = 4.8 Y X 10 Kcal. .
BMR = (4.8 x Y x 10 Kcal) / A, where A is surface are in sqm.
Normal values:
BMR is expressed in Kcal/ square meter body surface area/hour (i.e. Kcal/sq.m/hr).
Adult male= 35-38 Kcal / square meter body surface area / hour.
Adult female= 32-35 Kcal / square meter body surface area / hour.
Children= 53 Kcal / square meter body surface area/ hour.
Note: For all practical purposes, BMR is expressed as Kcal / Kg body weight/ day.
Normal BMR of an adult male is calculated to be about 24 - 26 Kcal/Kg body weight/ day
and for an adult female, it is about 22 - 24 Kcal / Kg body weight/ day.
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Nutrition 309
Factors affecting BMR:
1) Surface area: BMR is directly proportional to the body surface area. Higher the body
surface area, higher is the BMR.
2) Age: BMR is higher in infants and young children as compared to adults. BMR is at
its highest during the first 5 years of age and then gradually decreases.
3) Gender: Males have higher BMR than females.
4) Climate: BMR is higher in cold climates compared to warm climates.
5) Starvation: BMR decreases during starvation.
6) Exercise: BMR increases during exercise.
7) Fever: BMR increases during fever. (10- 12% increase is noted per degree centigrade
rise in body temperature).
8) Hormones: Thyroxine increases BMR. So BMR increases in hyperthyroidism and

decreases in hypothyroidism.
Significance of BMR:
1) Planning balanced diet:

Determination of BMR is important in calculating energy requirement by a person and
planning a balanced diet.
2) Diagnosis of thyroid hormone disorders:

In hypothyroidism BMR decreases (up to 40% decrease). In hyperthyroidism, BMR
increases (up to 70% increase).
Answer hint for BMR - 5 Marks

a) Definition - 0.5 Mark
b) Requirement - 0.5 Mark
c) Calculations and normal values -1 Marks
c) Factors influencing - 2 Marks
d) Significance - 1 Marks

Nutrition 310
b) Specific dynamic action (SDA) or thermogenic effect of food:

Definition:
SDA may be defined as the"extra energy" produced over and above the actual calorific
value of a food, when the food is metabolized by the body.
For instance, when 25 grams of protein is consumed by a person, the expected energy
release is 100 Kcalories (25 x 4 = . 100 Kcal). But, in actual practice when 25 grams of
protein is utilized by the body, 130 Kcal of heat is produced. Th is extra 30 Kcal is called
the SDA of protein.
Values:
SDA for protein = 30 %
Carbohydrate is = 05 %
Lipids = 13 %
Generally, a mixed diet containing variable proportion of carbohydrates, fats and

proteins is consumed. SDA for a mixed diet is calculated to be about 10 %.
Reason for SDA:

Exact cause of SDA is not known yet. SDA is believed to be the energy required for the
utilization of food . (i.e. digestion, absorption, transport, metabolism and storage of
foods in the body)
Significance of SDA:
Determination of SDA is important in calculating energy requirement of a person and
planning a balanced diet.
Note:
Certain amount of energy (10% for a mixed diet) is lost from the body stores towards
SDA. So, while calculating energy requirement of a person, an additional 10 % calories
should be added to the total energy requirement of the body to account for this loss
towards SDA.
Answer hint for SDA - 5 Marks

a) Defini tion -1 Mark
b) Values -1 Mark
c) Reason - 1 Marks
d) Significance -1 Marks

Nutrition 311
c) Physical activity:
The amount of energy required for physical activity depends on type and duration of
activity. For convenience, the activity level may be divided in to 3 categories; Sedentary/
light, Moderate and Heavy.
i) Sedentary / light work:

Office workers, teachers, doctors, accountants etc.
These persons require an additional 30 - 40 % of BMR for physical activity.
ii) Moderate work:

Students, house wives, indus try workers etc
iii) Heavy work:

Agriculture workers, miners, factory worker etc
• Lumberjacks, blacksmiths, porters and construction workers etc. do very heavy work
and they may require an addition of more than 100 % of BMR.
Additional energy requirement in pregnancy and lactation:

Extra energy is required d uring pregnancy (as fetus gets all the energy and
nutrients from the mother) and lactation (for milk production).
Additional requirement of energy during pregnancy is calculated to be + 300
Kcal; lactation is +550 Kcal d uring first 6 months and + 400 Kcal during next 6
months. This is in addition to their total energy requiremen t calculated.
Calculation of energy requirement :

The energy is expended by an individual mainly for 3 processes, such as,
a) Basal metabolic rate (BMR)
b) Specific Dynamic Action (SDA)
c) Physical activity
Physiological conditions such as pregnancy and lactation are also considered suitably.
Calculation of Energy requirement of an individual (Simplified method):

Energy requirement of a person can also calculated by a simple method. In this method,
energy requirement can be calculated with the help of BMR, physical activity and SDA.
Da ta regarding of sex, weight, type of physical activity are required to calculate total
energy requirement by this method.

Nutrition 312
Total energy requirement can be calculated in 5 steps:

Step 1: Calculate the energy required for BMR
Step 2: Calculate the energy required for physical activity
Step 3: Add step I and step 2
Step 4: Calculate the energy required for SDA of food (i.e. 10% of step 3)
Step 5: Add step 3 and step 4 and rounded to nearest multiple of 25.
Sample 1: Energy requirement of an adult man (60 Kg, light work):

Step 1: Energy required for BMR (At 26 Kcal/ Kg body weight).
= 60 X 26 = 1560 Kcal
Step 2: Energy required for physical activity (Additional 30 - 40 % of BMR)
= 1560X 40% = 624 Kcalories
Step 3: Sub total = 1560 + 624 = 2184 Kcal.
Step 4: Energy required for SDA (An additional IO % of the energy).
= 2184 X 10 % = 218 Kcal.
Step 5: Total energy requirement = 2184 + 218 = 2402 Kcal
Rounded to nearest multiple of 25 = 2425 Kcal/ day.
So, the energy requirement of a s tudent of 60 Kg, engaged in sedentary work is
calculated to be around 2425 Kcal / day.
Sample 2: Energy requirement of an adult woman (50 Kg, light work):

Step 1: Energy required for BMR (At 24 Kcal/Kg body weight).
= 50 X 24 = 1240 Kcal
Step 2: Energy required for physical activity (Additional 30- 40 % of BMR)
= 1240 X 40% = 496 Kcalories
Step 3: Sub total = 1240 + 496 = 1736 Kcal.
Step 4: Energy required for SDA (An additional IO % of the energy).
= 1736 X 10 % = 174 Kcal.
Step 5: Total energy requirement = 1736 + 174 = 1910 Kcal
Rounded off to nearest multiple of 50 = 1900 Kcal/ day.
So, the energy requirement of an adult woman (50 Kg, light work) is calculated to be
around 1900 Kcal / day.
During pregnancy, an additional + 300 Kcal of energy should be provided.
ICMR table of Energy requirement of different categories of people:

In Indian context, dietary guidelines recommended by ICMR (Indian Council of Medical
Research) are more relevant. ICMR Recommended Dietary Allowances of Energy is
given in RDA table later in the chapter.

Nutrition 313
Recommended dietary allowance (RDA)

Definitio n:
Recommen ded dietary allowance (RDA) is defined as average daily dietary intake of
nutrients that is sufficient to meet the nutritional requireme nt of nearly all (up to 98%)
the healthy individual s in a particular life stage or gender group.
Features of RDA:
• The RDA for a country's population is designed on the basis of current scientific
knowledge of the nutritional requireme nt of different age and sex groups, country's
food and dietary habits. In India, Indian Council of Medical Research (JCMR) is
responsible for setting up, reviewing and revision of RDA.
• RDA values are designed to meet the nutritional requiremen t of practically all (almost
98%) the healthy people. To ensure this, a safety factor (except for energy requiremen t)
is added to the estimated average minimum requiremen t of nutrients. This safety factor
is equal to two standard deviations (2 SD) of the average minimum requiremen t of the
subjects (individual s) measured. Thus,
RDA values (except for energy)= Average minimum requireme nt+ 2 SD
This safety factor ensures that the RDA values cover the requiremen ts of almost 98 %
of the individuals in a population .
• RDA of energy (unlike other factors of nutrition) represents only the dietary
requiremen t correspond s to the average daily expenditur e of individual. No safety
factor allowance is provided for RDA for energy, because both - 'inadequat e intake'
and 'excess intakes' are harmful.
• RDA is the amount of nutrients to be provided to meet the daily physiologi cal needs
and to maintain optimal health of individuals . It is not merely the minimum amount of
nutrients to prevent deficiency disease. It also takes into account a margin of safety for
nutrients (except energy) of most individuals . For e.g., the amount of vitamin C to
prevent scurvy is just about 10 mg/ day, but RDA of vitamin C is 40 mg/ day.
Factors influencin g RDA:

Sex, age, physical activity, pregnancy and lactation etc.
Significan ce of RDA:
It is essential to know the RDA of various essential nutrients for the formulatio n of
balanced diet for different categories of people.
Answer hint for RDA - 5 Marks

b) Features & Calculations - 2 Mark
c) Factors influencing - 1 Marks
d) Significance - 1 Marks
e) RDA of some important nutrients - 1 Mark
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Nutrition 314
ICMR Recom mended RDA table for some import ant nutrien ts:
Retinol
Group Particula rs Energy Protein Fat Calcium Iron VitA VitC
(Kcal) (g) (g) (mg) (mg) (µg) (mg)
Referenc e Sedentar y work 2425

Man Modera te work 2875 60 20 400 28 600 40
(60 Kg) Heavy work 3800
Referenc e Sedenta ry work 1875

Woman Moderate work 2225 50 20 400 30 600 40
(50 Kg) Heavy work 2925
Pregnan t
woman +300 65 30 1000 38 600 40
Lactatio n
(0- 6 months) +550 75 45 1000 30 950 80
(6 -12 months +400 68
Infants 0 to 6 months 108/ Kg 2.05/ Kg 500 350 25

6 to 12 months 98/ Kg 1.5/ Kg
Children 1 to 3 years 1240 22 12 400

4 to 6 years 1690 30 25 400 18 400 40
7 to 9 years 1950 41 26 600
Adolescents
Boys 10 to 12 years 2190 54 600 34

13 to 15 years 2450 70 22 600 41 600 40
16 to 18 years 2640 78 500 50
Girls 10 to 12 years 1970 57 600 19

13 to 15 years 2060 65 22 600 28 600 40
16 to 18 years 2060 63 500 30
Note: RDA's of different nutrients are given in appropri ate chapters.

75
Nutrition 315
Balanced diet
Definition:
A balanced diet is defined as a diet, which contains variety of foods in such quantities
and proportion s that the need for energy, amino acids, minerals, fats, carbohydra tes
and other nutrients is adequately met for maintainin g health, vitality and general well-
being. It also makes a provision of extra nutrients to withstand short duration ofleanness .
A balanced diet is a diet that provides all 6 nutrients; carbohydrates, proteins, lipids,
minerals, vitamins, water in proper amounts and proportion s to maintain good health.
Importance:
A balanced diet provides all the six nutrients; carbohydra tes, proteins, fats, minerals,
vitamins and water in proper amounts and proportion s to meet all the nutritional
requiremen t of the body. Each nutrient has its specific role in the body.
• Carbohydrates (sugars and starch) provide energy. Fibers provide roughage action.
• Proteins are vital for growth (body building), maintenan ce and tissue repair.
• Lipids provide energy as well as act as carriers of fat soluble vitamins.
• Vitamins and minerals are required for growth and also for regulation and control of
metabolic processes.
• Water provides the medium for all body metabolisms. Water is also required to flush
out the waste products and remove the toxins. It is a total cleanser.
A balanced diet should meet the energy requirement of the body and also need for
growth, maintenance and tissue repair. Balanced diet contributes towards good health.
Principles and Points to be considered while planning balanced diets:

• A balanced diet should ensure the proper amounts and proportion s of all the six
nutrients to maintain optimal health. Minimum RDA of all nutrients should be met.
• Factors like age, sex, physiologi cal states (pregnancy , lactation), growth, physical
activity, dietary habits, individual likes & dislikes, economic status and food budget
of the family, family compositio n etc. are considered while planning a balanced diet.
Guideline s for calculating individua l nutrient amounts of balanced diet:

Main purpose of food is to supply energy. But, while planning the balanced diet, body's
need for growth, maintenan ce, repair, protection, regulation must be fulfilled first (by
proteins & fats) & then the total energy requireme nt is made up by carbohydra tes. In a
balanced diet, the ratio of proteins, fat & carbohydrates would be 2:2:6 (or 1:2:7).
• Proteins: First and foremost, the daily requireme nt of proteins (both qualitative and
quantitativ e) should be met. This amounts to about 10-20%of total energy need.
• Lipids: Lipid (mainly fats) need is considered next. It is recommen ded that the lipids
provide about 20% of the total energy requiremen t. Lipids should not exceed 30% of
the total energy need & minimum should be about 10%. Saturated fat should not exceed
10% of total energy need and at least 10% of the total energy requiremen t must come
from unsaturate d fats (poly & mono).

Nutrition 316
• Carbohydrates: Carbohy drates ( rich in natural fiber) should make up the remainin g
energy. Tt is recomme nded that the carbohyd rates provide about 60-70% of the total
energy requirem ent, making it the chief source of energy. At least 40% of the total
energy requirem ent must come from carbohyd rate to prevent ketosis & muscle wasting.
• Vitamins and Minerals: The RDA of all vitamins and minerals should also be met.
When the energy and protein requirem ent is met and the food is selected from all food
groups, the requirem ent of vitamins and minerals are automatically fulfilled. However ,
RDA's of vitamin B12, iron, calcium and sodium are carefully considered.
Dietary Goals (Recom mendat ions for a balance d diet):

• Main food: Cereals form the main sources of energy in diets. At least 40% of energy
comes from cereals in a balanced diet. Energy from cereals should not be more than
75%. At least 60g of pulses should be taken. Ratio of cereal & pulse should be at 4:1
to ensure the better protein quality (by complem entary action). For non vegetaria ns
50% of pulses are replaced with one egg or 30 grams of meat or fish. If there are no
pulses in the diet, amount of fish, meat or eggs are doubled.
• Milk: There should be a minimum of 100 ml of milk included in every day's diet. 300
ml of fat-free or low-fat milk or milk products is recomme nded.
• Water: Diet should be containin g adequate water to ensure the passage of 1.5 liters
to 2 liters of urine per day.
• Fibers & antioxidants: Diet should be contain adequate dietary fibers and an tioxidants.
At least 1 medium sized fruit & green leafy vegetabl es are recomme nded per day.
• Energy: Total energy supply suggeste d to be± 50 of RDA.
• Protein: Protein should account for about 10 to 15 % of the total energy.
• Balanced diet must be low in saturated & trans fats, cholester ol, added s ugars, salt:
Total fat intake should not exceed 20 to 35% of calories. Lesser than 10% of calories
should come from saturated fatty acids. Cholesterol intake should be less than 300
mg/day and trans fatty must be restricted. Total calories from refined carbohyd rates
like sugars should be about 5% of total energy requirem ent. It should not exceed 20%
of total energy requirem ent. Salt intake should be limited to less than 5 mg/ day.
• Alcohol: Do not consume more than one alcoholic drink per day for women, two per
day for men. Certain individua ls should abstain from alcohol completely.
• Junk foods: Colas, ketchups & other foods that supply empty calories must be reduced.
Answer hint for Balanced diet question - 5 Marks

a) Definition -1 Mark
b) Importanc e -1 Mark
c) Principles , Guideline -1 Mark
d) Recomme ndations, Dietary goals - 2 Marks
Nutrition 317
ICMR recommended balanced diets:

In India, Indian Council of Medical Research (ICMR) is responsible for setting up,
reviewing and revision of balanced diet.
Balanced diet is prescribed by Nutrition expert committee of ICMR based on daily
total energy requirement and RDA for all the essential n utrients.
Balanced diet for reference man and reference woman based on portion size & exchange
system (ICMR values) is given here.
This accommodates daily total energy requirement & RDA for all the essential nutrients
for the reference group.
ICMR Balanced diet table

for Adults (Reference man (60 Kg) & Reference woman (50kg):
Number of portions (ICMR values)

Food groups Portion Sed entary Moderate Heavy
(ExchanS?e lists) size (g) M an Woman Man Woman Man Woman
Cereals and millets 30 14 10 16 12 23 16
Pulses 30 2 2 3 2.5 3 3
Milk (ml) 100 3 3 3 3 3 3
Roots and tubers 100 2 1 2 1 2 2
Green leafy vegetables 100 1 1 1 1 1 1
Other vegetables 100 1 1 1 1 1 1
Fruits 100 1 1 1 1 1 1
Sugars 5 5 4 8 5 11 9
Fats I oils (Visible) 5 4 4 7 6 11 8
For non vegetarians:

Exchange 1 pulse portion with 1 egg or 1 portion (50g) of meat/ chicken / fish (or both
pulse servings can be exchanged with 2 eggs or 2 portions (100g) of meat/ chicken /
fish).
An Indian reference man and reference woman:

• Age is between 20- 39 years, weighs 60 Kgs and surface area is 1.62 m 2 •
• Engaged in 8 hours of moderate work, sleeps for 8 hours, 6 hours of light activity.
• Engaged in 2 hours of active recreation.
An Indian reference w oman:

• Age is between 20- 39 years, weighs 50 Kgs and surface area is 1.4 ~ 2 •
• Engaged in 8 hours of moderate work, sleeps for 8 hours, 6 hours of light activity.
• Engaged in 2 hours of active recreation.

Nutrition 318
• Nutritional role of Carbohydrates, Lipids and Proteins:

1. Carbohydrates in diet :
Major dietary carbohydrates are polysaccharides (starch) and disaccharides (sucrose,
lactose). Small amounts of monosaccharides (fructose, glucose & pentoses) & dextrins
are also present in the food. Diet also contain dietary fibes (undigested carbohydrates).
Carbohydrates are the principal sources of energy (mainly starch and sugars).
Dietary carbohydrates are of two types:

A) Available (digestible) carbohydrates
B Unavailable (Indigestible) carbohydrates or dietary fibers
A) Available carbohydrates (Digestible carbohydrates)

Dietary carbohydrates th at can be digested and utilized by the body to give energy are
called available (digestible) carbohydrates.
Examples:
Starch, Glycogen, Sugars like lactose, maltose, sucrose, glucose, fructose etc.
Dietary sources of digestible carbohydrates:

• Starch: Cereals, pulses and tubers are the major sources.
• Glycogen: There is practically no dietary source of glycogen; any glycogen stored in
animal organ is quickly converted to lactic acid at the time of slaughter.
• Sucrose: The common table sugar. It is present in fruits, sugar cane, beet etc.
• Maltose: Malt, Germinating seeds.
• Lactose: Milk and milk products.
• Glucose: Mostly present as starch. But free glucose is present in some fruits, such as
grapes, oranges, water melon etc.
• Fructose: Present in some fruits and honey.
Clinical Significance:
1) Sucrose (table sugar) is largely used as a sweetening agent. Sucrose constitutes
empty calories and has no other nutritional value. Consumption of sucrose and sucrose
rich foods are the major risk factors for dental caries and obesity. Obesity predisposes
to diabetes, hypercholesterolemia and coronary heart diseases.
2) Fructose does not require insulin for its cellular uptake; so it is recommended for
diabetics.

Nutrition 319
B) Dietary fibers (Unavailable carbohydrates):
Definition:
Dietary carbohydrates that cannot be digested and assimilated by the body are called
Uflavailable carbohydrates or d ietary fibers.
Examples:
Cellulose, hemicellulose, lignin, pectin, gum and mucilage etc
Sources:
They are generally present in all vegetables, brans of cereals and hulls of legumes.
Dietary fibers are plant cell wall materials.
Significance of dietary fibers:
1. Prevents constipation: Dietary fibers add bulkiness to the food content and help in
peristalsis and normal motility of G.I.T and thus prevent constipation (Roughage action).
2. Easy elimination of stool: Dietary fibers absorb water and form soft feces, which is
easier to eliminate. So, dietary fibers are recommended for the treatment of piles.
3. Prevention of colon cancer: Dietary fibers reduce the risk of colon cancer and
diverticulosis.
4. Hypocholesterolemic effect: Dietary fibers absorb bile salts, cholesterol and increase
their fecal excretion, thus help in reduction of serum cholesterol level.
5. Increase glucose tolerance: Dietary fibers decrease the rate of glucose absorption .
So they are useful in diabetes patients.
6. Satiety value: Dietary fibers add to the bulk to the food and give a feeling of stomach
fullness and hence increase the satiety value of food.
7. Weight reducing diet: Dietary fibers provide a feeling of fullness without actually
providing calories. So, dietary fibers are useful in planning weight reducing diet for
obese persons.
Answer hint for question on Dietary fibers - 5 Marks

a) Definition, - 1 Mark
b) Examples - 1 Mark
c) Functions - 3 Marks
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Nutrition 320
Nutritional functions (Dietary role) of carbohydrates:

Carbohydrates form. major bulk of the food. Major functions of carbohydrates are,
1) Chief sources of energy: The dietary carbohydrates are the chief source of energy.
Each gram of carbohydrate provides 4 Kcal of energy. Carbohydrates provide up to 60-
70 % of the total daily energy requirement of the body. The energy from carbohydrates
is readily available for utilization.
2) Prevents ketosis and muscle wasting: When the diet does not contain enough
carbohydrates, body derives energy from alternate energy sources like fats and proteins
leading to ketosis and loss of muscle protein (wasting).
A minimum daily intake of 50 -100 grams of carbohydrates is recommended to prevent
ketosis and muscle wasting.
3) Protein-sparing action: When the diet contains enough carbohydrates, proteins are
spared for specialized functions like growth, repair (& not utilized for providing calorie).
4) Absolute requirement by brain: Brain has an absolute requirement for glucose. So,
even though proteins & fats can also provide energy, a minimum amount (About 50-
100 grams) of carbohydrates must be present in diet to provide glucose to brain.
5) Production of non-essential amino adds: Carbohydrates provide carbon skeletons
for the synthesis of non-essential amino acids.
6) Sweetening agents: Sucrose is used as a sweetening agent.
7) Taste: Starch which forms the main source of carbohydrate in the average diet has a
bland non-irritating taste and hence it can be consumed in large amounts to provide
major part of energy requirement of the body.
8) Dietary fibers: Dietary fibers have roughage action. They help in peristalsis and
easy elimination of foods. (Dietary fibers is already been explained in the earlier section).
Glycemic Index:
Glycemic index is defined as the area covered under the blood glucose curve after the
ingestion of test meal (50g carbohydrate-rich food), compared with the area covered under
blood glucose curve after taking the same amount of reference meal (50 g glucose).
Glycemic Index = Area under glucose tolerance curve after 50 g test meal X 100
Area under glucose tolerance curve after 50 g of reference meal (glucose)
The Glycemic index of the simple or refined carbohydrates (Glucose, Sucrose) is more than
the complex carbohydrates. This is because the digestion and absorption of the complex
carbohydrates are slow. When carbohydrates present in combination with proteins, fats or
fiber, glycemic index is low. So, ice cream has lower glycemic index alhough it contains
sugar (Ice-cream contains fat, which delays carbohydrate absorption).
Significance: Food low in glycemic index is considered good for health, especially for diabetic
patients. Nutritionists opine that the food with low Glycemic index and high fiber content
(E.g.: whole grains, fruits, vegetables) are preferred.

Nutrition 321
2. Lipids in diet:
Fats constitute upto 95 % dietary lipids. Rest 5% mainly is phospholipids and cholesterol.
Dietary Sources:
Vegetable oils, Vanaspati, Milk & milk products (like Ghee, butter), Meat, fish, poultry,
egg etc. Fats are present in almost all food articles (Except sugars) in small amounts.
Dietary role of different lipids:
A. Role of fats / oils in diet:

• Source of energy: Dietary fats provide energy. They contribute up to 20 to 30% of
daily energy requirement.
• Taste: Oils / Fats increase the taste and palatability of the food. They absorb flavors
and aromas of ingredients to improve overall taste.
• Satiety: Fats increase the satiety value of the food because of their slow digestibility
and increase in emptying time of stomach.
• Smooth mouth feel: Fats / oils provide creamy and smooth mouth feel.
• Dietary fats are the only sources of essential fatty acids: Absolute requirement of
fats in the diet is not for energy, but for the provision of essential fatty acids. Essential
fatty acids are required for eicosanoids synthesis and are important for epidermal
growth, reproduction and promoting normal growth in children and also critical for
cell membrane formation as a part of phospholipids. Dietary fa ts are the only sources
of essential fa tty acids.
• Digestion and absorption of fat soluble vitamins: Fats act as vehicles for the digestion
and absorption of fat-soluble vitamins and thus they promote the digestion and
absorption of fat-soluble vitamins.
B. Role of phospholipid in diet:

• Phospholipids also provide almost 9 Kcal/ gram of energy, like fats.
• Phospholipids are not essential in diet as they are synthesized in the body.
C. Role of Cholesterol in diet :

•· Cholesterol also forms a part of a balanced diet. Cholesterol does not provide energy.
But cholesterol is required for synthesis of bile sa lts, vitamin D and variety of steroid
hormones. They are also important constituents of cell membranes.
• Cholesterol is not an essential nutrient as it can be synthesized in the body. A human
being requires around 800 milligram of cholesterol per day. Out of this, about 300 mg
is generally provided by diet and rest can be synthesized in the body.

Nutrition 322
3. Proteins in diet :
Dietary proteins are essential for growth (body building) , maintena nce and tissue repair.
Even tho ugh proteins provide energy, the actual requirem ent of die tary proteins are
not for the provision of energy, but for su pply of essential amino acids.
Dietary role of Protein :

Dietary proteins are required for,
1) Provision of essential amino acids
2) Provision of energy (10% of total energy requirem ent of body)
3) Provision of nitrogen, phospho rous and sulfur
Note: The essential amino acid content and digestibil ity of proteins (i.e. quality of
proteins) is importan t in a balanced protein diet and n ot the quantity of proteins.
Quality of dietary protein s:

Q uality of dietary proteins is determin ed by their essential amino acid content and
digestibi lity. Biological Value of Proteins (BVP) and Net Protein Utilizatio n (NPU) are
two importan t indices of quality of proteins.
1) Biologic al value of protein (BVP):
Definitio n:
It is the ratio between the amounts of nitrogen retained to the amount of nitrogen
absorbed .
BVP = Nitrogen retained X 100
Nitrogen absorbed
Importance: BVP reflects the essential amino acid content of the proteins. But it does
not take into the account of digestibility of proteins.
2) Net protein utilizat ion (NPU):

Definitio n: Net protein utilizatio n is the ratio between the amounts of nitrogen retained
to the amount of nitrogen intake.
NPU = itrogen retained X 100
N itrogen intake
Importance: NPU accounts for both essential amino acid content and digestibil ity of
proteins. So NPU is a better index of protein quality than BVP.
75
Nutrition 323
3) D igestible coefficien t (DC):

It is the percentage of food nitrogen which is absorbed from alimentary canal.
DC= Food nitrogen - (fecal nitrogen - metabolic fecal nitrogen) X 100
Food nitrogen
Importance: DC of a protein gives an idea about the digestibility of a protein.
Legumes and seeds have lower DC than egg and milk proteins. This is because of
presence of indigestibl e cellulose cell wall of plant cells and presence of anti-trypsi n in
many plant proteins. Cooking improves the DC of plant proteins. This is due to the
denaturati on of anti-trypsi n and break in the cellulose cell wall and thereby allowing
the proteases to digest proteins inside plant cells.
4) Chemical score:
It is the ratio between the content of the most limiting amino acid in a test protein to
the content of the same limiting amino acid in egg protein.
Chemical score of a protein is a measure of essential amino acid content of a protein as
compared with the reference protein (egg protein). Since egg protein contains all the
essential amino acids in adequate amounts, it is assigned the chemical score of 100.
Chemical score = Mg of the limiting amino acid / g of protein X 100
Mg of the same amino acid / g of egg protein
Significance: By comparing the chemical score of different proteins with egg proteins,
the essential amino acid content of different proteins can be assessed. Here quality of
a dietary protein is based on the extent to which it deviates from reference protein.
5) Protein efficiency Ratio (PER) :

It is the weight gain per gram of protein taken.
PER = Gain in body weight (g)
Protein ingested (g)
Significance: PER is the indicator of efficiency of protein to promote growth.
Com12arison Qf qualiti~:i of ~ome 12roteins

Sources of proteins BVP NPU DC Chemical score PER
Egg 90 91 100 100 4.5
Milk 84 75 93 65 3.0
M eat 80 76 90 70 2.8
Fish 85 72 92 60 3.0
Rice 64 57 91 60 2.0
Wheat 58 47 92 42 1.7
Note: Egg protein is considered to be the best quality protein having highest nutritional
value, because it has all essential amino acids in ·proper proportion s and also have
good digestibility. It is used as a reference protein for protein quality.
Nutrition 324
• Dietary sources of proteins:

There are two main dietary sources of proteins.
a) Animal sources:
Animal proteins generally contain all the essential amino acids in sufficient amounts
and have good digestibility. So animal proteins are considered to be good sources of
proteins and are called biologically Complete proteins / First class proteins. Examples
are egg, milk, meat, fish etc. (An Exception is gelatin, which is an incomplete animal
protein. It lacks tryptophan).
b) Vegetable sources:
Vegetable proteins generally lack one or more essential amino acids. Vegetable proteins
also have low digestibility and low protein content. Therefore, vegetable proteins
(except, soy protein) are called biologically Incomplete proteins or Second class proteins
or poor quality proteins. Examples are cereals, pulses, nuts, beans etc. (An exception
is soy protein, which is the only complete plant protein).
In developing countries like India, cereals and pulses are the main sources of dietary
proteins as they are cheap, easily available and consumed in bulk.
Nutritional classification of proteins:

Proteins are required for growth, maintenance and tissue repair. Based on the nutritional
quality (essential amino acid content and digestibility), proteins can be classified into
complete proteins, partially complete proteins and incomplete proteins.
i) Complete proteins (First class proteins):

• Complete proteins contain all the essential amino acids in sufficient amounts and
have good digestibility .
• They promote proper growth.
• E.g.: Proteins obtained from egg, milk and meat etc.
ii) Partially complete proteins:

• They lack one essential amino acid.
• They promote moderate growth.
• E.g.: Vegetable proteins which partially lack one eessential amino acids. (Like
Gliadin, a wheat protein lacks amino acid tryptophan).
iii) Incomplete proteins :

• They comp letely lack more than one essential amino acids.
• They do not p romote growth at all.
• E.g.: Zein of com and Gelatin of animal source.

Nutrition 325
Proteins from different food sources differ from each other in their quality. Based on
this, proteins from different food sources are placed in 3 groups.
a) Sources of high quality proteins: Egg protein, Milk protein, meat, liver etc.
b) Sources of good quality proteins: Poultry, fish, muscle proteins, Soya bean.
c) Sources of poor quality proteins: Nuts, legumes, cereals.
Vegetable proteins (except, soy protein) are generally incomplete or partially complete
proteins as they lack one or more essential amino acids. So, they are not of good quality.
Limiting amino acids:
Definition:
Some vegetable proteins lack one or more essential amino acids. These deficient amino
acids are called limiting amino acids.
Example:
Pulses are deficient in methionine and cereals are deficient in lysine.
Supplementary action of proteins (Complementary proteins):

• Generally, proteins from animal sources (like egg proteins, milk proteins), provide all
essential amino acids and hence are considered good quality proteins. Vegetable proteins
are considered poor source of dietary proteins as they generally lack one or more
essential (limiting) amino acids (E.g. Pulses lack Methionine and Cereals lack Lysine.).
when the proteins lack one or more essential amino acids, they do not promote proper
growth and maintenance of body tissues.
• This problem can be overcome by judicious combination of vegetable proteins, so
that the lack of an essential amino acid in one vegetable protein is made up by its
presence in another vegetable protein. It will cancel out each other's deficiency and
provide a first class protein diet.
• For e.g., proteins of pulses and cereals (E.g. Dal plus rice or chapatti) have mutual
supplementary action if consumed together. This combination provides all essential
amino acids. Such proteins are described as complementary proteins. This is called
Mutual supplementation of proteins (or Complementary or supplementary action of
proteins).
Note:
Good quality proteins are very essential to maintain the nitrogen equilibrium of the
body, because they are the only sources of essential amino acids to the body. Deficiencies
of any one of the essential amino acids in the diet lead to nitrogen imbalance and
malnutrition. (Nitrogen balance is explained in amino acid metabolism).

Nutrition 326
Protein Deficiencies
• Adults: Protein deficiency in adults lead to loss of body weight, anemia, susceptibility
to infection, general lethargy, edema, and delay in wound healing etc.
• Children: Deficiencies of dietary proteins mainly cause Proteins Energy Malnutrition
(PEM) in children.
Protein energy malnutrition [PEM]

PEM is the most common nutritional disorder of the developing countries including
India. Kwashiorkor and Marasmus are two extreme forms of PEM.
Kwashiorkor and Marasmus are in two ends of PEM spectrum. One end is Kwashiorkor,
which is a predominant protein deficiency with relatively adequate calorie/ energy intake,
and other end is Marasmus, which is a predominantly energy deficiency and also a
lesser protein deficiern;:y (Primary calorie deficiency and secondary protein deficiency).
a) Kwashiorkor
Definition:
Kwashiorkor is caused chiefly by protein deficiency in the presence of adequate calorie/
energy. The word Kwashiorkor is derived from Ghana, which means " the sickness the
older one gets when the next child is born".
Cause:
Kwashiorkor is usually seen in children of 1-5 years, when the child is weaned from
mother's breast milk and is replaced by starchy gruels, which are rich in carbohydrates,
but deficient in proteins.
Symptoms:
• Hypoalbuminemia (decreased serum albumin level, sometimes less than 2 g / dl)
• Edema: Plasma proteins (mainly albumin) is equired to maintain the colloidal osmotic
pressure (COP) of blood. In kwashiorkor, there is reduction in synthesis of proteins,
resulting in reduction of COP & fluid retention in the extracellular spaces causing edema.
• Pot (swollen) belly due to swollen abdomen
• Fatty liver (enlarged liver because of failure of fat mobilization due to protein deficiency
which is required for VLDL formation and consequent and fat accumulation in liver).
• Growth retardation ·
• Skin lesions, skin rash
• Discolored hair (due to lack of melanin formation)
• Anorexia, Diarrhea (anorexia! diarrhea)

Nutrition 327
b) Marasmus
Definition:
Marasmus (means wasting) is characterized by predominant energy deficiency and a
lesser protein deficiency. (Primary calorie deficiency and secondary protein deficiency).
Cause:
Marasmus is generally seen in children under one year of age, when the mother's milk
is weaned and replaced by a low cost native cereal diet, which is poor in both calorie
and protein. (So, marasmus is also called protein calorie malnutrition).
Symptoms:
• Extreme muscle wasting (emaciation)
• Marasmic patients do not show edema & decreased serum albumin as in Kwashiorkor.
• Face of the patient shows apathy (no interest)
• Growth retardation
• Weakness
• Anemia
• Diarrhea
Treatment/ Management of PEM:

PEM is a complex syndrome of nutritional disorder coupled with infection. So the
treatment of PEM should ensure both these factors are well addressed.
a) Provision of well digestible (liquid diet initially for a week) and well balanced diet
that contains required amounts of calories, high nutritive value proteins and all other
essential nutrients.
b) Diagnosis and treatment of any bacterial and / or parasitic infection present.
ote:
Though a nutritional disorder, the root cause of PEM is socio-economic factors like
poverty, illiteracy, ignorance, cultural practice, customs, feeding practice etc. So, along
with the dietary management, health and nutritional education should also be given to
the families especially to pregnant and lactating women. They should be explained
about the signs, symptoms, causes and treatment measures of PEM.
In addition to this, the socio-economic condition of the country needs to be improved
through various government measures to effectively tackle PEM.
Answer hint for question on Protein Enegry Malnutrition - 5 Marks

a) Definition, Types - 1 Mark
b) Kwashiorkor - 1.5 Mark
c) Marasmus - 1.5 Marks
d) Management - 1 Marks
For queries and suggestions, contact the author at prasad_te:xt@yahoo.com or 9986449575

Nutrition 328
Obesity:
Definition:
Obesity may be defined as an abnormal excess deposition of fat in the body. Obesity
is caused due to increased size (hypertrophy) or increased number (hyperplasia) of
adipocytes or both.
Obesity is the major cause of overweight. Obesity & overweigl:-Vaccording to reference

height-weight table have to be carefully reviewed in terms of the lean body mass and
body muscle to fat. An athlete will have highly developed muscle mass and therefore
may be overweight according to reference height- weight table. But a thletes body has
more muscle mass than fat in proportion. Therefore, obesity is not only a matter of
weight. One must be able to distinguish between weight due to well developed muscle
mass (athletes) and due to excessive fat deposition.
In simple words, overweight is usually due to obesity but also can arise due to excess
muscle development. It can also be caused due to abnormal fluid retention.
Causes:
• Obesity results from a chronic imbalance between energy intake and energy
expenditure. Major contributing factors are excessive calorie consumption more
than the body need and / or lack of physical activity.
• Genetic (hereditary) factors also contribute to the condition.
• Obesity can also result from hormonal disorders like h ypo thyro idis m ,
hypogonadism, Cushing's syndrome and hypopituitarism.
Excessive fat intake is more fattening than excessive carbohydrate I protein intake.
Possible reasons are,
• Fat is more concentrated forrn of energy than carbohydrates and proteins. Fats give 9 Kcal/
gram compared to 4 Kcal/gram for carbohydrate or protein. So, fats are denser in calories t'1an
either carbohydrates or proteins.
• Body has the capacih; to increase utilization (oxidation) ofcarbohydrate and protein whenever
their intake is high. But, body can not do so during '1igh fat intake.
• Priority is given to maintain carbohydrate and protein balance over limiting fat storage.
When the energy intake is excessive, the body burns the excess carbohydrate and protein to
maintain balance. As the oxidation of these nutrients increases, fat oxidation decreases and
consequently,fat storage increases.

Nutrition 329
Assessment of obesity:
1. BMI (body mass index) or Quetelet index or Obesity index:
BMI = W (Where W =Weight in Kg and H =Height in meters)
H2
BMI is a simple index to assess obesity. Normal persons have the BMI in the range of
18.5 to 25. If the BMI is above 25, the persons is said to be obese.
• Classification of obesity (Based on BMI): BMI is also used to classify obesity.
BMI Type of obesity

These BMI values are
25 to 29.99 Obesity grade I age-independent and
30 to 39.99 Obesity grade II same for both sexes.
~40 Obesity grade III
2) Skin fold thickness (SFT): A large proportion of total body fat is situated subcutaneously,
most of it is accessible. So, measurement of skin fold thickness is used as a rapid and non-
invasive m ethod for assessment of body fat. This method cannot be standardized. So, this
method is not a reliable method in assessment of obesity.
Complications of obesity:
Obese persons are more prone to diabetes mellitus (Type II), coronary heart diseases
(like atherosclerosis, hypertension) and conditions like fungal infections etc.
Treatment (Management) of obesity:

• Reduction of intake of calories and regular exercise.
• Frequent small meals with lots of dietary fibers.
Answer hint for Obesity question - 5 Marks

b) Causes - 1 Mark
c) Assesment (mainly BMI) - 2 Marks
d) Complications - 0.5 Mark
e) Treatment/ Management - 0.5 Mark
Other nutritional disorders:

Include Night blindness, Rickets, Osteomalacia, Beri Beri, Pellagra, megaloblastic anemia,
Pernicious anemia, Scurvy, Iron deficiency anemia, etc. Refer related chapters for details.
Nutritional deficiency anemias:

Anemia is a clinical condition characterized by low blood Hb concentration. Anemia due to deficiency
of nutrients that are necessary for Hb synthesis are termed as nutritional deficiency anemia.
• Deficiency of Iron causes Iron deficiency anemia (Microcytic hypochromic anemia)
• Deficiency of Folic acid (Vitamin BJ causes mega loblastic anemia
• Deficiency of Caba/amine (Vitamin B11) causes pernicio11s anemia

Nutrition 330
Question Bank on Nutrition
Short Essays (5 Marks)

1) Basal metabolic rate (BMR)
2) Specific dynamic action (SDA) or thermogenic effect of food.
3) Balanced diet
4) Dietary fibers
5) Respiratory quotient (RQ)
6) Protein quality
7) Protein energy malnutrition - Kwashiorkor and Marasmus
8) Write a short note on calorific values (energy content) of foods?
9) Limiting amino acids. Add an ote on Complementary proteins
Multiple Choice Questions (1 Mark):.

1) Energy expenditure in resting state depends on (AI)
a) Lean Body Mass b) Adipose tissue c) Resting Heart Rated) Exercise
2 Which of the following about Recommended Dietary Allowances (RDA) is true (AI)
a) RDA is statistically defined as two standard deviations (SD) above Estimated Average
Requirement (EAR)
b) RDA is defined as being equal to the Estimated Average Requirement (EAR)
c) RDA is defined as being equal to Adequate Intake (AI)
d) RDA is defined as the recommended minimum requirement
3) Dietary fiber is rich in (AIIMS)

a) Starch b) Pectin c) Collagen d) Proteoglycan
4) Which of the following has greatest SDA?
a) Proteins b) Fats c) Vitamins d) Carbohydrates
5) The calorific value of fats in the body is
a) 9.0 Kcal b) 5.6 Kcal c) 6.0 Kcal d) 4.0 Kcal
6) The reference protein for the calculation of chemical score is

a) Meat protein b) fish protein c) Milk protein d) Egg protein
7) SDA of proteins is
a) 10% b) 15% c)30% d)05%
8) The following parameters is used for the nutritional value of a protein
a) itrogen balance b) Chemical score c) Respiratory quotient d) Specific dynamic action
9) The calorific value of carbohydrates is
a) 9.0 kca l/g b) 4.0 kcal/ g c) 12.0 kcal/g d) 1.0 kcal/g
10) Respiratory quotient of an average mixed diet is about
a) 0.65 b) 0.7 c) 0.75 d) 0.80
Answers for MCQ: 1) a 2) a 3) b 4) a 5) a 6) d 7) c 8) b 9) b 10) d
For queri~s and suggestions, contact the author at prasad_text@yahoo.com or 9986449575

Vitamins 331
Vitamins
Contents:
• General Considerations :
• Definition, Classification according to solubility, Properties
• Individual Vitamins :
• Chemistry
• Sources
• Digestion, Absorption
• Storage and Transport
• Functions
• Deficiency
• Hypervitaminosis (Toxicity)

Vitamins 332
Vitamins
Definition: Vitamins are defined as the organic compounds. They are categorized
as essential nutrients, because they are either not synthesized at all or not
synthesized in sufficient amounts and therefore must be supplied in the diet.
Vitamins (and minerals) are categorized as micronutrients, as they are required in small
amoW1ts. Vitamins do not provide energy, but are required for the proper utilization
of oth er nutrients (carbohydrates, proteins and lipids) to yield energy.
Classification:
Vitamins are mainly classified into 2 groups based on their solubility
i) Fat-Soluble {lipid-soluble) vitamins and ii) Water soluble vitamins
I) Fat-soluble vitamins:
They are soluble in fa ts and fat solvents (alcohol, acetone e tc). They include,
• Vitamin A
• Vitamin D
• Vitamin E
• Vitamin K
II) Water soluble vitamins:

Water soluble vitamins are soluble in water. They include,
1) Vitamin C
2) B complex vitamins
• Thiamine (B1), Riboflavin (82), Niacin (B3), Pantothenic acid (85), Pyridoxine (B6),
Biotin, Felic acid (B9), Cobalamine (B12)-
Provitamins: Provitamins are the precursors of vitamins. They can be converted to vitamins.
E.g.: Carotenes (mainly ~-carotenes) are the provitamins of vitamin A. Ergosterol and 7-
dehydrocholesterol are the provitamins of vitamin D.
Antivitamins (Vitamin antagonists): Antivitamins are the antagonist compounds o.f

vitamins. These inhibit the actions of vitamins in the body.
E.g.: Warfarin and Dicumarol are antivitamins of Vitamin K; Avidin is the antivitamin of
Biotin; Sulphonamides and Methotrexate are antivitamins of Folic acid.
Answer hint for Essay question on Vitamins(lO Marks):

a. Chemistry, if asked (1 Marks)
b. Sources (1 Marks)
c. RDA (1 Mark))
d. Functions (4-5 Marks)
e. Deficiency (2-3 Marks)
f. Hypervitaminosis, if any (1 Marks)

Vitamins 333
Fat - soluble vitamins
General information:
Vitamins in this group are fat -soluble (lipid-soluble). They are soluble in fats and fat
solvents like alcohol, acetone etc.
These include,
• Vitamin A
• Vitamin D
• Vitamin E
• VitaminK
Properties:
All fat soluble vitamins share the following features:
1. Digestion and absorption of fat-soluble vitamins require bile salts and the formation
of micelles. Fat-soluble vitamins can be only absorbed efficiently when normal fat
absorption is taking place.
2. Transport of these vitamins in the blood requires specific carrier proteins.
3. Body has the ability to store surplus fat-soluble vitamins.
• In cholestatic liver diseases, fat soluble vitamins are not properly absorbed: This
is because, bile salts which are required for absorption of fat soluble do not reach
intestine due to a block in bile ducts (cholestasis).
• If fat-soluble vitamins are not supplied regularly in the diet, the deficiency does
not manifest rapidly, as they are stored in the body.
• Hypervitaminosis (toxicity) develops during excessive dietary intake of fat-soluble
vitamins. This is because the body has the ability to store surplus fat-soluble vitamins
and excess storage causes toxicity.
Note:
If a question on difference between fat soluble and water soluble vitamins are asked,
write the properties of both.

Vitamins 334
Water soluble vitamins

General information:
Vitamins in this group are water-soluble.
They include vitamin C and v itamins of B complex group.

B complex group of vitamin are a group of chemically diverse molecules originally
grouped together on the basis of shared solubility in water and isolation from same
dietary sources.
B -complex group of vitamin are,
• Thiamine (Vitamin B1 )
• Riboflavin (Vitamin 8 2 )
• Niacin (Vitamin 8 3)
• Pantothenic acid (Vitamin B5)
• Pyridoxine (Vitamin B6)
• Biotin
• Folic acid (Vitamin B)
• Cobalamine (Vitamin B12)
Note: B -complex vitamins are not chemically related to one another. They are
grouped together because all of them are water soluble, isolate from same sources
and function as coenzymes.All vitamins of B complex series function as coenzymes
either as such or after getting converted to active form.
Properties:
Water soluble vitamins (except vitamin B12) share the following features:
l. Intestinal absorption is simple (by diffusion).
2. Transport of these vitamins (Except vitamin B12) in plasma does not require any
carrier proteins.
3. No appreciable storage (Except vitamin B12) as excess is excreted. Therefore,
• These vitamins must be supplied regularly in the diet,
• Deficiency manifests rapidly,
• Hypervitaminosis (vitamin toxicity) does not develop.
B complex vitamins are generally supplemented during antibiotic therapy:

B complex vitamins like biotin, pantothenic acid, vitamin B12 are produced by intestinal
bacteria. Antibiotics destroy the intestinal flora resulting in B complex deficiency.

Vitamins 335
Vitamin A:
Chemistry:
Vitamin A is a collective term for 3 compounds showing Vitamin A activity, namely
retinal, retinol and retinoic acid. Together these 3 are collectively called as 'Retinoids'.
The term retinoids also includes synthetic vitamin A compounds.
They have a cyclohexenyl ring (or p -ionone ring) with a polyisoprene side chain.
C H3 CH3
R
In retina l R =CHO
In retinol R=C1"½0H
In retinoic acid R = COOH
Provitamins of vitamin A :
Carotenes are provitamins of vitamin A. Carotenes yield vitamin A in the body.
Plants contain different types of carotenes, designated as a.., p and y carotenes.
p -carotene is the important and most common provitamin of vitamin A present in
plants. This is converted to vitamin A in the intestine.
(One molecule of P -carotene consists of 2 molecules of vitamin A).
Sources:
1. Fish liver oils like cod liver oil, shark liver oil, are very rich sources of vitamin A.
Milk, Uver and egg yolk are good sources. (Vitamin A is present in animal food only.)
2. Vegetarians get the vitamin A through provitamins of vitamin A (mainly as
P-carotenes). P-carotene is present green leafy vegetables and also in mango, carrot,
papaya, sweet potato (Yellow vegetables and frui ts), tomatoes etc.
Recommended daily allowance (RDA):

• Adult man, Adult woman = 600 µg retinol = 2400 of µg f3 carotene
• Pregnant woman = 600 µg retinol = 2400 of µg f3 carotene
• Lactation = 950 µg retinol = 3800 µg of p carotene
• Infants = 350 µg retinol = 1400 µg of p carotene
• Children = 400 to 600 µg retinol = 1600 to 2400 µg of P carotene
Vitamins 336
Digestion and Absorption:

Vitamin A ester is digested by pancreatic hydrolases to give vitamin A and fatty
acid. Vitamin A is absorbed in small intestine along with fats with the help of bile
salts. Fat content of foods stimulate vitamin A absorption.
Transportation and storage:

In the intestine, vitamin A is incorporated into the chylomicrons and transported
(through the lymph) to the liver, where it is stored as retinol palmitate. In its full
capacity, vitamin A stored in liver can last up to 6 to 9 months.
From the liver, vitamin A is transported to other tissues with the help of retinol
binding protein (RBP).
Functions of vitamin A:
Retinal, Retinol and Retinoic acid have specific biological functions.
• Retinal for vision
• Retinol for reproduction
• Retinoic acid for maintaining normal epithelium
1. Role of Retinal in vision:

Retinal form of Vitamin A is required for vision. Rod cells and cone cells present
in retina are responsible for vision.
Rhodopsin present in rod cells is responsible for vision in dim light and photopsin
present in cone cells is required for vision in bright light and color vision.
Both these visual pigments contain vitamin A in the form of 11 -cis-retinal.
2. Normal reproduction:
Retinol plays an important role in normal reproduction.
3. Iron transport:
Retinol promotes the synthesis of transferrin (Iron transfer protein) and thus helps
in iron transport, and hence hemoglobin synthesis.
4. Normal epidermal growth:

Retinoic acid is essential for normal differentiation & mucous secretion of epithelial
tissues and it also prevents keratinisation of epithelial tissues.
5. Growth:
Retinoic acid is also required for growth, especially skeletal growth.
6. Anti-carcinogenic effect:
P-carotene has anti-carcinogenic effect. P-carotene functions as antioxidant and
reduces the risk of cancers caused by free radicals.

Vitamins 337
1. Role of retinal in vision in dim light:

Rod cells of retina are responsible for vision in dim light. They contain the visual
pigment called rhodopsin (11-cis-retinal + Opsin) or visual purple. When the light
falls on retina, a series of biochemical changes takes place and rhodopsin is converted
to meta-rhodopsin (all-trans-retinal + opsin), which is unstable. Subsequently, all-
trans-retinal dissociates from opsin. This triggers a nerve impulse, which is carried to
the brain through optic nerve. (This is the mechanism of vision in the dim light.)
Rhodopsin
(11-,;,.,etiool + 0, ¥
'-
Metarhodopsin
(All -trans• retinal + Opr,in)
_A .
i
Opr,in ...___
Isomerase (slow)
11 · cis- retioaJ - - - - All• trans - retinal
(Retina)
RetioaJ reductase l RetinaJ reductase
( Retina)
11- cis- retinol

Jsomera~e (Liver)
1 (Retina)
All -trans- retinol
Wald's visual cycle:

Now all-trans-retinal has to be converted back to 11-cis-retinal. This process of
regeneration of 11-cis-retinal is called Wald's visual cycle or Rhodopsin cycle or
Vitamin A cycle. .
• First, all-trans-Retinal is converted to all-trans-Retinol by retinal reductase (in the
retina).
• All-trans-Retinal is then transported to the liver.
• In the liver cells all-trans-Retinal is isomerised to 11-cis-Retinol by the enzyme
isomerase.
• Then 11-cis-Retinol is transported back to retina.
• In the retina, 11-cis-retinol is converted to 11-cis-Retinal by the retinal reductase
enzyme. Now 11-cis-Retinal can bind with opsin to form rhodopsin.
This process of forming back rhodopsin is called Wald's visual cycle.

Vitamins 338
Deficiency of vitamin A:
• Night blindness or Nyctalopia: Vitamin A is required for the vision in dim light.
So deficiency of vitamin A causes night blindness.
• Xerophthalmia (dryness of eyes): Keratinisation occurs in cornea and conjunctiva

of eye causing dryness of these tissues. Dryness of conjunctiva is termed as
conjunctival xerosis, which is the first clinical sign of vitamin A deficiency. In
certain areas of conjunctiva, white triangular patches known as Bitot's spots are
seen. Dryness of cornea is termed as corneal xerosis.
• Keratomalacia: If untreated Xerophthalmia results in corneal ulceration and

degeneration, a condition called as keratomalacia, leading to total blindness.
• Renal failure: Keratinisation occurs in renal tract causing renal failure.
• Respiratory infections: Keratinisation occurs in respiratory tract. With reduced

mucous formation, there is atrophy of respiratory epithelium, increasing the
susceptibility to respiratory infections.
• Reproductive failure
• Growth retardation
• Microcytic anemia, because vitamin A is required for iron transport and hence
required for hemoglobin synthesis.
Toxicity of vitamin A (Hypervitaminosis A):

Excess consumption of vitamin A leads to the toxicity condition called vitamin A
toxicity or hypervitaminosis A. However, excess of ~-carotene is not toxic.
Symptoms:
• Hepatomegaly (Enlargement of liver)

• Skeletal deformities
• Anorexia
• Irritability
• Dermatitis
• Headache
• Drowsiness
• Peeling of skin

Vitamins 339
Vitamin D:
Chemistry:
There are two forms of vitamin D, Vitamin 0 2 (Ergocalciferol), Vitamin 0 3
(CholecaJciferol). Both have equal activity.
Sources:
a) Exogenous sources:
• Ergocalciferol (Vitamin 0 2) is found in plants.
• Cholecalciferol (Vitamin 0 3 ) is obtained from fish liver oils (like cod liver oil,
shark liver oil), shrimps, milk, eggs etc.
b) Endogenous sources:
Vitamin 0 3 (Cholecalciferol) can be synthesized from 7-dehydrocholesterol (which is
a derivative of cholesterol) present in the skin, by the action of UV rays of sunlight. So,
vitamin Dis also referred to as sunshine vitamin.
(Skin)
7- dehydrocholesterol • • • • • • • Cholecalciferol (vitamin D:J
Ultraviolet rays of sunlight
*** Similarly ergosterol is converted to vitamin 0 2 (Ergocalciferol).
Provitamin D :
7-dehydrocholesterol and ergosterol are caJled provitarnins of Vitamin D.
RDA:
The RDA for adults is 10 µg (400 I.U.)of vitamin 0 3 •
During pregnancy and lactation, RDA increases to 15 µg.
Absorption, Transportation and Storage:

• Vitamin D is absorbed in small intestine along with fats with the help of bile salts.
• In intestine, vitamin Dis incorporated into the chylomicrons & transported through
the lymph and enters the circulation. In the circulation, vitamin D binds to the vitamin
D binding protein (DBP, a plasma a2- globulin) and transported to different tissues.
• Vitamin D is stored in liver and other tissues (kidney, adrenal glands etc) to a
significant extent.
Biochemical functions of vitamin D:

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

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

Vitamin D3 (and D2) are not biologically active, but are converted to active form
1,25-dihydroxy cholecalciferol or calcitriol
Cholecalciferol (vitamin D3)
l 25-hydroxylase (liver)
2S-hydroxy choral:::::roxylase (kidney, bone etc)
1, 25 -dihydroxy cholecalciferol (Calcitriol)
,.,.,. Both the enzymes require cytochrome P 4501 molecular 0 2, NADPH
Calcitriol plays an important role in bone formation and calcium metabolism.
1) Bone mineralization and formation:

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

Vitamin D plays an important role in calcium and phosphate homeostasis. Calcitriol
is a hypercakemic hormone. The sites of action are intestine, kidney and bones.
a) Intestine: Calcitriol increases the intestinal absorption of calcium and phosphates

and thus increases blood calcium and phosphate level.
Action of calcitriol is similar to steroid hormones. It binds to a receptor in the cytosol
and this complex is then transported to the nucleus, where it acts on DNA to stimulate
the synthesis of calcium binding protein (CBP), which increases calcium absorption.
b) Kidney: Cakitriol promotes the reabsorption of calcium and phosphate by renal

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

Vitamins 341
Deficiency of vitamin D:
Vitamin D deficiency causes Rickets in children and osteomalacia in adults.
Rickets:
Rickets is caused due to the deficiency of vitamin Din children.
Symptoms:
Rickets is a disease of growing bone. There is insufficient mineralization of new bones.
Bones become soft and pliable (easily bent). Rickets is characterized by,
• Bowlegs,
• Knock knees,
• Pigeon-chest,
• Rickety-rosary (beaded appearance) of ribs
Basis: Rickets is a disease of growing bones. ln vitamin D deficiency during the stage of bone
growth, the depositions of minerals (calcification) fail to occur in the newly formed matrix, but
matrix formation continues. This results in the soft, pliable (easily bent) bones. Deformities occur
because cartilaginous structure cannot withstand the weight of the growing body. This results in
bowlegs, knock-knees, and pigeon-breast and rachitic-rosary a beaded appearance of the ribs.
Osteomalacia:
Osteomalacia is caused due to the d eficiency of vitamin Din adults.
Symptoms:
Osteomalacia is characterized by
• Insufficient mineralization of bones
• Softness of bones
• Bone pain and aches
• Easy fracture of bones
Renal rickets (Vitamin D resistant rickets):

This is seen in patient with chronic renal failure. Kidney is required for the formation
of calcitriol (active form of vitamin D) that is required for calcium absorption and bone
mineralization. So, chronic renal disorders lead to poor bone mineraJization and rickets,
which is referred to as renal rickets. These do not respond to vitamin D provision, but
respond to provision of calcitriol. So, some regards this as vitamin D resistant rickets.
Toxicity of vitamin D (Hypervitaminosis D):

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

Vitamins 342
Vitamin E
Chemistry:
Chemical name is tocopherol.
About 8 tocopherols have been identified; among them a - tocopherol is the most
active. Tocopherols have tocol ring system.
Sources:
Vegetables oils, (cotton seed oil, sunflower oil e tc) are rich sources of vitamin E.
Meat, egg, Milk and butte r are good sources.
Note that fish liver oils are deficient in vitamin E.
(Vitamin E requirement is proportional to PUFA intake. This is because vitamin E
protects PUFA from oxidative damage. So, more PUFA intake require more Vitamin E)
RDA:
8 mg (12 IU) to 12 mg (18 IU).

• Vitamin Eis absorbed in small intestine along with fats with the help of bile salts.
• From the intestine, vitamin E is transported as chylomicrons to the liver. From the
liver, vitamin Eis transported to other tissues, mainly to adipose tissues and muscles.
• Adipose tissues muscle and liver store vitamin E to a significant amount.
Biochemical functions:
• Vitamin Eis a powerful, natural antioxidant.
It prevents the peroxidative damage of PUFA of cell membranes caused by free radicals.
Free radicals are continuously produced in the body. They attack and oxidize PUFA of
cell membranes and destabilize the integrity of cell membranes, thus affecting the normal
functioning of cells. (Mainly RBC's). Vitamin Eis a powerful antioxidant, which destroys
the free radicals & thus prevents their peroxidative damage on cell membranes.
• Vitamin E has selenium sparing action.
The mineral selenium also has antioxidant property. Selenium is an integral part of
glutathione peroxidase enzyme, an enzyme which destroys free radicals and protects
RBC and other tissue membrane from peroxidative damage caused by free radicals.
Thus selenium and vitamin E act synergistically with each other against per oxidative
damages. Thus selenium decreases the requirement of vitamin E and vice-versa. Thus
selenium has vitamin E sparing action.
Deficiency:
Rare. Deficiency may cause anemia, as RBC's are prone to peroxidative damage.

Vitamins 343
Vitamin K:
Chemistry:
Vitamin K is naphthoquinone derivative compound.
Sources:
Green leafy vegetables like cabbage, lettuce, spinach etc.
Lntestinal bacterias also synthesize Vitamin K.
RDA:
50 - 100 µg / day (if synthesized by the intestinal bacteria)
1 - 2 mg/ day (if not synthesized by intestinal bacteria).

Vitamin K is absorbed in small intestine along with fats with the help of bile salts.
From the intestine, vitamin K is transported to liver by chylomicrons. Vitamin K is
mainly stored in the liver. From liver, vitamin K is transported to other tissue by LDL
Biochemical functions :
Vitamin K helps in coagulation process:
Vitamin K is required for the synthesis of active form of some blood clotting factors
(factor II, VII, IX and X). All these blood clotting factors are synthesized in inactive
precursor forms in the liver. These inactive blood clotting factors are converted to
active form by y -carboxylase enzyme (by the process of y carboxylation of some
glutamic acid residues). Vitamin K acts as a co-factor for these carboxylase enzymes.
Since vitamin K is helps in coagulati on, it is referred to as anti-hemorrhagic vitamin.
Inactive blood clotting factors y Carboxylase 11
Active blood clotting factors
(Factor II, VII, IX, X) (Vitamin K) (Factor 11, VII, IX, X)
Dicumarol and warfarin are 2 antivitamins (antagonists) of vitamin K: These are

structural analogues of vitamin Kand competitively inhibit y-carboxylation system of
blood coagulation (i.e. they decrease the availability of vitamin K for blood coagulation).
So, they act as anticoagulants. Warfarin is used in the treatment of thrombotic diseases.
Deficiency of vitamin K:
• Dietary deficiency of vitamin K lead to hemorrhage as vitamin K is required for
coagulation. This forms the basis of internal bleeding in vitamin K deficiency. It causes
prolonged prothrombin time, delayed clotting time and bleeding time.
• Vitamin K deficiency also seen in cholestasis (due to block of bile salts transport to
intestine, which is required for vitamin K absorption) and prolonged antibiotic therapy
(as it kills the intestinal flora, which produces vitamin K).

Vitamins 344
Vitamin C:
Chemical name of vitamin C is ascorbic acid. It has reducing property.
Sources:
• Rich sources: Citrus fruits (Orange, lemon etc), gooseberry (Amla), guava.
• Good sources: Green leafy vegetables, tomatoes, potatoes (particularly skin).
• Poor sources: Meat, milk, fish.
RDA:
• Adults require 40 mg/day
• Requirement increases to 80 mg/ day during pregnancy and lactation.
1. Collagen formation:
Vitamin C is required for the conversion of inactive protocollagen to active collagen.
Vitamin C is required as a cofactor in lysyl hydroxylase and prolyl hydroxylase enzymes,
which converts (post-translational modifications) certain lysine and proline residues of
protocollagen to form hydroxylysine and hydroxyproline which are required for the
formation of active collagen. Hydroxylysine and hydroxyproline are essential for the
collagen cross linking and tensile strength of fibre.
Protocollagen H ydroxylase
Vitamin C
.. Collagen
Collagen is the most abundant structural protein in the body. It is present in ground
substances of connective tissues, bone, dentine, capillaries and scar tissues. Thus,
collagen is required for the maintenance of connective tissues, proper development of
bone, teeth and wound healing process. So, vitamin C is supplemented along with
proteins in post-operative patients to facilitate wound healing & tissue repair.
2. Norepinphrine synthesis: Vitamin C is required for the synthesis of norepinphrine

from dopamine by dopamine beta-hydroxylase.
Dopamine !3--hydroxylase
Dopamine - - - - - - - - - - - -Norepinephrine
Cu•2, Vitamin C
3. Bile acid synthesis: Vitamin C is required for the initial 7 a-hydroxylase step in the
synthesis of bile acids from cholesterol.
7 a-hydroxylase
Cholesterol 7a-hydroxycholesterol --.--.--. Bile salts
Vitamin C
4. Role in steroid hormone formation in adrenal cortex: Vitamin C is found in large
amounts in adrenal cortex, believed to be required for the synthesis of steroid hormones.

Vitamins 345
5. Degradatio n of tyrosine: Vitamin C is required in the degradatio n of tyrosine for the

oxidation of p-hydroxy phenylpyru vate by p-hydroxyphenylpyru vate oxidase enzyme.
p-hydroxyphenylpyruvat e oxidase
p-Hydroxyp renylpyruva te Homogentis ate
6. Antioxidan t function: Vitamin C is a natural antioxidant. Vitamin C reduces the

formation of nitrosamine (a carcinogenic oxidant substance) in the intestine.
7. Reduces Cataract formation: Vitamin C reduces the risk of cataract formation.
8. Role in iron absorption : Iron can only be absorbed in fe+2 form. Vitamin C facilitates
iron absorption by reducing fe+3 form to f e+2 form. Thus vitamin C is required for iron
metabolism and thus erythropoiesis.
9. Regeneration of hemoglobin: Vitamin C facilitates the reconversion of methemoglobin
to hemoglobin.
10. Beneficial effect on common cold : Vitamin C, in high doses, appears to decrease
the severity and duration of common cold.
Deficienc y disorder:
Deficiency of vitamin C causes 'Scurvy'.
Symptoms:
Scurvy is mainly due to defective collagen synthesis. It is characterized by,
• Sore, Spongy, Swollen and bleeding gums, Loose painful teeth
• Fragile capillaries and leading to tendency to bleed under slight pressure.
• Hemorrhag e
• Microcytic anemia
• Delayed wound healing
• Aching muscles
• Weakness
• Swollen joints
• Bone fragility, easy fracture of bones
Reasons:
• Vitn111i11 C deficiency lends to nb11or111nl collagen formatio11 and wenk interce/111/nr cement materin/.
So, capillnries become Jrngile, leading to tendency to bleed eve11 under slight pressure. Tltis is tlze
reason why g11111s bleed d11 ri1Lg scurvy. Gu111s beco111e pai11f11l, swollen, purple and c;pongy. The pulp
is separnted Jro111 the dentine nnd fi11nlly teeth are lost.
• ln the bones, vitamin C deficiency results i11 thefailure of osteoblasts to form the 11or111nl i11tercell11/ar
cement material. This result in frngile bones and bones frncture ensily.
• Vitnmin C deficiency nlso causes microcytic anemia. This is /Jecn11se vitamin C is required for iron
1flbsorpt1011 by reducing Fe+3 to Fe+2 a11d also iron loss due to excessive bleeding.
• He111orrlinle in vitamin C deficienclJ is due to excessit•e /Jleedi11R.

Vitamin s 346
Thia mine (Vita min B1) :
Chemistry:
joined
Thiami ne consists of substit uted thiazole ring and substit uted pyrimi dine ring
by methyl ene bridge. Thiami ne is a sulfur contain ing vitamin .
Sources:
Rich source s: Dried yeast, wheat germ, unpoli shed whole grains of rice.
Good sources: Legum es (beans, peas), liver and whole grains of wheat,
oats.
Fair source s: Meat, eggs, fish, milk, vegetables and fruits.
Note:
of cereal
• Thiami ne is presen t in outer aleuron e layer (bran) of cereals. So, refining
g of paddy
grains in mills destroy s thiamin e during polishing. But, in parboi led (boilin
with husk), the thiamine is not lost in polishing.
• Thiami ne is lost d uring washin g of cereals.
results
• Boiling of rice in excess water and discard ing the water kanjee (or Conjee) also
in loss of thiamine.
• Thiam ine is also destroy ed during baking (Cooking with baking soda).
Recom mend ed daily allow ances

1 to 1.6 mg/ day for adult male.
0.9 to 1.2 mg/ day in adult females.
Requir ement increases during pregna ncy and lactation (upto 2 mg/ day).
.
Thiam ine require ment is dependent on calorie intake (carbohydrate intake)
metabo lism (as TPP
TPP, a coenzy me of thiamin e plays a central role in carboh ydrate
ogenas e,
is the coenzyme require d for pyruva te dehydrogenase, cx-ketoglutara te dehydr
metabo lism). So, thiamin e require ment
transketolase, the enzymes required for glucose
increases when carboh ydrate is intake is increased.
(0.5 mg of thiamin e is require d for every 1000 Kcal of energy).
Functions of Thiamine:
Coenzy me form of thiamin e is thiami ne pyroph osphate (TPP).
lism
TPP plays a central role in energy yieldin g metabo lism, especially in the metabo
ne is
of carboh ydrates . Thus, more the carboh ydrate is consum ed, more the thiami
n of HMP shunt pathwa y.
require d. TPP is also require d in transketolase reactio
m or 9986449575
For queries and suggestions, contact the author at prasad_text@yahoo.co
Vitamins 347
TPP act as a coenzym e in two types of reactions . These are;

1) Oxidativ e decarboxylation of ex -ketoacid s
2) Transke tolase reactions .
1) Oxidative decarboxylation of a -ketoacids:

Examples:
a) ex- Ketoglut arate dehydrog enase complex
a-Ketoglutarate dehydrogenase complex

a-Ketoglu rate - - - - - - - - - - - - - -• Succinyl CoA + COi
TPP, NAO, FAD, CoASH, Lipoic acid
b) Similarly, pyruvate dehydro genase (PDH) complex
2) Transketolase of pentose phosphate pathway requires TPP:
Xylulose 5-phosp hate Transke tolase Sedohep tulose 7-phosp hate

+ +
Ribose 5-phosp hate (TPP) Glyceral dehyde 3-phosp hate
3) TPP is also required for transmis sion of nerve impulses:
Thiamin e is particularly essentia l in the maintenance of 3 systems ,
• Nervous system: Thiamine is required for the proper functioni ng of nervous

system (Both periphera l and central). Rememb er brain derives all its energy from
carbohyd rates and utilizatio n of carbohyd rates requires thiamine. Thiamine is
also required for the transmiss ion of nerve impulse.
• Cardiovascular system: Thiamine is essential for the normal functioni ng of heart

muscles.
• GIT: Thiamine is essential for the normal maintena nce of GIT. It maintain s good
appetite and normal digestion .

75
Vitamins 348
Deficie ncy of thiamine:

Thiamine deficiency results in beri beri.
• Beri-beri:
Deficiency of thiamine leads to beri beri.
Four types of beri beri are seen; dry, w et and infantile beri beri.
i) Dry beriberi :
Symptoms:
• Neurolog ical manifesta tions are main features.
• Periphera l neuritis with burning and tingling sensation in the leg and feet,
• Anorexia (loss of appetite)
• Muscle wasting and loss of body weight.
• No edema is seen in dry beriberi.
ii) Wet beriberi:

Symptoms:
• Besides the above symptom s, edema and cardiovas cular manifesta tions are seen.
• Edema of legs, face and trunk are the main features.
• Enlargem ent of heart, palpitatio n, tachycard ia and death occurs due to heart failure.
iii) Infantile beri beri:

It is seen in children (between 2 to 5 months) born to thiamine deficient mothers. Breast
milk of mothers has low thiamine content and mother's milk is the only source of
thiamine for infants.
Symptoms:
• Restlessness and sleeplessness,
• Convulsi ons, vomiting
• Aloud, piercing cry that changes into a thin weak and almost inaudible voice.
• The affected baby develops cyanosis, dyspnoea and tachycard ia. If not treated, death
may occur suddenly due to cardiac failure.
• Wemick e's encepha lopathy (Wemic ke-korsa koff syndrom e):

This d isorder is seen in chronic alcoholics. Alcoholics generally consume less food and
derive energy from alcohol, which lead to insufficie nt provision of thiamine . Alcohol
also inhibits thiamine absorptio n.
Symptoms:
• Periphera l and central neurolog ical defects like severe loss of memory, nystagm us
(paralysi s of eye moveme nts), insomnia , cerebella r ataxia, abnorma l gait, mental
confusion , psychosi s, depressio n etc.
This condition is also called Cerebral beri beri.

Vitamins 349
Riboflavin (Vitamin B} :
Chemistry :
Riboflavin consists of an isoalloxazine ring attached to ribitol.
Sources:
• Rich sources: Dried yeast, milk powder, liver.

• Good sources: Milk, meat, egg, fish, Whole cereals, legumes.
• Fair sources: Milled cereals, meat, fish, roots, tubers and fruits.
Even though cereals and pulses are relatively poor sources of riboflavin, they provide
about 75% of riboflavin requirement of the body because they are consumed in large
amounts.
Recommended daily allowances:
1.5 mg / day. (i.e about 0.6 mg of riboflavin is required for every 1000 Kcal of energy).
1.9 mg / day in pregnancy and lactation (additional 0.4 mg/ day is needed).
Functions of riboflavin:
Riboflavin has two coenzyme forms;
a) FMN (Flavin mononucleotide)

b) FAD (Flavin adenine dinucleotide)
• Riboflavin coenzymes participate in many redox reactions in energy production

reactions of carbohydrates, lipids and proteins.
• Riboflavin coenzymes participate in tissue protein building and cell respiration.
• Riboflavin coenzyme also help in the conversion of tryptophan to niacin.

Vitamins 350
a) FMN dependent enzymes:
i) L- Amino acid oxidase

L - ex Amino acid ---/""'\:-""?""~---+• ex- Keto acid + NH3
FMN FMNH2
ii) Other examples are NADH dehydrogenase (Complex I of ETC) and cytochrome C
reductase of ETC.
b) FAD dependent enzymes:
i) D-amino acid oxidase:
O-ex Amino acid

______..,'\
____•
D - Amino acid oxidase
ex-Keto acid +NH3
7 FADH2
FAD
ii) Succinate dehydrogenase:

Succinate d ehydrogenase
Su ccinate ___ __::::--~:::--------+• Furn era te
FAD
7
iii) Other examples are Pyruvate dehydrogenase, Acyl CoA dehydrogenase etc.
Note: FAD is converted to FADB:i during the reaction. FADH2 gives two ATPs in
electron transport chain.
Deficiency:
Deficiency of riboflavin causes ariboflavinosis.
Symptoms are;
• Glossitis (Soreness of tongue and magenta colored tongue)
• Cheilosis (Fissuring of the lips)
• Angular stomatitis (Fissuring at the corners of mouth)
• Seborrhic dermatitis (Inflammation of skin)
• Corneal vascularization (Reddening of eyes)
• Photophobia (Redness and burning sensation in eyes)

Vitamins 351
Niacin (Vitamin B) :
Chemistry:
Niacin is the generic term to represent nicotinic acid and its amide nicotinamide.
Both have equal biological activity. They have pyridine ring.
Sources:
Peanuts, liver are the rich sources. Yeast, legumes, whole grains, green vegetables,
meat are good sources.
Amino acid tryptophan can produce niacin in the body.
1 mg equivalent of niacin can be generated from 60 mg of tryptophan.
Recommended daily allowances:

Adult 20 mg/ day. Requirement increases during pregnancy and lactation (25 mg).
(6.6 mg of niacin equivalent is required for every 1000 Kcal of energy).
Since, tryptophan can give niacin in the body, total niacin available from food is
given by the formula,
Niacin equivalent = Niacin content (mg) + Tryptophan content (mg)

60
Niacin has two coenzymes forms:

a) NAO+ (Nicotinamide Adenine Dinucleotide)
b) AD p+ (Nicotinamide Adenine Dinucleotide Phosphate)
Both NAO and NADP function as coenzymes in many oxidoreductase reactions.
• Niacin coenzyme forms participate in redox reactions in energy metabolism

(carbohydrates, lipids and proteins). NAO+ receives the reducing equivalents
from energy rich compounds and transfers it to electron transport chain of
mitochondria in the prod uction of ATP (Oxidative phosphorylation).
• Reduced NADP+(i.e. NAOPH + H+) is required for the biosynthesis of fatty
acids, cholesterol, steroid hormones etc.
• Niacin coenzymes also promote normal functions of skin, gastro intestinal tract
and nervous system.
• NAD is also required for repair of UV -light damaged DNA in areas of exposed
skin. (This function is not through redox reactions).

Vitamins 352
a) NAD• dependent enzymes: E.g.: Glyceralde hyde 3-phospha te dehydroge nase

Glyceraldehy de 3 - P - dehydrogena se
Glyceraldehy de 3-phosphate • l , 3 Bisphosphoglycerate
NAO-+ ADH + H +
Other examples are malate dehydroge nase, pyruvate dehydroge nase etc.
b) NADP+dependent enzymes: E.g.: Glucose 6 -phosphate dehydroge nase

Glucose 6 -phosphate
dehydrogena se
Glucose 6 -phosphate • 6 -phosphogluconolactone
NADP• NADPH+ H•
Other examples are 6-phospho gluconate dehydroge nase, malic enzyme etc
Fates of NADH + H• and NADPH + H+: During the reactions, NAO• is converted to
NADH + H +, whereas NADP is converted to NADPH + H +
. a) NADH + H•enter electron transport chain and give 3 ATP's.
b) NADPH + H + are required for biosynthes is of fatty acid, cholesterol etc.
Deficiency:
iacin deficiency leads to the clinical condition called Pellagra.
Symptoms :
Pellagra is commonly referred to as disease of three D's, Dermatitis, Diarrhea, Dementia.
If not treated, it may lead to 4th D i.e. death.
i) Dermatitis : Erythrema is skin exposed to sunlight - feet, ankle, neck and fingers.
Increased pig mentation around the neck is known as Casal's necklace.
ii) Diarrhea: Occurs m a inly due to the inflammati on of muco us membrane of the GIT.
Diarrhea may often contain blood & mucous. Prolonged diarrhea leads to weight loss.
iii) Dementia: Neurologic al symptoms like depression , delirium, irritability and memory
loss is frequently seen in chronic cases. Ataxia and spasticity are also seen.
• Pellagra is common among maize eaters, because in maize niacin is present in bound
form & unavailabl e for absorption . Also tryptophan content is very low in maize
proteins (Zein) and leucine content is high, which depresses the synthesis of tryprophan .
• Pellagra like symptoms is also seen hartnups disease & carcinoid syndrome (due to less
availability of tryptophan for niacin coenzyme formation) and in , itarnin 8 0 deficiency &
isoniazid (INH) treatment in tuberculosis (due to failure of PLP formation, which is required
to covert tryptophan to niacin coenzymes.
Vitamins 353
Pantothenic acid_(Vitamin B5)
Chemistry :
Pantothenic acid consists of pantoic acid and ~-alanine held together by an amide bond.
Sources:
Widely distributed in nature as the name suggests (pantothene means everywhere).
Liver, meat, milk, dried yeast, whole cereals, legumes are good sources. It is also
synthesized by bacterial flora in the intestine.
RDA:
10 mg / day.
Pantothenic acid has two active forms;
1) Coenzyme A (CoASH) and 2) Acyl Carrier Protein (ACP)
Both contain 4· -phosphopantetheine, which is formed from p antothenic acid.
1) Coenzyme A (CoASH):
Pantothenic acid is the constituent of Coenzyme A, which is central molecule involved
in all metaboli sms (Carbohyd rate lipid and amino acid metabolism ). Some of the
important CoA derivatives are acetyl CoA, propionyl CoA, succinyl CoA, H MG CoA,
malonyl CoA and fatty acyl CoA.
CoASH functions as an acyl carrier. Acyl groups are linked to CoASH by a thioester
linkage to give acyl CoA.
2) Acyl Carrier Protein (ACP):

Acyl carrier protein carries 4'-phosphopantetheine formed from pantothenate.
ACP is a component of fatty acid synthase complex and is involved in the synthesis
of fatty acids.
Deficiency:
Deficiency of pantothenic acid is rare because it is widely distributed in foods.
Burning foot syndrome in prisoners of war has been ascribed to pantothenic acid
deficiency.
Pantothenic acid deficiency leads to decreased de-novo synthesis of fatty acids. This is
because, pantothenic acid is required for formation of ACP, a component of fatty acid
synthase complex.

Vitamins 354
Vitami n B6 (Pyridoxine, Pyridoxal and Pyrido xamine ):

Chemistry:
Vitamin B6 is a collective term to represent three related pyridine derivativ es;
a) Pyridoxine b) Pyridoxal c) Pyridoxamine. All have equal biological activity.
Sources :
Rich sources are dried yeast, rice polishing, wheat germs, cereals, liver, legumes
(pulses), oilseeds, egg, milk, meat, fish and vegetables.
RDA:
About 2 mg / day. Requirem ent increases in pregnanc y, lactation and infancy.
Biochemical function s :
Pyridoxa l phosphat e (PLP) is the coenzym e of Vitamin B6. PLP requiring enzymes are,
1) Transamination reactions of amino acids (by transaminases):
Transam inase enzymes require PLP as coenzym es. E.g.: ALT, AST etc.
Alanine+ a-KG Alanine transamin ase (ALT) Pyruvate + Glutamate
(PLP)
2) Decarboxylation of amino acids (by amino acid decarboxylase enzymes:

PLP acts as a coenzym e for amino acid decarbox ylaseenzy mes, w hich decaroxy lates
various amino acids to form biogenic amines (such as Histamin e, Serotonin , GABA etc).
For example, histidine decarbox ylase enzyme produces histamin e from histidine.
Histidine Decarboxylase Histamine
PLP °'4
COi
3) Muscle glycogen phosphorylase requires PLP.

4) Non-oxid ative deamination of serine, threonine cysteine & histidine requires PLP.
5) ALA synthase, cystathione P-synthase, Kynureninase, cystathione y-lyase also require PLP.
6) PLP is also required for the synthesis of sphingol ipids and formatio n of myelin.
Deficien cy of pyridoxine (Vitamin BJ

Neurolog ical symptom s such as depressio n, irritabilit y, periphera l neuritis (due to
decrease d neurotran smitter synthesis like GABA, serotonin , catechola mines & myelin
forma tion failure), microcyt ic anemia (due to failure of heme synthesis ). Pellagra like
symptoms are also seen (as PLP is needed for niacin coenzyme synthesis from tryptophan).
• lsoniazid (I H), an antituberculosis drug, inhibits pyruvate kinase required for PLP
formation from vitamin 8 6 and causes vitamin Bb deficiency (causes pellagra like symptom s)
Vitamin B6 requirement is dependen t on protein intake: Coenzyme of vitamin 86, PLP is

required for transamination, deamination, decrboxylation, thus, plays a central role in amino
acid metabolism. So, vi tamin Bi; requirement increases when protein is intake is increased.
Vitamins 355
Biotin (Vitamin B7) :
Chemistry:
Biotin is an imidazole derivative. lt is a sulphur containing vitamin.
Sources:
Yeast, Liver, legumes (pulses), egg, milk, meat, fish.
Intestinal bacterias also synthesize biotin.
RDA:
20 to 30 µg / day
Coenzyme form of biotin is biotin itself.
Reactions requiring biotin as coenzymes : Carboxylation reactions require biotin as

coenzymes .
Example: Acetyl CoA carboxylase

Acetyl CoA+ COi ---~----::=---sc:.::::::::---=--------.~ Malonyl CoA
/ Mn++, Biotin........._.
ATP ADP+Pi
Other examples are propionyl CoA carboxylase and pyruvate carboxyla e etc.
Deficiency:
Rare, as they are widely distributed in foods and synthesize d by intestinal bacteria.
In experimen tal animals, deficiency produces anorexia, depression , insomnia, muscle
pain, dermatitis etc.
High consumpti on of raw eggs also can lead to biotin deficiency.
Note:
The egg white contains a glycoprotein avidin. Avidin is an antivitamin of biotin. It
,combines very tightly with biotin and prevents its absorption and inducing biotin
deficiency. Eating large amount of raw eggs for a long time can cause biotin deficiency,
which is called egg white injury.
Heating denatures avidin (as avidin is a protein), eliminating its biotin binding capacity.
But, it has been estimated that 20 raw eggs per day would be required to induce a
deficiency syndrome. Inclusion of an occasional raw egg in diet does not cn11se biotin
deficiency.
Vitamins 356
Folic Acid (Vitamin B9) :
Chemistry:
Folic acid consists of 3 components: Pteridine ring, PABA and glutamic acid.
Sources:
Green leafy vegetables, yeast, liver, eggs, whole grains etc.
Milk is a poor soUice of folic acid.
RDA:
200 µg/ day. Requirement increases during pregnancy and lactation.
Coenzyme form of folic acid is tetrahydrofolate (THF or FHJ
Tetra hydro folate (THF) is required for one carbon metabolism:

One carbon compounds are substances that contain only one carbon atom. Eg, methyl,
methylene, Metheny}, Formyl and formimino. Tetrahydrofolate (THF) acts a carrier
of these one carbon compounds and serves as an acceptor or donor of these 1-carbon
compounds in various reactions, which are collectively called 1- carbon metabolism.
Many important compounds are synthesized in 1 carbon metabolism.
1 Purine (C2 and C8 of purine rings), which are incorporated in to DNA and RNA
2. Deoxy thymidylic acid (dTMP)- a pyrimidine nucleotide present in DNA
3. Synthesis of Glycine, Serine, Ethanolamine, Choline, Methionine, SAM
4. Synthesis of N-formylmethionine, the 1st amino acid added dUiing protein synthesis.
Other functions of folic acids:

1) Folic acid reduces the level of homocysteine in blood and therefore helps in
preventing heart diseases.
2) It prevents birth defects (fetal malformations such as spina bifida).
Deficiency:
Deficiency of folic acid causes megaloblastic anemia.
Explanation: Folic acid is required for the synthesis of D A required during cell division.
Deficiency of folate causes impaired cell division. Cells which undergo rapid cell division
(like RBC's, Intestinal cells) are particularly more sensitive to folate deficiency.
Folate deficiency delays DNA synthesis, but hemoglobin synthesis is continued in RBC
precursors. The cytoplasm is welJ developed, but nucleus is not developed, resulting in the
formation of large and immature RBC called the macrocytes or megaloblasts. These abnormal
megaloblasts are released into the circulation and are rapidly destroyed in spleen resulting in
anemia, which is referred to as megaloblastic anemia.

Vitamins 357
Role of Folic acid in One carbon metabolism:

• Synthesis of some compounds involve receiving or donating one-carbon groups such
as methyl (-CH), methylene ((=CH2), methenyl (-CH=), formyl (-CH=O), and
formimino (-CH=HN) etc. The reactions involving one-carbon groups are collectively
known as one-carbon metabolism.
• Tetrahydrofolate (THF or FH4) is actively involved in one carbon metabolism; it acts a
carrier of the one carbon groups. They can receive and donate these one-carbon groups
in various reactions. Different forms of THF carrying one-carbon units are,
i) N 10 -Formyl THF or N s -Formyl THF
ii) Ns, N 10-Methenyl THF
iii) N5, N 10 -Methylene THF
iv) N s -Methyl THF.
• Methionine and cobaJamine are also involved in one-carbon metabolism.
• Some compounds donate 1-carbon groups to the one-carbon pool and some
compounds accept one-carbon groups from one carbon pool.
One-carbon donors: Tryptophan, Histidine, glycine, serine, choline, and formate
donate 'one carbon' groups to THF
One-carbon utilization: These one-carbon units are utilized in the formation of
C2 and C8 of purine, formyl methionine, TMP, Glycine, Serine, Methionine etc.
Sources of One-carbon carriers Compounds formed from

One-carbon units one-carbon utilisation
Formyl-methionine
Formate 10-Formyl THF --------- Purines (C2)
CO:i
l!
I listidine -+ N5 Formimino THF-+ Ns, Nto-Methenyl THF --+ Purines (CS)
Serine l! / Serine
Glycine
Choline
} N5, N HLMethylene THF
----=:::; ~ine
l!
N5-Methyl THF Methionine
Schematic representation of 1-carbon metabolism SAM
Antagonists of folic acid:

Aminopterin & amethopterin (Methotrexates) are structural analogues of folic acid.
They inhibit dihydrofolate reductase enzyme & formation of tetrahydrofolate (FH4 ),
which are required for cell division and multiplication. So, aminopterin & amethopterin
(antagonists of folic acid) are used as anti-cancer drugs, especially leukemia.

Vitamins 358
Cobalarnin (Vitamin B12) :
Chemistry:
Vitamin 8 12 consists of a corrin ring with a central cobalt atom. It is found as
methylcobalamin, adenosylcobalamin and hydroxocobalamin in animals.
Sources:
It is not present in vegetables. It is found only in foods of animal origin.
Milk, pork, chicken, liver is the richest source. Meat, fish, egg are good sources. Curd
is a good source, because lacto bacillus can synthesize B12.
RDA:
1-1.5 µg /day.During pregnancy and lacta tion it is 2 µg / day.
Absorption, transport and storage of vitamin Bu

• The absorption of Vit B12 requires the mediation of intrinsic factor (IF).
• IF is a glycoprotein secreted by the gastric parietal cells. One molecule of IF can
combine with 2 molecules of Vit B12..
• This IF- 8 12 complex gets attached to specific receptors on intestinal mucosa! cells and
is then internalized. The IF is digested inside the mucosa] cell and 8 12 is con verted to
methylcobalamin. This is then transported in the blood bound to a glycoprotein
transcobalamin -II.
• When in excess, m e thylcobalamin is taken up by the liver, converted to
deoxyadenosylcobalaminand stored in combination with transcobalamin-I (transcorrin).
Liver can store about 2 mg of vit. B12, which is sufficient to meet the body requirement
for 2-3 years (making it the only water soluble vitamin that can be stored).
Biochemical Functions :
There are 2-coenzyme forms of Vit 812
1) Deoxyadenosykobalamin
2) Methylcobalamin
i) Reaction requiring Deoxyadenosylcobalamin as coenzymes:

Conversion of L-meth ylmalonyl CoA to succinyl CoA (a TCA cycle intermediate), by
methylmalonya CoA mutase (or isomerase) requires deoxyadenosykobala.min.
methy lmalonyl CoA mutase
L-methylmalonyl CoA- - - - - - - - - - Succinyl CoA - - - TCA cycle
deoxya denosylcobalamin
Methylmalonyl CoA is obtained from propionyl CoA (from~ -oxidation of odd chain
fatty acids), va line, isoleucine, threonine, m~thionine, thymine, uracil etc.

Vitamins 359
i) Reaction requiring methylcobalamin as coenzymes:

Conversion of hornocysteine to methionine by methionine synthase (Homocysteine
methyl transferase) requires methylcobalamin as cofactor.
NS-methyl THF
'>-<THF
Cobalamin (VitB12) Methykobalamin
Methionine • Methionine synthase

Homocysteine
• This reaction explains the link between functions of folic acid and cobalamin.
• This step involves NS-methyl tetrahydrofolate and liberates tetrahydrofolate, which is
required for synthesis of purine, pyrimidine and nucleic acids and thus cell division.
• This reaction also produces methionine, which is required for myelin sheath formation
and proper functioning of nervous system.
Cobalamin deficiency causes functional folate defieciency - Folate trap:

Cobalamin is required for the conversion of N s-methyl tetrahydrofolate to
tetrahydrofolate by methionine synthase enzyme. In cobalamin deficiency, there is failure
to conversion of N 5-methyl tetrahydrofolate to tetrahydrofolate & entire body folate is
trapped as N 5-methyl tetrahydrofolate. This is known as folate trap.
Tetrahydrofolate pool is reduced a nd this lead to functional folate deficiency.
Deficiency symptoms:
1) Pernicious anemia (Megaloblastic anemia with neurological symptoms)
• Megaloblastic anemia: Cobalamin deficiency results in the folate trap and secondary
folate deficiency, which affects the cells that are dividing rapidly. Clinically this affects
bone marrow and erythrocyte formation, leading to megaloblastic anemia.
• Neurological symptoms: accumulation of Methylmalonyl CoA, which interferes with
myelin formation results causing demyelination of nervous system. Failure to methionine
formation, which is required for the synthesis of SAM and phospholipid synthesis in
myelin shea th formation also contribute to neurological symptoms.
2) Methylmalonyl aciduria
3) Homocysteinuria

Vitamins 360
B- Complex Vitamins, their Coenzymes and Deficiency symptoms

VITAMINS COENZYME FORMS DEFICIENCY
Berl Beri
I
! i l i
rhiamine Dry Wet Infantile Cerebral
Vitamin B1) TPP
(Thiamine pyrophosphate) General manifestations:
• Peripheral ne uritis with
burning, tingling sensation
in the leg and feet.
• Anorexia, Muscle wasting
• ausea, vomiting, headache
Ariboflavinosis
• Glossitis
• Cheilosis
1iboflav in i)FMN
• AnguJar stoma ti tis
Vitamin B2) ii) FAD
• Seborrhic dermati tis
• Corneal vasculariz.ation
• Photophobia
Pellagra
'1iacin i)NAD • Dementia
Vitamin 8 3) ii) NADP • Diarrhea
• Dermatitis
>antothenic add Coenzyme A Burning Foot Syndrome

Vitamin 85)
Microcytic anemia
'y,idorine }
>yridoxal Vitamin B6 PLP (Pyridoxal phosphate) and
.,yridoxamin Neurological symptoms
3iotin
Vitamin 8 7)
Biotin --------
~olic Acid
Tetra hydro fol ate (FIL) Megaloblastic anemia
Vitamin 8 9)
:yano cobalamin i) 5-deoxyadenosyl cobalamin Pernicious anemia and

Vitamin Bu) ii) Methyl cobalamin Methyl rnalonyl aciduria

Vitamins 361
Question Bank on vitamins

Long Esays (10 Marks):
1) Describe the sources, RDA, functions, deficiency manifestation, toxicity of vitamin A
2) Describe the sources, RDA, functions, deficiency manifestation, toxicity of vitamin D
3) Describe the sources, RDA, functions, deficiency manifestation of vitamin C
4) Describe the sources, RDA, functions, deficiency manifestation of thiamine
Short Essays (5 Marks):

1) Write a short essay on Vitamin E
2) Write a short essay on Vitamin K
3) Coenzymes & functions of any of B-complex vitamin
Short Answers (3 Marks) :

1) Provitamins / Antivitamins
2) Any one of the the vitamin deficiency and toxicity d iseases
3) Vitamin E has selenium sparing action. Explain

1) Which of the following is a Water Soluble Vitamin? (AI)
a) Folic Acid b) Vitamin A c) Vitamin K d) Linolenic Acid
2) Which Vitamin is required for carboxylation of clotting factors? (AI, AIIMS)
a) Vitamin A b) Vitamin D c) Vitamin E d) Vitamin K
3) Consumption of raw egg may lead to deficiency of (COMEDK)
a) Biotin b) Riboflavin c) Thiamine d) Avidin
4) The activity of the following enzyme is affected by biotin deficiency (AIIMS)
a) Transketolase b) Dehydrogenase c) Oxidase d) Carboxylase
5) Vitamin necessary for CoA synthesis (AI)
a) Pantothenic acid b) Vit C c) Thiamine d) Biotin
6) Vitamin required for maturation of collagen is
a) Ascorbic acid b) Folic acid c) vitamin A d) vitamin K
7) Lack of intrinsic factor causes deficiency of
a) Cobalamine b) Folic acid c) Biotin d D) Vitamin D
8) Which is the following vitamin is required for DNA synthesis?
a) Vitamin C b) Thiamine c) Riboflavin d) Cobalamine
9) VVhich of the vitamin is responsible to keep blood homocysteine levels within normal limits?
a) Cobalamin b) Folic acid c) Pyridoxine d) All of these
10) Pyridoxal phosphate is a cofactor in all the following reactions EXCEPT
a) Decarboxylation b) Deamination c) Transamination d) Dehydrogenation
Answers for MCQ: 1) a 2) d 3) a 4) d 5) a 6) a 7) a 8) d 9) d 10) d

Minerals 362
Minerals
Contents:
• Definition, Classification
• Discussions of minerals in terms of

• Recommended daily allowances (RDA)
• Dietary sources
• Functions
• Absorption, synthesis, metabolism, storage and excretion
• Deficiencies
• Overconsumption and toxicity

Minerals 363
Minerals
Minerals are inorganic substances obtained from earth. Minerals account for about 4%
of body weight. Minerals (like vitamins) do not yield energy, but are required for growth,
maintenance, repair and regulation of vital body functions.
Essential minerals: Out of 54 minerals found in earth, only a few minerals (about 25)
are present in the body. These minerals are called essential minerals.
Based on the daily requirements, the essential minerals are divided into 2 groups,
Major elements (or macro minerals):

If the daily requirement of a mineral is more than 100 mg per day, then those minerals
are called major elements or macro elements. Macro elements include,
• Calcium
• Magnesium
• Phosphorus
• Sodium
• Potassium
• Chloride
• Sulphur
Trace elements (or micro minerals):

If the daily requirement is less than 100 mg per day, then those minerals are called trace
elements or micro minerals. Trace elements include,
• Iron
• Copper
• Manganese
• Zinc
• Iodine
• Molybdenum
• Selenium
• Cobalt
• Fluoride
Trace contaminants with no known functions:

These are included under the trace elements, but their exact functions are not known.
These include lead, mercury, barium, boron, aluminum, bromine etc.
Electrolytes: Sodium, potassium, magnesium, calcium, chlorine etc. are single mineral
elements that function as electrolytes. Electrolytes are inorganic substances (either single
mineral elements or compounds), that can dissociate and exists as charged particles
(either anions or cations) in the solution.

Minerals 364
Biological role of minerals (General functions of minerals):

Minerals have diverse physiological and diverse biochemical functions.
1) Structural components:
• They the structural components of the bones and teeth.
E.g.: Calcium, phosphorous, magnesium, fluorine etc.
• They are also structural components of soft tissues such as liver, muscle, nerve cells
E.g.: Phosphorous, potassium, Iron, sulphur etc.
• Sulfur is a fundamental constituent of skin, hair, nails etc.
2) Fluid balance:
The volume and distribution of water in body and various body compartments are
determined largely by minerals like sodium, potassium, chloride and all the major
minerals (Calcium, phosphorous, magnesium)
3) Acid base balance:
Minerals like sodium, phosphorous are involved in acid base balance of the body.
4) Nerve cell transmission and muscle contraction:
The exchange of sodium and potassium across the nerve cell membrane causes the
transmission of nerve impulses. Calcium stimulates the muscle contraction. Sodium,
potassium and magnesium are involved in muscle relaxation.
5)Blood coagulation:
Calcium is invoived in blood coagulation and hemostasis.
6) Vitamin, enzyme and hormone activity:
Minerals are integral part of many important compounds of biological importance
• Iron is the constituent of hemoglobin and many heme containing compounds.
• Cobalt is the constituent of vitamin B12 (Cobalamine).
• Iodine is the component of thyroid hormones
• Minerals like zinc, magnesium etc are components of many metalloenzymes.
• Minerals also function as enzyme activators.
• Chromium is involved in insulin production.
Note: Different minerals have specific physiological and biochemical functions. The
individual min era ls are discussed in detail in the following segment.
Answer hint for Essay question on Minerals (10 Marks):

a. Distribution, if asked (1 Marks)
b. Sources (1 Marks)
c. RDA (1 Mark))
d . Absorption (1-2 Marks)
e. Functions (4-5 Marks)
f. Deficiency (2 Marks)
g. Excess, if any (1 Marks)

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

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

Minerals 366
Factors decreasing calcium absorption:

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

3) Muscle contraction: Calcium is required for the excitation and contraction of m uscle
fibres.
4) Transmission of nerve impulse: Calcium is required for the transmission of nerve
impulses in the synapses (pre-synaptic to post-synaptic region).
5) Neuromuscular excitability: Calcium decreases neuromuscular excitability.

Minerals 367
6) Hormone action: Calcium acts as a secondary messenger to some hormones.

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

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

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

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

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

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

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

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

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

Minerals 370
• Phosphorus (P)
Distribution :
Adult body has about 1kg of phosphorus. About 85% of this phosphorous present in
combination with calcium in the bones and teeth; remaining 15 % is distributed in
various chemical compounds of the body. So, utilization of phosphorous is closely
associated with calcium.
Sources:
Milk is a good source. Eggs, fish, cereals, pulses, nuts, oil seeds, leafy vegetables, meat
and soft drinks are good sources.
Bioavailability of phosphorous from plant sources is much lower as they contain
phytates, which decreases phosphorous absorption. In cereals, phosphorous is present
as components of phytin, which is not available to body.
RDA:
Adults - 800 mg
Children - 1000 mg
Pregnancy and lactation-1200 mg
Absorption:
Phosphorous is mainly from jejunum. Calcitriol increases phosphorous absorption.
PTH also facilitates phosphorous absorption.
Phosphorous absorption is influecnce by calcium: Phosphate ratio in diet:
The optimum ratio of calcium to phosphate for maximum absorption is 1:2 to 2:1 (as present
in milk). If the amount of either calcium or phosphate exceeds double the amount of the
other, then insoluble complex will be formed which will not be absorbed. Ideal ratio is 1:1.
Storage:
Phosphorous is mainly stored in bones, teeth and muscles.
Excretion:
Abou t 500 mg of phosphate is excreted in the urine per day. A sma ll amount of
phosphate is also excreted in feces.
Functions:
l. Formation of bone and teeth (along with calcium) as a structural component.
2. Production of high energy phosphates such as ATP, GTP, creatine phosphate etc.
Energy is released when these compounds are hydrolyzed.
3. Synthesis of nucleoside coenzymes such as NAO, NADP.
4. Nucleic acid synthesis i.e. DNA and RNA, where phosphodiester linkages form
the backbone of the structure.
5. Formation of phosphate esters such as glucose 6-phosphate, phospholipids and
phosphoproteins etc.
Minerals 371
6. Enzyme activation: Many enzy m es get activated and inactivated by

phosphorylation and dephosphorylation (reversible covalent modification).
(E.g.: Glycogen p hosphorylase enzyme is active in phosphorylated form and
inactive in dephosphorylated form).
7. Phosphate buffer system is an important buffer system of blood and cells.
Dietary deficiency of phosphorous:

Phosphorous is present in practically all the foods. So, dietary deficiency of
phosphorous rarely occurs.
Deficiency of phosphorous can occur during vitamin D deficiency, consumption of
large amounts of antacids (which interfere with phosphorous absorption).
Deficiency of phosphorous may produce osteomalacia, growth retardation etc.

Dietary excess is unlikely to reach toxic amounts. However, individuals receiving
high phosphorous intake than calcium, may develop bone resorption.
Blood phosphorous level and factors regulating it:

Normal serum level in adults is 3 - 4 mg/ dl. Children is 5 - 6 mg / dl.
Haemolysed blood specimens are not used for phosphate estimation. Because RBC has
high intraconcentration of phosphate. RBC lysis releases phosphate into plasma and
gives a false higher value than the true value.
Factors affecting the blood level:
1) Vitamin D causes release of phosphate from bones (by activating alkaline phosphatase),
which increases serum phosphate level.
2) PTH decreases reabsorption of phosphorous from the renal tubules, and hence
increases phosphorous excretion and decreases serum phosphate level.
Clinical aspects:
i) Hypophosphatemia:
• Definition: When blood phosphate level decreases less than 3 mg/ dl condition is
called is called h ypophosphatemia.
• Causes : Deficie ncy of Vitamin D (Due to decreased demineraliza ti on),
Hyperparathyroidism (d ue to increased excretion of phosphate by kidney).
ii) Hyperphosphatemia:
• Definition: When blood phosphate level Increases more tha n 4 mg/dl condition is
called is called hyperhosphatemia.
• Causes: Hypervitaminosis D, Hypoparathyrodism, Renal failure.

Minerals 372
• Magnesium (Mg)
Distribution :
Total magnesium content of body is about 25 g, about 60 % of which is present in
bones. Magnesium is one of major intracellular cation. ormal level of magnesium
ICF is 5 mEq/ L. Normal p lasma level of magnesium is 2 - 3 mg/ dl.
Sources:
Green leafy vegetables, milk, meat, sea foods, cereals, nuts, beans, fruits.
RDA:
Adult male - 350 mg per day.
Adul t female - 300 mg per day. Requirement increases during lactation.
Absorption:
Magnesium is absorbed from intestine with the help of a specific carrier. Increased
amounts of calcium, phosphate decrease the absorption of magnesium.
Net absorption of magnesium in a typical diet is about 50%.
Function:
• Constituent of bone and teeth: Magnesium is an importan t constituent of bone and
teeth (About 16 gm present in bone).
• Magnesium serves as a cofactor for many enzymes: E.g. Hexokinase, fructokinase,
PFK, enolase, alkaline phosphatase, adenylate cyclase, ALA synthase etc.
• Magnesium is required for proper neuromuscular function: Magnesium decreases
neuromuscular excitability. Magnesium along with calcium acts as acts as a relaxant
during activity, after contraction.
• Magnesium has a role in insulin sensitivity. So, magnesium deficiency leads to
decreased insulin dependent uptake of glucose. Magnesium supplementation
improves glucose tolerance.
• Magnesium activates the amino acids for protein synthesis and facilitates the
synthesis and maintenance of genetic material, DNA.
Dietary deficiency of magnesium:

Magnesium is widely distributed in foods. So, dietary deficiency of magnesium is rare.
Even in chronic low intake of magnesium, the deficiency of magnesium manifested
only in combination with protein energy malnutrition, alcoholism, persistent diarrhea.
Deficiency symptoms include muscle weakness, cramps, hypertension etc.

Dietary excess of magnesium is unlikely to reach toxic amounts. However, individuals
with renal insufficiency receiving supplements may experience toxicity symptoms like
nausea, vomiting and diarrhea.

Minerals 373
• Iron (Fe)
Iron is the one of the important trace element in the body. Total iron content of the body
is about 3 - 5 grams.
Sources:
• Rich sources: Organ meats (Liver, heart, kidney), Jaggery.
• Good sources: Leafy vegetables, pulses, cereals, fish, dried fruits.
• Poor sources: Milk, Wheat, Polished rice, Potatoes etc.
Note: Tron in food exists in two forms; heme iron and non heme iron. Heme iron is rich in animal
sources (meat, fish, and poultry) and nonheme iron is predominantly found in plants (grain,
vegetables, legumes and nuts).
Indian subcontinent diet which is predominantly vegetarian diet has iron in non-heme form & has
iron absorption inhibitory substances like phytates, oxalates & phosphates etc. So, absorption of
iron is only about 5%. Western diet which is predominantly non-vegetarian diet has iron in heme
I form. So, iron absorption is >10%. These factors need to be considered while planning Iron RDA .
Daily requirement of Iron:

Man and post-menopausal woman : 10-20 mg / day
Premenopausal woman : 20-40 mg / day
Children : 15-30 mg / day
Pregnancy and lactation : 20-40 mg / day
Actual requirement of Iron is only 1-2 mg/ day. Since, only 5 -10% of the dietary Iron
is absorbed depending on the food sources, this allowance is made in the diet itself.
D igestion and absorption of iron:

Iron is absorbed mainly in the upper part of duodenum in the ferrous (Fe •2) form.
Factors influencing Iron absorption:
• Amount of Iron: More the amount of Iron in the food more is the absorption.
• Chemical form of iron: Iron is absorbed in Fe •2 form and not in Fe •1 form.
• Physical form of Iron: Herne iron is better absorbed than non heme Iron.
Dietary irons are two types.

i) Heme iron absorption: It is absorbed as such into intestinal mucosa) cells.
ii) Non-heme iron absorption: HCl in stomach releases the iron from non-heme proteins.
Cooking also facilitates this release. This iron is in Fe·3 form. But, iron can be absorbed
only in Fe-2 sta te and not in Fe•3 form. Fe•3 is reduced to Fe•2 form by vitamin C, cysteine,
glutathione and also by HCI. Fe· 2 is then absorbed into intestinal mucosa! cells.

Minerals 374
• Factors stimulating iron absorption:

1) HCI: Stimulates the iron absorption by releasing iron from Iron containing proteins.
3 2
HCl also help in the conversion of Fe + form to Fe + form.
2) Vitamin C, Cysteine, Glutathione : These stimulate Iron absorption by reducing
3 2
Fe+ form to Fe+ form.
3) Vitamin C, Amino acids: These stimulate Iron absorption by forming soluble salts
like iron ascorbate and Iron amino acid complexes with Iron.
4) Calcium: Calcium helps iron absorption by forming complexes with oxalates,
phosphates which are inhibitors of Iron absorption.
• Factors inhibiting iron absorption:

1) Phytates (present in cereals), Oxalates (present in green leafy vegetables) and
phosphates inhibit iron absorption by forming insoluble iron complexes.
2) Fatty acids also inhibit iron absorption.
3) Tannie acid of tea decrease iron absorption by forming insoluble Iron tannate.
Regulation of iron absorption:
Mucosal block theory:

Iron metabolism is unique as the iron homeostasis is maintained at the level ofabsorption and
not at the level of excretion. Iron is probably the only nutrient regulated in this manner.
Amount of Iron absorbed depends on iron status of the body. During iron deficiency conditions,
more iron is absorbed and during Iron overload conditions, Iron absorption decreases. This is
referred to as "mucosa[ block" of regulation of iron absorptio11.
Explanation: According to this, iron absorption is regulated by the availability of a protein,

apoferritin, in the intestinal mucosa/ cells. lron is absorbed in the form of fe+ 2• Inside the
mucosa/ cells, fe+ 2 is oxidized to Fe•3 and combines with apoferritin to form ferritin, which
functions as a temporary storage form of iron in intestinal mucosal cells.
From ferritin, iron is slowly released into the plasma as per the requirement of the body. (In
anemia, iron is completely transferred within 24 hours). As long as iron is within cell as
ferritin ,further absorptio11 is decreased due to unavailability of apoferritin.
If transferrin is saturated with iron, any iron accumulation in the form of ferritin in the
mucosa! cells is lost when intestinal cells are desquanzated. Thus, iron status of the body
decides the rate of iron absorption. In iron deficiency, more iron is absorbed and when iron is
in excess, absorption is reduced. This is the basis of mucosa[ block .

Minerals 375
Transport:
Transferrin :
Transferrin is the transport form of Iron in the plasma. It has 2 Iron binding sites which
can bind to only Fe•3 form of Iron. So, Iron which enters the plasma as Fe· 2 form is first
converted to Fe•3 forrn by ceruloplasmin (ferroxidase enzymes).
Iron absorbed from small intestine is mainly delivered to bone marrow for hemoglobin
synthesis, which is then incorporated into developing RBC's. Transferrin also delivers
iron to other tissues like liver, spleen, muscle etc for the synthesis of other iron containing
proteins.
Iron Utilization:
Iron is required by all the tissues of the body for heme, heme protein and non-heme
iron protein synthesis. This requirement of iron is more in bone marrows, where
develpoing erythrocytes draw iron for hemoglobin synthesis.
Storage:
Ferri tin and Hemosiderin are the storage proteins of Iron.
i) Ferritin: Iron is stored mainly in liver (96 %); spleen, bone marrow in the form of
Ferritin. (Also in mucosa I cells, where ferritin acts as a temporarily).
ii) Hemosiderin: Iron storage protein in liver and spleen, mainly in Iron excess
conditions. Hemosiderin accumulation is a sign of iron overload.
( Iron absorption, transport and storage )
Lumen of GIT Mucosa( cell s of GIT Plasma Tissues
Food Iron
¥"
on-heme heme Stora~e
iron iro n Apoferrtin Apotransferrin _,,..,,.- __. Liver & spleen
lc~~~ing \ rF%r!~~ l Ferro-

as Fe rritin &
Transferrin Hcm osid eri n
f -
( Fe '- ) , ~
_ .::..U..::.ti;;.;.Iiz;:;.;a::..:cti;.. ;;o.:. :.n_ _
fel + Fe1- reductase Fe'• Bone m a rrow &
l
Ascorbic acid
Cy!>teine
Glutathione
tr e rroxida se Fe:i\._
l Ceruloplasmin
or ferroxidase JI
o ther tissues for
synthesis of Hb, l\llb,
cy tochromes etc
Fe z• - ----w..,
. Fe z, _ _ _ _ _......;:,---+• Fe:i •
Minerals 376
Functions of Iron :
Physiological functions of iron are performed as iron containing proteins. In the body
there are two types of iron containing proteins.
1) Heme proteins ( Heme Iron )
2) Non-heme proteins (Non-heme Iron)
1) ) Heme proteins :
In h eme proteins, Iron combines with protoporhyrin IX to form heme, which then
combines with different globin molecules to form heme proteins .
a) Hemoglobin: Accounts for about 70% of total body Iron. Hemoglobin is an
important respiratory and buffer protein of blood.
b) Myoglobin: Accounts for about 05% of total body Iron. Mb is a Oxygen storage
protein, present in muscle.
c) Cytochromes ( b, c, c1 , aa3): Present in ETC, have a role in oxidative phosphorylation.
d) Cytochrome P450: Present in liver ,which has a role in detoxification of Xenobiotics.
e) Catalases: Destroy H 20 2.
f) Peroxidases like Myeloperoxidases of neutrophils which help in phagocytosis.
g) Heme contining enzymes like tryptophan pyrrolase, xanthine oxidase etc.
2) Non-heme proteins:
These proteins do not contain heme, but contains iron, which is tightly bound to
non-heme proteins.
a) Ferritin : Storage form of iron in spleen, liver. Contains upto 24% of total body Iron.
b) Hemosiderin : Storage form of iron in liver ( Mainly in excess conditions)
c) Transferrrin : Transport form of iron in plasma.
d) Iron Sulphur (FeS) Proteins/ enzymes: NADH dehydrogenase (complex I of ETC),
Succinate dehydrogenase, Iron-sulphur proteins of ETC, Choline dehydrogenase,
Glycerophosphate dehyrogenase, Ribonucleotide reductase etc.
e) Aconitase of Krebs Cycle.
Iron excretion:
Iron is a one-way element. It operates in a closed system. Once iron enters the body, it
is effecti vely utilized and reutilized in the the body.
Only a little of Iron is excreted (less than 1 mg / day). Iron is excreted through
desquamation of intestinal mucosa! cells, exfoliation of skin, sweat, bile. Almost no
Iron is excreted through urine.
Menstruation is a major cause of iron loss in pre-menopausal women (about 30 mg/
cycle).

Minerals 377
Dietary deficiency of Iron: Iron deficien cy anemia

Dietary deficiency of iron causes iron deficiency anemia, which affects about 70% Indian
populatio n, common ly seen in children, adolescen t girls, pregnant & lactating women.
Causes:
a) Decreased intake of Iron as in inadequa te dietary iron, lack of absorptio n (as in
steatorrh ea, tropical sprue, gastrecto my, gastric carcinom a and achlorhy dria (due to
lack of HCI required for iron absorption), lead poisoning (lead decreases iron absorptio n)
b) Increased loss of Iron as in hook worm infestation (1 hookwor m will cause about
0.3 ml of blood loss per d ay), rep eated pregnanc y (about lg of iron is lost from the
mother during 1 delivery), bleeding in urinary or genital tract, peptic ulcer, hemorrho ids
(due to blood loss) & nephrosis (causes proteinur ia. The iron and heme binding proteins
like transferri n, hemopexin, haptoglo bin are lost in urine, with conseque nt loss of iron).
Manifestations:
1. Microcyt ic hypoch romic an em ia: Due to decreased synthesis of hemoglo bin, RBC's
are small (rnicrocytic), under pigmente d (hypochr omic), which are fast destroyed in
reticuloe ndothelia l system causing rnicrocytic hypochro rnic anemia.
2. A tropic glossitis 3. Oesopha gial web 4. Dysphag ia
These 4 symptom s together constitut e the condition 'Plummer Winson syndrome'.
Other manifest ations are,
• Koilonyc hia: Spoon shaped appearan ce of finger nails, Pica (Craving for Starch, Clay,
lee etc), apathy (due to decreased s upply of oxygen to tissues), The pallor of skin and
tissues, fatigue, weaknes s, giddines s, blurred vision, anorexia, impaired immune
functions , bone deformiti es, h ead ache are the other symptom s.
Iron overload or Iron excess (Iron toxicity): Hemochromatos is

Hemochr omatosis and hemoside rosis are 2 related terms used to describe iron over
load condition s. Now, Hemochr omatosis is used as a generic term for Iron overload
condition s. Hemochr omatosis is two types; Primary and Secondary.
i) Primary hemochromatosis: Caused due to a genetic defect in Iron absorptio n which
results in increased Iron absorptio n from the intestine.
ii) Secondary hemochromatosis: May be caused due to increased lrpn absorptio
n,
repeated blood transfusio ns, thalassem ia. Bantu tribes of Africa de, clop hemoside rosis
(called bantu siderosis ), due to low phosphat e in their staple diet com, which fa\'ors
iron absorptio n and habit of cooking foods in and brewing alcohol in iron vessels.
Complications: Iron overload condition s lead to an excessive depositio n of hemoside rin
in tissues like li\ er, spleen, pancreas heart etc (Hemosi derosis). Depositi on of
hemoside rin in Ii,er cells causes death of li\'er cells and liver cirrhosis . In pancreas , it
causes death of pancreati c cells resulting in diabetes. In skin, it causes bronze coloratio n
of skin. The triad of liver cirrhosis, bronze coloratio n of skin and diabetes are referred
to as Bronze diabetes (more common ly associate d with primary hemochr omatosis ).
Minerals 378
• Copper (Cu)
Total body copper is about 100mg.
Sources:
Liver, fish, meat, nut, lenticels are good sources.
Milk is a poor source.
RDA:
2 -3 mg / day.
Absorption of Copper:
• Copper is absorbed from the duodenum .
• Metallothionein is a transport protein that facilitates copper absorption.
• Phytates, zinc and molybdenu m decrease copper uptake.
Functions:
Copper plays an important role in the formation of many enzymes

E.g.: Serum ferroxidase, ALA synthase, monoamin o oxidase, cytosolic superoxide
dismutase (SOD), tyrosinase, lysyl oxidase, cytochrom e oxidase, Dopamine oxidase,
catalse, uricase, monoamin o oxidase etc.
Some of the important reactions requiring these copper containing enzymes are,
1. Mobilizat ion of iron (Iron transport) :

Copper is an integral part of ceruloplasmin (serum feroxidase), which catalyses the
conversion of Fe2+ to Fe3+. Iron can be transported only in the Fe • form by transferrin .
3
Fe 2+ Fe 3+
Cerulopl asmin (Cu +) 2
2) Synthesis of Hemoglob in (Copper is a constituen t of ALA synthase)

3) Formation of collagen, elastin (Copper is a constituen t of lysyl oxidase)
4) Synthesis of melanin (Copper is a constituen t of tyrosinase)
5) Catechola mine synthesis (as a constituen t of dopamine ~-hydroxylase enzyme)
6) Antioxida nt function: Used in scavenging free radicals (as a constituen t of SOD).
7) Role in ETC: Copper is a constituen t of cytochrome oxidase (Complex IV) of ETC..
Minerals 379
Dietary deficiency of copper:

Dietary deficie ncy of copper causes microcytic anemia , as copper is require
d for
mobiliz ation and utilizat ion of iron. Dietary deficie ncy of copper result in
ineffici ent
utilizat ion of iron, resultin g in microc ytic anemia .
Coppe r is an integra l part of enzym e lysyl oxidase , which is require d
for collage n
synthes is. Coppe r deficie ncy causes decrea sed lysyl oxidas e activity and
defecti ve
collage n format ion and associa ted disorde rs like osteopo rosis, fragile capilla
ries etc.

Coppe r toxicity may occur from contam ination of food cooked in copper
vessels.
Sympt oms are vomitin g, diarrhe a etc.
1. Wilson 's diseas e or hepatolenticular degeneration:

Defect
This autoso mal recessive disease results from defect in the synthe sis of Cerulo
plasmin.
There's a defect in the hepatic excreti on of copper into bile and also renal reabso
rption
of copper in the kidney .
Characteristics:
This disease is charac terized by low blood copper and excessi ve deposi tion
of copper
mainly in liver and brain resultin g in cirrhos is of liver and neurological disorde
rs (Hence
the name hepato lenticu lar degene ration) .
There is also excess deposi tion of copper in kidney and cornea resultin g in renal
damag e
and kayser fleisure ring a t the margin of cornea .
Treatment:
It can be treated by penicil lamine , a copper chelati ng agent, which binds
with copper
and brings about the urinary excreti on.
2. Menk e's diseas e ("Kinky" or "steely" hair syndrome):

Defect:
This X-linke d disord er results due to the defect in the intestin al absorption
of copper.
It is possibl e that copper may be trappe d by metallo thionei n in the intestin
al cells.
Symptoms:
The sympto ms include decrea sed copper in plasma and urine, anemia , growth
failure,
mental retarda tion, depigm entatio n of skin and hair, and "kinky " or "steely
" hair.
It affects only male infants . It's a fatal disease, child usually dies in infancy
.
For queries and suggestions, contact the author at prasad_ text@yahoo.co

m or 9986449575
Minerals 380
• Fluorine (F)
D istribut ion:
Mostly found in teeth and bones as calcium hydroxy fluoro apatite.
Sources :
Drinking water, fish (sardine, mackerel), tea etc.
RDA:
Adults: 2-3 mg. Safe limit of fluorine in drinking water is about 1 ppm in water.
(ppm = parts per m illion; 1 ppm = 1 gm of fluoride in million gram s of water, this is
equal to 1 mg / 1000ml).
Function:
1) Fluoride is required for proper teeth develop ment:

Fluoride prevents dental ca ries (dental decay) by strengthe ning the enamel of teeth.
Mechani sm:
Fluoride ions get incorpora ted into the hyd roxyapat ite to form fluoroapa tite of the
enamel and dentine. This fluoroapa tite is more resistant to destruction by bacterial
acids and plaques, making the enamel harder and resistant to bacterial acid attack and
thus p reventing the dental caries and tooth decay. ( ote that bacterial acids are main
cause of d ental caries and tooth decay).
Further, the fluoride inhibits bacterial enzymes which produce acids that cause dental
caries.
2) Fluorine is also required for bone develop ment.

Fluoride is also necessa ry for the proper developm ent of bones.
Note:
Fluoride is the inhibitor of glycolytic enzyme enolase. So, d uring the estimatio n of
blood glucose level, fluoride is used in the form of sodium fluoride as an anti-glycolytic
agent.
75
For queries and suggestions, contact the author at prasad_te xt@yahoo.com or 99864495
Minerals 381
Deficiency:
Consumption of drinking water with less than 0.5 ppm of fluorine leads to dental caries
and also osteoporosis.
Topical application of fluoride will result in the formation of fluoroapatite layer on the
enamel, which prevents the enamel from the decay by bacterial acid.
Excess:
Consumption of excess fluorine causes fluorosis.
Dental fluorosis:
An intake above 2 ppm (particularly above 5 ppm) in children causes mottling of enamel,
discolorization of teeth. The teeth become rough with yellow patches on the surface.
These manifestations are collectively called dental fluorosis.
Skeletal fluorosis:
An intake above 20 ppm is toxic and can cause pathological changes in bones also.
Hypercalcification, increasing density of bones of limb, pelvis and spine are seen. Even
ligaments of spine and collagen of bones gets calcified.
These manifestations are collectively called skeletal fluorosis.
In advanced stages, Ligaments of spine get calcified, ultimately crippling the individual
due to stiff spines. This condition is referred to as Genu Valgum.
• Cobalt (Co)
Body contains about 1.1 mg coba lt.
Function:
• Cobalt is a constituent of corrin ring system of Vitamin B12 and their coenzymes.
• Cobalt activates glycyl- glycine peptidase and ALA synthase.
• It stimulates the synthesis of erythropoietin, which promotes erythropoiesis.
Deficiency:
Deficiency of cobalt causes pernicious anemia (as it leads to cobalamin deficiency).
Over consumption and toxicity:

Increased cobalt in food can cause polycythemia (Increased RBC in blood).

Minerals 382
• Iodine (I)
Body contains about 25 mg of iodine. 80% of this is stored in thyroid glands.
Sources:
Drinking water, sea foods, iodized salt are rich sources.
Iodine content of food depends on soil where it is grown.
Vegetables and fruits grown on seash ore are rich in iodine.
Regions of high altitudes are deficient in iodine content in soil (due to soil erosion) as
well as in water. There will be decrease in iodine content in the food cultivated in this
soil. Thus people living in mountain region have more chances of developing iodine
deficiency. Such areas are called goiterous belts, e.g. Himalayan region.
( Goiterogens )
Certain foods like cabbage, cassava, sweet potatoes, maize, cauliflower etc. contain
goiterogens (substances, which interfere with iodine utilization). For example, cabbage
contain thiocyanate, which inhibits iodine absorption).
Consumption of these foods in large amount will lead to goiter.
RDA:
• Adults: 100 to 150 p g / day
• Pregnant woman: 200 µg / day
Absorption:
Iodine is absorbed from small intestine. Normally about 30% of the dietary iodine is
absorbed . Iodine also gets absorbed through skin and lungs.
Storage:
About 80% of the body iodine is stored as iodothyroglobulin in the thyroid gland.
Functions:
Th e only biological function of iodine is in formation of thyroid hormones.
Iodine is required for the synthesis of thyroid h ormones namely thyroxine (T4) and tri
iodothyronine (TJ
T3 is fw1etionally more active than T4 •

Minerals 383
Synthesis of thyroid hormones:

Triiodothyronine (T.J and tetraiodothyronine (T4 or Thyroxine) are the two thyroid
hormones. They are synthesized from tyrosine and iodine in the thyroid gland.
There are two steps in the synthesis of thyroid hormones,
Step 1: Iodination of tyrosine residue present in the protein thyroglobulin to produce

MIT (3 - Monoiodotyrosine) and DIT (3, 5 Diiodotyrosine).
Thyroid peroxid.ase
Tyrosine+ h MIT
Thyroid peroxidase
MIT+ h DIT
Step 2: Coupling of MIT and DIT to give T3 and coupling of DIT and OJT to give T4
DIT + MIT ------ [TJ (Triiodothyronine) ]
DIT + DIT - -- --- [ T4 (Tetraiodothyronine) ]
Synthesis takes place when tyrosine is still a part of thyroglobulin protein. Finally
T3 and T4 are released from thyroglobulin by proteolytic breakd own.
General functions of thyroid hormones:

• Thyroid hormones are required for the proper functioning of almost all the cells. They are
necessary for general development, tissue differentiation, genetic expression, cellular
rnetabolism of every cell of the body.
• Calorigenic effect or thermogenesis is the main effect of thyroid hormones. 1 mg of T4
produces more than 100 Kcal ofenergy. This Thermogenic effect is due to the uncoupling
property of thyroid hormones in oxidative phosphorylation.
• Thyroid hormones increase the basal metabolic rate.
• Thyroid hormones are hyperglycemic hormones; they increase the blood glucose level by
facilitating the glucose absorption in the intestine.
Deficiency:
Simple endemic goiter: Deficiency of iodine in the food causes goiter. Mainly seen in
geographical areas away from sea-coast, where water and soil are low in iodine content.
Iodine deficiency disorder (IDD): To denote iodine deficiency, the term iodine deficiency
disorder (IDD) is nowadays used instead of goiter, because iodine deficiency leads to a
group of disorders like hypothyroidism, cretinism, deaf-mutism, goiter & subnormal
intelligence. Intake of iod ized salt is advised to prevent iodine deficiency disorders.
Excess:
Toxic goiter: Increased iodine uptake may lead to toxic goiter or Exophlthalmic goiter.

Minerals 384
• Seleniu m (Se)
Sources:
Sea food, liver, kidney and grains grown in selenium rich soil.
Requirem ent:
50-100 µg per day
Function s:
1) Antioxidant property:
Selenium is an integral part of metalloenz yme glutathion e peroxidase, which destroys
hydrogen peroxide (which otherwise destroys the cell membrane by oxidative damage).
Thus, selenium acts as an antioxidan t and provides protection against peroxidatio n of
cell membrane s.
2) Selenium has vitamin E sparing action:
Vitamin Eis also an antioxidan t which destroys hydrogen peroxide. Since the actions of
selenium and vitamin E are complimen tary, the availability of selenium reduces the
requiremen t of vitamin E (and vice versa) in preventing peroxidativ e damages.
Thus selenium has said to have vitamin E sparing action.
3) Selenium is a constituen t of 5'-deiodin ase (enzyme that converts thyroxine (TJ to
triiodotyro nine (T3) .
Selenocyste ine: Selenium is incorporated into protein as selenocysteine. It is coded by UGA

(termination codon). Hence, Selenocysteine is considered as 21st amino acid. There are few
proteins identified with the existence of selenocysteine in their structure (e.g.: Glycine
reductase, thiorcdoxin reductase, 5'-deiodinase, Glutathionc peroxidase).
Clinical significan ce:

'Keshan disease', an endemic cardiomyopathy is attributed to selenium deficiency.
(Name is derived from Keshan province in china, where soil is deficient in selenium).
Symptoms include nausea, dizziness, loss of appetite, muscle dystrophy, enlargeme nt
of heart leading to cardiac failure.
Toxicity:
Sdenium toxicity is called selenosis, caused due to excessive intake of selenium.
Selenium is present in metal polishes and anti-rust compouns , so, people handling
these, generally deveop selenosis.
Symptoms include loss of hair, weight loss, irritability, diarrhea, garlic odor to breath.

Minerals 385
• Zinc (Zn)
Total zinc in body is about 2 mg. Prostate gland is particularly rich in zinc.
RDA:
10 to 15 mg/ day
Source:
Meat, milk, egg, shellfish, beans and nuts
Abs orption:
• Zinc mainly absorbed in duodenum. Zn from animal sources is better absorbed than
the vegetarian sources. Metallothionein facilitate the absorption of zinc.
• Fiber, phytate, calcium, copper, iron inhibits Zn absorption.
• Amino acids promote Zn absorption.
Excretion:
Zinc is excreted through feces. Small amounts of Zinc are also excreted through bile.
Normally Zinc is not excreted through urine.
Function:
• [n the formation of certain metallo-enzyme: Zinc is a component of certain metallo-
enzyrnes like Carboxy peptidase, Carbonic anhydase, RNA polymerase, Alkaline
phosphatase, Alcohol dehydrogen ase, Cytosolic Superoxide dismutase (SOD), retinal
reductase, Glutamate dehydrogenase etc.
• Participate in visual cycle: As a componet of re tinal r eductse enzyme, Zinc plays an
essential role in regeneration of rhodopsin in visual cycle (Rhodopsin cycle).
• Antioxidant role: As a componet of SOD, Zinc acts as a n antioxidant.
• Heme synthesis: Zinc is a componen t ALA dehydratase, required for heme synthesis.
• DNA and RNA formation: Zinc plays an essential role in DNA and RNA formation,
cell division and growth.
• Zinc finger motif: These zinc fingers facilita te the binding of certain transcription
factors with specific regions of DNA during transcription. Zinc is an .i ntegral component
of zinc finger motif of an these transcription factor proteins.
• Insulin secretion: Zinc is required fo r storage and secretion of insulin from pancreas.
• Immunity: Zinc is essen tial for maintaining integrity of immune system.
• Taste sensation: Zinc containing protein Gusten, present in saliva, p lays an important
role in taste sensation.
• Wound healing: Zinc plays an important role in wound h ealing. (Exact mechanism
unknown).
• Normal reproduction: Zinc plays an important role in normal reproduction. It is
required for sexual maturation.

Minerals 386
D ietary Deficiency:
Causes:
Zinc deficiency is common in children of developing countries due to lack of
consumption of non vegetarian food, high phytate content, inadequate food intake and
increased fecal losses during diarrhea. Severe zinc deficiency of pregnant mothers has
been associated with spontaneous abortion and congenital malformations.
Symptoms:
Deficiency of zinc causes poor wound healing, growth failure, anemia, loss of taste
sensation, diarrhea, loss of sensation and hypogonadism.
• Acrodermatitis enteropathica:
It is a autosomal recessive condition, where zinc absorption is defective.
It is characterized by acrodermatitis (inflammation around mouth, finger, nose etc) and
diarrhea. Ophthalmologic disorders, hypogonadism are also seen.
• Zinc toxicity is seen in welders due to inhalation of Zn-oxide fumes.
Symptoms include nausea, gastric ulcers, pancreatitis, pulmonary fibrosis, vomiting,
anemia and excessive salivation .
• Molybdenum (Mo)
Body contains about 9 mg of molybdenum.
Daily requirement:
About 45 µg per day.
Sources:
Widely distributed, rich in cereals, legumes, green leafy vegetables, meat are rich in
molybdenum.
Functions:
Molybdenum plays a role in red blood cell synthesis.
Molybdenum is a component of many enzymes, like xanthine oxidase, aldeh yde
oxidase, sulfite oxidase, nitrite reductase (nitrogen fixing enzyme of plants) etc.
Molybdenum works with riboflavin to incorporate iron into hemoglobin.
Deficiency:
Rare. Deficiency of molybdenum causes neurological symptoms, mental retardation,
mouth and esophageal cancer etc.

Minerals 387
• Manganese (Mn)
Total body mangan ese is about 15 mg.
Sources:
Cereals, nuts, leafy vegetables, fruits. Tea is a rich source of mangan ese.
RDA:
Adults: 5 - 6 mg / day.
Function:
1) Mangan ese is required as an activator for various enzymes:
Exampl es are Acetyl CoA carboxy lase, Pyruva te carboxy lase, Mitoch ondrial
Superoxide dismutase (SOD), Glycosyl transferase, Arginas e, squalen e synthas e,
isocitrate dehydr ogenase etc. As a part of enzyme s, mangan ese is involve d in
• Glucon eogenes is: Mangan ese is an integral part of Pyruva te carboxy lase (A key
gluconeogenic enzyme ).
• Fatty acid synthesis: Mangan ese is an integral part of Acetyl CoA carboxy lase (Key
enzyme in denovo synthes is of fatty acids).
• Glycopr oteins and chondrotin sulphat e synthesis: Mangan ese is an integral part of
glycosyl transferase responsible for synthesis of glycoproteins and chondro tin sulphat e.
• Cholesterol synthes is: Require d for squalen e synthas e activity.
• Manganese inhibits lipid peroxidation (Superoxide dismutase enzyme requires Mn).
• Mangan ese is needed for RNA polyme rase activity.
• Mangan ese is required for the skeletal develop ment, proper reproduction, blood
clotting and normal function ing of nervous system.
Deficie ncy:
Dietary deficiency of mangan ese is rare in humans , but it has been reported in case of
protein energy malnutr ition, diabetes and pancrea tic insufficiency.
Deficien cy manifests as impaire d growth and skeletal deformities. Impaire d chondro tin
sulphat e product ion, which leads to defective organic matrix of bone and cartilage.
Raised serums ALP levels, nervous system disorde rs, abnorm al reprodu ctive function s
are also observe d.
Overco nsump tion and toxicity:

Overco nsumpt ion of mangan ese is unlikely to cause toxicity. Toxicity can be caused
on prolong ed exposu re to mangan ese dust in miners. The excess mangan ese
accumu lates in liver and CNS, produci ng severe neurom uscular sympto ms similar to
those of Parkins on's disease.
For queries and suggestions, contact the author at prasad_ text@yahoo.com

or 9986449575
Minerals 388
Questio n bank on Minerals
Essays (10 Marks) :

1. Mention the sources, daily requireme nts, functions and deficiency symptoms of calcium.
Short Essays (5 Marks) :

1. Explain the digestion absorptio of Calcium. Add a note on factors affecting calcium absorption ?
2. Explain the digestion, absorption of Iron? Add a note on factors affecting Iron absorptio n
3. How plasma calcium level is regulated (Calcium Homeosta tsis)?
5. Functions of copper / Functions of selenium / Functions of manganes e

l. Tetany / Wilson's disease / Fluorosis / Iron deficiency anemia
2. Heme proteins / Non heme proteins
3. Hemochro motosis or Hemoside rosis
4. Transferri n and Ferritin
Multiple Choice Question s (1 Mark):

1) Glutathio ne peroxidas e contain- (Al)
a) Zn b)Mo c)Mn d)Se
2) Copper is a constituen t of which enzyme (COMED K)

a) Lysyl oxidase b) Glucose oxidase c) Xanthine oxidase d) Transketo lase
3) Iron carrier protein of plasma

a) Hemoside rrin b) Transferri n c) Ferritin d) Hemopex in
4) Protein gusten of saliva contains

a) Cu b) Zn c) Mn d) Fe
5) Antioxida nt trace element

a) Selenium b) Chromium c) Cobalt d) Tron
6) Normal serum calcium level is

a) 9-11 mg / d i b) 6-8 mg/ di c) 11-13 mg/ dl d) 4-6 mg/ dl
7) The mineral required for wound healing is

a) Selenium b) Copper c) Zinc d) Cobalt
8) Intracellu lar storage form of iron is

a) Transferri n b) Cerulopla smin c) Ferritin d) Hemoglob in
9) Major source of fluoride is

a) Liver b) Milk c) Ground water d) vegetable s
10) Absorptio n of calcium is increased by all EXCEPT

a) Calcitr iol b) Lysine & arginine c) PTH dD) Alkalinity
Answers for MCQ: 1) d 2) a 3) b 4) b 5) a 6) a 7) C 8) C 9) C 10) d
Water and Electrolyte Balance 389
Water and Electrolyte Balance
Water Balance
Contents:
• Functions of water in the body
• Distribution of body water, daily requirement, water turnover
• Regulation of water metabolism: Role of ADH and Thirst centre
Electrolyte Balance
Contents:
• Distribution of electrolytes
• Electrolyte balance: Role of Aldosterone, Rennin Angiotensin system, role of

Atrial Natriuretic Factor (ANF).

Water
Water is the solvent of life. It is the most important nutrient, even more important than
the food. Without food human beings can survive up to 6 weeks or long, but without
water, they cannot survive one week also.
• Functions of water:
1. Medium for biochemical processes:
Water provides the aqueous medium for all the biochemical reactions in the body.
2. Participate in the chemical reactions:
Water directly participates as a reactant in several biochemical reactions.
3. Transport of nutrients:
Water serves as a vehicle for transport of different compounds (nutrients, metabolites,
secretions, and oxygen etc) in the body.
Water circulates throughout the body in the form of blood (about 92% of blood plasma
is water) and various other secretions and tissue fluids. In these circulating fluids,
nutrients, metabolites, secretions, oxygen and other substances are transported.
Thus, the food we eat reaches all part of the body with the help of water.
4. Regulation of body temperature:
Water absorbs heat slowly and large amount of water in the body helps in maintaining
the body temperature homeostasis despite fluctuations in the environment
temperature. Also, heat produced in chemical reactions in the body will be distributed
throughout the body causing only a slight change in body temperature. Formation
and evaporation of water in the form of sweat has a cooling effect on the body.
This property of water is made use in giving cool sponge bath to patients with fever.
5. Provides shape and structure to cells, tissues, organs and body:

Water is structural component of cell (approximately two third of body water is
located within cells) and hence provides shape and structure to cells. Muscle cells
have about 75% water, compared to adipose tissues (about 25%). Water also gives
structure and form to the body by filling spaces within body tissues.
6. Elimination of waste products:
Water is required for excretion of waste products through urine, feces, sweat.
7. Lubricating effect:
Water as a major component of ground substances, mucus and other lubricant fluids
h as a lubricating effect.
For example, synovial fluid within the joint reduces the friction and helps to provide
smooth movement. Saliva and mucus acts as lubricants in mouth and oesophagus.

• Water distribution in the body:

Water is the chief constituent of body. 60% of the body weight is water. Body water
is distributed mainly in two compartments.
1) Intracellular fluid 2) Extracellular fluid
1) Intracellular fluid (ICF):

The fluid found within the cell is called intracellular fluid.
2) Extracellular fluid (ECF):

The fluid found outside the cell is called extracellular fluid. The extracellular fluid is
further divided into intravascular fluid (blood plasma within the blood vessels) and
exb·avascular fluid, which includes interstitial fluid (fluid that found in the interstitial
space) and fluids like lymph etc.
Fluid distribution in a 70 kg normal person (42 L water)
Total body water (42 L) i.e. 60 % of body weight (of 70 kg)
!
Intracellular fluid Extracellular fluid
l
(28 L, 40 % Body weight) (14 L, 20 % Body weight)
I
! l
lntravascular fluid (2.8 L) Extravascular fluid (11.2 L)
(4 % Body weight) (16 % Body weight)
Osmolarity and osmolality:

These 2 factors influence water distribution.
• Osmolarity: It is the number of moles solute particles/ liter of solution.
• Osmolality: It is the number of moles solute particles/ kg of solution.
It is a measure of the solute particles present in the fluid medium.
Osmolality of plasma is 280-300 millimoles/kg.
Osmolality of plasma is mainly contributed by sodium & its associate anions.

• Sources of water:
Water is supplied to the body by exogenous and endogenous sources.
• Exogenous source: Drinking water, milk and beverages (about 1500 ml/day) and
water content of solid foods like fruits, vegetables etc. (about 700 ml/ day).
• Endogenous source: The metabolic water (about 300 ml/ day) produced within the
body during oxidation of foodstuffs.
• Water turnover:
In a healthy person, the water intake should nearly be equal to water out put and the
body water content is maintained fairly constant.
A. Water intake:
Normal water intake in a healthy individual is about 2500 ml / day.
Water is supplied to the body by exogenous and endogenous sources.
1) Exogenous source:
• Ingested water and beverages (about 1500 ml/day)
• water content of solid foods (about 700 ml/ day) etc.
2) Endogenous source:
• Metabolic water (about 300 ml/ day) produced within the body during oxidation of
food stuffs.
B. Water output:
Normal water output in a healthy individual is about 2500 ml / day.
There are 4 distinct routes of water elimination.
• Urine: About 1500 ml/ day
• Skin (sweat): About 500 ml/ day
• Lungs: About 350 ml/day
• Feces: About 150 ml/ day
[ Water turnover]
Daily water intake Daily water output

• Ingested water and • Urine - 1500 ml/day
Beverages -1500 ml / day • Skin - 500ml / day
• Water from d solid foods - 700 ml/ day • Lungs - 350 ml / day
• Metal:olic water - 300 ml / day • Faeces - 150 ml / day
Total = 2500 ml/ day = 2500 ml/ day
There is an inverse relationship between water Jost through skin & urine according to the
climatic. In a dry temperate climate more perspiration and less urine is excreted, whereas
in cold climates less perspiration {due to closure of sweat pores) & more urine is excreted.

• Water balance:
The term water balance is defined as the maintenance of water content of the body.
Water balance is achieved by balancing the water intake and water output.
Regulation of w ater balance:

Water balance is regulated by following mechanisms;
1) Antidiuretic hormone (ADH) : ADH is secreted from posterior pituitary gland.
ADH promotes water reabsorption from kidneys and thus reduces the loss of water
from the body. ADH secretion increases in water deficient conditions. (ADH deficiency
causes excretion oflarge amounts ofwater through urine, condition known as diabetes i11sipidus).
2) Aldosterone: Aldosterone is a hormone produced in adrenal cortex. It increases Na~
reabsorption by renal tubules. Along with sodium ions, chloride ions are also absorbed.
As a result of sodium and chloride reabsorption, water also reabsorbed along with
them by osmotic pressure.
3) Thirst center: Thirst centre, located in hypothalamus of brain regulates the intake of
water. When there is water loss, the body reaches condition of dehydration. The
dehydration stimulates the thirst centre, which causes us to drink water.
4) Urine formation: If excess water is injected the kidney responds to it and excess
water is excreted in urine and water balance is maintained.
• Overhydration and water toxicity:

• Definition: Overhydration or water intoxication is caused by excessive retention of
water in the body.
• Cau ses: Normally, there is no dietary cause for over hydra tion. Fluid over load from
excessive fluid intake is rarely seen in healthy persons. Excess water consumed is
promptly excreted through urine within few hours.
Overhydration generally occurs due to abnormal decreased urine excretion or abnormal
sodium retention, such as in renal failure, congestive heart failure, overproduction of
ADH, corticosteroid therapy, cirrhosis. It may also happen in excessive intake of salt
free fluid after excessive sweatin g in hot climates (due to water and electrolyte
imbalance).
• Symptoms: Water intoxication is associated with the dilution of ECF and ICF w ith a
decrease in Osmolality. The clinical symptoms include head ache, lethargy and
convulsions.
• Treatment: Suggested treatment is stoppage of water intake and administration of
hypertonic saline with glucose.

• Dehydration:
• Definition:
Dehydration is a condition characterized by water depletion in the body (loss of 10% or
more water from the body).
Types: Dehydration is of two types,

i) Due to combined deprivation of water and electrolytes ii) Due to water loss alone
• Causes:
Dehydration may be caused due to insufficient intake or excessive water loss or both .
It can be caused as a result of reduced water intake for a prolonged period, diarrhea,
vomiting, excessive sweating, fluid loss in bums, illness, kidney diseases, adrenocortical
dysfunction, ADH deficiency (diabetes insipidus), diabetes mellitus etc.
• Complications of dehydration:
1. Dehydration causes a decrease in volume of ECF (like blood) and concomitant
increase in levels of electrolytes, urea, plasm a protein & osmolality. So, water is drawn
from ICF (from cells) resulting in shrunken cells and disturbed cellular metabolism.
There is also an increased protein breakdown finally resulting in loss of weight.
2. Decrease in plasma volume will reduce cardiac output leadingto circulatory failure
3. Dehydration is often accompanied by a loss of electrolytes (Na, K) from body.
4. Decreased ECF also stimulate ADH secretion, which results in the reduction in
urine volume to effect the water retention in the body. Reduction of urine excretion
can cause problem, as minimum of 500 ml of urine must be produced to excrete the
waste products like urea. Reduced urine volume cannot excrete all waste products.
• Signs of dehydration:
The effects are thirst, fatigue, loss of app eti te, decreased skin turgor, dry and flushed
skin, dry mouth and tongue, sunken eyeballs, heat intolerance, tachycardia, dark
scanty urine a nd loss of weight etc. The effects are progressive and cumulative over
time. Dehydration leads to delirium, coma and finally death, when water loss exceeds
10% of body weight.
• Treatment:
Intake of plenty of water is advocated in the treatment of dehydration. Incase of
combined water & electrolyte depletion, electrolytes are also supplied along with
water and glucose.
ORT (Ora l Rehydration Therapy): ORT is an effective method of treating dehydration by providing
tfle patient with fluids & electrolytes. WHO recommended formula oforal rehydration fluid con ta ins
NaCl (3.5g), NaHCO3 (2.5g), KC/ (1.5g) & glucose (20 g) dissolved in 1 liter of water.
Preparations such as electral, electrose, pedit ral etc are readily available for ORT.

Electrolytes
Electroly tes are inorganic substances (either single mineral elements or compound s),
tha t can readily dissociate and exists as charged ions (either anions or cations) in the
solution.
The major electrolytes in the plasma are,
a) Cations (Positively charged ions):

Sodium (Na +>, Potassium (K +), Calcium (Ca 2•), Magnesium (Mg 2+)
b) Anions (Negatively charg ed ions):

Chloride (Cl -), Bicarbonate (HCO3 -), Sulfate (SO4 2• )
• Composition of electrolytes in the body fluids:

The concentration of electrolytes are expressed as milliequivalents/Liter (mEq/L)
Composition of electrolytes in ECF (plasma/ serum electrolyte levels):

Concentration of electrolytes are expressed as milliequivalen ts/Liter (mEq / L)
CATIONS ANIONS
Na+ 142 mE.q/L 0 · 103mEq/ L
K+ SmEq/ L HCOr 24 mE.q/ L
Ca+2 SmEq/L HP()42· 2mEq/L
Mg+2 3mEq/ L S()4 2· l mEq/ L
Proteins 15 mE.q/L
Organic acids lO mEq/L
Total 155mEq / L Total 155mEq/L

Composition of electrolytes in ICF (Muscle cells):
CATIONS ANIONS
150 mEq/ L HPQ42 • 140 mEq/ L
Na + 10 mEq / L HCO3· 10 mEq / L
Mg+2 40 mEq/ L Cl ·· 2 mEq/ L
Ca+2 2 mEq / L SO4 2• 5 mEq / L
Proteins 40 mEq/ L
Organic acids 5 mEq/ L
Total 202 mEq/ L To tal 202 mEq / L
Note:
• The total concentration of cations and anions in each compartment (ECF or ICF)
is equal to maintain the electrical neutrality.
• Movement of water across the membrane is dependent on the osmotic balance
between the ICF an d ECF. In a healthy state, the osmotic pressure of ECF (mainly
due to Na+) and ICF (mainly due to K+) is equal. So, as such, there is no net
movement of water takes place in and out of the cells, due to this osmotic balance.
• Regulation of electrolyte balance:

Electrolyte balance is closely related to water balance. Electrolyte balance is affected
by water balance, NaCl intake and intake of other minerals in the diet. Thus, electrolyte
balance is closely related to water balance.
Electroly te balance is achieved through the hormones such as aldostcrone, ADH
and renin - angiotensin system. Kidneys play an important role in electrolyte balance
as th ese hormones act through kidney.
a) Aldosterone:
Aldosterone is a rnineralocorticoid produced in adrenal cortex. They increases Na+
reabsorption by the renal tubules in exchange of K• & H · ions. Along with sodium ci-
is also absorbed. As a result sodium & chloride are reabsorbed ; K• & H • are excreted.
when sodium & chloride are reabsorbed, water is also reabsorbed by osmotic pressure.

b) Renin angiotens in system:

Renin-angi otcnsin system regulates aldosteron e secretion.
• Renin is secreted by kidneys Uuxtaglom eular cells of nephron) in response to the
decrease in ECF volume, low BP, low a+ and H igh K+.
• Renin converts a ngiotensino gen to angiotensin I. Angiotensi n I is then converted to
angiotensin II by angiotensin converting enzyme (ACE). Angiotensin II stimuJates adrenal
cortex to secret aldosteron e. Aldosteron e promotes Na reabsorption (and also CI· &
water abosrption ); K• and H• excretion.
ECF D ecrease, Low BP,

Low N a, High K
®l Angiotensinogen
Kidney --+ Renin

-1
Angiotensin I
l
ACE
Angiotens in II
Aldos terone stimulates
the sodium reabsorptio n
and reabsortio n of
@l 0
chloride and water;
excretion of K• and H•.
This res ult in the
Aldosterone secretion - - • elevation of ECF & BP).
Angiotensinase convert angiote11si11 11 to angiotensin TII, which also stimulate aldosterone secretion.
Angiotensi nogen II is a vasocontrictor. So, it also contributes to elevation of ECF and BP.
c)ADH:
ADH also has role in electroly te balance (indirect). When plasma osmolality is
increased (Mainly due to Na increase), ADH is released.
ADH simulates water reabsorptio n by renal tubules.
Ald osterone and ADH coordinate with each other to maintain the normal water
and electrolyte ba lance.
d) Atrial natriureti c factor (ANF):

Atrial natriuretic factor is a polypeptid e hormone secreted by right atrium of the heart.
ANF increases the urinary sodium excretion.
A NF has opposite action of aldosterone, (So its an a11ti-a/dosterone hormone).
Spironolac tone, a synthetic aldosterone agonist is used as a diuretic and liypertensive drug.
lt is also used in the treatment of hyperaldosteronism.

• Sodium (Na)
Distribution:
Total sodium content of body is about 120 mg. Sodium is the most important
extracellular cation. Normal level of ECF sodium is 135-145 mEq/L.
Sources:
Table salt (NaCl, sodium chloride), as used in cooking, seasoning and processing of
food is the main dietary source of sodium. By weight, 39% of NaCl is sodium.
Eggs, meat, fish, milk, vegetables (carrots, spinach, beats etc) are good sources.
Processed foods are generally very rich in sodium.
N ote:
Sodium present in foods may not be adequate to meet the requirement. Hence table
salt (sodium chloride, NaCl) need to be included in the diet, not only to increase the
taste, but also to provide required amount of Na for the body.
RDA:
Exact RDA for sodium is not known. Requirement of sodium mainly depends on sodium
loss from body (through urine and sweat).
On hot conditions the requirement increases. For most individuals the minimum daily
requirement is about 500 mg (i.e. about 1200 mg of NaCl). Average intake of sodium as
NaCl is about 10-lSg in India.
It is recommended that sodium intake must be limj ted to abou t 2.3 g/ day (about 5 mg
or one teaspoon of salt).
Low sodium diets are prescribed for patients suffering from high Bigh pressure and
cardiac proplems.
Absorption:
Sodium is absorbed in small intestine. Almost all the dietary sodium is absorbed .
Excretion:
On an average Indian diet, about 3 to 4 grams of sodium (equivalent to 8 to 12 grams of
NaCl salt) is excreted in the urine. If the sodium intake increases, the excretion of
sodium a lso increases.
Aldosterone increases sodium reabsorption in the kidneys and causes its retention.
Sodium is also excreted through sweat (about 20 g of NaCl in tropical countries).

Sodium balance:
Normally, the amount of sodium intake equals the amount of sodium excreted. A
healthy kidney is required for maintaining normal sodium balance.
• Sodium balance is achieved by eliminating excesses in urine. A sodium rich diet
causes a temporary increase in serum sodium, which stimulates thirst. Drinking
fluids dilutes the sodium in the blood to normal concentration, even though the
volume of both sodium and plasma volume are increased. The increased plasma
volume stimulates the kidneys to excrete more water and sodium together to restore
normal sodium and plasma volume level.
• Conversely, low sodium or plasma volume level stimulates the aldosterone secretion,
which increases the sodium (and water) reabsorption by kidneys. Urine volume
also decreases.
Functions:
• Maintenance of resting membrane potential: Sodium (along with potassium)
maintains the resting membrane potential by maintaining the concentration gradient
of Na - K across the membrane. The high extracellular sodium concentration is
maintained by Na-K pump, which transports 3 sodium ions out of the cell and 2
potassium ions into the cell.
• N erve impulse transmission: Sodium (along with potassium) plays important role
in transmission of nerve impulses.
• Muscle contraction: Sodium plays important role in muscle contraction.
• Maintenance extracellular osmotic pressure and water balance: Sodium along
with chloride maintains extracellular (plasma) osmotic pressure. Retention of water
in the ECF is directly related to the osmotic effects of mainly Na and CJ·. Thus
sodium has a very important role in regulation of water balance.
• Regulation of acid base bal ance: Sodium also plays an important role in regulation
of acid base balance.
• Glucose, galactose and amino acid absorption: Sodium ions play an important
role in glucose and galactose absorption in the intestine.
• Cell permeability: Sodium ions play an important role in cell permeability.
• Origination of heart beat: Sodium ions play an important role in originating and
maintaining heart beat.
D eficiency of sodium:
Dietary deficiency of sodium is very rare because, generally body's requirement is
low and intake is high. Deficiency may be seen incase of heavy sweating (during
heavy labor or athletes during strenuous exercise or in hot environment etc). Sodium
deficiency may also be seen in prolonged diarrhea, vomiting or from certain renal
disorders. Such persons may need additional sodium supply to replace the losses.
Symptoms: Muscle cramps, acid base problems, weakness, head ache etc.

Sodium deficiency may also happen in excessive intake of salt free fluid after excessive
sweating in hot climates. During excessive sweating, both fluid and salt are lost causing
their deficiency.
Drinking pure water without salt aggravates the sodium (and chloride) deficiency,
due to fluid electrolyte imbalance.

Excess sodium intake is generally associated with hypertension. Excess consumption
of sodium may result in abnormal water balance leading to edema. (Acute excessive
intake of dietary sodium accumulates in extracellular spaces. To balance the osmotic
pressure, water is pulled into extracellular spaces, which result in edema).
But, in most of the normal persons with healthy kidney and adequate water intake,
the excess sodium is excreted in urine.
ormal level of blood sodium is 135-145 (Average 142) mEq / L.
a) Hyponatremia:
• Definition: Decreases in serum sodium level falls below normal level is termed as
hypona tremia.
• Cause: Severe vomiting, diarrhea, burns, sweating, Addison's disease (adrcnocortical

insufficiency), renal tubular acidosis etc
• Symptoms: Severe muscle cramps, headache, lethargy, drowsiness and nausea. Long
term manifestations of hyponatremia include reduced blood pressure and circula tory
failure.
b) Hypernatremia:
• Definition: Increase in serum sodium level more than normal level is termed
H ypernatremia.
• Cause: Cushing's syndrome (adrenocortical hyperactivity), prolonged cortisone

therapy, decreased intake of water, pregnancy (where steroid hormones cause sodium
retention), dehydration, excess intake of salt etc.
• Symptoms: Dry mucous membrane, fever, thirst and restlessness. Long term clinical
manifestations include increased blood volume and blood pressure.

• Potassium (K)
Potassium is the most important cation of intracellular fluid (ICF). Normal level of
ICF potassium is about 155 mEq/L. Normal level of ECF potassium is 3.5-5 mEq/L.
Sources:
Green leafy vegetable, meat, fish, poultry, certain fruits (banana, orange, peach),
vegetables (potatoes, carrot etc) are good sources. In plant sources, potassium is present
in more concentration than sodium (10 to 50 fold).
RDA:
Exact RDA for potassium is not known. Average intake is about 2 to 3 gm per d ay.
Excretion:
Potassium is mainly excreted through urine. Aldosterone increases K excretion.
Functions:
• Maintenance of resting membrane potential: K is the major intracellular cation. Along
with sodium, potassium maintains the resting membrane potential.
• Nerve impulse transmission: K plays important role in nerve impulse transmission.
• Maintenance intracellular osmotic pressure: Intracellular potassium maintains
intracellular osmotic pressure.
• Enzyme activation: Intracellular potassium is required for activation of certain enzyme
E.g.: Pyruvate kinase.
• Cardiac muscle activities: Extracellular K is a key fac tor for cardiac muscle activities
• Neuromuscular excitability: Controlling neuromuscular excitability.
Deficiency of potassium:
Potassium is present in practically all foods. So, dietary deficiency of potassium is rare.
Clinical significance: Normal level of blood potassium is 3.5 to 5 mEq/ L.

a) Hypokalemia: Decrease in serum potassium (K+) level is below 3 mEq/ L.
• Causes: Cushing syndrome (hyperactivity of adrenal cortex that produce aldosterone, which
increase K• excretion), Vomiting, Diarrhoea (due to loss of K-), prolonged use of diuretics
{due to excretion of K· along with fluid), treatment of diabetic coma with insulin (Insulin
promotes glycogenesis, which requires K+ and it is withdrawn from ECF. It is fatal), alkalosis
{due to redistribution of K- ions, where extracellular K• moves into the cell in exchange for
intracellular H+ ). Other reasons include intravenous admin istration of K• free fluids,
prolonged cortisone therapy etc.
• Symptoms: Muscle weakness, spasms, tachycardia, cardiomegaly & cardiac arrest.
b) Hyperkalemia: Increase in serum potassium (K•) level is above 5 mEq/ L.
• Cause: Addison's disease, renal failure, severe dehydration, uritreated diabetes etc
• Symptoms: Depression of CNS, mental confusion, anorexia, bradycardia, peripheral vascular
collapse, and finally cardiac arrest.

• Chlorine (Cl)
Chloride is the chemical form of chlorine as it appears in the body. Chloride is the
major anion in extracellular fluid. Normal level of ICF chloride is about 98-107 mEq/ L.
Chloride shar.es a parallel relationship with sodium in terms of dietary sources,
excretion, conditions causing deficiency etc.
Sources:
Common salt (NaCl) as cooking medium, whole grains, leafy vegetables, eggs, milk.
RDA:
Adequate intake of sodium satisfies the chloride requirement also. For most individuals
the minimum daily requirement is about700 mg (i.e. about 1200 mg of NaCl). Average
intake of chloride as NaCl is about 15g in India.
Absorption:
Chloride is almost totally absorbed in GIT.
Excretion:
Chloride is excreted in urine along with sodium in the form of NaCL (About 15 g /
day).
Functions:
• Regulation of acid base balance, water balance and osmotic pressure: Chloride
plays an important role in regulation of water balance, acid base balance and
maintenance of osmotic pressure. These functions are carried out the combined
actions of sodium, potassium and chlorides.
• Formation of HCl: Chloride is essential for formation of HCl in the gastric juice.
• Chloride shift (A mechanism in the transport of 0 2 in hemoglobin), requires chloride.
• The enzyme salivary amylase is activated by chloride.
Deficiency of chlorine:
Chlorine is present in practically aU the foods. So, dietary deficiency of chlorine is
rare. The normal intake and output of chloride from body share a parallel relation
with sodium. Conditions leading to sodium deficiency also lead to chlorine deficiency.
Vomiting is the primary cause of chloride deficiency.

• Sulfur (S)
Sulphur does not function as independent entity, but it is mostly present as components
of biotin, thiamine and amino acids like methionine, cysteine, cysteine e tc.
Sources:
Sulfur is widely available in egg, milk, meat, cheese, legumes and nuts etc.
RDA:
No specific RDA for sulphur . Adequate protein diet conta ining proper sulfur
containing amino acids provides adequa te sulfur.
Excretion:
Sulfur from d ifferent compounds is oxidized liver to form sulfate. So inorganic sulfate
{80%) is the m ain excretory form of sulfur. Organic sulfur in the form of ethereal
sulfate is also excreted in small amounts.
Functions:
• Components of sulfur containing amino acids: Sulfur is the constituent of sulfur
containing amino acids like methionine, cysteine, cystine, which are essential for
the structural conformation and biological functions of proteins. Sulfur containing
amino acids contributes to the disulfide link (-S-S-) and sulfhydryl groups required
for protein structure. Keratin (Structural protein present in hair, skin and nails)
has high sulfur concentration, which makes these more r igid.
• Sulfur is the constituent of many compounds like biotin, thiamine, lipoic acid,
coenzyme A, many glycosarninoglycans etc.
• Sulphur is a component of PAPS (Phosphoadenosine phosphosulphate): PAPS is
the active sulfate required for several sulfation reactions (in the synthesis of
glycosaminoglycan s, detoxification mechanisms e tc).
• Sulphur containing amino acid methionine is an integral component of SAM
(S-ad enosylmethionine), which is involved in several methylation reactions (in the
synthesis of creatine, epinephrine etc).
Dietary deficiency of sulfur:

Dietary deficiency of s ulfur is not known. A s ulfur deficiency may occur in severe
protein energy malnutri tion cases and absen ce of sulfur containing amino acids.

Question bank on Water and Electrolyte balance

1) Discuss water balance
2) Dehydration
3) Plasma electrolytes (serum electrolytes) and Discuss their balance

1) Water distribution in the body
2) Water turnover
3) ADH / Aldosterone / Rennin angiotensin system/ Atrial Natriuretic Factor
4) Thirst centre
5) Overhydration

1) Which of the following has no effect on fluid and sodium balance
a) ADH b) A ldosterone c) Oxytocin d) Renin-angiotensin system
2) Thirst centre is located in

a) Brain b) Liver c) Spinal cho rd d) Mouth
3) Most important intracellular cation is
a) Sodium b) Potassium c) Chloride d) Bicarbonate
4) Most important extracellular anion is
a) Sodium b) Potassium c) Chloride d) Bicarbonate
5) Rennin is produced in
a) Brain b) Liver c) Kidney d) Muscle
6) Which of the following drug is Aldosterone agonist?
a) Ameloride b) Triamterine c) Frusemide d) Spironolactone
7) Normal p lasma K• levels is
a) 135-145 mmol/L b) 95-105 mmol/L c) 3.5-5 mmol/ L d) 9-11. mmol/L
8) The metabolism of sodium is regula ted by the hormone

a) Insulin b) Aldosterone c) PTH d ) Somatostatin
9) Deficiency of ADH results in
a) Diabetes insipidus b) Hyponatremia c) Diabetes mellitus d) None of these
10) The enzyme responsible for the conversion of Angiotensin I to Angiotensin III is
a) Rcnin b) Angiotensinase c) Amino peptidase d) Angiotensin con verting enzyme
Answers for MCQ: 1) c 2) a 3) b 4) c 5) c 6) d 7) c 8) b 9) a 10) b

Nucleotide Metabolism 405
Metabolism of Nucleotides
Contents
• Purine nucleotides
• Denovo synthesis of purine nucleotides
• Salvage pathways of purine nucleotides
• Catabolism of purine nucleotides
• Disorders of purine metabolism
• Pyrimidine Nucleotide Metabolism

• Denovo synthesis of pyrimidine nucleotides
• Salvage pathways of pyrimidine nucleotides
• Catabolism of pyrimidine nucleotides
• Disorders of pyrimidine metabolism

Nucleotide metabolism
• Nucleotides are either Purine nucleotides or Pyrimjdine nucleotides.
• Nucleotides are the building blocks of nucleic acids (ONA and RNA).
• Nucleotides consist of a nitrogenous base (purine or pyrimidine), a pentose sugar

(ribose or deoxyribose) and one or more molecule of phosphates.
• These nucleotides are synthesized in the body at a rate consistent with physiologic
need.
Biosynthesis of Purine nucleotides:

Purine nucleotides consist of a purine base, a pentose sugar (ribose or deoxyribose)
and one or more molecule of phosphates.
Purine bases:
• Major purine bases: Adenine and guanine
• Minor purine bases: Hypoxanthine and xanthine
There purine bases combine with pentose sugar and phosphates to form purine
nucleotides.
Purine nucleotides:
• Nucleotides of Adenine are AMP, ADP, ATP (Similarly deoxy adenine n ucleotides)
• Nucleotides of guanine are GMP, GDP, GTP (Similarly deoxy guanine nucleotides)
• N ucleotide of hypoxanthine is IMP (lnosine Mono Phosphate)
• Nucleotide of xanthine is XMP (Xanthine mono phosphate)
There are 2 processes that contribute to biosynthesis of purine nucleotides,

1. De novo synthesis
2. Salvage pathways
a) Phosphoribosylation of purine bases
b) Phosphorylation of purine nucleosides

1. De novo synthesis of purine nucleotides:
Purine nucleotides are,

AMP, ADP, ATP, deoxy AMP, deoxy ADP, deoxy ATP
GMP, GDP, GTP, deoxy GMP, deoxy GDP, deoxy GTP
Site:
• Tissue site: Most of the tissues, Liver is the major sHe
• Intracellular site: Cytoplasm
Sources of individual atoms in purine ring:

Many compounds contribute to the purine nucleotide ring formation.
GI~
i l Sources C and N:
• Aspartate provides Nl of purine
/6 C
/7 • N 10 Formyl FH4 provides C2
Cs • N 5, N 10 methenyl FH~provides CB
~, 'I
Asparble - N I
• Glutamine provides N3 and N9
I 2 ,c - N•,N"m..,m; ru, • Glycine provides C4, CS & N7
N" formyl FH. - C J • CO2 provides C6
N N
Clulamine
Reaction pathway:
Purine bases are not synthesized as such, but they are formed as ribonucleotides i.e.
the purines are built upon a pre-existing ribose-5-phosphate to form ribonucleotides
directly and not as purine base. Ribose-5-phosphate, produced in the HMP shunt
pathway is the starting material for purine nucleotide synthesis.
IMP (Inosine monophosphate) is the first nucleotide synthesized; AMP and GMP are
formed from IMP.
De novo synthesis of purine nucleotides can be studied under 3 steps:

Step 1. IMP formation: Ribose-5-phosphate undergoes a series of reactions to form IMP.
IMP (lnosine monophosphate) is the firs t nucleotide synthesized.
Step 2. Conversion of IMP to AMP and GMP: IMP is then converted to AMP & GMP.
Step 3. Formation of ADP, ATP, GDP, GTP:

Step 1. IMP formation:

Ribose-5-phosphate reacts with ATP to form Phosphoribosyl pyrophosphate (PRPP).
This reaction is catalyzed by PRPP synthetase. The purine ring is built on the ribose-5-
phosphate moiety of PRPP. Then PRPP undergoes a series of reactions to form IMP.
IMP (Inosine monophosphate) is the first nucleotide synthesized.
Ribose-5-phosphate
h
ATP
Complete reaction PRPP Synthetase
steps in next page AMP
Phosphoribosyl pyrophosphate (PRPP)
Glutamine
PRPP Glutamyl amido transferase
Glutamate
PPi
5-Phosphoribosylamine
j,
!- (Series of reactions)
j,
IMP
Step 2. Conversion of IMP to AMP and GMP: IMP is then converted to AMP & GMP.
IMP
Adenylo succinate sy nthas e / "-.. IMP deh y drogenase
(GTP} /
!
Adenylosuccinate XMP
Aden y lo succinase (ATP)l GMP synthetase

AMP GMP
Step 3. Formation of nucleoside di phosphate & triphosphates (ADP, ATP, GDP, GTP):
Di & tri nucleotide phosphates are formed from these mononucleotide phosphates.
Adeny la te kin ase
• AMP • ADP
ATP ADP
• GMP
Guanylate kinase
.. GDP .. GTP
7'\- 7'\-ADP
ATP
ATP ADP
Note: Conversion of ADP to ATP is achieved p rimarily by oxidative phosphorylation
& secondarily by substrate level phosphorylation reaction of glycolysis and TCA cycle.

Complete pathway of IMP formation (Step 1 of denovo synthesis purines):
Ribose-5-phosphate
PRPP Syn thetase ik::-AMP

ATP
Phosphoribo syl pyrophospha te (PRPP)

Glutamine
PRPP Glutamyl amido transferase
Gluta mate
PPi
5-Phosphori bosylamine
Synthetase y Glycine, ATP
G lycinamide ribosr5-phosphate
: (GAR)
NS,1omethcnyl FHi
Formyl transferase
m
Formyl glyci namide ri bosyl 5-phosphate (FGAR)
Glutamine
Synthetase ATP
Glutamate
Fonnylgl:::::::••c::1:-Jho,phat,
y
r - . ADP + Pi
Aminoi mid azole ribosyl 5-phosphate (AIR)
Ca,bo,yla,e CO,
y
Aminoimida zole carboxylate ribosyl 5-phosphate
Syntheta,e Aspa,tate, ATP
Aminoimida zole s uccinyl carboxamide ribosyl 5-phosphate
Adenylo sucdn,,,. Fum,rnte ·
Aminoimida zole carboxamide ribosyl 5-phosphate

l--- 10 formyl Fl Li
Formyl transferase r-. m
N-formyl aminoimidazole carboxamide ribosyl 5-phosphate
IMP cydohydrnla ,e H-0
Inosine mono phosphate (IMP)
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Regulation of purine synthesis:

1. The intracellular concentration of PRPP regulates purine synthesis to a great extent.
This, in turn, is dependent on the availability of ribose-5-phosphate and the activity
of the enzyme PRPP synthetase. The end products AMP, ADP, GMP, GDP inhibit
PRPP synthetase by feedback mechanism.
2. When AMP and GMP are available in sufficient amounts, their synthesis is turned
off by inhibiting Glutamine PRPP amido transferase (feed back inhibition).
3. Another important stage of regulation is in the conversion of IMP to AMP & GMP.
a) AMP and GMP cause end product feed back inhibition of adenylo succinate
synthase and IMP dehydrogenase respectively. Thus, each nucleotide prevents
its own overproduction by inhibiting its own synthesis from IMP.
b) The formation of AMP from IMP requires GTP; similarly formation of GMP
requires ATP. Cross regulation between the pathways of IMP metabolism thus
ensures that both GTP and ATP are available in sufficient quantities.
Inhibitors of purine synthesis (Anticancer agents):
There are a number of compounds that inhibit purine synthesis. These inhibitors have
wide applications in the control of purine nucleotide synthesis and cell multiplication
in certain cancers like leukemia (As anti cancer drugs).
1. Compounds that are structural analogs of purine bases or nucleosides interfere at

specific metabolic sites in the biosynthesis of purine nucleotides.
E.g. Mercaptopurine is a structural analogue of adenine. It inhibits the conversion
of IMP to AMP and GMP.
2. Methotrexates (Aminopterin, amethopterin) are structural analogs of folic acid.

They competitively inhibit folate reductase thus competitively inhibiting the synthesis
of tetrahydrofolate (THF), the coenzyme of folic acid. THF is required in step 4 and
10 of purine synthesis.
3. Glutamine antagonists like azaserine and diazonoleucine inhibit the enzymes that
utilize glutamine as amino group donor in the biosynthesis of purine nucleotides
(step 2 and 5).

2. Salvage pathways of purine nucleotides:

Purine s that are formed when the nucleic acids are catabol ised or that are
obtaine d
from diet can be directly converted to the corresp onding nucleo tides. This
process is
called salvage pathwa y. Purine nucleos ides can a lso be salvaged.
There are two mecha nisms in salvag e reactions,
a) Phosp horibo sylatio n of free purine bases:

This pathwa y require s 2 enzym es.
• HGPRT or Hypoxa nthine -Guanin e phosph oribosy l transferase
• APRT or Adenin e phosph oribosyl transferase
APRT
• Adenine
• AMP
PRPP PPi
HGPRT
• Hypoxa nthine • IMP
PRPP PPi
H GPRT
7~
• Guanine
• GMP
PRPP PPi
b) Phosp horyla tion of purine ribonu cleosid e:

Nucleoside phospho rylase
Purine ribonucleoside ----~- --:::,, 0...--- --.11 Purine ribonucleotide
ATP ADP
Examples:
Adenosine kinase
• Adenosine + ATP AMP+A DP
Guanosin e kinase
• Guanosin e +ATP GMP..-A DP
Signif icance of salvag e pathway:

• The salvage pathwa y is pa rticularly import ant in certain tissues such as brain
and
RBCs where de novo synthes is of purine nucleotides is not operati ve.
• This cycle ensure s the recycling of purine bases formed by nucleotide degrad
ation.
• This pathwa y utilizes far less energy than denovo synthesis.
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Cataboli sm of purine nucleotid es:

Uric acid is the end product of purine metabolism in humans.
AMP dearninase
AMP .. IMP GMP
Nucleotidase Nucleotidase Nucleotidase

Pi Pi Pi
Adenosine deaminase
Adenosine .. Inosine Guanosine
Pi Purine
nucleoside
Ribose- phosphorylas e
1-phosphate Pi Purine
nucleoside
Hypoxanthin e phosphorylas e
Ribose-
1-phosphate
Xanthine
oxidase
G uanase
Xanthine .. Guanine
Xanthine
oxidase
Uric acid
Normal level of uric acid in blood and urine:
• The normal level of uric acid in blood ranges from 2 to 6 mg/ dl in females and 3 to
7 mg/ dl in males.
• The daily excretion of uric acid is about 500 to 700 mg / day.
Disorders of purine metabolism:
A. Gout:
• Gout is a disorder characterized by increased uric acid level in blood (Hyperuricemia).
• At physiological pH, uric acid exists in a more soluble form monosodium urate
(MSU). Hyperuricemia results in the deposition of sodium urate crystals in soft
tissues, particularly joints. This is referred to as "tophi'.
• This causes inflammation in the joints resulting in painful gouty arthritis.
• Uric acid may also precipitate in kidney tubules and ureters that result in stone
formation (calculi) and renal damage.
The most common abnormality in purine metabolism is Hyperuricemia. Note that
gout is associated with hyperuricemia, but hyperuricemia may not always associate
with gout. Hyperuricemia may or may not be associated with increased excretion
of uric acid in urine (Uricosuria).
Causes:
Hyperuricemia and gout can be caused by;
l. Increased production of uric acid (Metabolic gout)
2. Decreased renal excretion of uric acid (Renal Gout)
3. Increased intake of purine rich diet
1. Increased production of uric acid (Metabolic gout):

This is the less common cause of gout. Increased production of uric acid due to defect
in metabolism of purine nucleotides is called metabolic gout.
Metabolic gout is of two types, primary and secondary.
a. Primary metabolic gout:

Primary metabolic gout is caused due to genetic defect in the enzymes of purine
metabolism leading to overproduction of uric acid. Important among these are,
• Overactivity of PRPP synthetase: Lack of feed back control of the enzyme by
purine nucleotides leads to increased production of purines.
• Overactivity of Glutamine PRPP amido transferase: Lack of normal allosteric
control of the enzyme by purine nucleotides leads to their elevated synthesis.
• HGPRTase deficiency: It causes the increase in purine nucleotide synthesis by 2
ways. First, decreased utilization of purines by salvage pathway results in their
accumulation and diversion of PRPP for purine nucleotide synthesis. Secondly, the
defect in salvage pathway leads to decreased levels of IMP and GMP causing
impairment in the feed back regulation of their production by denovo synthesis,
leading to overproduction of purine nucleotides.

• Glucose-6-phosphatase deficiency (Von-Gierkes disease): Glucose 6-phosphatase

deficiency blocks the conversion of glucose-6-phosphate to glucose. Glucose -6-
phosphate thus accumulated is channeled into HMP shunt pathway resulting in
elevated levels of ribose-5-phospha te, then PRPP and ultimately purine
overproduction.
Note that van Gierkes disease also leads to the increase in lactic acid, which reduces the renal
excretion of uric acid. So, van Gierkes disease can also be included under prirnary renal gout.
b. Secondary metabolic gout:

This results due to secondary defects of various diseases causing overproduction of
uric acid. These disorders causes enhanced turnover rate of nucleic acids and hence
increased production of uric acid.Examples are,
• Various cancers (leukemias, polycythemia, lymphomas etc)
• Starvation (raised rate of catabolism)
• Trauma (increased tissue damage)
2. Decreased renal excretion of uric acid (Renal gout):

This is the most common ca use of hyperuricemia and gout. Decreased excretion of uric
acid, due to impairment in renal function is called renal gout.
Renal gout is of two types; primary and secondary.
a. Primary renal gout:

It is caused due to some inherited defect in renal excretion of uric acid.
b. Secondary renal gout:
It is caused due to secondary defects of other known diseases that affect renal excretion
of uric acid (such as lactic acidosis, use of drugs such as thiazide diuretics, or exposure
to lead etc). So, uric acid is not excreted properly leading to their accumulation in the
blood.
3. Increased intake of purine rich diet:

Increased intake can occur due to increased ingestion of purine rich foods, such as
sweetbreads, red meat e tc. This ca uses increased catabolism of purines and
hyperuricemia. This is thought to be the least contributor in hyperuricemia & gout.
Alcoholism and gout: Intake of alcohol precipitates uric acid. Ingestion of ethanol can
lead to increased lactic acid, which inhibits the secretion of uric acid in renal tubules.

Treatment:
1. Uricosuric agents:
Uricosuric agents increase the renal excretion of uric acids by decreasing their
reabsorption from kidney tubules. E.g. Probenecid, sulfinpyrazole, salicylates and
steroids etc.
2. Allopurinol:
Allopurinol is a structural analog of Hypoxanthine. So, allopurinol competitively
inhibits of xanthine oxidase, thereby decreasing uric acid production. Hypoxanthine
will be accumulated, which can be easily excreted in urine as it is more soluble form.
3. Restriction of high purine diet and alcohol:
Gouty arthritis attacks may be precipitated by high purine diet and increased intake
of alcohol. Therefore these must be restrkted in the diet.
4. Anti-inflammatory drugs:
Acute attacks of gout are treated with anti-inflammatory agents. Colchicine, steroid
drugs like prednisone, and nonsteroidal drugs like indomethacin are used.
B. Lesch-Nyhan syndrome:
Cause:
It is an X-linked inherited disorder due to the deficiency of HGPRTase enzyme.
Characteristics:
• Hyperuricemia, Gout develops in later stages.
• Uric acid lithiasis (stone).
• Neurological abnormalities such as mental retardation, self-mutilation.
Biochemical basis:
• HGPRTase deficiency results in decreased utilization of purines by salvage pathway.
This causes the increase of PRPP, ultimately leading to increased denovo synthesis
of purine nucleotides.
• Neurological symptoms suggest that the brain is dependant on the salvage pathway
for the requirement of purine nucleotides.
Severe Combined Immuno-Deficiency (SCIO) or ADA deficiency:

Deficiency of Adenosine deaminase (ADA) enzyme causes Severe Combined Immuno-
Deficiency (SCID). It involves T-cell and B-cell dysfunction. ADA deficient children
usually die before 2 years from severe infections associated with immune suppression.

Biosynt hesis of Pyrimidine nucleoti des :

Pyrimidine nucleotid es are,
• Nucleotide s of cytosine are CMP, CDP, CTP, deoxy CMP, deoxy CDP & deoxy CTP
• ucleotides of thymine are TMP, TOP, TIP (sugar in these are Deoxyribos e) TIP
• Nucleotide s of uracil are UMP, UDP, and UTP
There are 2 processes that contribute to biosynthesis of pyrimidine nucleotides,

1. De novo synthesis
2. Salvage pathway
Denovo synthesis of pyrimidine nucleotides is the important pathway for the production
of pyrimidine nucleotides .
De novo synthesi s of pyrimidi ne nucleotid es:
• The Pyrimidine ring is synthesized as free pyrimidine ring and then it is attached to
ribose 5-phosphat e to form pyrimidine nucleotide. (This is unlike the purine
synthesis, where the new purine ring is built on a p reexisting ribose 5-phosphat e).
• UMP is the first pyrimidine ribonucleo tide to be synthesized .
• All other pyrimidine nucleotides are synthesized from UMP.
• UMP is converted to UDP, which serves as precursor for the synthesis of dUMP,
dTMP, UTP and CTP.
Sources of individua l atoms in pyrimidin e ring:

Many compound s contribute to the pyrimidine nucleotide ring formation.
,C
Sources C and N:
Clulamint. _
Asparute • Aspartate provides C4, CS, C6 & 1
CO, - C
JI • Glutamine provides 3
• CO2 provides C2

Pathway of denovo synthesis of pyrimidi ne nucleotid es:
Dihydroorot ate
Dihydrooro tate d ehydrogena se
Orotate
k NAD
NADi l +H•
Orota tc phosphoribo syl transferase
r PRPP
PPi
(OMP (Orotidire monophosp hate)
OMP Decarboxyla se L
!~CO2
UMP (Uridire monophO!>phatc)
UMP is the first pyrimidine nucleotide to be produced. UMP in tum yields all other
pyrimidine nucleotide s.
Synthesis of UDP, COP, UTP, CTP and deoxy pyrimidin e nucleotide s:

UMP
l,--ATP
Kinase
~ADP
UDP
Rlbonucleo tide reductase
ATP
Kinase
ADP
UTP dUMP
Glutamine, N 5,to Methylene FH4

2ATP Thymidylate
CTP synthase synthase F8i
Glutamate,
2ADP+ Pi
TMP
CTP
Regulation:
• Carbamoyl phosphate synthetase II (CPS-II) is the regulatory enzyme of pyrimidine
synthesis. It is activated by PRPP and ATP. It is inhibited by UTP.
• Apartate transcarbam oylase is allosterically inhibited by CTP and activated by ATP.
Inhibitors (Anticancer agents):

There are a number of compound s that inhibit pyrimidine synthesis. These inhibitors
have wide application s in the control of pyrimidine nucleotide synthesis and cell
multiplicat ion in certain cancers like leukemia (As anti cancer drugs).
• 5-Fluorour acil and 5-iodo-2-d eoxyuridin e competitiv ely inhibit thymidyla te
synthase. So, these dugs are used as anti cancer drugs.
• Methotrexates (Aminopte rin, amethopterin) inhibit folate reductase enzyme and
thereby reduces the formation of FH4• FH4 is required for pyrimidine synthesis.
Thus, Methotrexates are used as anticancer drugs.
2. Salvage pathway of pyrimidi ne nucleotides:

Pyrimidine bases can also salvaged be to produce pyrimidine nucleotides in human
body. These pathways use PRPP as sources of ribose phosphate. This pathway forms
the basis of using uridine in the treatment of orotic aciduria.
Catabolism of pyrimid ine nucleot ides:

• Catabolis m of pyrimidi ne produces water-sol uble metabolit es like CO , and NHy P-
alanine and B-aminoiso butyrate. 2
• Catabolis m of Cytosine and Uracil yields P-alanine, CO , and NH

2 3
• Thymine on catabolism produces P-aminoi sobutyrat e, CO and NH •
2 3
• Pseudour idine obtained from tRNA is excreted unchange d.
02
Cytosine '::::::,, Uracil
....... Thymine
r=:
IJi
NHJ
ADPH + H•
NADPH ,H
r== ADP+ NADP+
rH~
Dihydrouracil
rDihyd rothymine
H,O
r
N - carbamoyl p - alanuu
H, O
'
r
carba moyl - P- aminoisobu tyrate
H, O
! l J
P-alanine ('() , 1 H, P-aminois obutyrate
Disorders of pyrimid ine metabuli:,uL

Clinically de tectable abnormal ities due to pyr111aJi ne overprod uction are few. This is
because the end products of pyrimidin e catabolism are highly water soluble compoun ds.
Orotic aciduria:
It is a hereditar y disorder associate d with pyrimidi ne nucleotid e biosynthesis.
• There a re two types of orotic aciduria. Type I reflects a deficiency of both orotate
p hosphori bosyl transfera se and orotidyla te decarbox ylase. Type II results from
deficiency of only Orotidyla te decarboxylase.
• In both the types, there is a defect in the conversio n of OMP to UMP that causes the
accumula tion of orotic acid in the blood and its excretion in urine. Other features are
severe anemia and retarded growth.
• Feeding diet rich in Uridine or Cytidine is an effective treatmen t for Orotic aciduria.
These compoun ds provide pyrimidi ne nucleotid es required for D A and RNA
synthesis . Besides this, UTP inhibits CPS-II and blocks synthesis of Orotic acid.
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Questio n Bank on Nucleot ide metabolism

ESSAY QUESTI ONS (10 Marks) :
1. Discuss the denovo synthesis of purine nucleotide s? Add a note on Gout
2. Discuss the denovo synthesis of pyrimidin e nucleotides?
SHORT ESSAYS (5 Marks) :

1. Catabolism of purine nucleotide s
2. Gout
3. Catabolism of pyrimidin e nucleotides
4. Salvage pathway of purine nucleotide s
SHORT A SWERS (2 to 3 Marks) :

1. Lesch Nyhan Syndrome / Orotic aciduria / ADA deficiency

1) The cause of hyperuric emia and gout in glucose-6- phosphata se deficiency is (AIIMS)
a) More formation of pentose b) Decreased availability of glucose to tissue c) Increased
accumulat ion of sorbitol d) lmpaired degradatio n of free radicals
(AIIMS)
2) Lesch Nyhan syndrome is caused due to
a) Total deficiency of HGPRT b) Partial deficiency of HGPRT c) Total deficiency of PRPP
d) Partial deficiency of PRPP
3) End product of purine catabolism is QIPMER)
a) Creatinine b) Urea c) Uric acid d) Phosphate s
4) Source of -1 nitrogen of purine nucleotide during denovo synthesis is
a) Aspartate b) Glycine c) Glutamine d) Glutamate
5) Severe Combined lmmuno Deficiency (SCID) is caused by the deficiency of

a) ADA b) HGPRT c) Xanthine oxidase d) PRPP syn thetase
6) Which of the following is NOT a source of purine ring?
a) Gluta mine b) Formyl TH FA c) Formimn o THFA d) Glycine
7) Conversio n of IMP to GMP & AMP is inhibited by

a) Mercaptopurine b) Methotrexate c) Azaserine d) None of these
8) Conversio n of adenosine to inosine is catalyzed by

a) IMP dehydrog enase b) Xa nthine oxidase c) adenylate cyclase d) adenosine deaminas e
9) Pyrimidine biosynthe sis begins with the formation from glutamine , ATP and CO2 of
a) Carbamoyl asp artate b) Orotate c) Carbamoyl Phosphate d} Dihydroor otate
10) Hyp eruricernia can result from deficiency of all the following enzymes EXCEPT
a) HGPRTas e b} APRTase c) Glucose-6 -phosphatase d ) Adenosine deaminas e
Answers for MCQ: 1) a 2) a 3) c 4) a 5) a 6) c 7) c 8) c 9) a 10) d
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Biochemical genetics (Part 1) - Replication, Transcription, Translation 421
Molecular Genetics and Protein Biosynthesis
Contents:
• Central Dogma of Molecular Biology
• Replication
• Definition
• Origin of Replication
• Salient Features
• Requirements (All enzymes, factors)
• Events
• Transcription
• Definition
• Requirements
• Salient features
• Requirements (AU enzymes, factors)
• Events
• Post-transcriptional modifications
• Promoter site
• Translation
• Definition
• Requirements
• Salient Features
• Requirements (All enzymes, factors)
• Events
• Post-translational modifications
• Inhibitors
• Protein targeting

Flow of genetic information:

Nucleic acids are required for the storage and expression of genetic information. DNA
is the carrier of genetic information. The genetic information present in DNA are not
only expressed in the organisms, but also carried from generation to generation.
This is by three processes, namely, replication, transcription and translation.
• RepJication:
Rc' pli,Mion means the synthesis of DNA. The genetic information stored in the DNA
" • ,,,-.. iPrl ,prl • rn nsmitted to daughter cells through DNA replication. DNA replication
~1 . ;.,Pd ,._ ~hp process of synthesizing two identical daughter DNA molecules from
.. .. ,. · ' ' \ ,nolecules.
• l t"'""~<Jp l1uu :
l lu11drt:!d S of different proteins are synthesized by the information stored in the DNA.
Transcription is the first stage in the expression of genetic information. Transcription
is defined as the process by which genetic information present in DNA is copied to
RNA. Simply, the process of synthesis of RNA from DNA is called transcription.
• Translation:
Next, the code present in the nucleotide sequence of messenger RNA molecules is
translated to the forma tion of proteins, thus completing the genetic expression.
Translation is defined as the process by which genetic information transcribed in
mRNA directing the synthesis of proteins.
Thus the genetic information flows from DNA to RNA and then to proteins. This is
calJed central dogma of genetic information.
Transcrip tion Tra nsla tion

RNA Protein
Cell cycle:
The cell cycle involves DNA replication followed by cell division to produce two
daughter cells from one parent cell. The whole cell cycle lasts for about 24 hours.
Cell cycle has 4 Phases, namely, Gl, S, G2 and M phase.
DNA replication takes place in S phase (synthesis) phase, where DNA completely
replicated (only once). M phase or Mitosis phase is the cell division phase.
These two phases are separated by two gap phases GJ and G2. In Gl phase, the cell prepares
for DNA synthesis. In G2 phase, cell prepares for mitosis, when proteins necessary for
daughter cells are synthesized.

DNA replication:
Introduction:
Synthesis of DNA from DNA is termed as replication.
DNA is the carrier of genetic information. During cell division, DNA is copied by
replication and transmitted to daughter cells. Replication results in the synthesis of 2
DNA daughter molecules identical to the parental D A. Replication is an important
aspect of inheritance as it ensures that each daughter cells get identical D A as
parent cell.
Definition:
DNA replication is defined as the process of synthesis od DNA from DNA.
It is the process of synthesizing two identical daughter DNA molecules from a parent
DNA molecule.
Origin of replication:
• The replication begins at a specific site on DNA called origin of replication (ori).
• In all the organisms, replication can occur only from a single stranded DNA (ssDNA)
templa te. So, first, DNA should unwind and separate to form 2 single stranded
DNA templates. This unwinding and separation is initiated at a site called the
origin of replication on DNA.
• Prokaryotes have a single origin of replication site and Eukaryotes have multiple
replication origin sites along the DNA.
• At the site of origin of replication, a short stretch of DNA unwinds to form a

replication bubble. A number of proteins (like DnaA protein in E coli and origin
recognition complex (ORC) proteins in yeast) h ave been identified as proteins
responsible for recognition and initiation of this process.
Salient features of DNA replication:
• Semi conservative
• Bidirectional
• Semi discontinuous

Biochemical genetics (Part 1) - Replication, Transcription , Translation 424
1. DNA replication is Semi conservative:

During replication, two strands of DNA molecule progressively unwind and separate
from each other. Each strand serves as a template against which a new complementary
strand is synthesized. The base pairing rule is always maintained. (i.e. Adenine always
pairs with thymine and cytosine always pairs with guanine). Consequently, each of
the 2 daughter DNA molecule synthesized contains one parent strand and one newly
synthesized strand.
Since the daughter DNA molecules consist of a parental strand and a newly
synthesized strand, the process of replication is known as Semi conservative.
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2. DNA replication is bidirectional:

At the 'origin of replication' sites, the 2 strands unwind and separate to produce a
replication bubble. Replication takes place in both the directions (Bidirectional) in
these replication bubbles. Each replication bubble has 2 "V" shaped ends called the
replication forks, which move in opposite directions as the DNA winds progressively.
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3. DNA replication is a semi-discontinuous process:
• DN A polymerases are the enzymes responsible for DNA replication. These en zymes
are able to read the parental template strand in 3'- 5' direction and synthesize the
new strand in 5' - 3' direction .
• When the small s tretches of DNA unwinds at replication forks, the two separa ted
template strands run in opposite directions - one in 3'- 5' direction and the other
in 5'-3' direction. (So, the two newly synthesized stretches of deoxyribonucleotide chains
should grow in opposite directions - one in the 5'- 3' direction towards the replication
fork and other in the 5' -3 ' direction aw ay from the fork. But, this synthesis takes place
by different mechanism on each strand. This is because the DNA poymerases can read the
parental template strand in 3'- 5' direction and synthesize the new strand in 5'- 3' direction.)
• The synthesis of new daughter strand is continuous in 5'-3' direction toward s the
replica tion fork. This strand is called the leading strand.
• Bu t in the other template strand, the synthesis of new daughter strand (away from
replication fork) is discontinuous in 5'- 3' direction in the form of m any short
segments (called the Okazaki fragments) . This strand is called the lagging strand.
• Such a replication, continuous in the leadin g s trand and discontin uous in the
lagging strand, is termed as a semi discontinuous process.
• Finally, when the replication is complete, these Okazaki fragments a re joined to
form a continuous daughter stran d.
[ DNA replication is semi-discontinuous)
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Okazaki fragments:
The small segment (or s tretch) of DNA molecule attached to its own RNA primer in
the lagging strand is called Okazaki fragments.
DNA synthesis by DNA polymerases can take pace only in 5'-3' direction. So, in
the leading strand replication takes p lace continuously in S'-3' direction. But in the
strand synthesizing away from the fork (lagging strand), the DNA is synthesized
discontinuously in 5'- 3' direction in the form of many short segments. These short
stretches of newly synthesized DNA along with RNA primer are called Okazaki
fragments.
DNA li.elica.se.
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• Each okazaki fragment is made up of 150-200 nucleotides.
• Eventually, RNA primers from each okazaki fragments are removed, the gaps are
filled by DNA polymerases and these pieces are ligated by ligase enzymes. Thus,
these fragments are joined to form a single continuous daughter strand (lagging strand).

Biochemical genetics (Part 1) - Replication, Transcription , Translation 427
Events in replication:
1. At the site of origin of replication, unwinding of a segment of DNA takes place to
form a replication bubble, which has two replication forks running in opposite
direction . A number of pro teins are responsible for recognition and initiation of
this process.
2. D NA helicase enzymes bind to the DN A near the replication fork and then moves
along the DNA, forcing the two strands to separate. This ca uses the unwinding and
progressive separation of DNA double strand .
3. Then, the Helix destabilizing proteins (SSB proteins) bind with these D A single
strands and maintain the separation of these two template strands.
4. DNA topoisomerase enzymes remove the positive supercoiling constraints that are
produced during the unw inding of D A by helicase enzymes.
5. The RNA primers (produced by RNA primase), complementary to a short stretch
of tem plate DNA are a ttached to the ssD A tem plates. Only one RNA primer is
required in lead ing strand to initiate D A synthesis. But lagging strand requires
many primers.
6. DNA polym erase enzymes are responsible for DNA synthesis. They are able to
read the parent strand in 3'- 5' direction and synthesize the new daughter strand
in 5'- 3' direction .
7. The leading strand is synthesized continuously (towards the replication fork) and
the lagging strand is synthesized d iscontinuously (away from the replication fork)
as Okazaki fragments. Both strands are synthesized in opposite d irections.
8. Once DNA synthesis is comp leted, the RNA primers are removed, the gaps are
filled by D A polymerases and these two pieces are ligated by ligase enzymes.
9. DNA polymerases also perform the proof reading to ensure the genetic fidelity.
Inhibitors of D NA replication:
• Nucleotide analogues like arabinosyl cytosine (cytarabine) and arabinosyl adenine
(vidarabine) inhibit DNA replication. They are used as anticancer and antiviral drugs.
• Antibiotics like Ciprofloxacin, novobiocin, Nalidixic acid inhibits bacterial DNA
gyrase; They have no effect on huma replica tion.
• Adriamycin, Etoposide, Doxorubicin inhibits human topoisomerase. They are used
as anticancer drugs.
• 6-mercaptopurine inhibits human DNA polymerase.

Requirements for replication:
• The four deoxy ribonucleotides (dATP, dGTP, dCTP, dTIP)

• RNA primer
• Proteins like Helix destabilizing proteins or Single Stranded DNA Binding (SSB)
proteins, proteins required for recognition and initiation of replication process (like
DnaA protein in E coli and origin recognition complex (ORC) proteins in yeast)
• Enzymes like DNA polymerases, DNA helkase, DNA topoisomerase, primase, ligases
etc.
DnaA protein in E coli and origin recognition complex (ORC) proteins in

yeast:
• DnaA protein in E coli and origin recognition complex (ORC) proteins in yeast have
been identified as the proteins responsible for recognition and initiation of replication.
These bind to specific nucleotide sequences at the origin of replication and break a
short AT rich region of DNA causing the unwinding of a small stretch of DNA to
form a replication bubble.
• In eukaryotes, similar proteins have been identified, but they are not yet precisely
defined.
RNA primer:
• DNA polymerases cannot initiate synthesis of a new complementary strand of DNA

against the template single strand. They require a RNA primer-that is a short stretched
RNA, which is attached to the DNA template, with a free -OH group on the 3' end of
the RNA strand. This free -OH end serves as the first acceptor of a nucleotide by
action of DNA polymerase.
• Only one RNA primer is required at the origin of replication on the leading strand. In
contrast, many such RNA primers are required at the replication fork on the lagging
strand.
RNA primase:
• A specific RNA polymerase ca lled primase, synthesizes the short stretches of RNA
that are complementary and antiparallel to the DNA template.
Primase binds to the DNA helicase form ing a complex called Primosome. Primosome complex
is responsiblefor creating RNA primers on a single stranded DNA during DNA replication..

DNA polymerases:
• DNA polymerases are the enzymes responsible for DNA synthesis. They are able to
read the parent strand in 3'-5' direction and synthesize the new daughter strand in
5'-3' direction.
• The synthesis of two new daughter strands takes place simultaneously, but in the
opposite directions - one continuously (leading strand) in the 5'-3' direction towards
the replication fork and other discontinuously (lagging strand) in the 5'-3' direction
away from the fork.
• DNA polymerase cannot initiate DNA synthesis against the template strand. Rather,
DNA polymerase adds the first deoxyribonucleotide to the 3' end of RNA primer
(short stretched RNA sequences which are attached at the beginning of DNA
template). Then, deoxyribonucleotides are added one at a time, the sequence of
this newly synthesized DNA strand is complementary to the sequence of template
strand.
• The nucleotide substrates are deoxyribonucleotide triphosphates (dATP, dGTP, dCTP,
and dTIP). As these deoxyribonucleotide triphosphates are added one at a time, to
the 3' end of growing chain, a pyrophosphate (PPi) is released and only dAMP, dGMP,
dCMP or dTMP are incorporated.
DNA polymerases have mainly three functions,

• DNA chain elongation in the 5'-3' direction by (5'-3' polymerase activity).
• DNA proof-reading (3'-5' exonuclease activity) to correct the mistakes of replication,
if any.
• DNA repair (5'-3' exonuclease activity) to remove RNA primer.
Prokaryotic DNA polymerases:

In prokaryotes, there are three types of DNA polymerases, I, II, III. Among them
DNA polymerase III is the most important one.
• DNA polymerase I (Pol I): Once the replication is completed, pol I removes the RNA
primers by 5' - 3' exonuclease activity. Pol I also fills this gap by synthesizing the
fragment of DNA (by 5' - 3' polymerase activity) and completes the chain synthesis.
Pol I also has proof reading activity (3' - 5' exonuclease activity).
• DNA polymerase II (Po1 II): This enzyme concerned with DNA proof-reading and
DNA repair.
• DNA polymerase III (Pol III): It is the most important polymerase synthesizing the
new strand in 5'- 3' direction (the 5'- 3' polymerase activity). This enzyme also has
proof reading (3'- 5' exonuclease) activity.

Eukaryotic DN A polymerases:
In eukaryotes, at least five types of DNA polymerases have been identified. They are
designated by Greek symbols (a , j3, y, 6, and £) rather than by Roman letters.
• Pol <X has prirnase activity (to synthesize RNA primer) and also by its 5'- 3' polymerase
activity, it initiates DNA synthesis in both leading and beginning of each Okazaki
fragments on the lagging strands.
• Pol j3 has DNA repair activity.
• Pol y replicates mitochondrial DNA.
• Pol 6 has 5'- 3' polymerase activity. It completes the DNA synthesis on the leading
strand and lagging strand. It also has proof reading (3'-5'exonuclease) activity.
• Pol£ has proof reading (3'- 5'exonuclease) activity and leading strand synthesis.
DNA helicase:
• DNA helicase enzymes are responsible for unwinding and progressive separation of
double helix. So, it is also called as DNA unwinding protein. It binds to ssDNA.
• DNA helicase enzymes bind to the DNA near the replication fork and then moves
along the DNA molecule forcing the two strands apart. ATP provides the energy for
this action.
Helix destabilizing proteins or Single Stranded DNA Binding (SSB) proteins:

• SSB proteins are responsible for maintaining the separation of the parent strands
(Maintain the unwinding of separated strands). They do not carry any enzyme activity.
• After the helicase enzymes separate the DNA strands, SSB proteins bind with these
DNA single strands and keep the two strands separated. They also protect the single
stranded DNA against degradation by nucleases.
DNA topoisomerases:
• As the two strands of the DNA helix unwinds and separated at the replication fork,
the DNA becomes positively supercoiled downstream away from the fork. These
supercoiling constraints are relieved by DNA topoisomerases, which remove
superhelicity and produce negative supercoiling and unwinding.
DNA gyrase is a type DNA topoisomerase present only in Prokaryotes.
DNA ligase:
• Once DNA synthesis is completed, the RNA primers are removed, the gaps are filled
by DNA polymerases and these two pieces are ligated by ligase enzymes. The energy
for this ligation is obtained by ATP hydrolysis.

Transcription:
Definition:
Transcription is defined as the process of synthesis of RNA molecules from DNA.
Requirements for transcription are:

• Template D NA strand
• Substrates: The four ribonucleotides (ATP, GTP, CTP, UTP)
• Enzymes: RNA polymerases (also known as DNA dependant RNA polymerases)
• Transcription factors
• Termination factors
Salient features:
• One of two strands of DNA acts as the template strand for transcription. This template
strand of DNA is referred to as non-coding strand or antisense strand. The other
DNA strand, which does not participate in the transcription, is called the coding
strand or sense strand.
• RNA polymerases are the enzymes required for transcription.
• Process of transcription begins with the binding of RNA polymerase and transcription
factors to the promoter region. Promoters are specific regions of DNA that binds
w ith RNA polymerase and initiates RN A synthesis. (Promoter site is not transcribed).
• In transcription the template strand is read in 3'-5' direction and synthesis of new
strand takes place in 5'-3' direction (Similar to replication).
• Only a small portion of DNA is transcribed into RNA (whereas entire D A genome
is replicated in Replication)
• Substrates for transcription are four ribonucleotides (ATP, GTP, CTP and UTP).
Replication substrates are four deoxyribonucleotides (deoxy ATP, deoxy GTP, deoxy
CTP, deoxy TIP). Note that uracil substitutes thymine as the complementary base
pair for adenine in transcription
• RNA polymerase does not have proof reading activity.
• RNA primer is not required to initiate RNA synthesis.
RNA polymerase (also called the DNA dependant RNA polymerases):

RNA polymerases are enzymes that cause transcription. They read the template
strand in 3'-5' direction and synthesize of new strand in 5'-3' direction.
• Prokaryotes have a single RNA polymerase that transcribes all 3 types of RNAs
(mRNA, tRNA, and rRNA)
• In Eukaryotes there are 3 different RNA polymerases that transcribe the 3 RN A's.
i) RNA polymerase I - Synthesizes rRNA
ii) RNA polymerase II - Synthesizes mRNA
iii) RNA polymerase Ill - Synthesizes tRNA.

Events in transcription:
1. Initiation:
• Process of transcription begins with the binding of RNA polymerase and
transcription factors to a specific region on the template strand known as the
promoter site (which is not transcribed).
• The binding of the RNA polymerase and initiation factor at the promoter site on
the DNA template results in unwinding and separation of a small segment of the
DNA double helix molecule. This produces a transcription bubble.
• Next, the first ribonucleotide (complementary to the first nucleotide of DNA
templa te) attaches to the RNA polymerase, followed by binding of the second
ribonucleotide to the polymerase. Now, a phosphodiester bond is formed between
the first and second ribonucleotide.
• RNA polymerase action continues by incorporation of ribon ucleotides, one at a time,
the sequence complementary to the DNA template strand.
• After 10- 20 nucleotides have been transcribed, the transcription factors are released.
Now the RNA polymerase enzyme leaves the promoter site and moves along the
template strand. This marks the end of initiation phase.
2. Elongation:
• During the elonga tion process, RNA polymerase then moves along the DNA
template adding nucleotides complementary to the template DNA, one by one. RNA
polymerase reads the template DNA strand in 3'-5' di rec ti on and newly synthesized
RNA chain elongates in 5'- 3' direction. The elongation process continues until the
termination signal is reached.
• As the RNA polymerase moves along the DNA template, DNA unwinding takes place
downstream nnd rewind at upstream area of template strand.
3. Termination:
• Termination of transcription is signaled by a unique sequence in the template DNA
strand called the termination site (or terminator). Termina tion signal is recognized by
a protein factor called the termination factor. In prokaryotes, this factor is called rho
(p) factor. In eukaryotes, it is not yet characterized .
• When termination factor attaches to DNA at the terminator, RNA polymerase cannot
move further. This marks the completion of transcription process and the newly formed
RNA (primary transcript) chain will be released.
Inhibitors of RNA synthesis:

• Actinomycin-D and Mitomycin binds with the DNA template and interferes w ith
the movement of RNA polymerase along the DNA, blocking the transcription. These
are used as anticancer d rugs.
• Rifampicin and Rifamycin inhibit the transcription by binding with prokaryotic RNA
polymerase. It is widely used in the treatment of tuberculosis and leprosy.
• cx-Amanitin (Source - poisonous mushrooms) inhibits eukaryotic RNA polymerase.

Biochemi cal genetics (Part 1) - Replicatio n, Transcrip tion, Translatio n 433
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Posttranscriptional modifications:
RNA synthesized from DNA is called the primary transcript. TI1en the primary transcript
undergoes certain covalent modification to form mature RNA. These modifications are
collectively known as posttranscriptional modifications. Essentially all RNA's are
covalently modified after transcription to form mature RNA.
a) Modification in mRNA:
Primary transcript of mRNA is known as hnRNA or heterogeneous nuclear RNA.
hnR A undergoes posttranscriptional modifications to become the mature mRNA.
These modification in volve,
• Removal of Introns and splicing: The primary transcript consists of sequences
which code for proteins called 'Exons' and sequences which do not code for proteins
called 'Introns'. A gene is made up of many exons and introns. In hnRNA, exons
are not continuous, but interrupted by introns. During the formation of mature
mRNA, the introns are removed and the exons are joined together by a process
called splicing. Thi? is catalyzed by small nuclear RNA or snRNA or ribozymes.
A multicomponent complex called the spliceosomes (which includes snRNA & specific proteins)
is involved in converting primary transcript to mRNA (see the box below).
• 5' Capping: The cap of 7- methyl GIP is attached to the 5' term inal nucleotide.
• Poly A tailing: Many Adenine nucleotides are added at 3' end.
• Methylation: Some of the internal nucleotides of mRNA are methylated.
• Endonuclease cleav age: Few nucleotides from 3' end are cleaved.
b) Modification in tRNA:
• Addition of CCA sequence at the 3' end.
• Modification of bases at specific positions to produce "unusual bases" like Pseudo-
uridine, Dihydrouracil etc.
• Intron removing and splicing of exons.
c) Modification in rRN A:
All rRNAs are synthesized from long precursor molecu les called preribosomal RN As.
The preribosomal RNA's are cleaved by ribonucleases to yield different intermediate
sized rRNAs, which are further modified to produce specific RNA species/ sub-units.
Spliceosomes:
Spliccosomes are special multicomponent systems that splice hnRNA to form active mRNA.
Spliceosomes consistes of a primary trascript (hnRNA), five snR As (Ul, U2, U4, US &
U6) and more than 60 proteins.
Formation and function: This takes place inside the nucleus. snRNA complexes with proteins
to form small nuclear ribonucleoprotein particles (SnRNPs, pronounces as "snurps").
SnRNPs associated with hnRNA at the exon-inron junction to form spliceosomes. Cuts
are made at both the ends of intron; then introns are removed and exon-exon ends are
ligated at G-G residues.

Biochemica l genetics (Part 1) - Replication , Transcripti on, Translation 435
Transcription factors:
• Transcripti on starts with the recognition of the template D A strand and the initiation
point of transcriptio n. This requires proteins factors known as transcriptio n factors.
Transcripti on factors recognize promoter sequence on DNA. They have high affinity
to promoter sequence. Once bound to DNA, they form strong association with RNA
polymeras e and initiate RNA transcriptio n.
a) In prokaryote s, transcripti on factors are called the sigma (6) factors.
b) In eukaryotes , at least 7 transcripti on fac tors (GTF's, TFIIA, B, D, E, F, and H) have
been recognized .
Promoters:
• Definition : Promoters are specific regions of DNA that binds with RNA polymeras e
and initiates RNA synthesis.
• Process of transcriptio n begins with the binding of RNA polymeras e and transcriptio n
factors to the promoter region. The binding of the RNA polymeras e and initiation
factor at the promoters on the DNA template resu lts in unw inding and separation of
a small segment of the D A double molecule.
Promoter sites are mostly situated towards S' side (upstream or left) of transcriptio n
start s ite wi th reference to the nontempla te strand. ( ote: By convention , promoter
sites are designated by the 5'-3' nucleotide sequence on the non template strand).
No te: A base in the promoter region is assigned a negative number if it occurs prior to (to tile
left of towards the 5' -end of or upstream of) the transcription start site. The first base at the
transcription start site is assigned the position of+ 1. Note that there is no base designated "O".
a) Prokaryotic promoters :
• Bacterial promoters are approxima tely 40 nucleotide s in length. In this region, there
are two short conserved sequence elements (which togeth er comprise the promoter).
i) -35 sequence: Approxima tely 35 bp upstream of the transcriptio n start site, there is a
consensus sequence [5'-TTGAC A-3'], to which the RNA polymeras e binds.
ii) Pribnow box: More proxima l to this site, about 10 nucleotide s up tream is a
consensus sequence of 6 nucleotide s [5'-TATAA T- 3'], known as Pribnow box
( ometimes referred to as TATA box).
b) Eukaryoti c promoters:
In eukaryotic promoters, there are two important conserved sequence elements.
• Goldberg Rogness box: There is a conserved sequence 5'- TATAAAA -3' (almost
identical to Pribnow box), located about 25 nucleotides upstream, known as Goldberg
Rogness box or TAT A box.
• CAAT box: Further upstream between -70 & -80 nucleotid es, another conserved
sequence of GGCCAATCT known as CAAT box is present.
• Few transcriptio n frequency signals, that determine how many times the RNAP should
work on that particular gen e, are seen further upstream in DNA.
• Enhancers & silencers increase or decrease the rate of transcriptio n. These are located
either upstream or downstrea m, about 1000 nucleotide s away from the start site.
Prokaryotic promoter region:
Promoter region • I .,__ Transcribed region _.

Start site of transcription
3' -------------....&--------- 5'

TTGACA TATAAT
5' 3'
-35 -10 +1
Pribnow box
Se::i.uences within the

Prokaryotic promoter region
Eukaryotic promoter region:
Promoter region • I .,__ Transcribed region _.

Start site of transcription
3'
CGCAATCT TATAAAA
5'
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CAATbox H ogness box
Se::i.uences within the

Eukaryotic Promoter region

Translation:
In protein synthesis, first, the genetic information stored in the DNA is transcribed in
the nucleus to the specific sequence of nucleotides of RNA molecule, which is then
translocated cytoplasm. In cytoplasm, the genetic information present on the mRNA
(in the form of genetic code) is translated into the synthesis of corresponding proteins
with specific sequence of amino acids.
Definition:
Translation is defined as the process of protein synthesis using mRNA as template.
Requirements:
• Amino Acids: (20 primary protein amino acids).

• Ribosomes: These are the protein synthesizing machineries of the cell.
• mRNA: These are the templates of protein synthesis
• tRNA: Transports amino acids to the site of protein synthesis.
• Energy (Both ATP and GTP)
• Proteins: Initiation factors (elFs), elongation factors (EF), releasing factors (RF).
• Enzymes: Amino acyl tRNA synthase, Peptidyl transferase
Salient features:
• Translation takes place in the ribosomes present in cytoplasm. Ribosomes are called
the factories of protein synthesis.
• The mRNA is read in the S' to 3' direction and polypeptide synthesis proceeds
from the amino terminal (N-terminal) to carboxy terminal (C-terminal).
• The N-terminal amino acid (First amino acid) is N-formyl methionine in prokaryotes;
Methionine in eukaryotes.
• Transfer RNA (tRNA) is the carrier of amino acids for protein synthesis. At least
one species of tRNA exists for each of the 20 amino acids.
• Translation can be studied under phases: Activation of amino acids, initiation,
elongation, & termination.
Translation consists of 4 processes,

1. Activation of amino acids
2. Initiation of protein synthesis
3. Elongation of polypeptide chain
4. Termination of protein synthesis

1. Activation of amino acid:
In this process, 20 different amino acids are charged with specific tRNAs.
The enzyme amino acyl tRNA synthetase attaches amino acids to specific tRNA by
ester linkage.
There are at least one species of tRNA to carry each of the 20 amino acids.
---7----,--~.,..------•-- Amino acyl tRNA

Amino acyl tRNA synthetase
Amino acid + tRNA
ATP AMP+ PPi
Amino acids remain attached to its specific tRNA until it polymerizes during translation.
Now, this aminoacyl tRNA transports the a ttached amino acids to the site of protein
synthesis (ribosomes).
2. Initiation of protein synthesis:
This requires mRNA, 40S and 60S ribosomal subunits, initiation factors (eIF), GTP and
methionyl tRNA (first amino acyl tRNA). To start with, two subunits (40S and 60S) of
ribosomes are in dissociated form and they associate at the end of initiation step to
form a complete 80S ribosome.
In this stage, me thionyl-tRNA is combines with mRNA on ribosomes to form initia tion
complex. Initiator Codon (AUG) of m.RNA interacts with anticodon (UAC) present in
methionyl tRNA. This step requires GTP and initiation factor.
Steps in detail:
• First, methionyl-tRNA, 40 S ribosomal subunits, GTP, and elF-2 are complexed
to form pre-initiation complex (43S). eIF-1, eIF-3, and eIF-5 are also required
in this process.
• Next, mRNA combines to this pre-initiation complex to form 48S initiation
complex. This requires eIF-3, eIF-4, and ATP. Initiator Codon (A UC) of mRNA
interacts with anticodon (UAC) present in methionyl tRNA.
• Then, the 60S subunit of ribosome combines with this 48S initiation complex
to form 80S initiation complex. This requires eIF-2, eIF-5, and GTP.
At the end of the initiation step, the formation of 80S ribosome is complete with mRNA
attached. The whole ribosomal unit encompasses two codons inside. This ribosome
has 2 sites called the P site (peptidyl site) and the A site (Aminoacyl site). P site contains
methionyl tRNA and A site is vacant.

3. Elongation step:
• A new (second) aminoacyl tRNA binds at A site. The second codon after AUG
determines the second amino acid. (Th is requires EF-1 and GTP).
• Peptidyl transferase enzyme forms a peptide bond between the first & second amino
acids, resulting in the formation of a dipeptide attached to tRNA in the A site. Then
the first tRNA is released from the site.
• Then the whole ribosomal unit moves along mRNA, by one codon in the 5' to 3'
direction so that the peptidyl tRNA is transloca ted from A site to P site. This
movement is called translocation. (This requires EF-2). '
• At this stage, the first initiation codon AUG is outside the ribosome, the 2nd codon is
directly opposite P site and third codon opposite the A site. Now, A site is vacant
and ready to receive the next amino acyl tRNA bearing appropriate anticodon.
The whole process is repeated a number of times till a "chain termination codon" is
approached. At this stage, P site contains tRNA with its attached polypeptide chain.
4. Termination of protein synthesis:
• After the successive addition of amino acid, the ribosome reaches termination codons
(UAA, UAG or UGA) on the mRNA. Since there is no tRNA with corresponding
anticodon sequence for the terminator codon on mRNA, the A site remains free.
• A releasing factor (RF) enters this site & binds with the nonsense codon on A site.
• The releasing factor along with peptidyl transferase enzyme brings about the
hydrolysis of the bond between the polypeptide chain and tRNA present in P site.
This results Jn the release of newly synthesized polypeptide (proteins) chain & tRNA.
• Ribosome in the same time is released from mRNA and finally this 80S ribosome
dissociates into its components 40S and 60S subunits.
Polyribosomes (Polysome):
Many ribosomes can attach to a single mRNA molecule at regular intervals and
translate the same mRNA simultaneously. As each ribosome moves along the
mRNA in 5' to 3' direction, it weaves a copy of the same protein. Multiple ribosomes
on a single mRNA molecule is called polyribosome or polysome.

( Events in translation)
( Initiation )
{,OS R.,eoS0'1E
A site
u
m RNA
5'
-+ Jnitio.tion -t ATP
f<>-dOT~ 6TP
40 S R. IBOSOMI:
(Elongation) Peptide bond
Pe pt ic!• Bo•d
fo-t"'o.tron
flon jo..tio11,
f"-dor~ uf ~·,2.
T,0-~$10,a.tion.
3
3'
(Termination)
0-0--0-0--0-0-0
0 0
Releo..sinj
f11d.0T Y~leo.<i"J
Pe pt;dyl :f"-ctoT
t v ll..l'\.Sfc.TO. Se
1
5' 5~3
mRNA
R.1 so10,u
Sv8vN1~.s

Inhibitors of translation:
In prokaryotes (antibiotics):
a) Tetracycline inhibits the binding of amino acyl tRNAs to the ribosoma l complex.
b) Chloramphenicol inhibits the peptidyl transfera se activity inhibiting peptide bond

formation and thus interferes with elongatio n of peptide chain.
c) Erythromycin prevents the transloca tion process.
d) Streptomycin - It binds to the smaller ribosoma l subunit of prokaryo tes and causes
misreadi ng of the genetic code (faulty translation).
In eukaryotes:
a) Cyclohex imide inhibits peptidyl transfera se by binding to the rRNA component of

60 S ribosoma l subunit in eukaryot es.
b) Diphther ia toxin: It prevents transloca tion in eukaryot ic protein synthesis by

inactivat ing elongation factor eEF-2.
c) Ricin: A toxic substanc e isolated from castor bean, inactivate s eukaryot ic 28S rRNA.
Note:
The process of translatio n in prokaryo tes differs from that in eukaryotes. This difference
is exploited for clinical purposes because many effective antibiotics specifically inhibit
only prokaryo tic protein synthesis . This results in growth arrest or death of the
bacterium. However, these antibiotics do not interact with eukaryotic ribosomal particles
and thus are not toxic to eukaryot es.
Puromycin:
Puromyc in has a structure simi lar to amino acid part of amino acyl tRNAs. So
instead of arninoacyl tRNAs, puromyc in binds to the A site on the ribosome and
causes the prematur e release of the polypept ide. It inhibits protein synthesis in
both prokaryo tes and eukaryot es. So, puromyc in (and also cycloheximide) are not
clinically useful, but have been importan t in understa nding translatio n process.

Biochemi cal genetics (Part 1) - Replicatio n, Transcrip tion, Translatio n 442
Posttranslational modifications:
Many of the polypept ide (protein) chains synthesiz ed in translatio n process are as
such not functiona l. They must be modified to become the active protein. These
modifications are collectively called posttrans lational modifica tions. These are,
a) Glycosyl ation (Attachment of carbohydrates to form glycoprotein / Proteoglycans):

For the formation glycopro teins and proteogly cans, carbohyd rate residues need to be
attached to proteins (glycosylation). E.g: Immungl obulin formation.
Carbohy drates are attached to serine, threonine or asparagin e residues of the proteins.
This is one of the most importan t types of post-tran slational modifica tion.
b) Proteolytic degradation (Trimming by proteolyt ic degradation):

Some proteins are synthesiz ed as precurso r proteins (proprote ins). Some amino acid
residues (generally from N-terminal) are removed from them to form the active protein.
E.g.: Formatio n of pepsin from pepsinog en, formation of insulin from preproins ulin.
c) Phospho rylation:
The hydroxyl groups of serine, threonine and/ or tyrosine are phosphor ylated resulting
in the modifica tion of the activity of the proteins.
d) Gamma carboxylation:
The prothrom bin is formed in the inactive form. y.-carboxylation of glutamic acid of
prothrom bin converts it to active form.
e) Hydroxylation:
Collagen is formed in the inactive form protocollagen. Activation of protocoll agen to
active collagen involves hydroxyl ation of proline and lysine residues.
Protein targeting (or Protein sorting):

The newly synthesiz ed proteins need to be transport ed to their target sites (either
inside the cell or outside the cell. This process is called protein targeting or protein
sorting. Golgi apparatus plays an importan t role in protein sorting.
Newly synthesiz ed proteins are two types:-
1) Proteins for external secretion: The secreted proteins, plasma membran e integral
proteins, lysosoma l enzymes etc belong to this group. These proteins are synthesiz ed
in membran e bound ribosome s of rough ER. They have signal sequence that targets
them to correct destinatio ns. Signal sequence s are usually short amino acid sequences
at the amino terminal of a newly synthesiz ed protein.
2) Proteins for internal part of the ceJl: Cy tosolic, mitoch ondri al, nuclear,
peroxisom al proteins belong to this group. They are synthesiz ed in free ribosomes.
They generally do not have signal sequences. They are readily available in cytoplasm.
75
Biochemical genetics (Part 1)- Replication, Transcription, Translation 443
Question bank on Biochemical genetics:
ESSAYS (5 Marks):
1) Explain the process of Replication in detail
2) Explain the process of Transcription. Add a note on posttranscri ptional modifications.
3) Explain the process of Translation in detail. Add a note on posttranslat ional modifications.
SHORT ESSAYS (5 Marks):

1) D A polymerase s
2) Posttranscr iptional modificatio ns
3) Posttranslat ional modificatio ns
SHORT ANSWERS (2 Marks):

1) Okazaki fragments
2) D A helicase / DNA topoisomera se / DNA ligase / RNA primer
3) Helix destabilizin g proteins or Single Stranded DNA Binding (SSB) proteins

1) Splicing Activity is a function of (AI)
a) mRNA b) snRNA c) r RNA d) tRNA
2) DNA supercoiling is done by (AIIMS)
a) DNA polymerase I b) DNA polymerase II c) A topoisomera se d) DNA gyrase
3) Inhibitor of DNA dependent RNA polymerase
a) a-amanitin b) Ciprofloxacin c) 5-flurouracil d) Actinomyci n
4) Translation occurs at (AI)
a) Mitochondr ia b) Ccntrosome c) ucleus d) Ribosome
5) Abnormal base in tR A is (MAHE)
a) Dihydroura cil b) Orotic acid c) Methyl Xanthine d) Cystine
6) A spliceosome consists of
a) Ribozyme & rho factor b) snR A & Rho factor c) snRNA & hnRNA d) Sigma & rho factor
7) Replication takes place during the following phase of the cell cycle
A) Gl B) G2 C) S D) M
8) Bacterial gyrase i inhibitor by
A) Ciprofloxacin B) ovobiocin C) alidixic acid D) all of these
9) Which of the following is not present in the replication fork and replication bubbles?
A) Primase B) RNA polymerase C) Single stranded binding protein D) Helicase
10) Synthesis of RNA molecule is terminated by
A) a-factor B) rho-factor C) y-factor D) P-factor
Answers for MCQ: 1) b 2) d 3) a 4) d 5) a 6) c 7) c 8) d 9) b 10) b

444
Biochemical genetics (Part 2) - DNA damag e, Repair, Mutation, Cancer
DNA damage, Repair, Mutation, Cancer
Conte nts:
• DNA Dama ge
• DNA repair
• Base excision repair
• N ucleotide excision repair
• Xeroderma Pigmentosa
• Mismatch repair
• Double-strand break repair
• Mutagens and Carcin ogens

• Definition
• Agents
• Mutation
• Definition
• Types
• Examples
• Consequences
• Biochemistry of cancer
• Oncogenes
• Proto-oncogenes
• Tumor suppre ssor genes
• Oncogenic viruses
• Reverse transri p tase
• Tumor marker s
• Growt h factors
hoo.com or 9986449575
Biochemical genetics (Part 2) - DNA damage, Repair, Mutation, Cancer 445
DNA damage and DNA reapir:

• DNA undergoes some extent of damage during the life cycle of the organism. DNA
damages are caused due to errors in replication, and variety of mutagens like physical,
chemical & biological agents (Refer next segments for mutagens). Some of the common
examples of DNA damage are deamination (of cystine to uracil, adenine to
hypoxanthine), depurination (ring opening of purines), two-base alterations (formation
of pyrimidine dimers by UV radiation), chain breaks (ionic radiations), cross linkages
(between the bases) etc.
• However, many damages are efficiently repaired through a number of repair
mechanisms. There are different repair pathways that occur in both prokaryotic cells.
a) Base excision repair:

This mechanism uses different enzymes to repair particular kind of DNA damage like
deamination of cystine to uracil and adenine to hypoxanthine etc.
3' 5'
4 steps are involved in this process; Normal C
Incision, Excision, Resynthesis, ligation. DNA G
5' 3'
1. Incision: In this step, the damage to
the DNA is recognized. DNA 1 DNA damage
glycosylase enzymes hydrolytically
remove such damaged bases; e.g.,
Deamination of C
3'
-.
u
5'
Uracil DNA glycosylase enzyme
!
removes uracil in DNA (by hydrolyzing G
Repair 5' 3'
13-N-glycosidic linkage between the base Removes U Uracil DNA
and deoxyribose). Similarly, other
damaged bases are removed by specific
0 glycosylase
glycosylases present in the cell. This 3' 5'

leaves an apyrirnidinic site (or apurinic, G
if a purine was removed), both referred 5' 3'
to as AP sites.
2. Excision: Specific AP-endonuclease
enzymes remove the deoxyribose with
0
AP-endonulcease
Removes sugar
3'
!
- - - - 5'
the missing base from the DNA (by
G
excising sugar-phosphate linkage). This 5' 3'
step leaves a gap in the DNA strand.
3. Repair synthesis (re-synthesis):
0 i DNA polymerase
3' - - - - . - - 5'
DNA polymerase repairs the effected C
stretch, by incorporating appropriate G
5' 3'
deoxyribonucleotide.
4. Ligation: Gap between the recently
3'
i Ligase
5'
added nucleotide and the existing one C
is joined by DNA ligase by linking the G
two ends with a phosphodiester bond. 5' 3'

b) Nucleotide excision repair:

Nucleotide excision repair mechanism corrects bulky damages to the DNA such as
UV light induced pyrimidine (mainly thymine) dirnerization.
3' - -- - - - -- - - 5'
Mechanism: - - - - - - Segment containg
• The damage to the DNA is

5' ------•N·N·H·Nl•J--- 3' damaged DNA
recognized and cleaved by UV-

specific endonuclease.
I Recognition of the defe:t
and unwinding of the
DNA strand
• The enzyme cuts and removes
a bout 12-nucleotide fragment
flanking the damaged bases. .....
• Then the missing segments are
r e-synthesize d by DNA
I The defective oligonucleotide
is excised by cutting
at its two ends.
.....
polymerase enzymes.
3' - - -- - -- - - - 5'
• Then this gap is joined by
5' 3'
ligase enzymes.
I
Degradation of the defective
Failure of nucleotide excision segment and resynthcsis of
repair mechanism causes a nc..-w fragment and ligation
genetic disorder called 3' - - -- - -- - - - 5'

"Xeroderma pigmentosa".
5' 3'
Xeroderma pigrnentosum:
It is a genetic disease caused due to the defective DNA repair mechanism (nucleotide
excision repair mechanism) required for UV-damage repair.
Exposure of skin to UV rays of sunlight can result in joining of two adjacent thymines
to form thymine dimer, which prevents DNA polymerase action. UV specific
endonucleases cleave the dimer and initiate the damage repair process. (The whole
process is called nucleotide excision-repair mechanism). Xeroderma pigmentosum
results from defects in genes required for nucleotide excision-repair process that repairs
the DNA damage caused by UV irradiation.
The clinical symptom includes marked sensitivity to sunlight with consequent formation
of multiple skin cancers and premature death.
Other d iseases associated with DNA repair are Fanconis anemia, Bloom's disease,
Ataxia telangiectasia etc.
Mismatch repair and double strand break repair are other two types of DNA repairs.
Mismatch repair corrects errors involving single base pair or a small region of unpaired DNA,
where as double strand break repair mechanism corrects the double-strand breaks.
Defect or failure in DNA repair mechanisms may lead to cancer.

Mutagens/ Oncogens (Carcinogens)

Mutagens: Any damage to DNA, which is passed to the subsequent generation,
is called mutation. Agents that trigger changes in DNA sequence leading to
mutation are called mutagens. Mutation of genes may lead to cancer.
Classification: Mutagens are classified into 3 groups; physical agents, chemical
agents & biological agents.
1. Physical agents:
Radiations such as ultraviolet rays, x-rays, a-rays are highly mutagenic or
carcinogenic agents. These radiations damage DNA in several ways. For
example UV radiation may trigger adjacent pyrimidine bases on the same strand
to dimers. It can also release pyrimidine/ purine bases from the sugar, leaving
vacant sites. They also cause DNA strand breaks.
2. Chemical agents:
Nitrous acid, dimethylnitrosamine, dimethyl-sulphate, aflatoxin B, arsenic,
asbestos etc are some examples for chemical mutagens. The chemical agents
cause damages to the bases of DNA by adding chemical groups or by removing
chemical groups (like amino groups). Chemically altered bases fail to obey
Watson-crick base pairing. During replication, altered base on the template
incorporate wrong nucleotide and causes altered DNA sequence (mutation) .
3. Biological agents:
Viruses such as DNA tumor viruses and RNA Tumor viruses cause mutations
in their hosts. Particularly damaging ones are RNA viruses. During their life
cycle, RNA tumor viruses make DNA copy of viral genome and insert the
newly formed viral DNA into the host DNA. They have powerful promoter
sequence. Hence insertion into the host DNA leads to elevated expression of
adjacent genes. Several proto-oncogenes are converted to oncogenes by viral
insertion. This mechanism is termed as insertion mutagenesis.
Mutation of genes may lead to cancer.
Oncogens (Carcinogens):
Oncogens or Carcinogens are agents that are capable of causing cancer in
humans. They do so by causing damages to the genetic material, the DNA. So,
all carcinogens are mutagens. (Remember all carcinogens are mutagens, but
all mutagens are not carcinogens (However these mutagens are potentially
carcinogenic). So, classification of carcinogens is similar to mutagens.

Mutation
Alterations in DNA structure that produce permanent changes in th em are called
mutations.
Types of mutation:
1. Point mutation:
It is defined as the type of mutation, where a single nucleotide is substituted by
another.
It is of 2 types,
a) Transition: Substitution of a p urine by purine or pyrimidine by a pyrimidine

b) Transversion: Substitution of a purine by a pyrimidine or vice versa
2. Frame shift mutations:

It is defined as the type of mutation where one or more nucleotides are added or
deleted from the DNA.
It is of 2 types,
a) Insertion: These occur when one or more nucleotides are inserted in the DNA
causing insertion mutations.
b) Deletion: These occur when one or more nucleotides are deleted from the DNA
causing deletion mutations.
Mutation
Point mutation Frame shift mutation
r
Insertion Deletion
Transition Transversion

1. Consequence of point mutations:
a) Silent mutation:
The p oint mutation is termed silen t m utation if it d oes not cause any ch ange in the
amino acid coded. This is because the m utation may change the codon for one amino
acid to a synonym for the same amino acid.
E.g. the codon UCA is m u tated to UCU; both code for serine.
H ence there is no change in the amino acid sequence of the gene product.
b) Missense mutation:
In this case, the changed cod on, codes for a different am ino acid . This mistaken
amino acid or missense, depending upon its location in the sp ecific protein, might be
acceptable or unacceptable to the function of tha t protein molecule.
Protein Codon Amino acid

molecule Changed Changed
Acceptable HbA, P-chain AAA or AAG 61 Lys

missense J. J. J.
Hb Hikari, P-chain AAU X A~C Asn
HbA, P-chain GAA or GAG 6Gln

Partially accepted missense J. J. J. J.
HbS, P-chain GUA or CUG Val
HbA, a-chain CAU or CAC 58 His
Unacceptable missense J. J. J. J.
HbM, a-chain UAU or UAC Tyr
c) Nonsense mutation:
Sometimes the codon with the altered base may become a termination (or nonsense)
codon resulting in premature termination of protein synthesis.
E.g.: Tyrosine codon UAC may be mutated to a terminator codon UAA or UAG.
The protein products of nonsense mutations are usua lly non functional.
2. Consequences of frame shift mutation:

The insertion or deletion of a base in a gene results in an altered reading frame of the
mRNA (hence the name frame shift). Since there are no punctuations in the genetic
code, the ribosome doesn' t recognize an insertion or deletion and translation
continues. This could resu lt in the synthesis of a distorted protein with altered biological
function (with altered amino acid sequence and/or a premature terminated protein).
Fo r queries and suggestions, contact the author at prasad_text@yahoo.com or 9986449575

Biochem istry of Cancer:

Cancer may be defined as any malignant growth or tumor caused by uncontroll ed
cell division. Study of cancer is called oncology. (Greek: Onco = tumor).
• All cancer cells usually originate from one single mutated cell, which undergoes
uncontroll ed cell division and produce tumorous mass.
• Cancers are multifactor ial in nature. They include genetic, hormonal, metabolic,
physical, chemical, biological (like viruses) and environme ntal.
• Oncogenes and antioncoge nes are 2 important regulatory genes that are involved in
cancer developme nt. owadays, a third category of genes that control apoptosis
(programm ed cell death) are also shown to be involved in carcinogenesis.
Oncogen es:
Oncogene s are defined as the genes that are capable of causing cancer. They drive a
normal cell to transform into a cancer cells. Eg, src, erb-B, erb-A, ras, myc, abl etc.
• Among these, the prototype, the oncogene "src" (pronounc ed as sarc) is present in
Rous sarcoma virus that causes sarcoma. (Hence the name src). The src gene
(oncogene ) produces a protein having tyrosine kinase activity. This protein is
responsible for the transforma tion of normal cell into cancer cell.
• It is noted that even normal animal cells also contain a normal gene quite similar to
the viral src oncogene. This was named c-src gene.
• Normal cellular counterpar ts of viral oncogenes are termed as proto-onco genes or
c-oncogenes. For example, c-src, c-myc, c-sis, c-abl, c-sis etc. are the correspond ing
proto-onco genes of src, myc, sis, abl, sis respectively.
Proto-oncogenes:
Proto-onco genes are defined as the genes that are potentially capable of becoming
oncogenes by mutation.
• Proto-oncogenes play very important role in normal cell, controlling proliferatio n and
differentiation. They are normal cellular genes, which produce normal proteins that
regulate growth and differentia tion of normal cells (like growth factors, receptors,
growth proteins, proteins involved in signal transductio n, transcriptio n factors etc).
These proto-onco genes are under the strict control of regulatory machinery of the
cell and expressed only when required.
• Proto-oncogenes can be converted to oncogenes by mutation. Oncogenes produce
proteins called the oncoprote ins. These proteins are the altered versions of growth
factors, receptors of growth proteins, proteins involved in signal transductio n etc.
These altered versions are less responsive to normal cellular control mechanism and
pushes the cell towards uncontrolle d cell division and malignancy .
• Proto-onco genes can be activated to oncogenes by various mechanism s like -
Promoter insertion, Enhancer inser tion, Ch romosoma l translocat ion, Gene
amplificati on, Point mutations etc.
Tumor suppress or genes (anti-oncogenes):

• Tumor suppresso r genes can be defined as the genes that normally protect the
ind iv id ual against cancer.
• Examples: Two most studied tumor suppressor genes arc p53 gene and RB (Retino
Blastoma) Gene.
• A tumor suppressor gene produces a protein product that normally suppresses cell
division and / or cell growth.
• Importanc e: When tumor suppressor gene is altered by mutation, the inhibitory
effect of its protein product on cell division is diminished or lost, hence leading to
increased cell division and/ or cell growth.
RB (Retino Blastoma) Gene :

• It is the first tumor suppressor gene discovered. This gene is located in chromosom e
13 and it produces a protein designated as plOS (Mol w t. 105 KD). This protein binds
and inactivates transcriptio n factor and thus suppress cell proliferation.
• Mutations in RB lead to cancer.
p53 gene:
• This is the most important tumor suppressor gene, considered the guardian of the
genome. This gene is located in chromosom e 17. This gene is so named because it
produces protein of 53 KD. This protein is expressed during cell damage and inhibits
cell division until the damage is repaired. If the damage is severe, p53 directs the cell
to apoptosis (cell suicide).
• Absence or mutations in p53 leads to cancer. Mutations of p53 gene are found in at
least one third of all cancers.
Apoptosi s and role of apoptosi s in cancer formation:

Apoptosis means the programm ed cell death. Failure of apoptosis may lead to cancer.
One of the important aspects of apoptosis is the role of apoptosis mediating genes
like p53, RB. Mutation in these genes inhibits apoptosis and may lead to cancer.
Oncogens, Oncogene s, Anti-oncog enes, Proto-oncogenes:

• Oncogen s are the ch emicals that produce cancer.
• Oncogenes are the genes that cause cancer.
• Anti-oncogenes are genes that suppress cancer.
• Proto-oncogenes are genes that are potentially capable of becoming oncogenes by mutation
Oncogenes are represented using lower case letters, whereas anti-oncogenes are designated
using capital letters.
Oncogenic viruses:
Viruses that can cause cancer are called oncogeni c viruses. Oncogen es were originally
discovere d in oncogeni c viruses. Their viral oncogene s were found to be closely similar
to certain genes present in the normal host cells, which are referred to as proto-oncogenes.
Oncogen ic viruses are of two types,

1. DNA viruses 2. RNA viruses
1. DNA viruses:
These contain DNA as genetic material.
Examples: Epstein-B arr virus (cause Burkitt's Lymphom a), Hepatitis B virus (cause
Hepatom a), Human papillom a virus (cause uterine cervical carcinom a) etc.
Mode of oncogenesis: During the infection, DNA of DNA viruses bind with host DNA
& causes alteration in gene expressio n leading to uncontro lled cell division and cancer.
2. RNA viruses:
These contain RNA as genetic material (Retrovir uses).
Examples: Rous sarcoma virus, leukemia sarcoma virus, Human immunod eficiency
virus etc.
Mode of oncogenesis: RNA gets copied by reverse transcrip tase in host cells to produce
single strand of vi ral DNA, which then produces its complem entary DNA s trand to
form the double stranded viral D A. This DNA gets integrate d into the host DNA and
becomes part and parcel of host DNA. The drive for multiplic ation by viral genome
overrule s the regulato ry mechani sms of host cell genome expressio n leadin g to
uncontro lled cell division and conseque nt developm ent of cancer.
Reverse transcriptase (also called RNA dependa nt DNA polymerase):

Reverse transcriptase enzyme uses RNA as template and produce single stranded D A
(called complementary DNA or cD A). This enzyme is present in retroviruses (Retroviruses
have RNA as genetic material).
These retroviruses (like HTV virus, RNA tumor virus like rous sarcoma) when infected, use
viral RNA as template and produce single stranded cD A, which then act as a template
to produce the double stranded cDNA. This double stranded cDNA is then inserted into
host DNA. Then the geneic informat ion flows in the conventi onal way.
Reverse transcriptase enzyme has 3 different activities.
1. Reverse transcription 2. DNA directed D A synthesis 3. RNA degradati on
Significance:
Reverse transcrip atse is a powerful tool in gene tic engineer ing for the productio n of
various proteins, homones and to construc t DNA libraries etc.
75
Tumor markers:
Tumor markers are biological indicators employed to detect the presence of cancers.
Tumor markers are biological substances (like proteins, surface antigens, enzymes,
hormones etc) that are abnorma lly synthesized & released specifically by cancer cells.
Measurement of these in plasma (or serum ) and/ or tissues are helpful in the detection
(diagnosis) of certain cancers. They are also used in localiza tion, differentiation and
prognosis of different cancers.
Tumor markers are not used primarily fo r diagnosis of cancer, but for prognosis.
Tumo11r markers are not specific only for cancer. Significant elevations of these biomarkers also
occur in variety of non-cancerous conditions. For e.g, elevations of Carcinoembryonic antigen (CEA)
are found not only in patients witlt vario11s types of cancer, but also in heavy smokers and people
with ulcerative colitis and cirrhosis. So, measurements of most of the tumor markers are not used
in primarily for diagnosis of cancer. Their main 11ses have been in following the effectiveness of
treatments and in detecting early recurrence. The entire clinical picture must be considered along
with other laboratory tests when interpreting the results of measurements of tumor biomarkers.
1) Alpha-fetoproteins (AFP):
AFP is oncofetal antigen, synthesized by yolk sac in fetal We.
AFP levels are elevated in liver cancer, germ cell tumors & teratoma of ovary. But, it
sh ould be noted that AFP is also elevated in liver cirrhosis, hepatitis and pregnancy, so
it is not specific for cancer. So, the result should be interpreted with care.
However, AFP is used in prognosis and monitoring cancer treatment. It provides an
index for tumor therapy and detection of recurrence.
2) Carcinoembryonic antigen (CEA):
CEA are also oncofetal antigens, synthesised by embryonic tissues.
CEA levels are elevated in colorectal, lung, breast, ovary and pancreatic cancers. But,
CEA levels are also elevated in alcoholic liver cirrhosis, smokers, ulcerative colitis and
diabetes mellitus. So, result should be interpreted with care.
However, CEA is used in prognosis and monitoring of cancer treatment. It provides an
index for tumor therapy and detection of recurrence.
3) Prostate specific antigen (PSA) used in the early detection of prostate cancer.
Normal level in blood : 1-4 ng/dl. Values above 10 ng/dl indicate prostate cancer.
PSA is more reliable marker than acid phosphatase level in detection of prostate cancer.
But, PSA levels are also elevated in prostatitis & benign prostatic hyperplasia (BPH).
4) Calcitonin (Hormone) levels are elevated in medullary carcinoma of thyroid.
5) Beta chain of human chorionic gonadotropin (beta-hCG) levels are elevated in the
ch oriocarcinoma, germ cell cancers, trophoblastic disease etc.
6) CA-125 (Secreted cancer antigens), a oncofetal antigen, is elevated in ovarian cancer.
7) Prostatic acid phosphatase (enzyme): Its level increases in prostate cancer.
8) Anti-thyroid peroxidase antibodies (Anti-tpo antibodies): Anti-tpo antibodies like
LATS and Anti-thyroid antibodies are elevated different thyroid cancers.

Growth factors:
• Definition: Growth factors are the substances that stimulate the cell proliferation
by stimulating mitosis and differentiation of target cells.
• Examples: There are more than 100 growth factor have been identified. Some of
them are Epidermal growth factor (EGF), erve growth factor (NGF), Platelet derived
growth factor (PDGF), Insulin like growth factors (ILF-I, ILF-II), Tumor necrosis factor
(TNF), Erythropoietin (EP), Fibroblast growth factor (FGF) etc.
• Action:
Growth factors are bind with receptors that may be present on the cell surface or
inside the cells (but, mostly cell membrane receptors).
Cell membrane receptors of growth factors are generally transmembrane proteins.
They have receptor activity on the outside of the cell and tyrosine kinase activity
inside the cell. (Refer group II D hormone action).
When the growth factors bind with these receptors, they activate the tyrosine kinase
activity inside the cell, which directly phosphorylates the target proteins (at the tyrosine
residues). These phosphorylated proteins promote growth and proliferation (hence
the name growth factors) .
Some of the growth factors that bind with cell surface receptors act through secondary
messengers, which then activates protein kinases that phosphorylates target proteins. Some
growth factors enter the cell directly and bind with the intracellular receptors. These receptor-
growth factor complexes promote growth and proliferation.
Some of the important growth factors and their functions are as follows,
Growth factors Functions
Epidermal growth factor (EGF) Stimulate growth of epidermal cells
Nerve growth factors (NGF) Stimulate the growth of sensory and
sympathetic neurons
Platelet derived growth factor (PDGF) Stimulate wound healing
Erythropoietin (EP) Stimulates erythropoiesis
Tumor necrosis factor (TNF) Stimulate the necrosis of tumor ceUs
Insulin like growth factors (ILF-I, ILF-m Stimulation of sulfation of cartilage
Significance:
• Sometimes proto-oncogenes can be mutated to oncogenes and produce proteins
called the oncoproteins. These oncoproteins could be the altered versions of growth
factors, receptors of growth factors, proteins involved in signal transduction of
growth factors etc. These altered versions result in uncontrolled cell division and
malignancy, causing cancer. (Read proto-oncogenes for detail).

Question bank on Biochemical genetics (Part 2)

1) Mutations
2) Oncogenes and Cancer. Add a note on proto-oncogenes
3) Growth factors
4) DNA repair
5) Tumor markers

1) Reverse transcriptase
2) p53 and RB genes
4) Xeroderma pigmentosa
1) Xeroderma pigmentosurn is caused due to defect in

a) Transcription b) DNA repair mechanism c) Translation d) Post-translational modification
2) The following is the defect in sickle cell anemia
a) Substitution of a base b) Insertion of a base c) Deletion of a base d) deletion of three bases
3) Ultraviolet radiations may cause formation of
a) Purine dimers b) Pyrimidine dimers c) Nucleoside dimers d) Nucleotide dimers
4) udeotide excision repair is defective in
a) Bloom's syndrome b) Gout c) Fanconi's syndrome d) Xeroderma pigmentosum
5) p53 Gene is
a) A proto-oncogene b) An oncogene c) A tumor suppressor gene d) An Oncogen
6) The type of mutation in hemoglobin is
a) [nsertion b) Deletion c) Missense mutation d) Non sense mutation
7 ) Alpha-fetoprotein is a tumor marker for
a) Colon cancer b) Liver cancer c) Ovarian cancer d) Prostate cancer
8) CA 125 is a tumor marker for diagnosis of
a) Gastric cancer b) Lung cancer c) Ovarian cancer d) Carcinoid syndrome
9) Ebstein Barr Virus is responsible for
a) Hepatoma b Burkitt's lymphoma c) Cervical cancer d) Sarcoma
10) Carcinoembryonic antigen level is elevated in the following carcinoma
A) Pancreas B) Protease C) Colon D) Ovary
11) Many cancer is associated with abnormal production of
A) Carbohydrates B) Fats C) Proteins D) Minerals
Answers for MCQ: 1) b 2) a 3) b 4) d 5) c 6) c 7) b 8) a 9) b 10) c 11) c

Biochemical genetics (Part 3) - Techniques 456
Techniques
Contents:
• PCR
• PROBES
• HYBRIDIZATION
• DNA LIBRARY
• SOUTHERN BLOTTING
• NOTHERN BLOTTING
• WESTERN BLOTTING
• RFLP
• HYBRIDOMA TECHNOLOGY

Polymerase Chain Reaction (PCR):

PCR is an in vitro method for DNA amplification. This is a much faster and more
sensitive method than cloning.
Requirements:
• Taq polymerase enzyme: Because the high temperature required (94°C) for heat-
denaturation of DNA (first step) inactivates DNA polymerase (third step), a
thermostable DNA polymerase is used (eg. Taq polymerase, isolated from the bacteria
thermus acquaticus that live in hot springs)
• Primer: Two synthetic DNA primers, which are complementary to the ends of each
strand of the target DNA to be amplified (called flanking sequences) are required.
• Primer construction requires the knowledge of nucleotide sequences (flanking
sequences) that flank the region of the target DNA to be amplified.
PCR comprises of repeated cylces of three Steps:

1) Heat denaturation of DNA
The DNA to be amplified is heated at 94°C to separate the double stranded target DNA
into two separate strands.
2) Annealing primers to "flanking regions" of single stranded DNA
The separated single stranded DNA are cooled and allowed to anneal to the two primers
(one for each strand).
3) Extending DNA with DNA polymerase (Taq polymerase)
To the above mixture DNA taq polymerase enzyme and deoxyribonucleotides are added.
DNA taq polymerase enzyme uses the two single stranded DNA as template and
synthesizes tvvo new strands, adding new nucleotides to the 3' end of primer.
At the end of this step, two copies of the original DNA are formed.
These three steps (one cycle) are repeated several tunes. Each cycle doubles the number
of target DNA molecule. Thus, PCR synthesizes millions of copies of a specific nucleotide
sequence in a few hours.
Applications of PCR:
PCR techniques are particularly useful when not enough D A molecules are present
in test samples for DNA analytical techniques.
1. Forensic Uses
When biological samples (blood, semen, or hair) obtained from a victim or suspect
is insufficient, PCR can be used to amplify D. A to get enough DNA material for
analytical techniques such as D A fingerprinting and sou thern blot technique.

2. Diagnostic uses
PCR may used to quickly detect microbial infections, especially when an insufficient
number of the microbes are present in the test sample.
Eg, Diagnosis of tuberculosis and AIDS by detecting DNAs of mycobacterium
tuberculi and HIV respectively, even when they are present in minute amounts.
3. Study of mutations
PCR is also used in the study of mutagenesis, particularly single gene mutation.
3 Original double- stranded DNA

~: I I I 11111111111111 ~,
l Step 1: Heat Denaturation

(Strand separation)
5' I I I I I I I I I I I I I I I 3'
Single-stranded template DNA
3' I I I I I I I I I I I I I I I 5'
Cycle I
l Step 2: Annealing Primers
5' • ········· Primer

5•~ 3·
3°JIIU I I I I I I I I I I I 5·
l Step 3: Extension
(By D A polymerase - Taq polymerase)
S' - - - - ~ - ~ ~ - 3'
3' 5
Two double-stranded DNA
5• ~ ......
3 5
l I. Heal denaturation
l 2. Annealing Primers
l 3. Extension
Four double stranded DNA
Repeated cycles
j
Millions of copies of target D A

Probes:
During studies on DNA and RNA, there's generally a task of detecting a particular
segment of DNA or RNA in a mixture. DNA or RNA probe are tools to locate DNA or
RNA of interest, where a labeled probe is made to base pair w ith the target nucleic acid
fragments (DNA/RNA). There are 2 types of probes; DNA probe & RNA probe.
Definition: Probe is a single stranded fragment of DNA or RNA, which is labeled

(mostly radio-labeled) & has the complementary base sequence to the target DNA
fragment or RNA.
Probes are used to detect the target DNA from a mixture. Complimentary sequences
help them to bind with the specific target DNA or RNA fragment a mixture, whereas
labeling helps them to be detected even if present in extremely small quantity.
Principle: The labeled probe is allowed to move freely with the mixture of fragment of
nucleic acid in search of a complementary sequence. If the target DNA fragment /
RNA are present in the mixture, the probe will detect the complementary nucleotide
sequence in the mi xture. The probe binds to the target fragment very specifically as per
the base pairing rule. It will bind tightly to the target fragment (hybridization), only if it
has a complementary sequence. Labeling of the probe helps them to be detected even
if they are present in extremely small quantity. Thus, the presence of the target DNA
or RNA is detected with the help of the probe.
Radioactive 32 P is used in preparing DNA probe. The radio labeled probes are detected
by using X-ray films. Even immunological labels are also used for labeling probes.
Application:
1) Detection of desired DNA fragments or R A in a mixture:
During studies on DNA and RNA, there's genera lly a task of detecting a particular
segment of DNA or RNA in a mixture. Probes are used to detect and identify DNA or
RNA fragment of interest.
2) DNA probes are used in blotting techniques:
Probes are used in Southern blotting, Northern blo tting.
3) DNA probes are also used in situ hybridization:
Probes can also be used to hybridize with the target DNA or RNA on tissue sections.
This technique is called situ hybridization.
Hybridization:
Definition: Hybridization refers to the formation of hybrids of nucleic acids (generally DNA &
DNA or DNA & RNA). This involves pairing of complementary base strands of these nucleic
acids.
Application: Different types of hybridizations are used in various blotting techniques like
Southern blotting, Northern blotting, and western blotting (Between DNA and DNA or between
DNA and RNA). In blotting techniques, specific probes are made to hybridize with desired target
DNA segments, which are then identified.

Southern Blotting:
Southern blotting is a technique to detect and characterize specific segments of DNA.
The technique employs restriction endonuclease (RE) to cut DNA fragments, which are
separated by agar gel electrophoresis to sort them according to length and then
transferred to a nitrocellulose membrane by blotting. (because agarose is very fragile and
cannot be used for auy procedure. So, DNA fragments have to be transferred to a sheet of
nitrocellulose membrane). DNA fragment of interest (i.e. DNA fragment to be tested) is
hybridized with labeled DNA probe and detected by using X-ray films.
Steps:
1. Digestion with Restriction endonuclease:
DNA from the suitable sou rce is purified and cut with a particular restriction
endonuclease. This cutting produces DNA fragments of different length.
2. Separation of D NA fragments by agarose gel electrophoresis:
Cu t DNA fragments are separated by agarose gel electrophoresis. This sorts out DNA
fragmen ts according to length and they occupy different positions on the track. Shorter
ones move faster than the longer fragments.
3. Transfer of DNA from agarose gel to nitrocellulose membrane:
First agarose gel is treated with dilute NaOH solution to convert DNA into single
stranded DNA (Nitrocellulose paper has a property to bind single stranded DNA).
Nitrocellulose paper is layered over agarose and pressed with absorbent paper on the
top. This step transfers separated DNA fragments without disturbing their position on
the track and thus producing exact replica of electophoretogram. DNA is fixed to
nitrocellulose paper by baking at 80°C or using ultraviolet light.
4. Hybridization and detection:
Specific DNA probes are used to detect target DNA fragments. The nitrocellulose
membrane is immersed in a buffer containing 32P labeled DNA probe. The DNA probe
will hybridize (pair) only with the desired DNA fragment if it is present. Excess probe
is washed and the X-ray film is placed over the membrane. The location of the DNA
fragment that was bound to the probe is revealed as a dark line on the X-ray film. This
is called autoradiography.
Applications:
1. Detection of particular gene from thousands of genes.
2. To characterize a clone of DNA and to compare its restriction map with that of
genomic DNA.
3. Locating the gene on the chromosome. This is called gene mapping on chromosome.
4. To study mutations (to assess any change in gene arrangement, deletion or insertion
in the genes.
5. Used in RFLP (restriction fragm ent length polymorphism) and DNA finger printing.
6. To study related genes in other species.

DNA DNA fragments Agar gel electrophoresis

Cut with
restriction
enzyme
Hybridization
-
& exposure Transfer
to X-ray film (Blotting) •
til Filter
Agarose gel
Nitrocellulose
Au toradi ograph Nitrocellulose
membran e
after blotting membrane
Diagrammatic representation of Southern blotting technique
Northern Blotting
orthern blotting is a technique to detect and characterize RNA. Northern blotting
uses the same principle as Southern blotting.
Steps:
l. Total RNA is purified from the suitable source. This contains all types of RNA. RNA
mostly present in secondary s tructure, which interferes with electrophoresis. Hence
the mixture of RNA is treated with formaldehyde to disrupt the secondary structure.
2. RNA sample with formaldehyde is separated on agarose gel by electrophoresis.
RNA with different size sorts out according to their lengths. Shorter ones move towards
anode, while longer ones move slower.
3. Agarose gel is blotted with nitrocellulose membrane to transfer separated RNA onto
the nitrocelJulose membrane. This step is similar to the Southern blotting.

4. Nitrocellulose paper with bound RNA is immersed in a buffer containing 32P labeled
DNA probe. The probe hybridizes to those RNA having complementary sequence. All
other RNA will not bind the probe. RNA hybridized with the probe is detected by
exposure to X-ray film.
Applications:
1. To detect and study tissue-wise distribution of RNA and estimate its level in the cells.
For instance, different tissues contain different mRNA; some of them are unique to a particular
tissue, e.g. Erythroblasts contain hemoglobin mRNA, liver contains albumin mRNA, P-cells
contain pro-insulin mRNA, etc.
2. To estjmate the size of R A and also to analyze its size variation.
Western Blotting
Western blotting is a method to detect particular protein in a sample. This is based on

electrophoretic separation of proteins & detection of target protein using specific antibody
as probe.
Steps:
1. Proteins in the test sample (serum, urine, cell extract etc) is separated on polyacrylamide
gel by electrophoresis. This separates proteins mainly based on size & charge properties.
2. The proteins on polyacrylamide gel are transferred to a sheet of nitrocellulose
membrane. Generally this blotting is done with the help of electric current.
3. Proteins bind to nitrocellulose with the same separation pattern. The Nitrocellulose
membrane is treated with a specific antibody probe that can bind to the protein to be
tested. The antibody binds only to the specific protein.
4. A second antibody conjugated with an enzyme (e.g. Peroxidase) is added to detect
the protein antibody complex formed in the previous step. It binds to the first antibody.
Excess antibody-peroxidase conjugate is removed by washing the membrane. Then
the peroxidase substrate is added. Peroxidase a ttached to the second antibody converts
the substrate to a colored product, which is measured colorimetrically. This detects the
position of protein on the membrane.
Applications:
1. This technique is widely used to detect even extremely small quantity of a protein in
cell extract or biological fluid.
2. It is used in the detection and confirmation of viral infections particularly AIDS.

Restriction Fragment Length Polymorphisms (RFLP)

Definition: Restriction fragment length polymorphism (RFLP) is defined as the variation
or polymorphism in the length (pattern) of restriction fragments resulting from
variations in the DNA that alters the restriction site of a restriction enzyme.
Principle: Genomes from any two unrelated people are 99.5% identical. (With 6 billion
bp in the human genome, 0.5% variation in genome represents variation in about 30
million bp). These genome variations are called polymorphism and are caused by
mutations. (Polymorphism occur mostly (98%) in the non coding region (inrons) of the
DNA or at sites that cause in change in the function of encoded protein). This variation
in the DNA sequences can result in either deletion or addition of restriction sites and
thus causes variation in the length of restriction fragments when cut by a specific
restriction enzyme. This is called Restriction fragment length polymorphism (RFLP).
RFLP is inherited. lt is used to detect human genetic variations.
Technique:
• Test DNA is cut into fragments using suitable restriction enzyme. This cutting generates
a few million fragments of different length. The fragments are then separated into
different bands by agarose gel electrophoresis. Longer fragments move slower and
shorter one move faster.
These fragments are transferred to nitrocellulose membrane (as in Southern blotting).
The DNA fragments are firmly immobilized on nitrocellulose.
• A radioactive DNA probe is introduced. The DNA probe binds to specific DNA
sequence on the nitrocellulose membrane. Several probes are available which are
known to detect polymorphic DNA sequences among human individuals. The excess
probe material is washed away leaving the unique DNA band pattern. Then the
position and location of DNA fragments is detected using by using X-ray films.
Applications of RFLP
J) DNA analysis is used in identifying individuals in medico-legal cases, paternity
disputes, identification of victims of accidents etc.
2) Analysis of genetic variations by RFLP provides information about the genetic makeup
and genetic diseases. This is employed in the detection and isolation of defective genes
in inherited disorders, such as sickle cell disease.
3) RFLP technique is also used in DNA finger printing:
No two persons have the same genome Qust like the finger print). Since restriction
endonuclease cut specific sequences, they can be used to make D A finger prints of
different samples of DNA. This material is used to identify an individual using RFLP
technique employing a collection of assorted probes. This kind of identification is
popularly known as DNA finger printing. DNA finger printing can be used to help
solve cri mes, disputed parenthood, identifying unclaimed bodies and various other
applications in forensic medicine.

Analysis of hemoglobin gene in sickle cell disease using RFLP.

In sickle cell disease, P-chain of hemoglobin has a defect; glutarnic acid at position 6 is
replaced by valine due to a point mutation in 6th codon. DNA from normal individual
produces two DNA fragments when cut with a restriction endonuclease called Mstll;
one fragment with 1.1 kilo base pairs (kb) and another shorter one with 0.2kb (see
figure). DNA from sickle disease patient produces only one fragment of 1.3 kb. This is
due to the mutation present at the 6th codon, which alters the base sequence and resulted
in the loss of one restriction site.
Individuals with sickle cell trait are heterozygous and have one normal and one variant
gene for P-chain, whereas sickle cell disease is homogygous, both gene is defective.
So, mixed pattern of restriction fragment sizes of DNA is obtained.
This is a simplified example where a restriction fragment variation has occurred due to
point mutation. This me thod is also used for the detection of sickle trait.
11 1 Norma l
Nonna! HbS HbS trait
0.2 1.1 kb 1.3 Kb

Kb
1.lKb
l l HbS Starting
0.2 Kb
\. J
Y'
13Kb
There are two types of DNA variation that commonly result in RFLP.
1) Single nucleotide polymorphism (SNP): SNP is defined as the genome variation

that involves just one-base change in DNA. This change in the DNA sequences can
result in either deletion or addition of restriction sites and thus causes variation in the
length of restriction fragments when cut by a specific restriction enzyme.
2) VNTR (Variable number of tandem repeats) polymorphism: Polymorphism in DNA
can also arise from the presence of VNTRs (variable number of tandem repeats).
VNTR are short sequence of DNA at dispersed locations in genome, repeated in
tandem (one after another, for example, GC-GC-GC-GC). The number of these repeat
units varies from person to person, but unique for any given individual and therefore,
serves as molecular finger print. C ut by restriction enzymes yields fragments that differ
in length depending on how many repeated segments are present be tween two
restriction sites in the fragment. D A finger printing largely exploit the presence of a
variable number of tandem repeats [VNTR].

Hybridoma technology (Production of monoclonal antibodies):

Hybridoma technique is a method for the production of monoclonal antibodies from a
clone of B-cells that secretes single kind (monoclonal) of antibody.
Differences between polyclonal and monoclonal antibodies:

Polyclonal antibodies: Polyc/onal antibodies are antibodies produced by more than one clone
(type) of cells. Antibodies produces in the body in response to antigens is polyclonal in nature,
because it is the mixture of antibody secreted by multiple copies of B-ly111phocytes.
When an anfige11 is injected i11fo an animal, the a11i111a/ produces different types of a11tibodies
against various epitopes of lite antigen. The antibodies th11s produced are polyclonal in nature. 111
all microbial infections, tlte body reacts with polyc/onal a11tibody production.
Monoclonal antibodies: Monoclonal antibodies are specific antibodies produced by a single clone
of immunoglob11lin producing cell. /11 nat11re, mo11oclo11a/ a11tibodies are produced in certain
disorders like multiple mye/0111a, where only one clone secrets a particular type of antibody. But in
multiple myeloma, these antibody produced is useless.
Principle:
Antibodies produced by a clone of specific lymphocytes are called monoclonal antibodies.
Hybridoma technology is based on somatic cell hybridization. Antibody producing B-
lymphocytes of spleen are fused with a myeloma cell (Cancer cell) to produce a
hybridoma cell.
Procedure:
• The antigen is injected into mice.
• After few weeks, spleen cells from the immunized mice are removed and fused with
mice myeloma cells to produce a hybrid cell. Polyethylene glycol (PEG) is used as a
fusion agent.
• Hybrid cells contain the gene of normal mice as well as the myeloma cells. They
acquire two important features from their parents; i) like myeloma cells, they can multiply
indefinitely in culture medium, ii) like B-lymphocytes of spleen cells, they can synthesize
and secrete a particular antibody.
• The culture contains three types of cells.

1) Normal spleen cells 2) M yeloma cells 3) Hybridoma cells. We require only hybridoma
cells. To differentiate them and to select only hybridoma cells, a special culture medium
called HAT medium (Hypoxanthine, Aminopterin and thymidine) is used.
1) Normal cells die within a week because they lack the ability to multiply.
2) Since myeloma cells do not contain HGPRTase (an enzyme of salvage pathway),
they lack the salvage pathway for DNA synthesis. Further, aminopterin, a folic acid
antagonist, in the culture media will inhibit the de novo synthesis of DNA. Since both
pathways are blocked, the non-fused myeloma cells also die.
3) Only fused hybridoma cells survive. In this case, normal cells provide the HGPRTase
enzyme and so DNA synthesis is possible from the hypoxanthine and thymidine
provided in the medium.

• These hybridoma cells formed retain the properties of both parent cells, that
is, antibody secretion property of B-lymphocytes and uncontrolled division
property of cancer cells. Clones of hybridoma cells always produce a particular
type of antibodies (monoclonal antibodies) that react with specific antigens.
• Then the hybrid cells are distributed to multi-well culture plates, such that
each well receives single cell. They are cultured for several days. Each culture
well contains cells derived from a single hybridoma cell. Each one is a clone of
cells. These clones are checked for the antibody specificity. Once, a clone is
obtained, it will serve as a continuous source of well-defined monoclonal
antibody.
Events:
1) Immunize animal (mouse or rabbit) with antigen.
2) Isolate spleen cells (containing antibody producing B cells)
3) Fuse spleen cells with myeloma cells using PEG (Polyethylene glycol)
4) Place the cells in culture medium containing HAT ((Hypoxanthine, Aminopterin and
thymidine).
5) Allow unfused normal B cells and myeloma cells to die. Select Hybridoma cells.
6) Clone the hybrid cells (Place 1 cell per well & allow each cell to grow into a clone of cells)
7) Screen supernatant of clone for the presence of the desired antibody (using ELISA)
8) Grow the chosen clone of cells in tissue culture indefjnitely
9) Harvest monoclonal antibody from the culture supernatant
Significance:
Monoclonal antibodies are highly specific and very useful reagents in immunological
detection of infectious agents, hormones, proteins, cytokines, etc by ELISA, RIA or
immunodiffusion. Specific monoclonal antibodies are also used as therapeutic agents.
Some of the specific applications are,
• Early detection of pregnancy
• Detection and treatment of cancer
• Diagnosis of leprosy
• Treatment of autoimmune diseases
• Hybridoma technology is used in the quantitative preparation of pure antigens.

Question bank on Biochemical genetics (Part 3)

1) PCR and applications
2) Southern blotting
3) RFLP
4) Hybridoma technology

1) VNTR
2) Nothem blooting
4) Xeroderma pigmentosa
1) Northern Blot test is used for analysis of (AIIMS/ AI)

a) DNA b) RNA c) Protein d) Enzyme
2) Southern blot technique is used for

a) DNA Identification b) DNA Amplification c) RNA Identification d) RNA Amplification
3) Technique for detecting particular protein by straining with specific antibody is known as
a) Western blotting b) Southern blotting c) Northern blotting d) Polymerase chain reaction
4) Jn Southern blotting, blotting is done on
a) Agarose gel b) Polyacrylamide gel c) Nitrocellulose membrane d ) Sephadex gel
5) The DNA polymerase used during PCR is isolated from
a) Escherichia coli b} Salmonella typhi c) Thermus aquaticus d) Microcoleus species
6) Which of these is not a s tep during PCR
a) Melting b) Annealing c) Blotting d) Chain extension
7) During preparation of monoclonal antibodies, the fusion of cells is brought about by

a) Amethopterin b) Polyethylene glycol c) hypoxa.nthine d) Calcium chloride
8) Hybridoma technology produces
a) Polyclona1 antibodies b) Monoclonal antibodies c) DNA fragments d) Antigen
9) Sickle cell d isease can be identified by

a) Southern blotting b) orthem blotting c) Hybridoma technique d) Western blotting
10) Which of the following is associated with western blotting?
a) Electrophoresis of DNA b) Used in diagnosis of HIV c) Electrophoresis of RNA d) RFLP
Answers for MCQ: 1) b 2) a 3) b 4) a 5) C 6) c 7) b 8) b 9) a 10) b

Regulation of Gene Expression 468
Regulation of Gene Expression
Contents:
• Definition
• Types of genes
• Constitutive genes
• Regulated genes
• Types of regulation
• Positive regulation
• Negative regulation
• Operon concept
• Definition
• Lac operon

Introduction: Genes are expressed in the form of proteins synthesized by them.

Synthesis of proteins under the influence of genes is called gene expression.
All the cells contain same set of genes, but, all the genes in a cell are not expressed.
Only a fraction of the genome is expressed. For eg, Insulin gene is expressed only in
cells of pancreas and not in other cells although all the cells have insulin genes.
Types of genes:
Not all genes are regulated. Based on whether the genes are regulated or not, the
genes can be divided into 2 types,
a) Constitutive genes (House keeping genes): Constitutive genes are not regulated.
Genes which are expressed almost always in all cells are called constitutive genes or
house keeping genes. The products of these genes are required all the time for the
basic cellular functions. For example, enzymes of glycolysis, enzymes, TCA cycle etc.
b) Regulated genes (Inducible genes): Inducible genes are regu lated by various
regulatory molecules. An inducer increases the expression of these genes and repressor
decreases. These genes are expressed only under certain conditions.
Eg, production of glucokinase is under the regulation of insulin enzyme.
Types of gene regulation:

There are two types of gene regulation, Positive regulation and Negative regulation.
i) Positive regulation: When the gene expression is quantitatively increased by the
presence of a specific regulatory element, regulation is said to be positive. The element
or molecule mediating positive regulation is called a Positive regulator or Inducer.
ii) Negative regulation: When the gene expression is quantitatively decreased by
the presence of a specific regulatory element, regulation is said to be negative. The
element mediating negative regulation is called a Negative regulator or Repressor.
Regulation of gene expression:

Genes are under the influence of va rious factors for their expression. Regulation of
gene expression is primarily performed at the transcription level in both prokaryotes
and eukaryotes. In eukaryotes, gene expression also performed at the replication,
posttranscriptional modifications, translation & posttranslationaJ modification stages.
A) Regulation of gene expression in prokaryotes:

Regulation of gene expression is primarily performed at the transcription level in
prokaryotes. The structural genes that code for proteins required in a particular
pathway are often found sequentially grouped on the DNA. These structural genes
are thus can be coordinately controlled (that is, turned on and off as a unit), This
entire package is referred to as an operon.
Regulation of gene expression by induction and repression can be explained by Operon
concept, which was first proposed by Jacob and Monad in 1961.

Operon concept:
Operon is a coordinated unit of gene expression in bacteria. It includes structural
genes, regulator/ inhibitor gene, promoter and operator genes.
• Structural gene: The structural gene codes for specific proteins or enzymes of a
pathway. They are often found toge ther and thus can be regulated as a unit.
• Promoter site: Promoter site binds RNA polymerase to initiate mRNA synthesis.
• Operator gene: A gene called operator gene, which is adjacent to the structural
genes, controls them . RNA polymerase enzyme first bind to promoter, then passes
over the operator gene and reaches the structural gene and transcribes it.
• Regulator gene: Operon is in turn controlled by a regulator gene. The regulator
genes codes for a specific mRNA, which directs the synthesis of a protein known as
repressor. The repressor then binds to the operator gene. Once this happens, the
RNA P cannot pass over operator gen e to reach structural gene, which is not
transcribed to give mRNAs.
• Inducer: When an inducer molecule is present, it binds to the repressor, making it
unable to bind to operator. Now, RNA polymerase can proceed to the structural
gene to produce mRNA.
Operon concept can be better understood with the help of a lac operon model.
Lac operon (lac for lactose) of E.coli bacteria:

Lac operon consist of structural genes (Z, Y, A), regulator gene (I), & operator gene (0). Besides
these genes, it also contains a promoter site (P), next to operator gene.
• Structural genes (Z, Y, A): The structural genes codes for enzymes ~-galactosidase, galactose
permiase, galactoside acetylase respectively. These enzymes are required for lactose metabolism
in E.coli.
• Promoter site (P): It binds to RNA polymerase to initia te the transcription..
• Operator gene (0): It lies adjacent to the structural genes and controls them. RNA polymerase
enzyme first bind to promoter, then passes over the operator gen e and reaches the structural gene
and transcribes it.
• Regulator gene (I): Regulator gene is constitutive. 0peron is in tum controlled by a regulator
gene. The regulator gene produces a protein called lac repressor. Lac repressor is a tetrameric
regulatory protein, which binds to the operator gene (0) and interferes w ith the progress of RNA
polymerase & blocks the transcription of structural genes. This is the example of negative regulation.
This is what happens when E.coli is grown on glucose medium (or when lactose is not there in
the medium). Thus when E.coli is grown on glucose, there is repression of enzymes.
When E.coli is grown on lactose (inducer):

In this case, lac operon is induced. Lactose binds to the lac repressor. The repressor-lactose
complex cannot bind to the operator gene and the negative effect of lac repressor is then lifted.
Now, RNA polymerase can proceed to the structural gene and transcribe it leading to the synthesis
of enzymes involved in lactose metabolism. Thus, lactose acts as an inducer and the mechanism
is said to be derepression (because lactose act by inactivating the repressor molecule). This is an
example of positive regulation. A regulatory protein known as catabolite activator protein (CAP),
bound to cAMP acts as n positive regulator (enhancer) of the lac operon.

Lac operon model: (In the absence of inducer):
Regulatory Promoter O perator Structural genes

gene site gene
A) p 0 z. y A
B) p
EB z ...,. A
l
-.....RNA
l
88
R.er,e uov
> ffi
Re~•essov
S... bll.,.(h tdY<1-mn'
In the presence of lactose (inducer):
C) p
0 z. y A
l l l l Polycistronic
mRNA
l
Q)
l·
®
l
@
I - '
'''"'
' \ \ I
"R.ep-reS~OY "Rep•eHo r Lo.cto~e
y
Su bu'..,.tis iet l'4.n,e,- 1. ~-galactosidase
2. Galactose permiase
3. Galactoside acetylase
In.o..c.ti ve. Rt.fYt HOY

B) Regulation of gene expression in eukaryotes:

Regulation of eukaryotic gene expression is performed at the transcription level and
also at th e replication, posttranscriptional modifications, translation &
posttranslational modification stages. It is a complex, multi-leveled complex process.
Some important ones are given here.
1) Gene amplification:
Gene amplification refers to production of multiple copies of gene by repeated rounds
of replication. This.results in the increased gene expression and increased levels protein
that is encoded by that gene.
Significance: Gene amplification plays a significant role during embryonic
developmental stages. For instance, in drosophila (fruit fly), gene amplification of
genes producing egg shell proteins during oogenesis.
2) Gene rearrangement:
Gene rearrangement refers to mixing of parental DNA from two different sources.
There are mainly three types of gene rearrangements, namely homologous
recombination, site specific recombination and transposition.
Significance: Gene rearrangement plays a significant role in generation of antibody
diversity. It is estimated that human beings can produce about 10 billion antibodies
in response to different antigen stimulations. The specificity of antibody is determined
by V region of the antibody. There are several V, D and J gene segments. One gene
from each of the variable domain gene combines with one gene of the segments to
produce a large number of rearranged variable domain genes leading to the generation
of diverse antibody specificity.
3) Post-transcriptional modification (RNA processing):
RNA synthesized from DNA is called the primary transcript. Then the primary
transcript undergoes certain covalent modifica tion to form mature RNA. These
modifications are collectively known as posttranscriptional modifications, which
provide a effective site for eukaryotic gene expression.
4) Alternate mRNA splicing:
Splicing is a process of removal introns and joining of exons to produce the desired
mRNA. Alternate splicing can produce different proteins that code for different
proteins. In this way, a single gene expression can produce diverse protein m o lecules.
5) Degradation of mRN A:
Gene expression can be regulated by adjusting the rate of degradation of mRNA.
Some hormones influence the degradation certain mRNA. For example, the hormone
estradiol prolongs the degradation of vitellogenin mRNA.
6. Regulatory elements:
Regulatory elements like promoter elements, repressors, inducers, Hormone responsive
elements (HRE), also play a vital role in regulation of gene expression. (These are
discussed in detail in hormones chapter).

Question Bank on regulatio n of gene expressio n

1. Lac operon

l. Constitutive genes
2. Regu la ted genes

1) CAP in Lac operon is an example of (AI)
a) Positive regulator b) egative regulator c) Constitutive expression d) Attenuation
2) Promoter site attaches
a) DNA polymerase b) RNA polymerase c) D A ligase d) Topoisomer ase
3) The structural A gene of lac operon codes for
a) Galactose permiase b) 13-galactosidase c) Galactose acetylase d) Lactase
4) Regulatory element, which decreases the gene expression quantitative ly
a) Repressor b) Allosteric inhibitor c) Inducer d) Operator
5) Regulatory element, which increases the gene expression quantitatively
a) Repressor b) Allosteric activator c) Inducer d) Operator
6) Lac Operon is a cluster of gene present in
a) Human beings b) EcoRI c) Lambda phage d) All of these
7) An example for an inducible gene is the gene coding for
a) Hexokinase b) Citrate synthase c) Glucokinase d) Aconitase
8) Operator site in a gene is a region which binds to
a) RNA polymerase b) Repressor c) Heat shock proteins d) Inducer
9) Promoter site
a) Present downstream of the gene b) Codes for repressor molecule
c) Binds to RNA polymerase d) Binds to repressor
10) Constitutive genes are those genes which are
a) Expressed only in the presence of certain stimulus b) Protect the genome
c) Expressed at a constant rate at ail times d) synthesize the repressor molecule
11) DNA element, not responsible for regulation of gene expression at transcriptional level
a) TATA box b) CAAT box c) GC box d) DHU box
Answers for MCQ: 1) a 2) b 3) c 4) a 5) c 6) b 7) c 8) a 9) c 10) c 11) d
For queries and suggestions, contact the author at prasad_tex t@yahoo.co m or 9986449575
Recombin ant DNA Technolo gy 474
Recom binant DNA techno logy (Gene tic engine ering)
Conten ts:
• Definition
• Overview of recombinant DNA Technology
• Various stages of recombinant DNA Technology
• Restriction enzymes
• Vectors
• Plasm.ids
• Bacteriop hages
• Cosmids
• Annealing
• Introduction of vector into host cells
• Transformation
• Transfection
• Electrop oration
• Selection of bacteria containing selected vector

• Multiplication
• Production of proteins using recombinant DNA Technology
• Gene therapy
• Application of recombin ant DNA Technolo gy
For queries and suggestio ns, contact the author at prasad_text@yahoo.com or 9986449575
Recomb inant DNA Technol ogy 475
Introd uction:The techniq ues involve d in prepara tions of a recomb inant D A

molecu le and cloning of specific D A segmen ts are collecti vely referred to as
'Recom b inant DNA technol ogy' or in commo n terms 'genetic engineering'.
Recombinant DNA:
DNA molecule contain ing segmen ts from two or more sources is called recomb inant
DNA or chimeric DNA.
Overvi ew of Recom binant DNA Techno logy:

Recomb inant DNA technol ogy involve s selectin g & separati ng the required DNA
segmen t from a large chromo some, incorpo rating it into a carrier DNA (vector DNA)
to produce a recomb inant DNA and then replicating this modified DNA several times
to produce multiple copies of the recomb inant DNA along with desired DNA fragmen t.
DNA cloning using recomb inant DNA technology is perform ed in two major steps.
1. Constru ction of a recomb inant DNA molecul e.
2. Amplification of recomb inant D A inside the bacteria to produce clones.
1. Construction of a recombinant DNA molecu le.

a) Cleavag e of D NA with a specific res triction endonu clease enzyme : Cut the
vector D A and the DNA of interest (DNA to be cloned or Desired D A) with
the same restriction endonuclease to produce complem entary sticky ends.
b) Anneal ing (Inserti on/ Integration of the des ired DNA fragment with the vector):
Then the two DNA (vector DNA and desired DNA fragmen t) are incubat ed
together. Since both the pieces of D A have complem entary sticky ends, they
anneal spontan eously. Then the DNA ligase enzyme s are added to this mixture ,
which covalen tly joins the vector D A & inserted D A of interest to produce a
recomb inant DNA.
2. Amplif ication of recombinant DNA molecu le inside bacteria.

a) Introduction of recombinant DNA into host cell: The recombinant DNA molecule
(vector) is introdu ced into the host cells. (This is by transfor mation, transfection,
electrop oration etc)
b ) Amplification of recomb inant DNA: Host cells containi ng the recombi
nant vector
DNA are then identified, sepa rated and then grown in a suitable culture medium .
As the host cells amplify , the recomb inant D A also replicates and multipli es to
produce large n umber of recombinant DNA along w ith the desired DNA fragmen t.
c) Release of the cloned D A molecu les from the host: Cloned D A fragmen
t are
released from the vector DNA molecule using the same restriction enzyme as
used in the first step.
For queries and suggest ions, contact the author at prasad_t ext@yah oo.com or
9986449575
Recombin ant DNA Technolo gy 476
Diagram atic Recomb inant DNA technol ogy
Pl u,..id. DNA
so....t.
(Cleavag e ) R"t.idio.,.,
er.-a~rnt.
( Annealin g )
Reco,..l,i,i, ,.t J>MA

(co,,to.:ni"J plo.s111id. Pl.IAucl d~1ircd .DNA f Tll.rt•~
Introduct ion into
the host cells l r... tvod..,Ho n. i..to host UII
11c,o,-binu,t V«lo1 !>WA < I (D ~ , ;> ci.,0 .,.010ma l ONA
(Multipli cation) 'l

Grown u nder condition s
required to D A amplificati on
l Isol ..t t plu1r>id .1
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Cteove .,:l1. u .. e.
l l.e.st"i,t1••'1\ •P\1~1'ftl
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Recombinant DNA Technology 477
Events in recombinant DNA technology (to produce DNA clones):
Various Stages of recombin ant DNA technology Protocol:
1. Isolation of desired DNA fragment to be cloned:

First step in rDNA technology is the isolation of the desired DNA fragment (DNA
to be cloned) from the cells. It involves digestion of cell membrane to expose the
nucleic acids, followed by separation of nucleic acids from other organelles and
then isolation of nucleic acids in pure form.
Then the desired DNA fragment of interest (D A to be cloned) is cut and separated
from the large nucleic acids with the help of restriction endonuclease enzyme.
Restriction endonucleases are tools th at cut DNA at specific sites within the
molecule. These fragments are separated by gel electrophoresis.
2. Cutting of vector:
Cut the cloning vector (carrier DNA) with the same restriction endonuclease enzyme.
Vectors are DNA molecu les into which the desired foreign DNA fragmen ts can be
introduced to form rDNA for the purpose of cloning. Commonly used vectors
include plasmids, bacteriophages, cosmids etc.
3. Insertion of the selected DNA into vector to produce a recombinant DNA:

The selected DNA fragment (DNA to be cloned) is then inserted into the vector
DNA to produce the recombinant D A (chimeric DNA). This process is called
annealing.
4. Transfer of recombinant DNA into host cell:

Then the recombinant DNA (vector) is transferred from test tube to the host cells.
(By various processes like Transformation, Transfection and Electroporation etc.)
5. Multiplication of recombinant vector in host cells to produce DNA clones:

The host containing recombinant vector is allowed to multiply. As the host cells
multiply, the recombinant vector is also replicated inside the host to produce multiple
copies of the recombinant vector along with the desired DNA segment.
6. Release of the cloned DNA from the host:

Finally, the cloned vectors are isolated and cloned DNA fragments are obtained
from the vector DNA by using the same restriction enzyme.
Clone is an identical copy. DNA cloning is the production of an iden tical copy of DNA
molecules from a common ancestor DNA using recombinant DNA technology. Clonin g
involves DNA amplification . (PCR is another method of amplification of DNA).

Applications of recombinant DNA technology:

1. Basic research of DNA study
Recombinant DNA technology has revolutionized DNA study, to understand the
structure & functions of DNA. It provides a means of isolation, characterization of
genes & to study how a specific segment of DNA works. rDNA technology is used to
prepare gene library (Refer last segment of the chapter). This technology made possible
DNA sequencing & complete study of human genome (Human genome project).
2. Application in forensic medicine
DNA technology is used for identification of the criminals or deciding the parent of the
child by analytical techniques like DNA finger printing, southern blotting etc.
When biological samples (blood, semen, or hair) obtained from a victim or suspect is
insufficient, DNA technology (by cloning or PCR) is used to amplify DNA to get enough
DNA material for analytical techniques such as D A fingerprinting.
3. Large scale production of proteins/ hormones (Details given in the next section):
Using recombinant DNA technique, several proteins can be produced in large quantities
for therapeutic purposes. (If the host cells containing amplified recombinant DNA (DNA
clones) are grown under conditions permitting expression (i.e. production of protein)
of the recombinant DNA, large amounts of the desired proteins can be produced).
Example: Insulin, growth hormone, tissue plasminogen activator, erythropoietin,
interferons, clotting factors, vaccines, enzymes like super oxide dismutase etc.
Proteins, thus produced can be used produce vaccines without the risk of contamination.
4. Gene therapy (Details given in the next section):
Gene therapy involves the treatment of diseases by the introduction of functional gene
into individuals having genetic diseases in place of mutated defective gene.
Some genetic disorders where gene therapy has been tried are SCID (severe combined
immune deficiency), sickle cell anemia, thalassemia, cystic fibrosis etc.
5. Prenatal diagnosis of disease
DNA probes are used for predicting the risk of genetic disorders like sickle cell anemia,
Phenylketonuria etc. by analyzing the DNA collected from amniotic fluid.
(Also, since a very small quantity of prenatal sample is available, rDNA technology or
PCR can be used to amplify the DNA to get sufficient amount of DNA for analysis.
6. Understanding and analysis of Molecular basis of disease
Recombinant DNA technology offers a rational approach to understand the molecular
basis of various diseases such as sickle cell anemia, thalassemia, cystic fibrosis, familial
hypercholesterolemia, haemophilia etc.
7. Laboratory diagnostic applications
Diagnosis of diseases like AIDS has become simple and rapid by this technology.
8. Transgenesis
It refers to the introduction of genes into the genome of fertilized ovum, which will be
found in somatic as well as germ cells and also passed on to the offspring's.

Tools and process of Recombinant DNA technology (In detail):

I) Restriction endonucleases (or Restriction Enzymes)
II) DNA ligases
III) Vectors - The cloning vehicles (eg, plasmids, bacteriophages, Cosmids etc)
IV) Annealing (Integration of DNA fragment to be cloned with the vector)
V) Introduction of vector into the host cell
VI) Selection of bacteria containing selected vector
VII) Multiplication of recombinant D A in host cells
VIII) Release of the cloned DNA molecules from the bacteria
I) Restriction endonucleases (or Restriction Enzymes):

• Definition: Restriction endonucleases are bacterial enzymes that cut DNA at specific
sites within the molecule (Hence the name endonucleases). These enzymes are also
called restriction enzymes, because they cut the viral DNA and restrict the growth
of certain bacterial viruses (bacteriophages).
• Examples : EcoRI and Haelll
• Nomenclature: Thousands of restriction enzymes have been discovered in different
bacterial species. Restriction endonucleases are named after the bacterium from
which they are isolated. For example, EcoRI is isolated from Escherichia (E) coli
(co), strain Ry13 (R) and first endonuclease to be discovered (I).
• Restriction sites (Recognition sites): Restriction endonucleases recognize specific
sequence of DNA at restriction sites and cut at a specific point. This short stretch
of DNA sequence that is recognized by a restriction enzyme is called a restriction
site. The restriction sites are usually 4 - 7 nucleotide base-pair long and they have
palindromic sequence.
• Palindromic sequences: Recognition sites have palindromic sequences (DNA
sequences which have same sequences in both DNA strands when read in 5' - 3'
direction are called palindromic DNA sequences).
• Sticky end/ blunt ends: Some restriction enzymes (Eg, EcoRI) cut D A in such a
way that they produce one to four unpaired nucleotides in each strand. These
unpaired strands are referred to as sticky ends because they can base pair with
each other or with complementary sticky ends (or cohesive ends) of other DNA
fragments Some restriction enzymes do not produce unpaired bases in DNA
fragments. These strands are referred to as blunt ends (Eg, HaeIII). Refer the figure
below.
The restriction enzymes that produce sticky ends are particularly useful in recombinant
DNA technology. The vector and the DNA to be inserted are cut with the same restriction
enzyme to produce complementary sticky ends, which are easy to anneal (join).

Example 1) Recognition site and action of EcoRI

EcoRI makes a cu t at each of the strand between 5'-G and A. So, the cut DNA
fragments have sticky ends.
(5') G ~ C (3')
(3')CTT AA .f (5')
1
(5') G AA TT C (3')
(3')C TT AA G (5')
Example 2) Recognitio n site and action of Haelll

Haem ma kes a cut a t each of the strand between 5'-GG and CC (3'). So, the cut
DNA fragments have blunt ends.
(5') G CICC (3' )
(3') C C G G (5')
1
(5') G G C C (3')
(3') C C G G (5')
Endonucleases and restriction endonucleases:

• Endonucl eases are enzymes that digest nucleic acids (DNA and RNA. They
hydrolyze nucleic acids within the molecule to form oligonucleotides. There are 2
types of endonuclea ses, ribonucleases (RNAase) and deoxyriboncleases (D Aase),
which act on RNA and DNA respectively. They cut non-specifically (without regard
to sequence) within the nucleic acids to form oligonucleotides.
Significance: Digestion of nucleic acid in intestine takes place by ribonuclea ses and
deoxyribo nclcases (pancreat ic endonucle ases), to form oligonucl eotides.
(Oligo11ucleotides are futher digested by phosplzodies temses, nucleotidases & nucleosidases).
• Restriction endonucl eases are also endonuclea ses that act on nucleic acids, but
are different from typical endonucleases in that these act at specific nucleotide sequences
(Restriction sites) within the nucleic acids. Restriction site are palindrome sequence of
about 4 to 6 nucleotide long. Restriction enzymes are isolated from bacterias. They cut the viral
DNA at specific points and restrict the growth ofiiirus (Hence the name restriction endonuclease).
Applications of restriction enzymes:
• DNA scissors: Genomic DNA is too long for analysis. Restriction enzymes provide
a very convenient means of cutting DNA into smaller, more manageable fragments.
• Restriction map: Restriction enzymes are used in the preparation of restriction maps.
Linear representation of DNA along with the information of restriction sites of various
restriction enzymes is called restriction map. Recognition sites of various restriction
enzymes on a given DNA fragment are quite characteristic.
Restriction maps provide valuable information abou t the cutting pattern of the DNA
by a given restriction endonuclease.
Restriction map is used to characterize a DNA during its study.
• Recombinant DNA technology: Restriction endonucleases (and DNA ligases) are

important tools in recombinant DNA technology. The vector and the DNA to be
inserted are cut with the same restriction enzyme to produce complementary sticky
ends. When these two DNA (vector D A and DNA fragment of interest) are mixed
with each other, the complementary sticky ends come together and they are joined
by DNA ligase to produce a recombinant DNA.
• Southern blotting: Restriction enzymes are used in the preparation of southern

blotting, where DNA samples are digested with restriction enzymes to produce
fragments. This technique is used to detect the presence of particular base sequence
in the sample DNA.
(Note tlznt Northern blotting is a technique for detection and isolation of RNA).
• Restriction Fragment Length Polymorphism (RFLP) and D NA finger printing:

Restriction enzymes are important tools in Restriction Fragment Length Polymorphism
(RFLP) and DNA finger printing. RFLP is a widely employed technique in identification
of individuals, and genetic diseases. DNA finger printing is used to differentiate
DNA of different sources, because the fragments of DNA from one individual will
have a characteristic pattern. This property is used in forensic applications.
• Genomic library: Restriction enzymes are also used in preparing a genome library.
A genome library is entire collection of DNA fragments from an organism placed
in a suitable vector for cloning.
II) DNA ligases:

After the fragments of DNA (one from human genome and another from vector
DNA) have base paired, the ends are covalently joined by the action of D A ligase.
Restriction enzymes in conjunction with DNA ligase produce vector containing
recombinant or chimeric DNA.
III) Vectors:
Vectors are DNA molecules into which the foreign DNA can be introduced for the
purpose of cloning. Vectors can replicate inside the appropriat e host cells.
Essential properties of a vector include:

• It must be capable of self replication within the host cell.
• It must contain at least one specific nucleotide sequence recognized by a restriction
endonucle ase.
• It must carry at least one gene that allows the selection of the vector, such as an
antibiotic resistance gene etc.
There are many vector used in recombina nt DNA technology. Commonly used vectors
include plasmids, bacteriophages, cosmids etc.
a) Plasmids
• Definition: Plasmids are small circular duplex DNA molecules present in some of
the bacteria in addition to their single, large circular chromosom al D A.
• Plasmid DNA confers antibiotic resistance: The natural function of plasmid is to
provide antibiotic resistance to bacteria. Plasmid DNA carries genes for antibiotic
resistance. Thus, those bacteria which have such plasmids have an advantage over
other bacteria to survive even in the presence of certain antibiotics.
• Plasmids replicate independe ntly of circular DNA in the bacterial cells. Since
the plasmids are relatively smaller DNA, they can be easily separated and isolated
from bacteria. The complete DNA sequence of many plasmids is known.
• Significan ce: Plasmids are used as carriers (vectors) in gene cloning. They can
carry foreign DNA segment from any other source to produce recombina nt DNA.
Such recombina nt plasmid DNA can be inserted back to bacteria and allowed to
multiply to generate large quantities of plasmid molecules along with inserted DNA
fragments.
b) Bacteriop hages
• Bacteriophages (or simply, phages) are viruses that replicate inside the bacteria.
• Advantage s of phages are that they can take up larger segments of D A than
plasmids. This makes them suitable to work with human genome.
• Lambda phage is the most common phage used in recombina nt DNA technology.
c) Cosmid s
• Cosmid s are plasmid s that contain DNA sequenc es (cos sites) of bacterio phage
lambda. (Cos sites are DNA sequenc es that are required for packagi ng lambda D A
into the phage particles). Cosmid s are constru cted by adding a cos site of phage
lambda to plasmid s. Cosmid s can grow in the bacteria just like plasmids. They contain
best features of plasmid s and phages.
• Cosmid s can carry larger segmen ts of DNA compar ed to plasmid s.
Nowada ys, artificially produce d vectors like bacterial artificial chromo some (BAC),
Yeast artificial chromo some (YAC) etc are used in recomb inant DNA technology. They
can carry very large fragmen ts of DNA.
Comparisons of cloning capacities of plasmids, bacteriophages, Cosmid s

Vector DNA Insert Size (kb)
Plasmid p BR322 0.01-10
Lambd a charon 4A 10-20
Cosmid s 35-50
BAC,P l 50-250
YAC 500-3000
IV) Annea ling (Integration of DNA fragment to be cloned with the vector):
• Anneali ng is the process of insertio n of the DNA fragmen t of interest (DNA to be
cloned) into the vector DNA to produce recomb inant DNA. Since the two pieces
of DNA are cu t with the same RE, both the DNA fragme nts will have
comple mentary sticky ends, so they anneal spontan eously.
• D A ligase enzyme s are added to this mixture which covalen tly joins the two
pieces of DNA to form a circular recombinant vector DNA.
V) Introduction of vector into host cells:

The recomb inant DNA (vector) should be inserted back to the host cell (bacteria). This
is done by various processes. Transfo rmation and Electrop oration are two commo nly
employ ed techniques.
• Transformation: Transfo rmation is the process by which plasmid s are introdu ced
into bacterial cells. In this technique, the host cell cells (eg, E. coli) and vector (eg,
plasmid DNA) are incubated together at 0°C in a calcium chloride solution, then subjecte d
to a heat shock by rapidly changin g the tempera ture to 37 - 43°C.
The calcium channel s are opened, through which the plasmid is imbibed into the host
cell. This method makes the bacterial membra ne permea ble to plasmids.
ote that the process of introdu cing the foreign D A into the eukaryo tic cells is called
Transfection.

575
Recombinant ONA Technology 484
• Electroporation: In electroporation, the host cells incubated with vector (eg, plasmid
DNA) are subjected to a high-voltage electric pulse. This method transiently renders
the bacterial membrane permeable to vectors.
VI) Selection of bacteria containing selected vector:

Introduction of vector into host cells is a complicated process & sucess rate is very low.
It should be noted that only 5% of bacterial colonies contain the desired recombinant
vector. Therefore, only the colonies containing desired vector should be selected and
isolated by specific techniques (eg, by antibiotic sensitivity techniques).
Then these colonies are allowed to multiply in a suitable culture to produce multiple
copies of recombinant DNA.
For eg, let's take the case of plasmid pBR 322,
• Plasmid pBR 322 is one of the commonly employed vectors in genetic engineering.
It is circular DNA containing 4361 base pairs. It contains ampicillin resistance
gene (AmpR) and tetracycline resistance gene (TetR).
• The restriction enzyme Pstl will cleave the plasmid in the middle of AmpR gene.
So, when the foreign DNA is inserted, the resistance against ampicillin is lost.
This insertional inactivation of AmpRgene is the marker for recombinant DNA.
• Then the bacteria are cultured in a suitable medium. There will be many colonies;
some containing recombinant vector and some do not. These colonies are replica-
plated onto another culture medium containing ampicillin. The colonies containing
recombinant vectors are killed in the replica-plate, because the insertion of foreign
DNA abolishes ampicillin resistance. (Whereas the colonies that do not con tain
recombinant DNA survive, as their AmpRgene is not cleaved).
• Colonies in the original plate, corresponding to the dead colonies in the replica-
plate are selected. These bacteria carry the recombinant DNA.
• The selected colonies of bacteria will serve as a source of recombinant DNA for
further use. The selected colonies are further cultured to produce multiple copies
of recombinant DNA (DNA cloning).
VII) Multiplication of recombinant DNA in host cells to produce DNA clones:

The selected colonies (host cells) are further cultured in an appropriate medium. As
the host cells multiply, the added recombinant vector is also replicated inside the
host using the enzyme machinery of host to produce clones of the recombinant vector
DNA along with the desired DNA segment. Thus all their progeny will contain clones
of the original (multiple copies) of recombinant plasmid.
VIII) Release of the cloned DNA molecules from the bacteria:

Cloned DNA fragment are released from the vector DNA molecule using the same
restriction enzyme as used in the first step.
Production of proteins using recombinant DNA technique:

Many proteins, especially human proteins, are produced in large quantities using
recombinant DNA technique. If the host cells are grown under conditions permitting
expression of the recombinant DNA, large amounts of the desired protein is produced
from this amplified recombinant DNA.
The preliminary steps involved are same as those for DNA cloning:
• For protein production, the DNA fragment (Gene that codes for the proteins to be
produced) is introduced into the vector.
• A bacterial promoter region is added to the upstream of human gene and then inserted
into a vector. Such a vector is called expression vector.
• Host (bacteria) containing the recombinant expression vectors are selected and isolated
by specific techniques and allowed to multiply in a suitable culture.
• The human proteins are produced and isolated from such a bacterial culture.
Insulin is the firs t protein to be produced by this technique.
Rcco,.,bio••t >"«lo, ll+JA c,,,,.

0 , 0 ...,..1 OoJA
Protein production:
DNA cloning: Grown under conditions
Grown under the required for the expression
conditions required for of cloned genes (to
DNA amplification produce proteins)
Proteins that are produced using this technique are used for:
Replacement therapy & other treatments (e.g. insulin, growth hormone, antihemophilic
factor, interleukins, interferon, etc).
Disease prevention (e.g. vaccines, such as hepatitis B antigen)
Diagnostic tests (e.g. monoclonal antibodies).
Merits of this technique:

1) It can produce large amounts of proteins that could not be obtained by conventional
purification methods. For eg, to get 1 unit of growth hormone, more than 1000 cadaver
pituitaries are required.
2) It can provide human proteins, which are not antigenic when administered to humans
(compared to animal proteins that are isolated by conventional methods).
3) Risk of contamination is eliminated while handling infectious samples.

Gene therapy:
• Definition: Introduction of a normal functional gene into the somatic cells of a

patient having defect in that gene is called gene therapy. So, in gene therapy a
normal gene is inserted into the genome to replace the defective gene.
• Rationale: Each genetic disease is due to defect in a particular gene. Gene therapy
attempts to supply a functional gene into the somatic cells. Currently, gene therapy
is still at experimental level.
• Types: Gene therapy can be of two types. The approach of introducing normal
gene into somatic cells is often termed as somatic cell gene therapy. Supplying the
functional gene to germ cells is termed as Germ line gene therapy, which is not
permitted as it is considered unethical.
• Gene therapy for severe combined immunodeficiency disease (SCIO): Gene

therapy was first successful in 1990, where a child suffering from SCID was cured
by transferring a functional gene into her bone marrow cells. SCIO is a genetic
disease characterized by the failure of immune system mainly due to the genetic
defect in the gene for enzyme adenosine deaminase (ADA). In gene therapy,
functionally actjve ADA gene is incorporated into the somatic cells of the patient.
• Other examples: Gene therapy is being tried in cystic fibrosis, hemophilia etc.
• Vectors: Different kinds of vectors are used in gene therapy to introduce genes into
humans. Eg, retroviruses, adenoviruses, plasmid-liposome complexes etc.
The procedure of gene therapy involves,

1) Isolation of the healthy gene
2) Incorporation of this gene into a carrier or vector
3) Delivering the vector into the target cells
The steps employed in gene therapy for SCID are as follows:-

• Functionally active and normal ADA gene is introduced into a suitable retrovirus
vector. This recombinant genetic material can be packed into a viral particle in an
appropriate cell culture.
• The virus particles are allowed to infect immune cells of patients in vitro. The retrovirus
incorporates the normal gene into the cell.
• These cells are allowed to multiply and introduced back into the patient's body.
These newly introduced cells produce active ADA enzyme.
• These cells multiply in the bone marrow and restore immune system function.

Antisense therapy
• The mRNA contains a message or "sense" to be translated into protein. If a nucleotide
having complimentary sequence to an mRNA is prepared, it is said to be "antisense".
When antisense oligonucleotide (either RNA or D A) is added, it will bind and trap
the normal mRNA and so protein biosynthesis can be stopped. These oligonucleotides are
designed to hybridize either with the target gene to prevent transcription or with mRNA to
prevent translation. This is the basis of antisense therapy.
• Generally, gene therapy is employed to introduce a normal functional gene to produce
the defective protein. But in some disease especially cancers, normal growth-stimulating
genes are either overexpressed or undergo mutation producing abnormal p roteins that
result in uncontrolled cell growth, leading to cancer. These genes are called oncogenes.
In principle, cancer growth can be stalled by inhibiting the expression of oncogenes in
cancer-affected cells. In the same way, viral diseases can be treated by inhibiting the
expression of certain viral genes.
• In antisense therapy, nuclease-resistant oligonucleotides (about 7-20 nucleotide length)
that are complementary to an unwanted mRNA are used as antisense molecules.
The antisense nucleotides are delivered into the cells by liposome encapsulation. They
hybridize with the mRNA and there by block translation.
Clinical trials on HIV and cancer are being conducted using antisense molecule.
DNA library:
A DNA library is a collection of DNA fragments from a particular species. The formation
of gene libraries is done by isolating the complete genome, which is then cut into
fragments and cloned in suitable vectors. Then the specific clone carrying the desired
DNA can be identified and isolated and a library of genes or clones (Gene bank) for the
entire genome of a species can be constructed. These are two types of DNA library,
a) Genomic DNA library
In genomic DNA library both intron and extrons are represented and thus, contains
every sequence from the genome of a specific organism.
The entire genomic DNA of an organism is cu t into small pieces by restriction
endonucleases. These cut pieces are then introduced into vectors and cloned.
A collection of these different recombinant clones is called genomic DNA library.
b) Complementary DNA (cDNA) library
In cDNA library only exons are represented. Thus, cDNA library is a more specialized
and exclusively DNA library. It is constructed so as to include only those genes that are
expressed in a given organism or even in cells or tissues.
First, complementary double stranded DNAs are prepared from mRNA by reverse
transcriptase. These DNA fragments are cut by restriction endonucleases and introduced
into the vector DNA and cloned.
Collection of these clones is Complementary DNA (cDNA) library.

Question Bank on Recombinant DNA technology:
Essay Questions (10 marks):

I. Explain recombinant DNA technology and applications of recombinant DNA technology
Short Essay Ques tions (5 marks):

1. Restriction Enzymes and significance
2. DNA cloning
3. Vectors
4. Gene therapy
Short Answers (2-3 marks):

1. Plasmid / Cosmids
1) The following are required for the formation of chimeric DNA molecules EXCEPT
a) DNA ligase b) Liposomes c) Plasmid d) restriction endonuclease
2) The first approved pharmaceutical product of recombinant DNA technology for human
a) Growth hormone b) Interferon c) Calcitonin d)Insulin
3) Restriction endonuclease
a) Cuts both strands of double-stranded DNA b) Removes nucleotide from interior of DNA
c) C uts single strand of double-stranded DNA d) Cuts single stranded D A
4) Function of restriction endonuclease is
a) To ligate cut ends of plasmid DNA c) To cleave animal DNA
d) To synthesize RNA from DNA d) Synthesis of DNA from R A
5) is termed as molecular scissors
a) Reverse transcriptase b) Restriction endonuclease c) Taq Polymerase d) Phosphodieterase
6) DNA of Kb can be inserted into a plasmid
a) 6-10 b) 10-20 c) 35-50 d) 50-80
7) The first genetic disease to be t reated by gene therapy was
a) Adenosine deaminase deficiency b) LDL receptor deficiency
c) Duchene muscular dystrophy d) Cystic fibrosis
8) Recombinant DNA molecule which can directly give rise to protein in bacteria are called
a) Plasmid b) Cosmid c) Expression vectors d) Hybridoma
9) The process of introducing a plasmid into the bacterial cell is called
a) Translocation b) Transformation c) Transduction d) Transvection
10) Plasmid D A is
a) Circular single stranded b) Linear single stranded c) Circular double stranded d) Linear single stranded
Answers for MCQ: 1) b 2) d 3) c 4) c 5) b 6) a 7) a 8) c 9) b 10) c

Techniques 489
Techniques
Contents:
• ELECTROPHORESIS
• CHROMATOGRAPHY
• RIA
• ELISA
• IMMUNOELECTROPHORESIS

Techniques 490
Electrophoresis:
Electrophoresis is a technique for the separation of charged particle under the influence
of electric field. The term electrophoresis literally means that "The migration of charged
particles under the influence of electric field."
Principle:
Many important biomolecules like amino acids, proteins, nucleic acids, nucleotides
have ionisable groups and can exist as cations (positively charged particles) or anions
(negatively charged particles) depending on pH of the medium.
Under the influence of electric field these charged particles migrate either to cathode or
to anode depending on the nature of net charge they possess i.e. cations move towards
cathode (Negative electrode) and anions towards anode (Positive electrode). The rate
of migration depends on net charge, size and shape. When an electric field is applied,
these charged molecules are separated based on (a) Net charge, (b) size (c) shape.
Electric field is removed before the particles reache the electrodes. (Thus electrophoresis
is incomplete form of electrolysis). The separated particles are then located by staining
with the suitable dye.
Note: A supporting medium is required to carry out electrophoresis. It can be filter

paper (paper electrophoresis) or glass slide coated with agar (agar gel electrophoresis).
Technique (of Agar gel electrophoresis):

A small amount of sample is applied on the supporting medium (paper or glass) near
one margin. The slide is kept on an electrophoretic chamber and connected to buffer
compartments by filter paper strips. Current is applied through electrodes dipped in
each buffer compartment/chamber. This causes separation of sample into fractions.
Their positions are visualized after staining with a suitable dye. The rate of migration
of each component depends on net charge, shape and size.
Paper/
Agar plate
Cathode Anode
Flller paper

Techniques 491
Application of electrophoresis:
1. It is a technique for the separation and quantitation of serum proteins:
In the separation of serum proteins, a buffer of pH 8.6 is used, which is greater than the
isoelectric pH of all the serum proteins. Hence all the serum proteins have negative
charges in this pH. So these serum proteins migrate to anode under the electric field.
This results in separation of serum into 5 fractions, namely albumin and al/ a 2, p, Y
globulins. These bands of proteins are visualized by staining with the stains like amido
black or coumassie blue.
The width and intensity of each band is the measure of concentration of each fraction.
In normal electrophoretogram, the proportion of the various bands are as follows:
Albumin 56%, a 1 globulin 6%, a 2 globulin 13.5 %, globulin 12.5%, r globulin 12%.
• o ••
( -) ( +)
1rn y a 2 Clt Al bumin

1
Globulins
2. Electrophoresis of serum proteins is useful in diagnosis of some diseases like,

a) Nephrotic syndrome: Albumin is decreased (because albumin is lost in urine) and a 2
band is increased and, also occasional decrease in Y globulin level.
b) Chronic liver disease: Albumin level decreases with increased production of some
unidentified proteins, which migrate into pand pr globulin region, causing a fusion.
c) Infective hepatitis: There is elevated Y globulins and reduction in a 1 & globulins.
d) Multiple myeloma: An extra (abnormal) band called M-band is seen in Y region or
between Y and p globulin bands.
e) Rheumatoid arthritis: There is usually a small to moderate increase in alpha fraction.
A similar increase is also seen in some cases of cancer.
f) Primary immune deficiency (hypogamrnaglobulinemia or agammaglobulinemia):
There is a marked decrease or absence of gamma globulins.
3. This technique is useful in separation of lipoproteins.
4. Electrophoresis is also useful in separation of isoenzymes.
Answer hint for Electrophoresis question (5 Marks):

a. Introduction (1 Mark)
b. Principles {1 Mark)
c. Technique/ Procedure (1 Mark)
d . Applications (2 Marks)

Techniques 492
Chromatography:
Chromatography is a simple technique used for separation of individual components
of a mixture based on differential distribution of these components between two phases;
1. Stationary phase
2. Mobile phase
There are many different types of chromatography techniques, which use various
principles (like adsorption, partition, gel filtration, ion exchange etc) for the separation
of different types of substances. Among these, paper chromatography is most common
type.
Paper chromatography:
In paper chromatography, principle of partition is employed to carry out separation.
Separation of individual components of mixture is achieved by differential relative
solubility (partition) of these components in two phases. The cellulose fibres of paper
act as a supporting matrix for stationary phase.
Technique:
Method consisting of applying a small amount of mixture on a strip of chromatographic

paper. The solvent mixture, which usually consists of water (stationary phase) and
organic solvent (mobile phase) is flown past the application point.
The water is held back by cellulose fibres of the paper and organic solvent moves over
the paper and the sample mixture. ·
As the mobile phase moves past the sample mixture, it carries different components of
the mixture at different rates, the rate being dependent on relative solubility of these
components in mobile and stationary phase. If the compound is more soluble in water
and less soluble in organic solvents it moves slowly and vice versa.
After the solvent has run for an appropriate distance, the paper is taken out from the
chromatography chamber and 'solvent front' is marked.
Then the paper is dried and individual components are visualized by spraying with a
suitable dye. Then the position of each component is marked and the distance from the
origin to these fronts is measured. Now the RF (relative front) value for each component
is calculated.

Techniques 493
RF value is ratio of distance moved by 'solute front' to the 'solvent front'.
RF= Distance moved by solute front

Distance moved by solvent front
RF value is a characteristic property of a compound for a solvent system. It is possible

to identify a unknown substance by their RF value in relation to known substance.
- ---@ Solvent front
• • 1. Alanine
2.Leucine
3. Glycine
4. Unknown Mixture
•
1 2
•
3
•
4
Linc of Application
Paper Chromatography
Application of paper chromatography in Biochemistry
1. Separation technique: Paper chromatography is a simple technique for separation

of sugars carbohydrates, amino acids etc.
2. Oinical significance: Usually very low amount of carbohydrate and amino acid
present in urine. However, in some diseased conditions, large amount of specific amino
acids, carbohydrates and rare metabolites may be excr eted in u rine. Paper
chromatography techniques can be employed in detection of these abnormal compounds.
Some of the compounds that are usually detected by chromatography are,
• Phenylketo acids in Phenylketonuria
• Alkaptone bodies in alkaptonuria
• Glucose (Glucosuria) in diabetes mellitus, renal glycosuria
• Pentoses in essential pentosuria
• Fructose in fructosuria
• Lactose (lactosuria) in lactose intolerance

Techniques 494
Radioimmunoassay (RIA)
Radioimmunoassay or RIA is a quantitative assay technique for the determination of
concentration of antigenic substances. It combines the specificity of immune reaction
with the sensitivity of radio-isotopic techniques.
RIA is used in the estimation of specific substances even when they are present in
small quantities (in nanograms or picograms) or mixed with other substances.
Principle:
RIA is based on competition between radio-labeled antigen and unlabelled antigens
(substance to be determined) to bind with the limited number of antibody available.
Outline of the assay:

1. Specific antibodies are adsorbed to a solid surface in a microtiter plate.
2. Then radio-labeled antigen is added and allowed to react (bind) with the antibody
attached to the solid surface. Any unbound antigen is removed by washing.
3. Then a sample containing unlabeled antigen is added to this mixture. The
unlabeled antigen competes with the labeled antigen for binding to antibody and
d isplaces bound labeled antigen. More the antigen in the sample, more the
displacement of labeled antigen. The amount of labeled antigen remaining in the
microtiter plate is determined by measuring the radioactivity.
4. The radioactivity measured will be inversely proportional to the concentration
of antigen present in the sample.
5. A standard curve is prepared by using different known concentrations (standards)
of unlabelled antigen and same quantities of labelled antigen and antibody.
6. Then the assay is performed with the sample of unknown concentration of antigen
(whose concentration to be measured) and compared with standard values.
Application:
• RIA is used in the assay of any compound that is immunogenic, available in pure
forms and can be radio labeled. It is useful in biomedical research .
• RIA is used in assay of hormones, tumor markers, vitamins, steroids, drugs etc.
Advantages:
• High sensitivity - compounds at levels of pg / ml also can be assayed
• High specificity
• High reprod ucibility
• Can be automated
Disadvantages:
• High cost of equipment and reagents
• Th e shelf life of reagents: Half lives of label being small
• Radiological hazards of using radioactive isotopes
• Long duration of assay (usually days are required)

Techniques 495
Enzyme Linked Immunosorbent Assay (ELISA)

ELISA is a qualitative and quantitative assay for the detection and determination of
concentration of antigens and antibodies.
Principle:
The assay is based on specific binding of antigen to antibody and competition between
labeled and unlabelled antigen for binding sites of antibody. In ELISA, an enzyme
(usually alkaline phosphatase or horse radish peroxidase) is used as a label instead
of radioactive label employed in RIA. So, there is no risk of radiation hazard in ELISA
as in RIA.
Out line of the assay:

1. Specific antibodies are adsorbed to a solid surface in a microtiter plate.
2. Then the enzyme labeled antigen (antigen bound to enzyme) is added to the
plate, which binds to the antibody fixed in the plate. The plate is then washed to
remove the unbound labeled antigen.
3. Then a sample containing unlabeled antigen is added to this mixture. The
unlabeled antigen competes with the enzyme labeled antigen for binding to
antibody and displaces bound labeled antigen in the proportions they are present.
More the antigen in the sample, more the displacement of enzyme labeled antigen.
4. Then the substrate (S) to the enzyme is added to the plate and the product (P)
formed is estimated.
5. Amount of product formed is directly proportional to the concentration of
enzyme labeled antigen bound to the antibody in the plate and hence the
inversely proportional to the concentration of antigen in the sample.
6. As in RIA, a standard curve is prepared by using known concentrations of
(standard) of unlabeled antigen and same quantities of antibody and enzyme
labeled antigen and measuring the product formed.
7. Then the assay is done with the sample containing unknown concentration of
antigen (whose concentration to be measured) & compared with standard values.
Application:
• It is used to measure hormones, such as insulin, estrogen and human chorionic
gonadotropin (hCG) in the pregnancy test.
• Used in the assay of immunoglobulins like IgG, IgE, and oncofetal proteins etc.
• It is used in the study of infections diseases for detection of bacterial toxins,
retroviruses, HIV antigens, tumor markers etc.
Advantages:
• As In RIA: High specificity, High sensitivity, High reproducibility.
• Advantages over RIA: Relatively cheap, Reagent shelf life longer, Safe (no risk of
radioactive hazards), can be performed in small diagnostic laboratories.

Techniques 496
RIA
..-- (Antibodies are fixed on a plate)
yyyy
.R
R R
r:I ./
R
' R • •R
R
(Radio labeled a ntigen)
Radioactivity
tttt l
••• (Unlabeled antigen)
Standard antigen concentration
R R
tttt
(Radioactivity of the plate is inversely proportional to t he
concentration of the unlabeled antigen)
IELISA I
..-- (Antibodies are fixed on a plate)
yyyy
(Enzyme labeled antigen)
Cone. of
Product
E E
••• (Unlabeled antigen)
l
t ttt
Standard antigen concentration
Substrates Products (amount inversely proportional to the

concentration or the unlabeled antigen)

Techniques 497
Immunoelectrophoresis:
• This technique combines the principles of electrophoresis & immunological reactions.
• This method is more sensitive and specific than ordinary electrophoresis.
• It is useful in the analysis of complex mixtures of antigens and antibodies.
Outl :ne of the assay:

1.The sa mple (For e.g. serum) containi n g antigen is subjected to agar gel
electrophoresis and the proteins are separated.
2.The antisera containing specific antibody is applied in a trough cut in the gel, parallel
to the direction of electrophoretic separation. The antibodies diffuse and when
they come in contact with antigen separated, precipitation occurs. This results in
the formation of precipitin arc, which can be identified. The development of
precipitin arc identifies the presence of specific protein of interest.
Well containing serum

I Step 1:
+Ve • -Ve Electrophoretic separation
0 of proteins, but they are
1) not visible
Step 2:
+Ve -Ve Antibody is added to the
0 trough and incubated
2) Precipitation arc is formed
A trough cut in the gel
Application:
Immunoelectrophoresis can be performed in patient's serum, urine or spinal fluid to
detect the presence of abnormal proteins and/or finding of abnormalities in the
concen trations of antigens, relative to the normal control sample analyzed in the
same time.
Autoanalysers: The large sample size in many cli11ical laboratories has lead to the development of
autoanalysers, nn instrument that cnn perform a large number of tests in a very short time. ThetJ
generally use less sample volume.

Techniques 498
Question Bank on Techniques:
Short Essay Questions (5 marks):

1. Electrophoresis
2. Chromatography
3. RIA
4. ELISA
5. lmmunoelectrophoresis
Short Answers (3 marks):

1. Applications of electophoresis
2. Applications of ch roma tography
1. Applications of RIA
2. Applications of ELISA
1) Rad ioisotope generally used for ELISA is

a) 6()Co b) 51Cr c) 3 ~P d) 1311
2) Possibility of Radio H azard is present in

a) RIA b) ELISA c) Sandwich ELISA d) Flame Photometry
Answers for MCQ: 1) d 2) a

Radioactive Isotopes 499
Radioactive Isotopes and their applications
Contents:
• Introduction
• Nature of radioactivity
• Applications of isotopes medicine

Introduction:
• An atom is made up of subatomi c particles like proton, neutron a nd electron.
• Proton (p) carries one positive charge and electron (e) carry one negative charge.
Neutron (n) carries no net charge.
• Atomic number of an atom is the number of protons present in it.
• Atomic weight of an atom is the sum of the numbers of protons & neutrons in it.
Representation: Atomic number is shown on the lower left corner and atomic weight
on the upper left corner of the symbol of the element. E.g. atomic number and atomic
weight of sodium are 11 & 23 respectively and it is shown as 11 a & 2., a respectively.
Isotopes:
Definitio n: Isotopes arc defined as the elements with the same atomic number
(protons) but different atomic weight (varying number of neutrons ). So, isotopes
occupy the same place in the periodic ta ble. Isotopes react similarly in chemical
reactions because they contain the same number of electrons.
Example: 1H is normal hydrogen w ith one proton, H, called deuterium , has Ip+ ln
2
and 1H, called tritium, and has lp + 2n. These three are isotopes of hydrogen .
Isotopes may be stable or unstable (radioactive isotopes).
Radioactive isotopes, radioactivity and radioactive decay:

The nucleus of a unstable isotope degrades spontane ously with consequent emission
of particles or rays and gets transform ed to another element. Such unstable isotopes
are ca lled radioactive isotopes; process is called radioactivity and the degradat ion
is called radioact ive decay . Radioac tivity can be detected or estima ted by
autoradiography, liquid scintillat ion counter and spectrometry.
Nature of radioactivity:
The radioacti ve elements emit 3 types of radiation - alpha (a), beta(~) and gamma (y).
These radiation s, in their path\vay , knock off electrons from surround ing atoms
producin g ions, & hence are called ionizing radiations. These radiations, if pass through
a living cell can cause physical and chemical changes leading to varied biological effects.
• Alpha radiation is particula te. The alpha particle has 2p + 2n and carries 2 positive
charges. Since it is particula te, alpha radiation has negligibl e penetrati on power
and is stopped by a few sheets of paper.
• Beta radiation is generate d by splitting of a neutron into one proton, one electron
(beta particle) and one nutrino. The electrons thus emitted become the beta rays.
So they a re negativel y charged. Since their mass is negligible they can penetrate
more d istance (few sheets of aluminum ).
• Gamma ray has no mass and no charge, so it penetrate s more. While alpha & beta
radiation are particles, gamma radiation is in the form of electrom agnetic waves.
• Only beta & gamma radiation s ( not alpha radiation s) are useful in clinical medicine.
Gamma radiation has more penetrati ng power, hence used in the treatmen t of cancer.
App lications of radioisotopes:

1) Research applications:
Tracer technique: Radioisotopes of an element will have same chemical reactions as
the normal isotopes. Hence when a radio-labeled compound is administered, these
molecules are metabolized by the body similar to normal molecules. This principle is
exploited in tracer technique. Almost all the metabolic pathways are discovered by
using such tracers. For e.g. N itrogen of heme is shown to be derived form glycine
using tracer technique. This is by feeding ra ts w ith 14N-labeled glycine and detecting
14
N in heme.
Determination of turnover rate, pool size of a substance in the bod y and volumes of
body compartments containing body fluids (e.g. plasma volume), and carbon dating
etc. are of the other research app lica tions of radioisotop es.
2) Diagnostic applications: There arc 2 types of diagnostic applications,
i) Procedures in which radioisotopes are administered to the patient: Radioisotopes
are administered for thyroid uptake studies and scanning of thyroid gland, bone,
kidney and heart etc.
ii) Proced ures in which radioisotopes are used as reagents (in vitro): As reagen ts,
radioisotopes are used in 1) Radioimmunoassay (RIA) to quantitative hormones,
tumor markers and other biological substances present in blood or other bod y fluids
in very small quantities and 2) DNA finger printing in DNA analysis
3) Therapeutic applications:
• Radioactivity is used mainly in the treatment of cancer:
This is based on the principle tha t the radiations w hen absorbed by the tissues
produce ionization in the p ath. This causes damage to nucleic acid in the cell so
that next cell division is halted. Radiotherapy mainly affects cells in the d ivision
phase. Cancer tissue is preferentially affected by radiation, it contains more divid ing
cell than normal tissues.
• Radioactivity is also used for treatment of hyperthyroidism.
Some commonly used rad ioisoto pes in Medicine:

Element Isotope Radiation Application
Carbon 14c Beta Carbon dating, Research in metabolism
Hydrogen 3H Beta Cell biology research
Phosphorus 32p Beta N ucleic acid research, treatment for
polycythem.ia
Iodine 125J Gamma Radio immunoassay
Iodine 131J Gamma Thyroid uptake studies, thyroid
scanning, treatment of thvroid cancer
Cobalt 60Co Gamma Cancer treatment
Caesium Cs
137 Gamma Cancer treatment

Question Bank on Isotopes

l. Applications of radioisotopes in Medicine
Short Answers (2-3 Marks) :

l. Isotopes
2. Radioisotopes

1) Radioisotope used for the treatment of p(?lycythemia is
a) J2p b) 1J1p c) 60 Co d) 1311
2) Which of the following emitter is more suitable for the diagnostic purpose?
a) a b) c) y d) o
3) Unit of radioactivity is
a) Bequeral b) Curie c) Rad (r) d) All the above
4) Tracers used to study nucleic acid metabolism
a) 51Cr b) ~o c) 51P d) 1311
5) Radioisotope used for the treatment of cancer is
a) 60Co b) 51Cr c) 32P d) 131l
6) Radioisotopes are
a) Elements having different mass number and different atomic number
b) Elements having same mass number and different atomic number
c) Elements having different mass number and same atomic number
d) Element having same mass number and same atomic number
7) The isotopes used as tracer in PET scan is

a) 90Sr b) 18F C) 99Tc d) 12SJ
8) The number of p rotons inside atom w ill determine

a) Atomic number b) Atomic weight c) Molecular weight d) Polarity
9) Which ionizing radiations has more penetrating power, so used in the treatment of cancer?
a) Alpha radiations b) Beta radiations c) Gamma radiations d) None of these
10) Which of the following radioactive isotope is used to treat thyroid cancer?
a) p 3 1 b) p is c) Tc99 D) p 32
11) Extracellular volume is determine by

a) 24 a labeled NaCl b) 3H labeled water c) 32P labeled erythrocytes d) 1311 labeled albumin
Answers for MCQ: 1) a 2) c 3) d 4) c 5) a 6) c 7) b 8) a 9) c 10) a 11) a

Hormones 503
Hormone Action
Contents:
• Introduction
• Classification
• Mechanism of hormone action
• Receptors and signal transductions
• Second messengers: cAMP, cGMP, Phosphatidyl inositols, Calcium
• Neurotransmitters

Hormones 504
Definition: Hormones are biochemical messengers, which carry signals to generate

some alteration at the cellular level.
Classific ation of Hormones:
1. Based on receptor site (or Based on the mechanis m of hormone action):

Hormone actions begin w ith the binding of hormones with their receptors. These
receptors are situated either on the cell m embrane or inside the cell.
Based on the site of receptors, hormones are classified into 2 groups,
Group I hormones:
These hormones bind to intracellula r (cytoplasm or nucleus) receptors. The hormone-
receptor complex alters genetic expression of the target cell.
E.g.: All steroid hormones (like g lucocortico ids, mincraloco rticoids, estrogen etc),
cakitriol, thyroid hormones (T3 and TJ
Group II hormones:
Th ese hormones bind to cell membrane r eceptors o n the target cells. They
communic ate inside the cell through intermedia tes, called second messenger s. These
second messenger s have intracellula r effect.
Based on the type of second messenger s, group II hormones are further classified
into 4 categories.
• Group II A hormones:
Hormones that have cell membrane receptors and second messenger in cAMP.
E.g. Glucagon, Epinephrin e, Calcitonin, ADH, LH, ACTH, PTH, FSH, TSH etc.
• Group II B Hormones:
Hormones that have cell membrane receptors and second messenger is cGMP.
E.g.: Atrial Natriuretic factor (ANF) and Nitric oxide (NO) etc.
• Group II C Hormones:
Hormones that bind to cell membrane receptors and second messenger is calcium or
phosphatid yl inositols or both.
E.g.: Acetyl Choline, Gastrin, TRH, Vasopressi n (ADH), Oxytocin, Cholecysto kinin,
Ang iotensin II, Gonadotro pin releasing Hormone (GnRH) etc.
• Group II D Hormones:
Hormones that bind to cell membrane receptors and act through receptor tyrosine
kinase activity.
E.g.: Insulin,, Growth hormone, Prolactin, Several growth factors like Insulin like growth
factors, epidermal growth factors etc.
Hormones 505
2. Based on Chemica l nature:
Hormones may be classified into 3 classes based on their chemical nature.
a) Proteins / peptide hormones

E.g. Insulin, Glucagon, TRH, LH, ADH, Oxytocin
b) Amino acid derivative hormones:

E.g. Epinephrin e, Norepinep hrine, Thyroid hormones etc.
c) Steroid hormones:
E.g. Cortisol, Aldosteron e, Progestero ne, Testosteron e etc.
Receptors and Signal transduc tion:
Hormone actions begin with the binding of hormones to their specific receptors.
These receptors are situated either within the cell (Group I hormones) or on the
plasma membrane (Group 11 h ormones). These two groups of hormones have d ifferent
modes of action.
Properties of receptors:
• They have high affinity for the hormone.

• The binding of hormone and receptor is reversible and it is saturable.
• Binding is highly specific.
• Receptors are proteins in nature. All receptors have at least two functional
domains. A recognitio n domain, which binds with the hormone and second
domain, which generates the signal that couples hormone binding with some
intracellul ar function.
Thus, receptors have two basic functions:
i) Bind with the hormone
ii) Couple hormone binding with signal transductio n.
In case of group I hormones (which have intracellul ar hormone receptors), the

hormone - receptor complex itself is the intracellula r signal. But, in case of group II
hormones which bind with cell surface receptors, the signal transductio n is through
the second messenger s, such as cAMP, cGMP, Ca 2 etc. These second messenger s act
as transducer s of signa ls from hormones to elucidate various metabolic effects inside
the cell.

Hormones 506
Mechanism of Hormones action:
Hormone actions begin with the binding of hormones to their specific receptors.
These receptors are situated either intracellularly (Group I hormones) or on the cell
surface (Group II hormones). These two groups of hormones have different modes of
action.
Mechanism of Group I hormones action:
• Group I hormones include

- Steroid hormones (like glucocorticoids, mineralocorticoids, estrogen etc), and
- Thyroid hormones (Ty TJ
• Steroid hormones are lipophilic hormones and can easily diffuse through the cell
membrane into the target cells. Thyroid hormones are carried inside the cell by
active transport. Inside the cell, steroid hormones and thyroid hormones have
slightly different mechanisms of action.
i) Steroid hormones bind to specific receptors in cytosol to form hormone receptor

complex (HR complex). The HR complex then undergoes a "activation reaction"
that results in change of size, conform ation and surface. This activa ted H R
complex is then translocated into nucleus. In the nucleus, the HR complex binds
to specific regions on the DNA called hormones responsive element (HRE).
ii) In contrast, thyroid hormones diffuse from the exterior of cell directly into the
nucleus. In this case, receptor is already bound to HRE.
• Binding of hormone receptor complex to HRE activates the expression of specific

genes and increase the transcription rate to produce sp ecific mRNA, which
produces specific proteins and thus influencing the metabolic process of the target
cell.
Hormone responsive element (HRE):

Activated hormone receptors complex (HR complex) binds to specific region on the DNA
called hormone responsive element (HRE).
HRE is situated in the 5' upstream of transcription initiation site and the promoter site
on DNA.
The binding of HR complex to HRE modulates the transcription rate of specific genes to
produce appropriate mRNA. Consequently, the amounts of specific proteins are changed
and the cellular metabolic processes are influenced.

Hormones 507
( Diagrammatic representation of group I hormon e action )
[!]
l
0iolofco.l •<--- Srecifc. m RNA
1'espo'l'LSt pTotein. TY-<l.n.sl(l..tion
Mechan ism of Group II hormon e action:
Group II hormone s are water soluble, so they cannot pass through the cell membran e.
They bind with the specific receptor s present on the cell membra ne. They
commun icate to the inside of the cell through the second messenge rs, whkh exhibit
metabolic response s inside the cell.
Mechani sm of Group II hormone s can be explained in terms of the second messenge rs

produced by them.
Hormones 508
Group II A hormones:
Group II B hormones bind with cell membrane receptors and act through second
messenger cAMP.
E.g. Glucagon, Epinephrin e, Calcitonin, ADH, LH, ACTH, PIH, FSH, TSH etc.
cAMP or cyclic AMP (3', 5' -adenylic acid) is a nucleotide derived from ATP by the
enzyme adenylate cyclase, which is embedded in plasma membrane . This process is
mediated by G proteins.
ATP - - - - - - - - - cAMP
Adenylate cyclase
• When stimulator y group II A hormones bind to the receptor, the adenylate cyclase
enzyme is activated, which results in increased cAMP production from ATP. This
process is mediated by G proteins.
• The increased cAMP activates protein kinase A (PKA), which phosphory lates many
specific target proteins (at their serine or threonine residues), and alters their biological
functions. For example, glycogen phosphorylase is active in its phosphory lated form,
where as glycogen synthase is inactive in its phosphorylated form.
• cAMP is degraded by phosphodi esterase enzyme to 5' AMP. Insulin activates
phosphodi esterase enzymes.
cAMP - - - - - - - - - 5' AMP
Phosphodiesterase
Diagrammatic representation of group II A hormone action
- S ti.,,uldoYJ
t-1ou,1o)le. Y«eft,.., 1--~K:p
Sti"lul,doYj • llu,.,------....
6, Pyotd"
ATP c l,HP+Pf>i

Hormones 509
Group II B hormones:
Group II B hormones bind with cell membrane receptors and act through second
messenger cGMP.
E.g. Atrial natriuretic factors (ANF) and Nitric oxide.
GTP cGMP
Guanylate cyclase
Guanylate cyclase enzyme has 3 domains in the same polypeptide chain -
• Extracellular domain which serve the role of hormone receptor
• Membrane spanning domain (Transmembrane domain)
• Intracellular (cytoplasmic) domain which has guanylate cyclase enzyme activity
When the hormones bind to the receptor site (extracellular domain), the guanylate
cyclase enzyme activity on cytoplasmic domain increases, which results in increased
cGMP production from GTP.
The increased cGMP activates protein kinase G (PKG) which in turn phosphorylates
many specific target proteins (at their serine/ threonine residues) and changes their
biological activities.
For example, ANF is a hormone that is released when GFR is increased. A.NF acts
through cGMP and protein kinase G which phosphorylates smooth muscle proteins
(myosin light chain), and causes relaxation of smooth muscle and vasodilatation.
Diagrammatic representation of group II B hormone action
t-lorf'!\.on.e.
.~ e:~t.,-o..Celll.ll&S.-< -, ec..epto,doma.~I\
1 - - - - - - #/,
&tua.nJ'o..h
Cycl.._$,_
s,st~.M
C,,T
l Adi...~tron
Ih.Q.c.t ive Ac. tive
f'vot.l!in. k:n.a.se Cr
l
f..,oteir1. l<inQ,sd,,
Phosph.crr,lo:tion.
Pvote~r"-.op-vote:rt

Hormones 510
Group II C hormones:
Group II C hormones bind with cell membrane receptors and act through second
messenger calcium or phosphatidyl inositols or both.
E.g.: Acetyl Choline, Gastrin, TRH, Vasopressin (ADH), Oxytocin, Cholecystokinin,
Angiotensin II, Gonadotropin releasing Hormone (GnRH) etc.
Binding of group II C hormones to the specific receptors causes activation of membrane

bound phospholipase C enzyme. This process is mediated through a G protein. The
phospholipase C enzyme cleaves phosphatidyl inositol 4, 5 bisphosphate (PIP2) to
two second messengers 1, 2 Diacylglycerol (DAG) & Inositol 1, 4, 5 triphosphate (IP).
G protein also activates a calcium channel to influx calcium into the cells from outside.
a)DAG :
• DAG is formed from by the hydrolysis of PIP2 by phospholipase C.
• DAG stimulates protein kinase C (PKC) activity, which intum phosphorylates
many specific target proteins (at their serine/ threonine residues) and alter their
physiological functions.
• PKC is a Ca dependant kinase. Activated DAG also increases affinity of PKC for
calcium.
b) IP3:
• IP3 is formed from by the hydrolysis of PIP2 by phospholipase C.
• IP3 diffuses into the cytoplasm and binds to membrane receptors of intracellular
Ca+2 stores (such as endoplasmic reticulum, sarcoplasmic reticulum, mitochondria
etc) and causes the release of Ca+2 • This calcium mediates many biochemical
processes like smooth muscle contraction, glycogen break down, exocytosis etc.
c) Calcium:
Ca+2 also can be termed as second messenger as intracellular calcium concentration
can be rapidly altered by hormones and also due to the presence of intracellular
targets of calcium.
Intracellular (cytosolic) calcium concentration is much lower than extracellular
calcium concentration.
Group II C hormones bind to specific receptors on cell membrane and can increase
the cytosolic calcium in 4 ways,
• By activating calcium channels into the cell, a process mediated by G protein.
• By production of IP3 from PIP2 by phospholipase C activity. IP3 releases calcium
from intracellular stores such as endoplasmic reticulum, mitochondria etc.
• By inhibiting the Ca+2ATPase enzyme (that extrudes Ca+2 from the cell).
• By inhibiting the Ca+2 / Na+ pump (that extrudes Ca+2 from the cell, in exchange
with Na·).

Honnones 511
( Diagrammatic representation of group II C hormone action )
Hormone
(Q. fc,\,. m
G>,o.r,nel
.. 0 -+.2.
oCc-
o o 0
p.,,ot<!:.ins
l
Other pyotei,is
l
Functions of intracellular calcium:
• Main action of calcium is through cal modulin, a calcium binding regulatory

protein. Calmodulin has four Ca•2 binding sites. Ca•2 - calmodulin complex then
can activate many calmodulin dependent protein kinases, which in turn
phosphorylate specific target proteins and alter their biochemical functions.
• Calcium can also directly activate proteins / enzymes like Troponin C, PKC,
guanylate cyclase, adenylate cyclase, myosin light chain kinase etc.
• Intracellular calcium can also mediate many biochemical processes like smooth
muscle contraction, glycogen break down, exocytosis etc.

Hormones 512
Group II D hormones:
Group II D hormones bind with cell membrane receptors and act through receptor
tyrosine kinase activity.
E.g.: Insulin, growth hormone, and growth factors such as insulin like growth factors
(ILF-1, ILF-II), Nerve growth factor (NGF), epidermal growth factor (EGF) etc. act through
receptor tyrosine kinase activity and do not have second messengers.
The receptor tyrosine kinases have 3 domains in the same polypeptide chain -
a. Extracellular domain that has the hormones receptor site
b. Transmembrane domain (transducer)
c. Cytoplasmic domain that has tyrosine kinase activity
The binding of hormones to the receptor site (present in the extracellular domain)
induces conformational changes that are transduced to the cytoplasmic domain. This
promotes the autophosphorylation on specific tyrosine residues on cytosolic domain.
Autophosphorylation switches on its tyrosine kinase activity, which in turn
phosphorylates certain tyrosine residues of target proteins and alters their biological
response. Usually, these target proteins induce growth and differentiation of cells.
Note: Receptor tyrosine kinase activity causes the activation of proteins that induce
growth & differentiation of cells. So, any mutations in the genes resulting in the synthesis
of the receptors with persistent tyrosine kinase activities lead to cancers.
Diagrammatic representation of group II D hormone action
E,11.tY'o. Cetl1,1,l4:<
Rt!ceptov dom4.r t\
Rt..c.,t.rtor ·t.f "e

K11\.A Se

Hormones 513
G proteins:
Different peptide hormones can either stimulate or inhibit the production of
cAMP by adenylate cyclase enzyme. This process is mediated by transducer
proteins called G proteins. G proteins are so named, as they are bound to GTP.
G proteins are either stimulatory (G.) or inhibitory (G), each of these has 3
subunits- a, p, and y. Among these, p and y subunits of c. and G; are same, but
a subunit is different. a-subunit is either stimulatory (a.), which is present in G.
or inhibitory (a), which is present in G;.
Whenever a stimulatory hormone binds to its hormone receptor, CX5 subunit of
G protein dissociates from p and y subunits and binds to adenylate cyclase
enzyme and activates it to produce cAMP. Similarly, inhibitory hormones inhibits
adenylate cyclase enzyme through a ; subunit of G protein.
G protein toxins: Certain toxins act through G proteins to cause diseases. Eg,
Cholera toxin and pertussis toxin causes cholera and pertussis respectively.
• Cholera is caused by continuous activation of G proteins by cholera toxin
(choleragen) by Vibrio cholera bacterium.
• Pertussis (Whooping cough) is caused by inactivation of G proteins by pertussis
toxin secrete by Bordetella pertussis bacterium.
Neurotransmitters:
Definition: Neurotransmitters are substances released by nerve cells and they are
responsible for transmission of nerve impulses through synapses.
Examples: Acetylcholine, norepinephrine, dopamine, histamine, GABA etc.
• The neurotransmitters synthesized in pre-synaptic neurons are stored in vesicles.

When an action potential r eaches the axon terminal, neurotransmitters are
released into the synaptic cleft and then combine with the receptor present in
postsynaptic membrane and execute its action. As soon as the action of these
neurotransmitters is over, they are quickly disposed.
• Neurotransmitters are thus almost similar to hormones in their synthesis and
mechanism of action. Some of the neurotransmitters act as hormones when they
are produced in glands.
For example, the epinephrine and norepinephrine (catecholamines) function as
neurotransmitters when secreted in central nervous system and act as hormones
when secreted from adrenal medulla.
Note:_Refer amino acid metabolism for the synthesis of dopamine and epinephrine.

Hormones 514
Question bank on Hormone s :

1. Classification of hormones
2. Receptors and signal transduction
3. Secondary messengers
4. Neurotrans mitters
5. Mechanism of action of individual hormones

1. cAMP
2. cGMP
3. Calcium as secondary messenger
4. Role of phosphatidy l inositol in signal transduction

1) Phosphoryl ase b is maintained in an inactivated state by (AI)
a) ATP b) cAMP c) Calcium d) Insulin
2) All of the following enzyme are regulated by calcium or calmodulin except (AI)
a) Adenylate cyclase b) Glycogen synthase c) Guanylate cyclase d) Hexokinase
3) What is known as the "second messenger" (AIIMS)
a) ATP b) ADP c) Cyclic AMP d) GMP
4) The fo llowing hormone does not have any intracellular receptor
a) Vitamin 03 b) Cortisone c) Adrenaline d) Thyroxine
5) Adenylate cyclase is system is mediated by (MAHE)

a) cAMP b) Phosphodie sterase c) GTP regulating proteins d) uclear receptors
6) The hormone that binds to intracellular receptor is

a) Epinephrine b) Insulin c) Thyroid stimulating hormone d) Thyroxine
7) cAMP is converted to 5' AMP by

a) Phosphodie sterase b) 5'-nucleotid ase c) Adenosine deaminase d) Ribonucleot ide reductase
8) The hormones that act through tyrosine kinase is

a) FSH b) ANF c) GH d) Somatomed in
9) Which of the following acts to increase the release of Ca " from endoplasmi c reticu lum?
2
a) 1,25 Dihydroxy cholecalciferol b) Inositol triphosphat e c) PTH d) Diacylglycerol
10) The hormone that lowers the cAMP concentratio n in liver cells is
a) Glucagon b) Epinephrine c) Thyroxine d) Insulin
Answers for MCQ: 1) d 2) d 3) c 4) c 5) c 6) c 7) a 8) c 9) b 10) d
Detoxifica tion (Biotrans formation } 515
Detox ificati on (Biotr ansfo rmatio n)
• Conten ts:
• Definition
• Different types of detoxification processes
For queries and suggestio ns, contact the author at prasad_te xt@yahoo .com or 99864495 75
Detoxification (Biotransformation) 516
Detoxifica tion is the process of converting toxic compound s into more water soluble
and more easily excretable form and thus non-toxic or less toxic compound s.
Note: The term 'detoxification' is not always appropriat e because in some cases
converted compound s are more toxic than the original substances. (e.g. conversion
of methanol to more toxic formaldehyde). Hence some prefer to use the term
'Biotransformation'.
The substances need to be detoxified are of 2 origins:
1. Endogenou s origin:
These toxic compound s are produced in the body itself.
Examples:
a) Toxic compound s that are produced in the body by normal metabolism .
E.g.: Ammonia, Bilirubin, Some steroid and phenolic compound s etc.
b) Compound s produced in the intestine by bacterial putrefactio n like indole and
skatole (from tryptophan ), histamine (from histidine), tyramine (from tyrosine) etc.
2. Exogenous origin:
These toxic compound s are substances administer ed into the body from outside.
(Detoxification of these foreign compound s is called metabolism of Xenobiotics).
Examples:
a) Drugs which are used for therapeutic purpose has to be eliminated after their metabolic
use. (Detoxification of drugs is called drug metabolism).
b) Industrial pollutant like CCl4 and compound s that are accidentally injected.
c) Food adulterants , insecticides.
Detoxification process in the body is performed by 4 processes:

I. Detoxification by oxidation
II. Detoxification by reduction
Ill. Detoxification by hydrolysis
IV. Detoxification by conjugation
Note: Sometimes compound s detoxified by oxidation, reduction or hydrolysis
may not be in readily excretable form. They need to be further detoxified by
conjugatio n reactions. So detoxification reactions by oxidation, reduction and
hydrolysis are referred to as phase I detoxification reactions and detoxification
by conjugatio n is referred to as phase II detoxifica tion reactions. Sometimes
toxic compound s need only phase I or phase II reactions.
Liver is the major site of detoxificati on. Kidney and intestine are involved to a smaller extent.
For queries and suggestions , contact the author at prasad_tex t@yahoo.co m or 9986449575
I. Detoxification by oxidation:
Here detoxification process involves oxidation process.
a) Detoxification by microsomal mono oxygenase enzymes systems:

These enzymes are also called mixed function oxidases. These enzymes require
molecular oxygen NADPH and cytochrome P450 for their activity.
Microsomal monooxygenase
i) Toluene .. Benzyl alcohol
__:::::----;yt P4so,~
NADP+ NADPH+H+
Microsomal monooxygenase
ii) Phenobarbital p-OH phenol barbital
P4so, .,
NADP+ NADPI I+H-
Similarly steroids, morphine, acetanilide etc. are detoxified by mixed function oxidases.
Cytochrome P450 is a family of cytochromes that absorbs light maximally at 450 nm.
These are conjugated proteins containing heme as the prosthetic group. Cyt P450 plays
an important role in detoxification of foreign substances. See previous section.
b) Detoxification of alcohols and aldehydes:
Alcoho1 Aldehyde
dehydrogenase dehydrogenase
Ethyl alcohol • Acetaldehyde /4 Acetic acid
/2.,~ Fe, Mo
NAO+ NAOH+H+ NAO+ NAOH+H+
Similarly, methanol to formaldehyde, formaldehyde to formic acid.
c) Detoxification by amine oxidases:

Amines are oxidatively deaminated by amine oxidases like MAO (monoarnino oxidase).
Amine oxidase [MAO]
i) Serotonin 5-hydroxy indole acetic acid+ Nfu
Amine oxidase [MAO]

ii) Histamine Imidazole acetic acid + Nfu
Amine oxidase [MAO]
iii) Adrenalin Vanillyl mandelic acid (VMA)

_j
II. Detoxification by reduction:

Here detoxification process involves reduction process. It is not common.
Usually nitro compounds are detoxified by this process.
i) N itrobenzene .. Amino benzene
NAOH + H • NAO+
ii) Picric acid Picramic acid

7 ..
NAOH + H + NAO +
Similarly prontosil, chloral etc. are detoxified by reduction process.
III. Detoxification by hydrolysis:

Here the detoxification process involves hydrolysis process.
Hydrolysis is brought about by esterases and amidases.
a) By esterases:
Esterase
i) Aspirin (acetyl salicylic acid) Acetic acid + Salicylic acid
Esterase
ii) Atropine Tropic acid + tropine
Similarly procaine, digitalis etc. are detoxified by esterases.
b) By amidases:
Amidase
i) Acetanilide aniline + acetic acid
Amidase
ii) Procainamide - - - - - - - - - - - PABA+ Diethyl amino ethyl amine
Similarly iproniazid, phenacitin etc. are detoxified by arnidases.
For queries and suggestions, contact the author at prasad_te:xt@yahoo.com or 9986449575

IV. Detoxification by conjugation process:

This is the common & most important type of detoxification process. 1n this process,
the compounds to be detoxified combine with conjugating agents and get converted to
a more water soluble & easily excretable form. There are a variety of conjugating agents
like glucuronic acid, glycine, glutamine, sulphate, methyl & acetyl groups etc. sometimes,
these conjugating agents should be in active form to bring about conjugation.
Example: Active form of glucuronic acid is UDP-glucouronic acid, active form of sulphate
is PAPS and active form of sulphate is SAM.
a) Conjugation with glucouronic acid:
Active form of glucouronic acid is UDP- glucouronic acid
i) Bilirubin + 2 UDP-glucuronate UDP glucu.ronyl han sferase Bil.irubin diglucuronide + 2 UDP
ii) Phenol+ UDP-glucuronate UDP glucuronyl transferase Phenyl glucuronide + UDP

•
Similarly, morphine, paracetamol, steroids are detoxified by conjugation with glucouronate.
b) Conjugation with Sulphate:

Active form of sulphate is PAPS (Phospho Adenosyl Phospho Sulphate).
i) Pherol + PAPS Sulpho transferase Prenyl sulp hate+ PAP
•
ii) Indoxyl + PAPS Sulp ho transferase .. Irdoxyl sulphate+ PAP
Similarly Skatoxyl, cresol, paracetamol are detoxified by conjugation with sulfate.
c) Conjugation with Glycine:

Be n zoic acid Benzoyl CoA +Glycine - - - • Benzoyl glycine+ CoASH
(Hippuric acid)
Similarly salicylic acid, nicotinic acid are detoxified by conjugation with glycine.
d) Conjugation with Glutamine:

Phenyl acetic acid+ glutamine - - - - - • Phenyl acetyl glutamine
Similarly, indole acetic acid is detoxified by conjugation with glutamine.
e) Conjugation with acetyl CoA:

Sulfanilamide + acetyl CoA - - - - - -Acetyl sulfanilamide + CoASH
Similarly, lsoniazide (INH) is detoxified by conjugation with acetyl CoA.
0 Conjugation with SAM:

Epinephrine + SAM Catechol-0-methy l transferase .,, Metanephrine + SAH
Similarly, norepinephrine, nicotinic acid are detoxified by conjugation with acetyl CoA.

Question bank on detoxification :

1) Detoxification / Biotransformation
3) Metabolism of xenobiotics
4) Drug metabolism
5) Detoxification by conjugation process
6) Role of liver in detoxification

1) H ow aspirin and benzoic acid detoxified?
2) am e four agents causing detoxification by conjugation
3) Microsomal monooxygenase system
4) Detoxification of alcoh ols
5) Name the organs where detoxification takes place in the body.

1) Which of the following is found in conjugation with bile acids (DELH I)
a) Cholic acid b) Pregnen olone c) Glycine d) Choly lacetyl CoA
2) Cytochrome that is required by microsomal monooxygenase is
a) cytoch rome a b) Cytochrome P450 c) Cytochrome a d) cytochrome a3
3) Detoxification of indoxyl requires
a) PAPS b) SAM c) MOMS d) MAO
4) Example of exogenous toxin is

a) Ammonia b) Bilirubin c) Indoxyl d) Carbon tetrachloride
5) Phase II detoxification prose involves
a) Oxidation b) Reduction c) Hydrolysis d) Conjugation
6) Major organ involved in detoxification is

A) Liver B) intestine C) kidney D) Pancreas
7) Important enzyme necessary for Xenobiotics

a) Cytochrome Pi50 b) Hexokinase c) LDH d)CPK
8) Coenzyme required for cytoch rome P4so is

a) ADPH b) FMN c) NAD d) FAD 4) Example of exogenous toxin is
9) Which one of the !ollowing compound is detoxified by hydrolysis reaction?

a) Aspirin b) Bilirubin c) Methanol d) benzoic acid
10) Monooxygenase are found in

a) Cytoplasm b) Microsomes c) Mitochondria d) Cytosol
Answers for MCQ: 1) c 2) b 3) a 4) d 5) d 6) a 7) a 8) a 9) a 10) b

Free Radicals, Reactive Oxygen Species and Antioxidants 521
Free Radicals, Reactive Oxygen Species and Antioxidants
Contents:
• Overview
• Free Radicals
• Oganiza tion
• Individual Complexes and Mobile carriers
• Inhibitors
• Anti-oxidants
• Mechanism
• Chemiosmotic hypothesis
• Inhibitors
• Uncouplers

Free radicals and anti-oxidants
Free radicals:
Free radicals are defined as an atom or molecule that contains one or more unpaired
electrons in the outer most orbital.
A free radical is represented by a superscript dot [X ·]
Examples:
• Super oxide anion [ 0 2· 1
• Hydroxy free radicals [ OH ·]
• Hydroperoxy free radicals [ HOO ·1
• Lipid peroxy free radicals [ ROO ·1
• Alkoxy free radicals
Reactive oxygen species or ROS:

Tlzese are oxygen derived reactive molecules produced from oxygen. ft includes both free radicals
and non-free radicals. Examples are,
Free radical ROS: Super oxide anion, Hydroxy free radicals, Hydroperoxy free radicals etc.
Non-free radical ROS: Hydrogen peroxide and Singlet oxygen.
H202, by definition is not a free radical (as it does not contain unpaired electron in the outer
orbital). However H202 is discussed along with free radicals because it is also reactive and
potential oxidants like free radicals and is an intermediate in free radical metabolism.
Effects of free radicals:

Free radicals are highly reactive and toxic. They can attack almost all biological macro
molecules.
1) Peroxidative damage of PUFA in membrane lipids:

Free radicals in the presence of oxygen can attack PUFA of cell membrane and cause
peroxidation of lipids of membrane and blood. The peroxidation of biological membranes
leads to loss of membrane functions like absorption and secretion. lntegrity of cell
membrane is lost. In erythrocytes, it lead to hemolysis.
Lipid peroxidation is a chain reaction process, producing a continuous supply of

free radicals like lipid radicals, lipid alkoxy radical, lipid peroxy radicals, which are
themselves very reactive and initiates further peroxidation (this autocatalytic reaction
is called propagation), which lead to extensive cellular damage.

2) DNA Damage:
Free radicals may attack purine and pyrimidine bases of DNA and cause lesions in
D A. This can cause inhibition of protein synthesis and may cause mutations which
are carcinogenic or lead to cell death.
3) Protein damage:
Free radicals may attack protein and damage them, mainly sulfhydryl group containing
proteins are oxidatively inactivated by free radicals.
4) Carbohydrate damage:
Free radicals may also cause polysaccharide depolymerisation.
Free radicals and diseases:

Free radicals have been implicated in the causation and progress of many diseases
like cardiovascular diseases, cancer, inflammatory diseases, respiratory diseases,
diabetes, aging, Parkinsonism, Alzheimer's disease etc.
Antioxidants (scavenging free radicals and other oxidants):

Free radicals are continuously produced in the body, which cause extensive damages
to the body tissues if not destroyed. Body has multiple mechanisms to scavenge these
free radicals. The compounds or enzymes which remove these free radicals and other
oxidants are termed as antioxidants. There are 2 types of antioxidants,
a) Preventive anti oxidants:

These prevent the initial production of free radicals.
E.g.: Catalases and Peroxidases like Glutathione peroxidase.
b) Chain breaking anti oxidants:

These interfere with chain propagation by destroying the free radicals.
E.g.: Super oxide dismutase enzyme, Glutathione peroxidase, Vitamin E, Vitamin C,
Urate, ~-Carotenes, and Caffeine etc.
Note:
• All the above mentioned antioxidants are natural anti oxidants.Glutathione peroxidase
falls into both the categories.
• Propylgallate, Butyrated h ydroxy anisole (BHA) Butyrated hydroxy toluenes (BHT)
are artificial anti oxidants, w hich are used as additives of food.
• Some authors consider SOD, Glutathione peroxidase & Catalases as cytoprotective
enzymes against free radicals and all others like vitamin E, Vitamin C, ~-Carotene,
Urate etc as anti oxidants. (Vitamin Eis the most powerful natural anti oxidant).

Generation of free radicals:
Leak in the electron transport chain (ETC) in mitochondria.

ln the electron transport chain, electrons are added to oxygen, which is finally reduced
to water. This is generally complete.
However partial reduction of oxygen occur in certain reactions due to leaks in
mitochondria, where sequential univalent reduction of oxygen takes place by one, two,
three and four electrons give 0 2·, H 20 2, OH, H 20 respectively.
Mitochondrial cytochrome oxidase systems
4e· +4H• / l
e-
• Oi
2H•
rH202ft ott·r
e- e- e-
2H20
This accounts for 1 - 5 % of total oxygen consumption of the body per day.
Respiratory Burst: During phagocytosis of bacteria by macrophages, there is a rapid

consumption of large amount of oxygen, which accompanies with the generation of super
oxide anion (which has bactericidal activity). This process is called 'Respiratory burst'.
Super oxide anion :

Super oxide anion is a very harmful free radical.
Generation of super oxide anion:

Super oxide anions can be produced in many ways.
1) Leakage in the ETC in mitochondria:

Refer the top section of this page.
2) Spontaneous non-enzymatic reactions of oxygen with ferrous iron (also copper).

0 2 + Fe2+
3) Aerobic enzymes like xanthine oxidase also can produce 0 2

• as a side product.
Enz - flavin - H2+ 0 2 Enz - flavin - H + 0 2· + H+

4) Super oxide are also formed as a side product in cytochrome P450 dependent reactions
(during metabolism of xenobiotics).
Cyt P450 Cyt P450 Cyt P450 CytP450

I I I I
Fe 2
5) NADPH oxidase enzyme can also produce super oxide anion.
2 O2+NADPH
6) Super oxide is also formed when FMNH 2 or FADH2 (the reduced forms of FMN or
FAD) of flavoproteins (FP) are reoxidised univalently by molecular oxygen.
Super oxide anions are very harmful free radicals. They can also produce hydroperoxy
(HOO") free radicals, which accentuate the harmful effects. .
Degradation of super oxide anions:
Super oxide anions are degraded by super oxide dismutase enzyme.
Super oxide dismutase (SOD):
SOD
Dismutation is a reaction in which 2 identical m olecules are converted to different

compounds.
SOD is present in both cytosol and mitochondria. The cytosolic SOD isoenzyme is Cu+2
and z n+2 dependent, whereas mitochondrial SOD isoenzyme is Mn+2 dependent. SOD
is a chain breaking anti oxidant, which protects the aerobic organisms against the potential
harmful effects of free radical super oxide anion.

H 2O 2 is itself is not a free radical. The harmful effect of H 2O 2 is due to its conversion to
one of the most reactive free radical, hydroxy free radical (OH), which have harmful
effects.
Generation of hydrogen peroxide:
H 20 2 is formed in several reactions.

1) In the decomposition of super oxide anion by super oxide dismutase.
2) In the action of L-amino acid oxidase, D-amino oxidase & Xanthine oxidase etc.
3) Leakage in the ETC in mitochondria
Note:
H 20 2 is itself is not a free radical. The harmful effect of 8i02 is due to its conversion to
h ydroxy free radical (OH '), one of the most potent reactive free radical.
Formation of hydroxy free radical (OH') can be by 3 reactions,
i) Fenton reaction:
Fe+3 + OH + OH ·
ii) Haber Weiss reaction:
O2+OH+ OH '
iii) Homolytic fi ssion of H 2 O 2 by radiation:
- - - - - - - • 2 OH .
Degradation of hydrogen peroxide

H 2O 2 are potentially harmfu l and need to be decomposed immediately by
H ydroperoxidases.
Hydroperoxidases are enzymes involved in decomposition of 8i0 2•

There are two types of hydroperoxidases - Cata lase and Peroxidase.

1) Catalases:
Catalases present in peroxisomes. They are mainly found in liver, blood etc. its function
is to destroy H 2O2 •
catalase
Catalases use one molecule of H 2O 2 as electron donor and another H 2O 2 as electron

acceptor.
2) Peroxides:
Peroxides reduce hydrogen peroxide using various electron acceptors.
Glutathione peroxidase: Selenium containing glutathione peroxidase is the most

important peroxidase, which catalyze the destruction of H 2O 2 to water.
During the decomposition of 1\02 to water, the reduced glutathione (GSH) of glutathione
peroxidase enzyme is converted to oxidized glutathione (GS-SG). This oxidized
glutathione is converted back to reduced glutathione by NADPH dependent glutathione
reductase enzyme.
Glutathione peroxidase
2GSH GS-SG
Glutathione reductase
NADP- NADPH+H+ HMP shunt pathway
Glutathione peroxidase enzyme can also destroy OH · free radicals.
2 OH · + 2GSH
Thus, glutathione peroxidase enzyme fun.ctions as both preventive anti oxidant and
chain breaking anti oxidant.

Free Radicals, Reactive Oxygen Species and Antioxidants
1) Free radicals and its effects

2) Antioxidants
1) Glutathione peroxidase
2) Lipid peroxidation
1) The subcellular organelle rich in catalase is

a) Lysosomes b) mitochondria c) Peroxisome d) Ribosome
2) Electron donor & electron acceptor used by catalase is

a) 0 2 b) Hp2 c) CO2 d) Hp
3) Following are antioxidant enzymes except

a) GSH peroxidase b) Catalase
c) Superoxide dismutase d) Lactate dehydrogenase
4) Free radicals are

a) Chemical species with unpaired electrons b) +vely charged ions
c) Ions with both +ve and - ve charges d) derived from hydrogen
5) Enzyme that serves both as chain breaking and preventive antioxidant

a) Glutathione reductase b) Glutathione peroxidase c) Catalase d) SOD
Ans wers for MCQ: 1) c 2) b 3) d 4) a 5) b

Connective 'T issues and Muscle 529
Biochemistry of Connective tissues
Contents
• Introduction
• Collagen
• Elastin
• Glycoproteins and Proteoglycans
• Bone composition and defects

Connectiv e Tissues and Muscle 530
Conne ctive tissues :

Cells are basic units of life. Cells are surround ed by a complex extracellular matrix
often referred to as extracellular matrix (ECM), often referred to as connectiv e tissues.
The connective tissues have many functions, mainly supportin g the cells they surround.
The major compon ents of ECM:

The ECM contains three major classes of biomolecules,
1) Structura l proteins like collagen, elastin, and fibrillin
2) Certain specialized proteins (like fibronectin, laminin)
3) Various proteoglycans.
Collag en
• Collagen is the most abundant protein in the animal kingdom . It constitut es about 25
% of total protein in the body.
• Collagen is formed by specialized tissues like fibroblasts in connective tissues and
osteoblas ts in bones.
Compos ition:
• Mature collagen has approxim ately 1000 amino acids.
• About 33 % of collagen is made up of glycine, that is, every third amino acid js glycine.
Proline and hydroxy praline form 21 %.
• Other major amino acids present in collagen are lysine, hydroxy lysine, arginine and
alanine.
The primary structure motif of mature collagen is (Gly- X - Pro/Hy p) n
Structure:
• Structura l unit of collagen is called tropocollagen. Each tropocollagen is made up of
three polypept ide chains.
• Each of three polypept ide chains wound around each other in a right handed helical
manner. They are held together by extensive hydrogen bondings and covalent cross
linkages (both inter- and intra chain).
• These Covalent cross-links are formed through the action of enzyme lysyl oxidase (a
copper containin g enzyme). It reacts with lysine and hydroxylysine residues producin g
reactive aldehyde s. These aldehyde s can form aldol condensation products w ith other
lysine of hydroxylysine produced aldehyde s or Schiff bases with unoxidized lysine and
hydroxy lysine residues.

Connect ive T issues and Muscle 531
Formation of collagen:
Each of three polype ptide chain of tropoco llagen is synthes ized as immatu re
Preprocollagen. Preproc ollagen is convert ed to procolla gen by hydroxy lation of certain
proline and lysine molecules. This is catalyze d by prolyl hydrox ylase and lysyl
hydroxy lase enzyme s. This step requires vitamin C.
Procollagen undergo es glycosylation at hydroxylysine residues, followed by assembling
of 3 polypep tide chain together to form triple helix. This procollagen is then secreted
from fibroblasts.
Then proteina ses (procoll agen aminop roteinas e & procolla gen carboxy proteina se)
remove peptide s from both N termina l and C-term inal of procoll agen to form
tropocollagen. Finally tropocollagen forms collagen by covalen t cross linking. The 3
chains are further twisted in a righ t handed manner to give a stable collagen structur e.
Functions of collage n:
1) Support: Collagen gives support to the organs.
2) Wound healing: Collage n is involved in wound healing.
3) Anchorage: Collage n provide s anchora ge to cells, which help in the prolifer
ation
and differentiation of cells.
4) Thromb us formati on: In blood vessels, when collagen is exposed , the
platelet s
adhere and thrombu s formatio n is initiated.
Abnormalities of collage n structure and function:

A number of diseases resuJ t from the abnorm alities in the synthes is of collagen.
1) Ehlers-Danlos syndrome:
Result due to defectiv e collagen formation.
Principa l features are hyperex tensibil ity of skin (loose skin), abnorm al tissue fragility
and increased joint motility.
2) Osteoge nesis imperfecta:
This is caused due to a mutatio n, which results in the replacem ent of a single glycine
residue by a cysteine residue.
This disrupts the triple helix, resultin g in extensiv e glycosylation and hydroxy lation
that causes the unfoldin g of helix and formation of imperfect collagen.
It is characte rized by brittle bones, multiple fractures and skeletal deformities.
3) Scurvy:
It is caused by the deficiency of vitamin C. (Vitamin C is required for the formation of
mature collagen). Sympto ms of scurvy are mainly due to defective collagen synthesis.
Refer vitamin C for details.
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Connective Tissues and Muscle 532
Elastin
• Elastin is a connective tissue protein present in elastic fibers. Elastin mainly present
in elastic fibers of lungs, large arterial vessels like aorta. .
• Major amino acids present are glycine (about one third), proline and alanine.
Hydroxy proline is present, but hydroxy lysine is absent. Elastin does not form triple
helix. It does not contain carbohydrates.
• Major cross linking formed in elastin is desmosin es, which are formed from the
condensa tion of three lysine derived aldehyde s with an unmodifi ed lysine.
• Elastin is responsible for extensibility and elasticity in tissues. Jt confers extensibility
and recoil on lung, blood vessels and ligaments.
Other minor connective tissue proteins :

Fibrillin: Fibrillin is a glycoprotein present in connective tissues of microfibrils.
Defect in the gene producin g fibrillin causes Marfan syndrom e.
Laminin: Laminin is an importan t compone nt of basal laminae.
Fibronectin: Fibronectin is a glycopro tein involved in cell adhesion and migration.
Glycoprotein (mucoproteins), proteoglycans:

Both glycopro teins and proteoglycans contain variable amounts of carbohyd rates and
proteins, which are covalently attached. The difference is relative amounts & type of of
carbohyd rates and proteins.
a) Glycoproteins (Mucoproteins):
Definitio n: Glycopro teins contain oligosacc harides (branche d or unbranch ed) as
carbohyd rates and the carbohyd rate content is less than 10% of the molecule.
E.g.: Almost all p lasma proteins (except albumin) are glycoproteins. Collagen, elastin
are also glycoproteins. Glycoproteins are also present in cell membran es, for example,
glycophorin of erythrocyte membran e
b) Proteogl ycans:
Definitio n: Proteogl ycans contain mucopol ysacchar ides (Glycosa minoglyc ans) as
carbohyd rates and the carbohyd rates content is more than 10% of the molecule.
E.g.: Syndecan, aggrecan, betaglyca n, decorin etc.
Proteoglycans are mainly present in connective tissues.
Note:
If we remove protein content from a proteoglycans, we get mucopol ysacchar ides and if
we remove protein content from glycoproteins, we get oligosaccharides.
Types of glycoproteins:
GJycoproteins are 3 types, based on nature of the linkage between the protein &
oligosaccharides.
a) O-glycosidic linked: Hydroxyl side chain of serine/threonine and oligosaccharides.

E.g.: ABO blood group antigens and m ucins are 0-glycosidic linked .
b) N-glycosidic linked : Amide nitrogen of asparagine and oligosaccharides.

E.g.: Most glycoproteins are N-glycosidic linked.
c) GPI (Glycosylphosphatidylinositol)-linked: Carboxyl terminal amino acids and

oligosaccharides.
E.g.: Cell membrane glycoproteins and are CPI-linked.
Structure of proteoglycan
• Pro teoglcyan molecules con sists of a core protein linked up to 100
glycosaminoglycan residues (except hyaluronic acids) covalently. This proteoglycan
structure resembles a 'bottle brush'.
• Many such proteoglycan monomers non-covalently associate with a long strand of
hyaluronic acid through link protein.
Keratan sulfate
.----- Chondroitin sulfate

Link protein
Core
protein
Hyaluronic acid
Diagramatic representation of a proteoglycan

Muscle:
Muscle tissue is otherwi se called the contractile tissue. There are 3 types of muscles;
skeletal muscle, cardiac muscle & smooth muscle. Only skeletal muscles are urtder the
control of will (Voluntary). Both skeletal & cardiac muscles are striated.
Protein s play an importa nt role in muscle contrac tion. Major contrac tile proteins
(Contrac tile elements) present in muscle are actin, myosin, troponin, tropomyosin. Role
of these contractile proteins are better studied with the structur e of skeletal muscle.
Individ ual contractile protein s:

1) Myosin:
filament.
Myosin constitut es about 55 % of the muscle protein by weight and it forms the thick
of myosin have been identifie d. Myosin II is the most common
There are at least 15 variety
variety.
of
Myosin is a hexamer (6 chains, 2 heavy chains and 4 light chains) with a molecula r weight
around 460 kDa.
head of
Myosin molecule s in thick fi laments are arranged in a linear order so that the globular
.
each myosin molecule projects out from the surface of the thick filament at regular intervals
site for
These heads are called actin binding sites. They have ATPase activity and binding
actin.
2) Actin:
s
Actin forms the thin filament. A thin filament is made up of two long chains of actin molecule
weight.
twisted around tropomy osin filament. It forms about 25 % of total muscle proteins by
r
At low ionic concentr ations and absence of Mg2•, it exists as water soluble globular monome
G-actin
called G-actin. ln the presence of Mg • and isotonic or hyperton ic concentr ations,
2
polymer izes to form insoluble , double helical fibrous fi lament called F-actin.
Each actin molecule has myosin binding site.
3) Tropomyosin:
osin
Thin filament s are made up of two long chains of actin molecules twisted around tropomy
arou nd 66
filament. Tropomy osins are rod like fibrous molecule s with a molecula r weight of
kDa. It is dimer containin g a and b chains.
strands
The double stranded tropomy osin molecules lie in the long groove between F-actin
intervals .
and remain bound to T subunit of troponin. Tropomy osin contains troponin at regular
4) Tropon in :
around
Tropomy osin contains troponin at regular intervals. Troponin has a molecula r weight of
Troponin T, Tropon.in I and Troponin C
80 kDa. Each troponin molecule i!, tripartite having
regions.
Troponin T (TpT): Binds Troponin I and Troponin C to actin filament.
binding
Troponi n I (Tpl): Inhibits interacti on between myosin and actin filaments and it has
site for both F-actin and TpC.
ions that
Troponi n C (TpC): It is the calcium binding proteins, having binding site for Ca •
2
initiates contracti on.

ely.
Troponin systems remain attached to F-actin and tropomy osin by Tpl and TpT respectiv
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Energy for muscle contraction:

ATP is the direct and constant source of energy for muscle contraction. In m uscle cells, ATP
is generated by following ways;
• From oxidative phosphorylation (mainly from oxidation of fa t and carbohyd rates).
• From ubstrate level phosphorylation of glycolysis
• From creatine pho phate (CP)
During muscle contraction, energy is first deri ved from ATP hydrolysis. The amount of ATP
present in muscle (about 4 mM) is sufficient to su tain the muscle contraction for about 1 to
4 seconds. So, ATP should be immediately regenerated du ring muscle contraction. Creatine
phosp hate serves as an immediate store of energy in muscle cells. ATP is regenerated by
hydrolysis of Creatine phosphate (Lohm ann's reaction).
Creatine kinase
Creatine phosphate 7" Creatine
ADP ATP
• Creatine ki nase (CK), particularly isoenzyme CK-MB, eleva ted in myocardial infarction.
Concentration of Creatine phosphate (CP) in resting muscle cells is about 25 mM, w/1icl1 will be
depleted approximately in 4 to 6 seconds. Thus, ATP level is maintained by combination of ATP+CP
for about 5 to 10 seconds. Once the CP stores are depleted, body resorts to stored glucose/glycogen
and fat for energy (by both aerobic and a11aerobic).
While all three energy systems (ATP + CP, anaerobic glycolysis, and oxidative phosphorylation)
are active all the time, which system provides the majority of energy will be dctermi11ed ln; a time
based and intensity based process.
• ATP + CP is primarily used for up to 10 seconds, glycolysis for up to a minute, and oxidative
system is the primary system for all long-term exercise.
• Generally, ATP + CP used in high intensity exercise, a11aerobic glycolysis used in moderate
intensity exercise and oxidative system used in low intensity exercise.
Creatine:
• Synthesis: Refer glycine under amino acid metabolism ch apter.
• Fate: Crea tine converted spontaneo usly to creatinine, a waste product.
• Clinical significance: ormal serum crea tinine level is 0.7-1.5 mg/ dl & urine crea tinine
level is 1-2 g/ d ay. These levels are d ependent on muscle m ass & more or less constant.
Serum creatinine level is an indicator of renal function & it increases in renal damage.
Both serum & urine creatinine level increases in m uscular dystrophics.
Muscle dystroplties: Muscle dystrophies are caused by mutation in gene coding dystrophi11 (a
specialized muscle protein). Level of creatine kinase (CK) in serum is elevated. They cause
progressive muscle weakness, 11111scle wasting, cramps & deterioration. There are different types
of muscular dystrophies that have been identified. Duchetme Muscular Dystrophy is tlze most
common & best characterized of all these. ThetJ primarily affect the skeletal muscles.
Creatine deficiency ca uses muscle cramps: In contracting muscle, creati11e phosplzate formed
from creatine, serves as an immediate source of energy. 111 creatine deficiency, energy is not properly
supplied to the contracting muscle, resulting in muscle cramps.
Question bank on Connect ive tissues:

1. Give a detailed account of collagen (compositio n, structure and functions). Add a note on
abnormaliti es in collagen structure and functions
2. Explain connective tissue proteinsl. Structure and functions of collagen
2. Compare and contrast glycoprotein s and proteoglyca ns
3. Explain contractile proteins

1. Elastin
2. Lohmann's reaction
3. Glycoprotei ns
4. Proteoglyca ns
5. Actin I Myosin / Tropomyos in / Troponin

1) In collagen synthesis, hydroxy praline is formed from (MAHE)
a) Praline b) Lysine c) Hydroxylys ine d) None of the above
2) Keratin of skin and nail differ because of (ABMS)

a) Disulphide bond b) Covalent bond c) Vander Waal bond d ) Hydrogen bond
3) Triple helix is found in (AI)

a) Cystine b) Collagen c) Pectin d)DNA
4) Hair is rich in amino acid- (AIIMS)

a) Cystine b) Lysine c) Glycine d) Leucine
5) The immediate source of energy for muscle contraction is

a) ATP b) Creatine phosphate c) GTP d) Glucose
6) Integral membrane glycoprotei n of human RBC

a) Porin b) Ankyrin c) Spectrin d) Glycophori n
7) Marfan's syndrome results from a mutation in the gene coding

a) CoUagen b Elastin c) Fibrillin d) keratin
8) Vitamin required for maturation of collagen is

A) Ascorbic acid B) Folic acid C) vitamin A D) vitamin K
9) Creatinine synthesis involves all the following EXCEPT

a) Cysteine b) Citrulline c) Arginine d) Glycine
10) Muscle dystrophies are ca used by mutation in gene coding

a) Dystrophin b) creatine kinase c) Collagen d) Elastin
Answers for MCQ: 1) a 2) a 3) b 4) a S) a 6) d 7) c 8) a 9) b 10) a
Plasma Proteins 537
Plasma Proteins
Content s:
• Types
• Functions
• Albumin
• Globulins
• Structure and functio ns of irnmunogl obulins
Plasma Proteins 538
Plasma proteins
Plasma contains hundreds of different proteins known as plasma proteins.
ormal total protein concentrat ion in plasma = 6 - 8 g / dl.
AJI plasma proteins, except immunoglo bulins, are synthesize d in the liver.
Most plasma proteins (except albumin) are glycoproteins.
Fractions:
Albumin, globulins and fibrinogen are three important classes of plasma proteins.
Albumin = 3.5 - 5 g/ dl
Globulins = 2.5 - 3.5 g/ dl
Fibrinogen = 200-400 mg/ dl
Albumin:
Albumin is the major plasma protein (Constitute up to 60% of plasma proteins).
Human albumin has 585 amino acids. It is synthesize d in liver. It is a simple protein.
Globulins :
Globulins constitute many different proteins, which are separated into 4 distinct
bands in electropho resis. Each band contains various globulin proteins.
i) a. 1 -globulins: a 1 -antitrypsi n, Retinol binding protein (RBP), Thyroxine binding

globulin (TBG), HDL etc.
ii) cx2 -globulins: a 2-macroglob ulin, Haptoglob in, Ceruloplas min, Prothromb in etc
iii) p - globulins: LDL, Transferrin, Hemopexi n, plasminog en etc.

iv) y - globulins: lmmunoglo bulins.
Fibrinogen:
Fibrinogen s are proteins that are responsible for coagulation.
Serum proteins are separated by electrophoresis:

Electrophoresis is the most common technique used for the separation of serum proteins.
In agar gel (or paper) electropho resis, the serum proteins are separated into 5 distinct
bands. These are,
• Albumin,
• a I-Globulin
• a 2-Globulin
• ~-Globulin
• y-Globulin.

Plasm a Protei ns 539
Functions of plasm a prote ins:

1) Main tenance of colloi dal osmotic pressure:
Plasm a protei ns, especially album in is responsible for the colloidal
osmotic pressu re
of plasm a, which is respon sible for mainte nance of plasm a blood
volum e and body
fluid distribution.
2) Transport functions:
Several plasm a protei ns serve as carrier s of variou s compo unds in
the blood.
Transport proteins Substance transp orted
Album in Bilirubin, free fatty acids
T ransferrin Iron
Thyroxine bindin g pre-al bumin T3 and T4
Retinal bindin g protei n Retinol
Thyroxine bindin g globulin T3 and 1'4
Hapto globin Hemo globin
Hemopexin Heme
Lipoproteins Triacylglycerol, Phosp holipi ds, Cholesterol
3) Buffering action :
Plasm a protei ns (mainly album in) act as buffer s in regula tion of
acid base balance.
They are the second most impor tant buffer after bicarb onate buffer
in the blood.
4) Protei n reserve (Nutritive role):
Plasm a protei ns, mainl y album in, serve as source of amino acids
especially during
s tarvation. Cells uptake album in by pinocytosis & degra de it to supply
amino acids.
5) Blood clotting:
Fibrinogen participates in blood clotting.
6) Defen ce role:
Immu noglo bulins have role in humo ral immu nity.
Clinical Signi ficanc e:

• Decrease total plasm a protei n concentration is an indica tor of liver
diseas e (due
defect in synthe sis of album in and other plasm a protei ns) or nephrotic
syndrome (due
to increa se in pore size leadin g to abn ormal excret ion of album
in in urine) .
• Album in (and total protei n) level is also decrea sed in nutriti onal
deficiency like
kwash iorkor , burns and in diseas es like tropical sprue.
• Edem a is the main consequence of hypop rotein emfa or hypoa lbumi
nemia .
(Plasma proteins, especially albumin maintains the colloidal osmotic pressur
e (COP) , which is
responsible for fluid distribution between the body compartments (ECF &
!CF). Decreased
albumin level causes fluid redistribution, water enters the tissues and causes
edema).
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Plasma Proteins 540
Albu min:
s).
• Album in is the major p lasma protein (Const itute up to 60% of plasma protein
• Human albumi n has 585 amino acids. It is synthesized in liver.
Functions of albumin:
1. Maintenance of colloid al osmotic pressure:

of blood.
Principal function of albumi n is the mainte nance of colloidal osmotic pressu re
n accoun ts for about 80%
(Colloidal osmotic pressu re of blood is 25mm Hg and albumi
of this.)
ed in
Note that colloidal osmoti c pressu re and blood pressu re are the forces involv
mainte nance of plasma blood volum e and microcirculati on in tissues .
2. Transport:
free fatty
Album in is involve d in the transpo rt of hydrop hobic substan ces (bilirubin,
acids), metal ions (Ca cu~+, 2n~+) and hormo
0
, ne, thyrox ine.
3. Buffering action :
as buffer.
The histidy l residue s in albumi n can accept or donate proton s and hence act
the second most import ant buffer after
Plasma protein s, includ ing, album in are
bicarbo nate buffer in the blood.
4. Suppl y of amino acids to cells:

essenti al
Cells take up albumi n by pinocy tosis and degrad e it, thus getting a supply of
and other amino acids from the liver.
conditi ons
This plays an import ant role in supply of amino acids to cells during the
of limited amino acid supply .
Answe r hint for Functi on of Pl asma protei n ques tion (5 Marks ):

a. Colloid al osmotic pressu re (1 Mark)
b. Transp ort functio n (2 Mark)
c. Other functio ns (4 more) (2 Mark) - 0.5 each)
If functio ns of album in is asked for 5 Marks , write all the functio ns of

plasma friteins except coagul ation and defenc e functions.
Note that answer looks almost similar to functio ns of plasma protein s
as albumi n is the most impora tnt plasma protein .
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Plasma Proteins 541
Globulins:
Globulins include many proteins, which separate into 4 distinct band in electrophoresis.
i) a.1 -globulins: a 1 -antitrypsin, retinal binding protein, thyroxine binding globulin, HDL etc
ii) a 2 -globulins: <Xi-macroglobulin, Haptoglobin, Ceruloplasmin, Prothrombin etc.
iii) - globulins: LDL, Transferrin, Hemopexin, plasminogen etc.
iv) y - globulins: Immunoglobulins.
Functions of globulins:
1) cx1-antitrypsin: It inhibits all serine proteases (i.e. proteases having serine at their active
site, like trypsin, elastase, plasmin, thrombin, cathepsin etc). Deficiency is implicated in
emphysema and certain liver d iseases. It is an acute phase protein.
2) a 2-macroglobulin: Inhibits protease activity, serves as anticoagulant. Elevated in nephrosis.
3) Haptoglobin: It is a glycoprotein. It binds with free Hb, which is released into the plasma
during hemolysis. This haptoglobin - Hb complex cannot be fil tered through glomerulus, so it
is not excreted in urine. The haptoglobin - Hb complex is taken up by hepatocytes and degraded.
Haptoglobin is also an acute phase protein.
4) Ceruloplasmin: Has ferroxidase activity, he lps in incorporation of iron into transferrin.
Level of ceruloplasmin decreased in Wilson's hepatolenticular degeneration.
5) Transferrin: Iron transport protein.
6) Retinol binding protein (RBP) transports retinal.
7) Thyroxine binding globulin (TBG) transports T3 and T4 •
8) Lipoproteins transport various lipids like triacylglycerol, phospholipids and cholesterol.
9) Hemopexin transports heme.
10) Immunoglobulins: Functions as antibodies.
Acute phase proteins:

Level of certain proteins increases markedly in various inflammatory states, infection, injury
& neoplastic conditions. These proteins are termed as acute phase proteins. These proteins
are believed to play an vital role in the body's response to inflammation.
E.g.: C-reactive proteins (CRP), 0'.1-antitrypsin, a 1-acid glycoprotein, haptoglobin etc.
C-reactive proteins (CRP):
1t is so named because it reacts w ith the C polysaccharide of pnuemococci. It is produced in
liver. CRP level is increased in acute, chronic inflammatory states and cancer.
CRP is involved in promotion of immune system through the stimulation of the classic
complement pathway during inflammation. It is used as a marker for tissue injury, infection,
and inflammation . The response of CRP to surgery is u sed in the evaluation of conditions of
surgery. During normal surgery, the level of serum CRP rises and come back to normal
level in 7-10 days after surgery. A continuous increase in CRP level reflects an associated
infection, which necessitates further treatment.
Recently, CRP is gaining importance as a possible indicator to certain types of coronary
heart diseases secondary to atherosclerosis.

Plasma Proteins 542
Immunoglobul ins
• Irnmunogl obulins are antibodies produced by B-lymphoc ytes (Plasma cells) in
response to the entry of antigens.
• lrnmunogl obulins are responsibl e for antibody mediated immunity (Humoral
immunity) .
• Irnmunogl obulins are glycoproteins.
Structure of immuno globulin s:

• lmmunogl obulins consist of 2 pairs of polypeptid e chains (Tetramer), i.e. one pair
of identical light chains (L chains) and another pair of identical heavy chains (H
chains).
• Light chains are either K (kappa) or 11. (lambda) in all classes.
• Heavy chains are of 5 types - y (gamma), µ (mu), a (alpha), 8 (delta) or £ (epsilon).
• Depending upon the type of heavy chains present, there are 5 m ajor classes of
immunoglo bulins.
Class H chain Molecular weight (Dalton)
IgG y (gamma) 1,50,000
IgM µ (mu) 90,000
IgA ex (alpha) 1,70,000
IgD o(delta) 1,80,000
IgE £ (epsilon) 1,90,000
About 80% of immunoglo bulins found in blood are of the IgG class. So, the structure
of IgG is used to study the general structure of an immunoglo bulin. IgG is responsinsible
for secondary immune response. IgG can cross placenta.
Structure of IgG:
• IgG has 2 heavy chains and 2 light chains. Heavy chains are gamma (y) and light
chains are either kappa (K) or lambda (A). Th us, the structure of IgG is either y2 ~ or 'YiA.i.
• Subunits are arranged in "Y" shape, which are held together by disulfide bridges.
Each light chain is held together with one heavy chain by interchain disulphide (S-S)
bridges. Similarly, two heavy chains are held together by interchain disulphide bridges.
Besides these are a number of intrachain disulphide bridges.
• Both heavy and light chains contain variable (V) regions- - VLand VHand constant (C)
regions - CLand C 11• Different immunoglo bulins vary from each other at their variable
region. There are millions of different structural types of immunoglo bulins, whose 3-D
structures vary at the variable regions from each other.
• Immunogl obulins bind to the antigens at the variable region and to the macrophag e
at the constant region, thereby fixing the antigen to macrophag es for phagocyto sis.
Plasma Proteins 543
..S...S .
.s .s
s..s
( Structure of IgG )
COOH COOH
Functions of immunoglobulins:
Immunogl obulins are antibodies that are responsible for antibody mediated immunity
(Humoral immunity) . These bind with antigens and degrade them. They protect the
body against bacterial and viral infections.
Individua l Functions of immunog lobulins:

Class Major Functions
IgG Mainly responsible for humoral immunity. Involved in secondary immune response
IgA Protects the body surface
IgM Humoral immunity, serves as first line of defense (Primary immune response)
IgD B-cell receptor (not certain)
IgE Humoral immunity & histamine release (allergy, hypersensitivity, anaphylaxis etc.)
Multiple myeloma:
The light chains of immunoglo bulins are produced in excessive amounts in multiple
myeloma. Serum electropho resis shows a distinct M band in multiple myeloma. lg
light chains are excreted in large amounts in urine. These constitute Bence Jones
protein. Excess excretions of these Bence Jones Proteins can damage renal tubules.
Bence Jones proteins:
These are light chain of immunoglo bulins that are excreted in the urine in cases of
multiple myeloma. Bence Jones protein precipitate s on heating the urine to 45 0 C,
maximally precipitate d at 600 C, but start redissolvin g at 600 C and form a clear
solu tion at 1000 C. The precipitate reforms on cooling. Presence of Bence Jones protein
in urine is diagnostic of multiple myeloma.
Plasma Proteins 544
Questi on bank on Plasma Proteins:

1) Plasma Proteins: Types and Function s
2) Classific ation of immuno globins
3) Structure of immuno globins
4) Acute phase proteins (ACP)
Short Answer s (3 Marks):

1) Functions of Albumin
2) Functions of Individu al plasma proteins
3) C-Reacti ve Protein (CRP)
3) Multiple Myelom a
4) Bence Jones Protein
Multipl e Choice Questio ns (1 Mark):

1 Protein that precipita tes on heating to 45°C and redissolv es on boiling is (AI)
a) Bence Jones Protein b) Gamma g lobulin c) Albumin d) Myosin
2) The plasma protein which moves fastest during electroph oresis is

a) Albumin b) Gamma globulin c) Alpha globulin d) Beta globulin
3) The most abundan t immuno globulin present in the body is

a)lgE b) IgG c) IgM d)IgD
4) The antibody class which can pass through the placenta to protect fetus is
a)IgE b)IgG c) Ig M d)IgD
5) Plasma albumin perform s all the followin g function s except;

a) Transpo rt b) utritive c) Antioxid ant d ) Mainten ance of osmotic pressure
6) Protein that are seen multiple myeloma is (AI)

a) Bence Jones Protein b) Gamma globulin c) Albumin d) Myosin
7) Which of the followin g is a negative acute phase protein?

a) CRP b) Cerulop lasmin c) Transfer rin d) Alpha-1- Antitryp sin
8) Allergic reactio ns (anaphyl actic) are mediated by

a) lgE b) lgG c) lg M d) IgD
weight
9) Out of different types of lmmuno globulin s, which pos es the highest molecula r
a) IgG b) lgM c) lgC d) lgA
10) Which of the following immuno globulin is responsi ble for primary immune
response ?
a) lgG b) lgM c) lgD d) IgA
2)a 3)b 4)b S)c 6)a 7)d S)a 9)a l0)b

Answer sforMC Q: l)a
9986449 575
Acid Base Balance 545
Acid Base Balance
Contents:
• Acids, bases and buffers
• Buffer systems of the body

a) Mechanism of buffering action
b) Bicarbonate buffer system
• Role of lungs in acid base balance
• Role of kidneys in acid base balance
• Acid base imbalance:

a) Metabolic acidosis
b) Metabolic alkalosis
c) Respiratory acidosis
d) Respirato ry alkalosis
• Diagnostic tests

Acids, Bases, Conjugate acid-base pairs and buffers:

Acids are proton donors i.e. substance s which can donate a proton (H·).
Bases are proton acceptors i.e. substance s which accept a proton (H+)
Acid + - - - - - Base+ H +
H2C03 HCQ3· + H•
For e.g, (Carbonic acid) (Bicarbonate) (Proton)
Carbonic acid can donate a proton, so it is an acid; bicarbon ate can accept a proton,
so it is a base. Conseque ntly, acids and bases exist as conjugate acid-base pairs.
Buffers :
Buffers are solutions , which resist changes in pH when an acid or alkali is added. Buff-
ers are generally a mixture of weak acid and its conjugate base (or sa lt of its conjugate
base, both are same). Buffer = Salt/ Acid or Base/ Acid
Examples: Bicarbonate buffer system is a mixture of carbonic acid, H 2C03 (weak acid)
and sodium bicarbon ate, NaHC03 (salt of its conjugate base).
Bicarbonate buffer = aHCO / H2CO3 [or HCO3· / H 2CO 3 ]
3
Similarly,
• Acetate Buffer = CH3C00Na / CH3COOH (Mixture of acetic acid and acetate)
• Phosphate buffer = Na 2HP04 / NaH2P04 (Mixture of disodium hydrogen phosphat e
& sodium dihydrog en phosphat e. Sodium dihydrog en phosphat e is a titrable acid.
• Ammonia buffer = NH3 / NH/ (Mixture of ammoniu m and ammonia )
Biological significa nce of buffers:

Buffers play an importan t role in acid base balance of the body. Buffers are the first line
of defense against the acid load. (Details given later).
Bufferin g capacity :
Capacity of the buffer to resist change in pH when an acid or alkali is added is called
the bufferin g capacity of buffers. It is defined as the amount of acid or alkali required
to change the pH of one litre of buffer by one unit. It depends on various factors.
1) pH of the buffer: Buffer acts best when pH=pKa. Its buffering range is best about
1
pH unit above or below pKa value of the buffer. When the pH is close to pKa, its buffering
capacity is maximum. Any deviation from this particular pH, buffering capacity also decreases.
2) Concentr ation of acid and base compone nt of the buffer: More the concentra tion
of
buffer, more the buffering capacity.
Acid Base Balanc e 547
How do buffer s act ?

Buffers resist changes in pH when an acid or alkali is added to them.
a) When a strong acid (like HCl) is added to the buffer, the salt of the buffer
acts on that acid
forming the weak acid.
b) When a strong base (like NaOH) is added to the buffer, the weak acid
of the buffer acts on
that base to form water and salt of the buffer.
Conseq uently the changes in the pH are minimi zed.
Example: Action of bicarbo nate buffer is explain ed here.

a) When a strong acid like HCl is added to the bicarbo nate buffer, the
salt of the buffer
(NaHCO :J acts on that acid forming the weak acid (H CO/
2
NaHCO J + HCI - - - H2C03 + aCl
Since a week acid is formed the changes in the pH is minimi zed.
b) When a strong base like NaOH is added to the bicarbo nate buffer, the H
2C03 of the buffer
acts on that base to form water and salt.
aHCOJ + H20
Since a salt is formed, the changes in the pH is minimi zed.
Henderson-Hasselbalch equati on (HH Equation):

The dissocia tion behavio r of a buffer is givenby HePder son-Has selbalch
equatio n.
pH= pKa + log [Base]
[Acid )
Signific ance of Henderson-Hasselbalch equation:

1) HH equatio n is useful in determi ning the status of acid base balance of
the body.
Knowin g any 3 parame ters, the 4r11 parame ter can be calculat ed using this
equatio n.
2) HH equatio n is also useful in prepara tion of buffers.
Signifi cance of Hender son-Ha sselbal ch (HH) equatio n in bicarbo nate buffer
system :
The normal bicarbo nate level in the plasma is 24 mmol / L. }\C0 concent
ration is given by
prod uct of pC02 (40 mm Hg) & solubili ty constan t of CO (0.03). Thus,
3
H C03 concent ration
is (40 X 0.03) = 1.2 mmol/L . pKa of bicarbo nate buffer is26.1 and pH of plasma
2
is 7.4.
Substit uting these values in HH equatio n,
7.4 = 6.1 + log 24
1.2
= 6.1 + log 20
= 6.1 + 1.3
= 7.4
Hence, the ratio of HC01• to 8iC0 at pH 7.4 is 20 urder normal healthy
3 cond itions.
So, it is evident that there is a high HC0 • concent ration (alkali reserve
3 ) in plasma. This is
s ignifica nt in maintai ning the acid base balance , because body produc es predom
inantly acids.
Refer bicarbo nate buffer system in next segmen t for more details.

hoo.com or 998644957
Regula tion of pH (Regul ation of Acid Base balance of the body)

The normal pH of plasma is 7.4 and it is maintain ed within a narrow range of 7.36 to
7.44. Even minute changes in the pH result in alteration of structure and functions of
biomolec ules (especially enzymes) , which in tum influence the normal metabolis m.
During the normal metaboli sm of the body various acids are produced , which poses
a constant acid threat. This acid challenge should be effectively met and the pH of
plasma should be maintain ed within normal limits.
Acid threat: During normal metabolis m, large quantitie s of acids are being produced
and added to plasma. These are,
a) Volatile acids: Carbonic acid formed from CO2
b) Non-vola tile acids: Lactic acid, keto acids, sulfuric acid, phosphor ic acid etc
The pH of plasma is maintained within normal limits by 3 mechanisms, namely,

1) Buffer systems of body
2) Respiratory mechani sm
3) Renal Mechani sm
1) Buffer systems of the body :

Buffers are the first line of defense mechanism against the acid load. Buffers respond
immedia tely within seconds against acid threat and neutraliz e them. They resist a
change in pH when an acid or alkali is added.
Buffer systems in the body are a mixture of weak acid and salt of its conjugate base.
There are,
a) The plasma buffers :

1) Bicarbon ate buffer (HCOONa I H2 C03 )
2) Phosphat e buffer (Na2HP04 / NaH2 P04)
3) Protein buffer (Protein / Proteinate)
b) RBC buffers :
1) Hemoglo bin buffer (Hb I HHb)
2) Phosphat e buffer (NaHP04 / NaH2P0 4)
3) Bicarbon ate buffer (HCOO a / HS03)
c) Urine buffers :
1) Phosphat e buffer (NaHP04 I aH 2P04 )
2) Ammoni a buffer (NH3 / NH/ )
queries and suggestio ns, contact the author at prasad_te xt@yahoo .com or 9986449575
Buffers act quickly, not permanently:

• Buffers are the immediate defense mechanisms against the acid load. Note that for
every molecule of acid detoxified by buffer, one molecule of base component of the
buffer is converted to acid component of buffer. Since body continuously produces
acids during metabolism, all the alkali would be exhausted very soon, if they are not
replenished. Buffers are unable to replenish the alkali reserve of the body. They also
cannot remove the H+from the body. So, buffers act as temporary defense mechanism
against acid load and they are effective as long as the alkali reserves are not exhausted.
• For the final elimination of acids and to replenish the alkali reserve, respiratory and
renal mechanisms are required. Therefore, buffer system is called the first line of defense,
respiratory mechanism is called the second line of defense and renal mechanism is
called third line of defense of acid base balance.
Bicarbonate buffer system is the most important buffer system of the body in
maintaining the acid base balance. It is discussed below,
Bicarbonate buffer system (HCO3 · / H2CO3 :

• Bicarbonate buffer is the most important extracellular buffer system of the body.
• Bicarbonate (HCO3) and carbonic acid (8iCO3) constitute bicarbonate buffer.
Importance of bicarbonate buffer :
1) Present in High concentration: Bicarbonate buffer system is present in higher concentration

than any other buffer systems in the body. It accounts for 40 to 50 % the total buffering capacity
of the whole body and about 65% of the buffering capacity in plasma.
2) High alkaJi reserve: Bicarbonate buffer system has a high alkali reserve (ratio of
concentration of HCO3• to H 2CO3 is 20 : 1). This is very significant, because the cellular
metabolisms mainly produce acids & hence the bicarbonate level should be sufficiently high
to meet this acid load. Thus, a high alkali reserve ensures the effective buffering of acids.
3) Concentration of components can be regulated: Concentrations of components of
bicarbonate buffer system can be regulated by lungs and kidney. Acid component (8iCOJ can
be regulated by the lungs (by respiratory mechanism), base component (bicarbonate) can be
regulated by kidneys (by renal mechanism).
Note: The acid component, H1CO1 (denominator) is regulated by respiration. So, it is called the respiratory
component of bicarbonate buffer system. Base component, HC01- (numerator) is regulated by metabolism in
kidney. So, it is called the metabolic compo11e11t of bicarbonate buffer system.
Phosphate buffer system (NaHPO4 I NaH2 POJ:

• Phosphate buffer system is an important intracellular and urinary buffer system.
• Disodium hydrogen phosphate ( a 2HPO.) and Sodium dihydrogen phosphate (NaH2 PO4 )
constitute phosphate buffer.
• Phosphate buffer system is an effective buffer system, as its pKa value is nearest to physiological pH.
But, it is of less important in plasma tha11 bicarbonate buffer system due to it's low concentration.
IProtein buffer system is given in amino acid & protein chemistry chapter.
Role of lungs (Respiratory mechanism) in acid base balance

Respiratory mechanism is called the second line of defense in acid base balance.
The acid component of bicarbonate buffer system (I\C03) is under respiratory regulation.
The respiratory regulation of pH is achieved by changing the concen tration of CO2
(pC02) as lungs can regulate elimination of CO2 • The rate of respiration (rate of elimination
of CO) is controlled by chemoreceptors p resent in respiratory center of brain, which is
sensitive to changes in pH of blood.
During acidosis:
Ra te of respiration increases (hyperventilation), which results in elimination of more
of CO2 thus lowering the pC02 (H2C03) level and increasing pH. Thus, pH is maintained
within the normal limits.
During alkalosis:
Rate of respiration decreases (hypoventilation), which results in the elevation of pC02
(8iCOJ level and decrease in pH.
Role of hemoglobin in respiratory regulation:

Tissues
(Role of hemoglobin buffer in acid base balance):
The Hb buffer in R.B.C plays an important role in respiratory
y
CO, H20
regulation of pH. It helps in the transport of CO2 produced in
the cells to the lungs for excretion. CO2 produced in the cells H , CO,
reach the lungs through blood with help of Hb buffer. A

HCO,· H• I-lb
In the RBC's: y
CO2+ H 20 _ . H2C03 HHb
H2C03 _ . H ... + HC03-

H+ + Hb _ . HHb Lungs
HHb and HC03• are then transported to lungs.
In the 1ungs: Hx
Reverse changes occur in the lungs during oxygenation.

y
HCO ,· H• Hb0 2
H Hb + 0 2 _ _ _.,. Hb02 + H+
H + + HCQs· _ _ _ .,. H2C0 3 H 2CO ,
H 2C03 _ _ _.,. H 20 + CO2

I CO2 H ,O
CO2 is then excreted through lungs by respiration. +
Expired through lungs
Chloride shift: It occurs during transport ofCO2 in RBC's. In RBC's, CO2 combines with Hp to give
H 2C03 (lnj carbonic anhydrase enzyme), which dissociates to produce H+ and HC03•• H· is immediately
buffered by hemoglobin to form HHb. As the concentration of HC03· increases in RBC's, it diffuses into
plasma in exchange with Cf· (to maintain electrical neutrality). This phenomenon is called chloride shift.

Role of kidney in acid base balance:

Renal mechanism is called the third line of defense in acid base balance.
The renal mechanism of acid base balance is achieved by 4 processes:

1) Excretion of H• ions and generation of HC03
2) Reabsorption of HCQ3-
3) Excretion of titrable acids (or buffering by phosphate buffers)
4) Excretion NH/ (ammonium) or (buffering by~ buffers).
1) Excretion of H• ions and generation of HC03 - (bicarbonates):
Plasma Tubular Cell T u bular lumen
Na+ Na+ Na+
H CO3- H CO3·
........__... H· - H•
I *
Excreted in u r ine
H2COJ
i
CA
H 2O +CO2
The CO2 generated by cellular metabolism combines with 8i0 to form carbonic acid,
which ionizes to form H• & HC03•• The H• is secreted into tubular lumen in exchange
with Na•. These Na• along with HC03• will be reabsorbed into blood. This mechanism
serves to excrete H• and generate bicarbonate, thereby increasing the alkali reserve.
2) Reabsorption of bicarbonates:
Plasma Tubular Cell T ubular Lumen

I
Na+ _ Na+ - Na+ i

y
+ H•
H COJ H CO3
...______ H• H CO3·
I
H2CO3 H2CO3
iCA
H ,O + CO2
i CA
CO2 + H2O
This mechanism reabsorbs HC03• filte red by glomerulus. The HCQ3- is almost
completely reabsorbed PCT, so urine is normally bicarbonate free.

3) Excretion of titrable acids (NaH2PO4) :
The major titrable acid present in urine is NaHzPO4 (Sodium dihydrogen phosphate).
Plasm a Tubular Ce ll Tubular Lum en

-
Na2HPO•
..---l..
y
a+ Na+ Na+ a H PO,
II CO 3 H CQ 3·
,___.. H • H •
H 2C O l NaH2PO,
t
CA
H 2O+CO 2
t
Excreted in urine
The major titrable acid present in urine is sodium acid phosphate (NaH,_PO4). The H+
which is released into the lumen, combines with Na2HPO4 to form NaH2PO4 (Titrable
acid) & excreted. Excretion of titrable acid enables elimination of acids from the bod y.
4) Excretion of ammonium :
-
Plasm a Tubular Cel l Tubular lumen
C lutaminas e
Glutamine 7 ~ " G lutamate
H 20 N II ,
•
-
H
•---y
Na+ Na+ Na+ N H,
H C 01 - HCO , H . ;::
I H 2C O l NH••
i cA
H 20 + CO 1 Excre te! in urine
This is one of the most important mechanisms of H• excretion. The NH3 is produced
in the kidney cells by the action of enzyme glutaminase. NH3 combines with H ' to
form NH/, which is excreted in urine.
During acidosis, the action of glutaminase enzyme is stimulated and excretion of
NH4 + is increased.

Acid Base imbalance (or acid base disorders):

Normal pH of ECF is 7.4. (7.36 to 7.44). There are 2 abnormal conditions,
Acidosis: If the pH falls below 7.36, the condition is called acidosis.
Alkalosis: If the pH exceeds 7.44 conditions is called alkalosis.
Note: Ratio of concentration of HC03- to the concentration of H 2C03 is 20:1 in a healthy

person. If this ratio is altered as a result of change of one component, the pH of the blood
changes leading to acidosis or alkalosis.
If the alteration is due to any change in HC03·, then the imbalance is termed as metabolic.
If the alteration is due to changes in pC02' tlzen the imbalance is tenned as respiratory.
There are 4 acid base imbalances, due to alteration in HCQ3- or pC02 level.
1. Metabolic acidosis (Decrease of pH due to deficit of HC03-)
2 . Metabolic alkalosis (Increase of pH due to excess of HC03- )
3. Respiratory acidosis (Decrease of pH due to excess of pC02)
4. Respiratory alkalosis (Increase of pH d ue to deficit pC02)
1. Metabolic acidosis :
Defined as the decrease in blood pH due to primary decrease in HC03- in blood.
Causes:
Metabolic acidosis may result from an increased production of acids (which are
buffered by HCQ3- resulting in decreased level of HCQ 3-) or loss of bicarbonates. These
cond itions are,
• Excessive production of acid: In diabetes & starvation, th ere is excessive production
of ketone bodies. In severe exercise, there is increased prod uction of lactic acid.
• Accidental ingestion of acids
• Ingestion of acids like NHSI: NH3 is detoxified to urea leaving behind H +(acid ).
• In renal tubular acidosis: Due to reduced elimination of H+due to renal failure.•
• Overdose or poisoning by aspirin, methanol, salicylates, ethylene glycol etc:
Oxidation of these compounds prod uces acids.
• Diarrhea: In diarrhea, there is excessive loss of HC03• in feces, resulting in the deficit
of bicarbonates.
Compensation:
• Primary compensation is by lungs. Hyperven tila tion takes p lace to decrease the
pC02 and increase of pH .
• Secondary compensation is by kidney. It involves increased excretion of H +, increased
p roduction of N~, increased generation of HC03 and complete reabsorption of HCOy

Anion gap
D efinition:
The anion gap is the difference between the concentration of measurable anions and
measurable cations routinely measured in serum.
Explanation:
The sum of cations and anions in ECF is always equal so as to maintain the electrical
neutrality. But, all the anions cannot be measured (such as protein anions, sulpha te,
phosphate and organic acids) and there is always a difference between measurable
anions and cations. The unmeasured anions in serum constitute the anion gap.
Normal l evel:
Normal anion gap is 12±5 mEq/ L.
Significance:
l. Anion gap helps in the differential diagnosis of me tabolic acidosis. There are two
types of anion gaps;
• High anion gap metabolic acidosis: Anion gap is increased (> 17 mEq/ L) in
conditions like diabetic ketoacidosis, lactic acidosis, starvation ketoacidosis, renal
failure, ethylene glycol and methanol poisoning.
• Normal anion gap metabolic acidosis: Anion gap is normal in diarrhea, renal
tubular acidosis and ureteric transplantation etc.
2. Anion gap is decreased in Hypoalbuminemia, hemodilution, hypercalcemia,
hypermagnesemia etc.
Relationship between pH with K• (potassium ion):

• Potassium ion (K+) is the major intracellular cation. When there is an increase in H+
concentration in ECF, there may be an exchange of H+with intracellular K+, net effect is an
apparent increase in ECF potassium level (lzyperkalemia). So, in general, acidosis is associated
with hyperkalemia and a/kalosis is associated with hypoka/emia.
• However, in renal tubular acidosis (a failure of kidney to excrete H+), K+is lost in urine
leading to hypokalemia (due to failure to exchange with H+).
• In correction of alkalosis, hypokalemia may occur due the sudden influx of K+ into the
cells. So, it is important to maintain K+level during the correction of alkalosis.
Alkaline tide:
It is the sudden rise in pHof the blood after having food due to rise in the serum HCO3• [evel. ft can be
explained by the mechanis111 of HCI production in tire parietal cells. HCQ3• is pumped into the blood
for exchange of CL· by parietal cells for HCI production dt1ring mealtime.

2. Metabolic alkalosis:
Defined as the increase in blood pH due to primary increase in HC03- in blood.
Causes:
It is caused due to excess of bicarbonate as in cases of,
• Violent vomiting: there is loss of HCl.
• Ingestion of antacids (NaHC03) in the treatment of gastric ulcer.
• Accidental ingestion of alkali
• Gastric suction
• Pyloric stenosis
Compensation:
• Primary compensation is hypoventilation.
• Secondary compensation is excessive release of HC03-by kidneys.
3. Respiratory acidosis:
Defined as the decrease in blood pH due to the primary increase of pC02 in blood.
Causes:
It occurs due to increase of CO2 due to hypoventilation as in,
• Obstruction to respiration due to pneumonia, emphysema, asthma, bronchitis etc.
• Depression of respiratory centers, as seen in morphine or barbiturate poisoning.
• Disease of diaphragm
• Tetanus
Compensation: Compensation is by reabsorption of HC03-by kidneys and increased

H+ secretion and NH4 production.
4. Respiratory alkalosis:
Defined as the increase in blood pH due to the primary decrease in pC02 in blood.
Causes:
It is caused due to excessive loss of CO2 due to hyperventilation.
• Hypeventilation occurs when respiration is stimulated as in fever, hot bath, high
altitude, hysteria, anxiety, excessive exercise, sepsis, diseases of the nervous system
like encephalitis, action of drugs like salicylates that stimulate respiratory centre,
and voluntary hyperventilation etc.
Compensation: Compensation is by increased secretion of HC03 - and decreased

secretion of H+ and NH/ .

Diagnostic tests in acid base balance:

• The analysis of blood gases like pCO2, pO2 and pH of blood is major diagnostic tool
in the assessment of acids base status of the body in health and diseases.
• The estimation of blood gases is done by a instrument called blood gas analyzer.
The Blood gas analyzer is designed to quantitatively determine the pH, pCO2, pO2,
and Hb etc by means of electrode.
Note: Arterial blood is used for blood gas analysis. Heparinized blood is collected
and directly introduced into the analyzer. The blood collected should be analyzed
within 30 minutes of collection. There should not be any contact with the air
during the collection or analysis.
Normal levels:
Normal pH of blood =7.40 (Average 7.36 - 7.44).
Normal partial pressure of arterial blood (pCO2) = 40 mm Hg (Range 34-46 mm Hg).
Normal bicarbonate level in blood= 24 mEq/1 (Range 22 - 26 mEq/1).
Significance:
1. Useful in assessment of acid-base status of the body

Measurements of pH, PCO2, bicarbonate are useful in assessment of acid-base status
of the body.
2. Diagnosis of acid base imbalance:

Measurement of pH, pCO2 and bicarbonate is useful diagnosis of acid base disorders.
There are 4 major acid base disorders in the body.
1. Metabolic acidosis:
• pH is decreased
• Bicarbonate level is decreased
2. Metabolic alkalosis:
• pH is increased
• Bicarbonate level is increased.
3. Respiratory acidosis:
• pH is decreased.
• pCO2 level is increased.
4. Respiratory alkalosis:
• pH is increased
• pCO2 level is decreased.

Question bank on Acid base balance

Long Essays (10 Marks):
1) What is the normal pH of Blood? How is it maintained?
2) Role of kidneys in acid base balance
Short Essays (5 Marks):

1) Different buffer system of acid base homeostasis
2) Role of Bicarbonate buffer system
3) Role of lungs in acid base balance
4) Metabolic acidosis / Metabolic alkalosis / Respiratory acidosis / Respiratory alkalosis
5) Anion gap

1) Glutamine formation and its importance
2) Hcnderson- Hesslebach equation (HH equation)

1) Which is called as the alkali reserve of the body
a) Protein b) NalIPO4 c) HCO1• d) Hb
2) The ratio of HCO3• to H 2CO1 in a person with normal acid-base status is

a) 10:1 b) 20:1 c) 1:10 d) 1:20
3) Which of these buffers is not present in the blood

a) Bicarbonate b) Hemoglobin c) Protein d) Ammonia
4) The pKa of bicarbonate buffer is

a) 4.5 b) 9.3 c) 6.1 d) 7.2
5) Titrable acidity refers mainly to the concentratio n of

a) N'½HPO4 b) NaH 2PO4 c) N~ d) NH.
6) A Cause of high anion gap metabolic acidosis is

A) Diabetic Ketoacidosi s B) Diarrhea C) Vomiting D) Renal tubular acidosis
7) Important buffer of extracellula r fluid is

a) Bicarbonate / carbonic acid b) Plasma proteins c) Phosphate buffer d) Organic phosphate
8) pH of buffer is determined by pH= pKa + log [salt]/ [acid] which is also known as equation
a) Anderson-J oule b) Henderson- Smith c) Henderson- Harris d) Henderson- Hasselbalch
9) Normal pH of blood is
a) 7.15 to 7.25 b) 7.25 to 7.35 c) 7.35 to 7.45 d) 7.45 to 7.55
10) Respiratory alkalosis occurs in

a) Bronchial asthma b) Collapse of lungs c) Hysterical hyperventil ation d) Pneumonia
Answers for MCQ: l) c 2) b 3) b 4) c 5) b 6) a 7) a 8) d 9) C 10) C
Hemoglobi n 558
Hemog lobin
Content s
• Hemoglobin: Structure and functions

• Abnormal Hemoglobins
• Heme synthesis
• Heme degradation
• Jaundice: Definition, Classification and Detection

Hemoglobi n 559
Hemogl obin and Heme

Hemoglob in is the red pigment present excl usively in erythrocytes.
Normal hemoglobi n level in males is 14 to 16 g/dl and in females 13 to 15 g/dl.
Structure of hemoglob in:

Hemoglob in is a conjugated protein containing globin (protein part) and heme (non-
protein conjugate part). Hemoglob in = Heme + Globin
i) Heme containing Protoporph yrin IX molecule with one Fe• a tom at the centre.
2
Iron + protoporphyrin IX = Heme.

(Protoporp hyrin IX has 4 pyrrole rings, which are linked by Methene Bridge.)
ii) Globin is the protein chain. It belongs to the class globulins.
Hemoglobi n is a tetramer containing 4 subunits, which are held together by non-covale nt
bonds. Each subunit has a globin chain and a heme group.
There are 3 types of normal hemoglob in, based on the nature of globin chains:
HbA1, HbA2 & HbF. Normal adults contains 97% HbA1, 2% HbA2 and 1% HbF.
a) Hemoglob in Ai (HbA1): HbA1 is the major form of hemoglobi n in adults. HbA1
contain 574 amino acids and has 4 subunits; 2 a (141 amino acids) & 2 (146 amino
acids). Each a & subunits possess primary, secondary & tertiary structures. 4 subunits
are associated by non-covale nt salt bridges to form functional HbA1 (quaternar y
structure). Refer Protein chemistry chapter for structural representation of Hb.
• Each globin chain contains 58 histidine residues. Among them, 87 ' IIistidine in a and 92
11 nd
histidine in are called proximal histidine (as the1; are close to iron atom) & 58 ' IIistidine in
11
a and 63 rd histidine in~ are called distal histidine (as they are far away from iron atom).
• Each subunit ofhemoglobin contains one heme as prosthetic group. Each heme contains one
Protoporphyrin IX molecule with one Fe+2 atoms at the centre. Iron can form 6 coordinate
bonds, Four are bound to the pyrrole of protoporphyrin IX; the 5 ' to proximal histidine of
11
globin (a chain 87 histidine or~ chain 92 histidine), and 6 is involved in binding to oxygen
th
in oxy-Hb. (Oxygen also forms a bond with distal histidine (a chain 58 I ~ chain 63). In
deoxy-Hb, this 611' valency is occupied by water molecule.
b) Hemoglob in A2 (Hb~) has 2 a subunits & 2 8 subunits.

c) Fetal hemoglob in (HbF) has 2 a subunits & 2 y subunits. (Life span 60-90 days)
Functions of hemoglob in:

i) Its main function is to transport respiratory gases (oxygen and carbon dioxide). It
carries oxygen from lungs to the tissues and carbon dioxide from tissues to lungs.
ii) Hemoglob in also has buffering capacity. The buffering capacity of hemoglobi n is
main ly due to histidine content (38 out of 574).

Hemoglobin 560
Binding of oxygen to Hb shows the phenomenon of cooperativity.

Hb can transport 4 molecules of oxygen, one in each subunit. The binding of oxygen to
one subunit increases the affinity of binding of oxygen to remaining subunits of the
same molecule of hemoglobin. This is called positive cooperativity. Affinity of oxygen
to fourth subunit of Hb is about 100 times greater than that of oxygen to first subunit.
The sigmoid shape of the oxygen dissociation curve (ODC) is due to the allosteric
effect or cooperativity. Cooperative interaction between the subunits leads to a marked
increase in the oxygen affinity of Hb with increase in oxygen concentration. Thus, oxygen
saturation curve of Hb (or oxygen dissociation curve of Hb) is sigmoidal (whereas,
oxygen saturation curve of Mb is rectangular hyperbola, as it doesn't show cooperativity).
C:
0
·.c 0 Substra te satu ration cu rve of M b :
Shows Rectnagular hyperbola curve
.E
OS
t/) G) Sub stra te s atu ra ti on c urve of Hb:
Shows Sigmoid curve
pO,(mm Hg)
Explanation: Hb can exist in two forms - T (Taut or tense) form and R (relaxed) form .
The quaternary structure of deoxy-Hb is T (taut) form results from presence of salt
bridges between the subunits; and that of oxy-Hb is described as R (relaxed) form
results from disruption of salt bridges.
The T form has less affinity for oxygen than R form. Binding of the first oxygen molecule
to one of the subunits of Hb produces a conformational change; salt bridges between
the subunits arc broken progressively. This facilitates uptake of oxygen by the other
subunits, a phenomenon known as cooperativity. T form gets converted to R form.
Factors affecting:
Factors which favor conversion of T form to R, i.e. favor O 2 loading (high oxygen affinity):
Increased pO2, decreased pCO2 , decreased 2, 3 BPG
Factors which favor conversion of R form to T, i.e. favor O 2 release (low oxygen affinity):
Decreased pO2, increased 2, 3 BPG; increased pCO2 and decreases pH (Bohr's effect).
Bohr effect:
Release of oxygen from
Decreased pCO2,
Increased pH, Hb is e nhanced when the
Increased pO2, pH is lowerer or when Hb
Decreased 2, 3 BPG is in the presence of
increased pC02• Both
Normal resu Its in decreased
Decreased pl 12 and
oxygen affinity of Hb,
lncrea!.Cd pC02 (Bohr effect), therefo r~ a shift in the
Decreased pO , oxygen disociation curve
50 100 Increased 2, 3 BPG to the right .
pO,(mm Hg) This is called Bohr effect.
IOxygen b inding curve of Hb !
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Hemoglobin 561
Hemoglobin Derivatives:
Hemoglobin derivatives are compounds formed by combination of substances with
hemoglobin. Besides major derivatives like oxyHb and deoxyHb, there are minor
derivatives like carbaminohemoglobin, methemoglobin (metHb) & carboxyhemoglobin.
Carbaminohemoglobin:
Carbaminohemoglobin is formed by the combination of CO2 with hemoglobin. It is
responsible for transport of about 2 -10 % of total carbon dioxide in the blood.
Methemoglobin:
Hemoglobin containing heme iron in the fe+3 form is called methemoglobin (MetHb).
Methemoglobin is formed when heme iron (Fe+2) is oxidized the ferric state (Fe+3) in
the living system by H 20 2 free radicals. Normally, the enzyme methemoglobin reductase
reduces the Fe3+ of methemoglobin back to Fe2+ .
Hemoglobin can carry oxygen only when iron is in the ferrous (Fe 2+) state.
Methemoglobin (with Fe3+), thus can neither bind nor transport 02.
Methemoglobinemia: Increase of methemoglobins in blood is methemoglobinemia.
Causes: Methemoglobinemia can arise by oxidation of hemoglobin as a side ~ffect of
drugs such as sulphonamide, by compounds such as nitrates, aniline, from hereditary
hemoglobin M (HbM) or due to reduced activity of enzyme methemoglobin red uctase.
(This is the reason why aniline dye workers are more prone to develop methemoglobinemia).
Clinical manifestations: The methernoglobins are characterized by "chocolate cyanosis"
(a brownish blue coloration of the skin and mucous membranes and brown colored
blood) as result of the dark colored HbM. Symptoms include anxiety, headache and
dyspnea, in rare cases, coma and death.
Treatment: Administration of methylene blue, which reduces the Fe3+ back to Fe2+.
Carboxyhemoglobin (CO-Hb):
It is a hemoglobin derivative that is bound to carbon monoxide. Affinity of CO to Hb is
200 times more than 0 2,- So, presence of CO in blood impairs the formation of OxyHb.
Normal level of Carboxyhemoglobin in blood is 0.16%. This level is increased in smokers.
An average smoker has around 4 to 5 % more CO-Hb.
Clinical manifestations: Clinical symptoms manifests when the CO-Hb level exceeds
20%. Symptoms are breathlessness, nausea, vomiting, head ache and pain in the chest.
Death can result at 40-60% saturation.
Treatment: Administration of oxygen is the best treatment.
Abnormal hemoglobins (Hemoglobin variants):

Abnormal hemoglobins are defined as those hemoglobins that are produced due to
mutations in the genes that code for alpha or beta globin chains of hemoglobin.
Mutation results in the formation of hemoglobin with altered structure and function.
Hemoglobinopathies: All the abnormal hemoglobins do not have clinical manifestations.
About 400 abnormal hemoglobins are known n ow. Of these, only few have clinical
manifestations; such conditions are known as hemoglobinopathies. (So, all hemoglobinopathies
are abnormal hemoglobins, but all abnormal hemoglobins are not hemoglobinoptl1ies).
Hemoglobin 562
Abnormal hemoglobins are of two types:

1) Caused due to altered primary structure
2) Caused due to altered quaternary structure or subunit makeup (Thalassemias).
1) Abnormal hemoglobins due to altered primary structure:

This type is generally caused due to point mutations which result in the replacement of
a single amino acid (altered primary structure) leading to formation of hemoglobin
with altered conformation and function. Some of the examples are,
a) Sickle cell Hemoglobin (HbS): (Refer primary structure section in protein chapter & RFLP
in molecular biology technique chapter, for basis, types & diagnosis of HbS).
• Defect: In HbS, the glutamic acid at 6th position of Pchain is replaced by valine.
• Basis: This results in the formation of a sticky patch on the surface of Pchain of both
oxy- and deoxy HbS. Deoxy HbS contains the receptor site complementary to sticky
patch. Binding between them results in the polymerization of HbS forming long fibres
that distorts the erythrocytes forming sickle shaped RBC. This sickle cell RBC are fragile
& causes hemolysis, resulting in anemia, which is referred to as 'sickle cell anemia'.
b) Hemoglobin C (HbC):
In HbC, the glutamic acid at 6th position of Pch ain is replaced by lysine.
c) Hemoglobin M (HbM):
In HbM, a 58 histidine is replaced by tyrosine (HbM Boston) or 13 92 histidine is replaced
by tyrosine (HbM Hydepark). HbM lead to the formation of methemoglobin.
d) Hemoglobin D (Hb D):
In HbD, the glutamic acid at 121 st position of p chain is replaced by glutamine.
2) Thalassemias (Abnormal hemoglobins caused due to altered subunit makeup):

Definition: Thalassemias result from absence of 1 or more a or 13 chains of hemoglobin,
caused due to the defect in the production of a or 13 globin chain. There are 4 copies of a
globin gene, 2 on each chromosome 16 & 2 copies of~ globin gene, 1 on each chromosome 11.
Thalassemia is classified into a thalassemia and 13 thalassemia on this basis.
i) a Thalassemia: Caused due to defect in production of a chain. Types are,
• a Thalassemia silent carrier: absence of one a globin genes. No clinical effects.
• a Thalassemia trait: absence of two a globin genes. Benign.
• a Thalassemia Minor (HbH)~absence of three a globin genes. Mild anemia.
• a Thalassemia Major (Hb Barts, Hydrops fetalis): absence of all four a globin genes.
ii) 13 Thalassemia: Caused due to defect in production of Pchain. Types are,
• pThalassemia Major: absence of both p globin genes. Homozygous condition.
• pThalassemia minor: absence of on e Pglobin gene. Heterozygous condition.
!Apart from marrow transplantation, treatment is symptomatic (Refer Physiology for details). !

Hemoglobi n 563
Heme synthesis:
• Heme is a prosthetic group present in heme proteins such as hemoglobin, myoglobin,
cytochrom es and enzymes like catalases, peroxidase s etc.
• Heme is an iron porphyrin compound (Fe 2 - protoporph yrin Ill).
• Protoporph yrin l1J is also referred to as protoporph yrin IX.
Tissue site:
Heme synthesis takes place mainly in liver and reticuloend othelial cells (erythrocy te
producing cells of reticuloend othelial cells).
Mature RBC's does not synthesize heme as they do not contain mitochond ria.
Intracellular site:
Partly in cytosol and partly in mitochond ria.
Starting compound:
Succinyl CoA and glycine
End product:
Heme
Reaction pathway:
Glycine combines with succinyl CoA to form ALA (&- Amino levulinic acid) by ALA
synthase enzyme. ALA synthase is present in mitochond ria and it is the key enzyme
of heme synthesis.
ALA then undergoes series of reactions to form porphyrin ring, which then combines
with iron to form heme.
ALA synthase
Glycine + Succinyl CoA - - - - - - - ALA • • • • Heme
Complete reaction of heme synthesis is given in the next page.
Regulation:
Heme synthesis is regulated by feedback mechanism (both feedback inhibition and
feedback repression. When heme is synthesize d in sufficient amou nts, it blocks its own
synthesis by regulating ALA synthase enzyme by feedback mechanism .
• Feedback inh ibition: When heme concentrati on is high, it blocks its own synthesis by
allosterically inhibiting ALA synthase enzyme.
• Feedback repression: When heme level is high, it acts as a co-represso r and binds
with an aporepress or to form holorepres sor. Holorepres sor then binds to the DNA of
ALA synthase and decreases the transcriptio n, thus decreasing ALA production . When
heme level is low, it can't bind with aporepress or, which alone can't bind repress ALA
synthase DNA, resulting in production of ALA synthase & heme synthesis.
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Hemoglobin 564
Complete pathway of heme synthesis:

Succinyl CoA + Glycine
(Cu·', PLP) j ALA synthase

CoASH, CO2 ..,.....-i
ALA
ALA9
M!f' ALA dehydratase
2H20
Porphobilino gen
J Uroporphyri nogen I synthase (or PBG deaminase)

4 NHJY!
Hydroxymet hylbilane (Enzyme bound)
Ring closure 1 Uroporphyri nogen Ill syn thase
Uroporphyri nogen Ill
4C02.lllt'!
J Uroporphyri nogen decarboxylas e
Coproporphy rinogen llI
_J
CO2
Oi
1 Coproporphy rinogcn oxidase
Protoporphy rinogen Ill
6H y!J Protoporphy rinogen oxidase
Protop~lhyr in Ill
Ferrochelatas e (Heme synthase)
1
Fe 2•
Heme
Note:
Step 1 takes place in mitochond ria. Step 2, 3, 4, 5 takes place in cytosol. Step 6, 7, 8
takes place in mitochond ria.
Heme can then combine with va riety of proteins to form heme proteins like Hb etc.
Hemoglobin 565
Porphyria:
Porphyrias are the group of disorders associated with heme synthesis. These are
characterized by increased production and excretion of porphyrins and / or porphyrin
precursors. They can be inherited or acquired.
Six major types of porphyrias have been described. Each type of porphyria results in
the accumulation of a unique pattern of intermediates (porphyrin and its precursors).
Porphobilinogen
Uroporphyrinogen J synthase Acute intemtittent po,phyria

(or PBG deaminase)
Hydroxymethylbilane
Uroporphyrinogen ill synthase CongenH•d e,.,,,hropoieti, po,phy,;a
Uroporphyrinogen TI!
Umpo,phyrinngen decruboxylase Po,phyria Cutanea Tania
Coproporphyrinogen Ill
Copropo,phy,inogen oxidase $-+ HeredH,uy Coprnpo,phy,ia
Protoporphyrinogen III
Prntopo'J'hyrinogen oxidase Variegate Po,phyda
$---
Protoporphyrin ill
Fen-ochelatase Hereditary Pn>topo,phyria
Heme
Types and enzyme deficiencies in Porphyrias

Hemoglobin 566
1. Acute intermittent porphyria (AIP):

Defect: Uroporphyrinogen I synthase (PBG deaminase).
Lab findings: Increased urinary excretion of PBG (porphobilinogen) and ALA.
Major symptoms: Abdominal pain, vomiting, constipation, neuropsychiatric
symptoms. These patients do not have photosensitivity. This is because these patients
do not accumulate porphyrins and porphyrinogens.
Significance: AIP is not expressed before puberty. Drugs and steroids that are
metabolized through cytochrome P450 (a heme protein) can aggravate the condition.
Administration of hematin can lessen the symptoms.
2. Congenital erythropoietic porphyria (CEP):

Defect: Uroporphyrinogen III cosynthase.
Lab findings : Increased urinary and fecal excretion of uroporphyrin I and
coproporphyrin I. This is due to the formation of large amounts of m:oporphyrinogen
type I (an unnatural isomer of uroporphyrinogen type III) & coproporphyrinogen I.
Major symptoms: Cutaneous photosensitivity.
3. Porphyria Cutanea Tarda (PCT):

Defect: Uroporphyrinogen decarboxylase. Most common type.
Lab findings: Increased urinary excretion of uroporphyrins of type I and III.
Major symptoms: Cutaneous photosensitivity.
4. Hereditary coproporphyria (HC):

Defect: Coproporphyrinogen oxidase.
Lab findings: Increased urinary excretion of ALA, PBG, coprophoporphyrin III and
fecal coprophoporphyrin III.
Major symptoms: Cutaneous photosensitivity, in addition to all the symptoms of
AIP (like abdominal pain, neuropsychiatric symptoms etc).
5. Variegate Porphyria (VP):

Defect: Protoporphyrinogen oxidase.
Lab findings: Increased urinary excretion of ALA, PBG, coprophoporphyrin III and
fecal coprophoporphyrin IX.
Major symptoms: Cutaneous photosensitivity, in addition to all the symptoms of
AIP (like abdominal pain, neuropsychiatric symptoms etc).
6. Protoporphyria (PP):
Defect: Ferrochelatase (Heme synthase).
Lab findings: Increased fecal and red cell protoporphyrin IX. But there is no increased
urinary excretion of porphyrins or porphyrin precursors.
Major symptoms: Cutaneous photosensitivity. Clinically there is urticaria on exposure
to sunlight.

Hemoglobin 567
Classification of Porphyria:
Porphyrias can be classified on the basis of the organs or cells that are most affected.
into a) Erythropoietic porphyria and b) Hepatic porphyria.
a) Erythropoietic porphyrias:
Eg, Congenital Erythropoietic Porphyria, Protoporphyria.
b) Hepatic porphyrias:
Eg, Acute intermittent porphyria, Porphyria Cutanea Tarda, Hereditary Coproporphyria
and Variegate Porphyria
Biochemical causes of major signs and symptoms of porphyrias:
• The symptoms in different porphyria depend on the site of enzyme defect. If the
enzyme lesion occur early in the pathway (like in AJP), the major symptoms are
abdominal pain, neuropsychiatric symptoms etc due to the accumulation of ALA
and PBG. These patients do not develop photosensitivity. This is because these patients
do not accumulate porphyrins and porphyrinogens.
• On the other hand, if the enzyme lesion occurs later in the pathway, the major
symptom is photosensitivity due to the accumulation of porphyrins. Porphyrins,
when exposed to visible light (of wavelength of 400nm), are believed to become
excited and react with oxygen to produce reactive oxygen radicals, which injure
lysosomes and other organeIJes. The damaged lysosomes release their degradative
hydrolases, which causes skin lesions.
• In Hereditary coproporphyria and Variegate porphyria, in addition to

photosensitivity, abdominal pain and neuropsychiah·ic symptoms are also seen. This
is because, in addition to the accumulation of corresponding porphyrins, ALA and
porphobilinogen are also accumulated. (Reason: Deficiency of heme stimulates the
ALA synthase activity, leading to elevated ALA and porphobilinogen). Accordingly,
these patients exhibit the signs and symptoms of A[P along with photosensitivity
due to coproporphyrin and uroporphyrin.
Acquired (toxic) Porphyria:

This result from exposure to toxic compounds such as hexachlorobenzene, lead, heavy
metals etc. Heavy metals inhibit several enzymes in the heme synthesis such as ALA
dehydratase, uroporphyrinogen I synthase and ferrochelatase.

Hemoglobin 568
Heme catabolism (or formation of bilirubin):

Bilirubin is the end product of heme catabolisrn. Herne obtained from heme proteins
(mainly h emoglobin) is first degraded to form biliverdin by heme oxygcnase enzyme.
Biliverdin is th en converted to bilirubin by biliverdin reductase enzyme.
This takes place in the reticuloendothelial tissues of liver, bone marrow and spleen.
Heme
l Heme oxygenase
Biliverdin
l Biliveniin reductase
Bilirubin
Transport of bilirubin in plasma: The bilirubin that is formed from heme is not soluble
in plasma. Hence it is transported in the p lasma after binding to albumin . This albumin
bound bilirubin is known as unconjugated bilirubin.
Fate of Bilirubin:
• Unconjugated bilirubin is taken up by liver. The bound albumin detaches from
bilirubin.
• In liver, u n conjugated bilirubin is conjuga ted with glucuronic acid to form
conjugated bilirubin (Bilirubin monoglucuronide and Bilirubin diglucuronide) by
UDP-Glucuronosyl transferase enzymes. This process is called conjugation.
UDP-Glucuronosyl transferase
Bilirubin - - - -- -7--=--=~~
- - •• Bilirubin monoglucuronide
UDP-Glucuronic acid UDP
Bilirubin monoglucuronide / <:...::::: •

UOP-Glucuronosyl transferase
Bilirubin diglucuronide
UDP-Glucuronic acid UDP
• This conjugated bilirubin is then secreted from liver into bile, which passes through
bile duct into the intestine.
• In intestine, bilirubin is converted to urobilinogen (UBG) by bacterial enzymes.
• Under normal conditions, most of these urobilinogens are oxidized to urobilins (colored
compounds) and excreted in the feces. (Some authors refer these as stercobilins).
• A part of the urobilinogen is reabsorbed and re-excreted through the liver to constitute
the enterohepatic urobilinogen circulation. A small amount of UBG is also excreted
in urine (About 4 mg / day).

Hemoglobin 569
Clinical significance: Hyperbilirubinemia and Jaundice (Icterus):

• Normal serum total bilirubin Level is 0.2 to 1.0 mg / dl.
• Normal serum unconjugated bilirubin Level is 0.2 to 0.8 mg / dl.
• Normal serum conjugated bilirubin Level is 0.0 to 0.2 mg / dl.
A rise in total serum bilirubin level above 1 mg/ di is termed as hyperbilirubinemia.
When the bilirubin exceeds beyond a certain limit (normally above 2-2.5 mg/ di), it
diffuses into the tissues like sclera, conjunctiva, cornea, skin, mucous membrane etc,
causing the yellow colorization of these tissues, a condition called as Jaundice or lcterus
Jaundice can be defined as a clinical condition characterized by hyperbilirubinemia and
yellow colorization of sclera, conjunctiva, cornea, skin, mucous membrane etc due to
the deposition of bilirubin in these tissues.
Classification of Jaundice:
Jaundice is not a disease, but a clinical condition or symptom. It is caused due to
multiple factors. It is complicated to classify jaundice because of its multifactorial causes.
However, for the sake of convenience, jaundice can be classified into 3 types.
Jaundice may be broadly classified into three types:
a) Prehepatic jaundice (Generally called hemolytic jaundice as hemolysis is the main cause):
Prehepatic jaundice is caused mainly by excessive destruction of erythrocytes.
Causes: Excessive hemolysis due to the conditions like incompatible blood transfusion,
malaria, sickle cell anemia. Also, Gilbert syndrome, Criggler-Najjar syndrome etc.
b) Hepatic jaundice:
Hepatic jaundice is caused by dysfunction of the liver due to the damage to liver cells.
Causes: Damage may be caused by viral infection (viral hepatitis), toxins (chloroform,
carbon tetrachloride, acetaminophen etc- causing toxic hyperbilirubinemia & Jaundice).
c) Posthepatic jaundice (Generally called obstructive jaundice as obstruction is the main cause):
Posthepatic jaundice is caused mainly due to the obstruction of bile duct that prevents
the passage of conjugated bilirubin from liver to intestine.
Causes: The obstruction of bile duct due to cholelithiasis (gall stone) and tumors in the
adjoining tissues/ organs (carcinoma of head of pancreas).
Intra-hepatic cholestasis can also cause obstructive jaundice.
Neonatal "Physiologic Jaundice":
This is a temporary condition seen all newborn infants, caused due to an increased hemolysis,
due to immature hepatic system for uptake & conjugation of bilirubin & b0cause low acti\ ity
of UDP-glucuronyl transferase in newborn<;. This condition becomes normal within 2 weeks
& it does not require any treatment. However, sometimes plasma level exceeds 20 mg/ dl
(most of it is unconjugated), which can penetrate the blood brain barrier. This result in
hyperbilirubinemic toxic encephalopathy or "Kernicterus" that causes mental retardation.
Phototherapy (exposure of neonates to blue light that converts unconjugated bilirubin into
easily excretable forms) & Administration of Phenobarbital (wh ich induces bilirubin
metabolizing system in liver) is effective in the treatment of this disorder.

Hemoglobin 570
Congenital hyperbilirubinemia
Elevation in serum bilirubin level due to genetic defects is termed as congenital
hyperbilirubinemia. It results from abnormal uptake, conjugation or excretion of
bilirubin due to inherited defects. Some of these are explained here,
1. Gilbert disease: It is an Autosomal dominant trait. The defect is uptake of bilirubin

by the liver or an impairment in conjugation due to reduced activity of UDP-glucuronyl
transferase. Bilirubin level may be up to 3 mg/ dl leading to mild jaundice. Otherwise,
patient is asymptomatic. lt is more common among males.
2. Crigler- Najjar syndrome: These are due to conjugation defects. There are 2 types,
Type 1 (Congenital nonhemolytic jaundice): It is due to deficiency of UDP gluconyl
transferase enzyme. Jaundice appears within first 24 hr of life & results are fatal due
to kernicterus, where bilirubin exceeds 20 mg / dl. Children die before the age of 2.
Type 2: It is due to partial deficiency of UDP gluconyl transferase. It is a milder form,
where bilirubin does not exceed 20 mg/ dl. These respond to large dose of phenobarbital.
3. Dubin- Johnson syndrome: It is an inherited autosomal recessive trait, resulting in

the defective secretion of bilirubin into bile and increased conjugated bilirubin in blood.
The conjugated bilirubin gets deposited in liver causing blackish discoloration of it (So,
this condition is also called black liver jaundice).
Diagnosis of jaundice:
Serum bilirubin (both conjugated and unconjuga ted) and urinary bilirubin (bile pigment)
and urobilinogen are used in the differential diagnosis of jaundice.
a) Prehepatic jaundice:
Elevation of unconjugated bilirubin in serum and increased UBG in urine.
b) Posthepatic jaundice:
Increase in conjugated bilirubin in serum and increased bilirubin & bile salt in urine.
c) Hepatic jaundice:
An increase in both the conjugated & unconjugated bilirubin in serum and increased
bilirubin & bile salt in urine.
Tests:
• Serum bilirubin levels (unconjugated & conjugated) are measured by van den Bergh reaction.
• Urinary UBG is measured by Ehlrich's test.
• Urinary Bilirubin is measured by Fouchet's test.
• Urinary bile salt is detected by Hay's test.

Hemoglobi n 571
Question bank on Hemoglo bin

Essa y ques tions (10 Marks) :
1. Discuss the formation of Heme. Add a note on porphyria
Short essa ys (5 Marks) :

1. Porphyria
2. Abnormal Hemoglobin
3. Catabolism of Hemoglobin
4. Define Jaundice and classify them. Add a note on differential diagnosis.
Short answers (2 to 3 Marks) :

1. Sickle cell anemia and Thalassemia
4. Crigler-Najjar syndrome / Dubin Johnson syndrome / Gilberts disease
5. Individual porphyria like ATP / PCT / CEP

1) Iron is complexed in hemoglobin to (AIIMS, AI)
a) Leucine b) Histidine c) Isoleucine d) Valine
2) Hemoglobin electrophor esis is based on (AIIMS)
a) Molecular weight b) Charge c) Solubility d) Calorimetri c properties
3) Which of the following is a precursor of protoporph yrin- (AIIMS)
a) Alanine b) Leucine c) Histidine d) Glycine
4) Number of heme groups present in hemoglobin (AI)
a)I b)2 c)3 d)4
5) The oxidation of ferric (fe+2) iron to ferrous (Fe· 3) iron in Hb results in the formation of
a) Methemogl obin b) Carboxyhem oglobin c) Carbaminoh emoglobin d) Oxyhemogl obin
6) The rate limiting enzyme in heme biosynthesis is
a) ALA Dehydrates b) UPG decarboxyla se c) ALA Synthase d) one of these
7) Iron in Hemoglobin is held by which bonds
A) Polar B) non polar C) Co-ordinate D) Covalent
8) Life span of embryonic hemoglobin is
A) 3 months B) 9 months C) 3 weeks D) 9 weeks
9) Normal serum level of unconjugate d bilirubin is
a) 0.2-0.6 mg/di b) 0.0-0.2 mg / d i c) 1.0-2.0 mg / di d) 0.2-0.4 mg/dl
10) Acute intermitten t Porphyria is due to deficiency of
a) CPG III oxidase b) UPG Decarboxyla se c) PBG deaminase d) Ferrochelata se
Answe rs for MCQ: 1) b 2) b 3) d 4) d 5) a 6) a 7) c 8) a 9) b 10) b
I
j
Clinical Biochemistry 572
Clinical Biochemistry
Contents:
• Table of normal blood levels of Glucose, Urea, Creatinine, Bicarbonate,
Bilirubin, Sodium, Potassium, Calcium, Cholesterol, Proteins, Albumin and
Enzymes like AST, ALT, ALP, ACP etc
• Abnormal constituents of urine
• Functional tests:
Liver Function Tests (LFT)

• Liver functions
• Various liver function tests
• Differential diagnosis of Jaundice
Kidney Function Tests or Renal Function Tests (RFT)

• Kidney functions
• Various kidney function tests
• Clearance tests
Thyroid Function Tests (TFT)

• Thyroid functions
• Various thyroid function tests
Gastric Function Tests (GFT)

• Gastric functions
• Various gastric function tests
Reference range:
Reference range is a set of values of biochemical analytes that are used as standard values
to interpret the results obtained during various clinical biochemical investigations. It is
usually defined as the values of analytes that 95% of the population fall into.
Reference range can be statistically obtained by measuring the analytes level in the
population and taking two standard deviations (SD) either side of the mean.

Normal levels of biochemical constituents in Blood /Plasma/ Serum
CONSTITUENT UNITS
Glucose (Fasting) (B) 60 - 100 mg/ dL
Glucose (Fasting) (P) 70-110 mg/dL
Total Bilirubin (S) Up to 1.0 mg/dL
Conjugated bilirubin Up to 0.2mg/d L
Unconjugated bilirubin Up to 0.8 mg/dL
Calcium, Total (S) 9-11 mg/dL
Urea (B) 12-36 mg/dL
Uric Acid (S) 2-6 mg/dL

Creatinine (S) 0.7 -1.5 mg/dL
Total O10lesterol (S) 150 - 220mg/d L
HDL cholesterol > 60 mg/dL
LDL cholesterol <130mg/dL
VLDL cholesterol < 40 mg/dL
ALTorSGPT Up to 35 U/L
ASTorSGOT Up to 40 U/L
Alkaline phosphatase (S) 3 -13 Units (KA units/dL)
Acid phosphatase (S) 1 - 5 Units (KA units/d L)
CK-MB (S) < 25 U/L
Lactate dehydrogenase (S) 230-460 U/L
Total protein (S) 6-8 g/ dL
Albumin (S) 3.5 - 5.5 g/d L
Bicarbonate (P) 22-26 mEq/L
Sodium (P) 136 -145mEq/L ..
Inorganic phosphate (S), -

2.5 - 4.5 mg/dL
Chloride (S) 98-107 mEq/L

Abnormal constituents of Urine:

Many substances such as glucose, proteins etc are present in trace amounts in normal
urine, but they escape detection due to the low sensitivity of the methods employed.
The concentrations of these constituents in urine are increased markedly in different
pathological conditions. When these compounds are present in urine in detectable
amounts, they are called abnormal constituents.
1. Glucose in urine (Glucosuria):

Excretion of detectable amounts of urine is called Glucosuria.
Glucosuria (excretion of gl ucose) mainly occurs in diabetes mellitus and renal glycosuria.
Glucose (and other reducing sugars) in the urine are detected by Benedict's test.
Benedict's test (Semi-quantitative):
To 5ml Benedict's reagent, add 8 drops of urine. Mix and boil for exactly 2 minutes.
Depending upon the amount of glucose present, the following colors will be obtained.
Color Blue Green Yellow Orange Brick Red
Amount of Nil 0.5 1 1.5 2 or more

Glucose (gm %) - + (trace) ++ +++ ++++
Note: Benedict's test for is not a specific test for detection of glucose in urine. Benedict's test gives
a positive response for nil other reducing sugars like fructose, galactose, lactose, pentose and reducing
substances like ascorbic acid, glutathione, uric acid etc. So, result should be interpreted wit/1 care.
2. Proteins (Albumin) in urine (Proteinuria / Albuminuria):

Presence of detectable amount of albumin / protein in urine is characteristic feature of
kidney diseases such as nephrotic syndrome (and also in some types of nephritis),
where albumin is filtered at the glomerulus and lost in the urine.
Albumin/ protein in urine is detected by heat and acetic acid test.
3. Ketone bodies:
Presence of ketone bodies in urine is a characteristic feature of Ketoacidosis as in the
case of diabetes mellitus & starvation. Bile salts in urine are detected by Rothera's test.
4. Bile salts:
Presence of bile salts is suggestive of hepatic and obstructive jaundice. Bile salts in
urine are detected by Hay's test.
5. Bilirubin (Bile pigment):

Presence of bile pigments in urine is suggestive of hepatic and obstructive jaundice.
Bile pigments in urine are detected by Fouchet's test.
6. Blood:
Presence of blood is urine is characteristic feature of glomerulonephritis. Blood in
urine is detected by Benzidiene test.

Liver function tests:

Liver plays an important role in various metabolisms. Liver function tests are the
group of tests, which are done to assess the functions of liver.
The liver function tests are classified based on the major functions of liver such as
excretory, secretory, metabolic, and detoxifying functions.
I. Tests based on excretory functions :
Measurement of bilirubin and urobilinogen in serum and urine:

Liver plays an important role in Bilirubin metabolism. So measurement of products of
bilirubin metabolism (i.e. bilirubin and urobilinogen) in serum and urine play an
important role in liver function tests.
a) Serum bilirubin :
Serum Bilirubin level is measured by van den Bergh Reaction.
Normal serum total bilirubin level is 0.2 to 1.0 mg/ dl

Normal serum unconjugated bilirubin level is 0.2 to 0.8 mg/ dl
Normal serum conjugated bilirubin level is 0.0 to 0.2 mg/ di
A rise in total serum bilirubin level above 1.0 mg/ dl is termed as hyperbilirubinernia.
Jaundice manifests only if the level goes above 2mg/ dl.
There are three types of Jaundice that can be diagnosed by serum bilirubin level.
i) Prehepatic jaundice:
Unconjugated bilirubin is elevated in prehepatic Jaundice (or Hemolytic Jaundice).
ii) Posthepatic jaundice:

Conjugated bilirubin is elevated in posthepatic jaundice (or obstructive jaundice).
iii) Hepatic jaundice:

Both conjugated and unconjugated bilirubin (biphasic response) is eleva ted in
hepatic jaundice.

b) Urinary bilirubin:
Note:
Only conjuga ted bilirubin can be excreted in urine because it is water-soluble. So
bilirubin is found in urine only when conjugated bilirubin level is elevated in the
blood, i.e. in obstructive jaundice and in hepatic jaundice.
Urinary bilirubin is measured by Fouchet's Test.
i) In prehepatic jaundice, unconjugated bilirubin increases, so it does not appear in

the urine.
ii) In posthepatic jaundice, the conjugated bilirubin increases, so it appears in the

urine.
iii) In hepatic jaundice, small amounts of bilirubin are excreted in urine.
c) Urinary urobilinogen (Urinary UBG):
Urinary UBG is measured by Ehlrich's Test.
Normal excretion is up to 4mg/ day.
i) Prehepatic jaundice: Bilirubin synthesis increases leading to increased UBG synthesis,

resulting in increased excretion of UBG in urine.
ii) Hepatic jaundice: Urinary UBG level is normal or decreased.
iii) Posthepatic jaundice: Bilirubin does not reach the intestine, and thus UBG is not
formed. So UBG is not found in urine.
d) Urinary bile salts:
Urinary bile salts detected by Hay's Test.

During obstructive jaundice and hepatic jaundice, bile salts are excreted in the urine.

Clinical Biochemist ry 577
Differntial diagnosi s of jaundice:

Performed by Serum and Urinary measurem ent of bilirubin and bile Salts.
IN BLOOD (SERUM) I URINE

Condition Total Unconjugated Conjugated UBG Bilirubin Bile Salt
Bilirubin Bilirubin Bilirubin
Normal 0.2 to 1.0 0.2 to 0.8 mg/di 0.0 to 0.2 Up to4
mg/di N IL NlL
mg/di mg/day
Prehepatic
(Hemolytic) i i Normal i NlL NIL
Jaundice
I lcpatic Jaundice (+) i
i i i ormal
i (+)
Po~t-Hepatic
(Obstructive) i Normal i(+-+)
i .J. i (+++)
Jaundice
II. Test based on the synthetic functions of liver :
a) Measurem ent of serum levels of proteins:

Almost all plasma proteins except immunoglo bulins are synthesize d in liver. So, the
measurem ent of plasma proteins forms a reliable index of liver functions.
i) Normal Total Serum Proteins Level is 6 to 8 g / dL.
ii) ormal Serum Albumin Level is 3.5 to 5.5 g / dL.
In liver diseases, these level decreases.
b) Prothrombin time (PT):

The Ii\ er synthesize s Prothromb in. In the case of liver diseases PT is prolonged .
(Prolonged PT can also occur due to vitamin K deficiency. This can be ruled out by
estimating PT before and after administra tion of Vitamin K.)
III. Tests based on the metabolic functions of liver:

Galactose tolerance test:
Substances like galactose are exclusively metabolize d in Liver. This property is used
in LIT. Galactose is converted to glucose exclusively in Liver. Galactosc has a half-life
of 10 minutes. In LFT, around 30g of Galactose are given and capillary blood is
tested for galactose at 10 minutes intervals for 1 hour.
Half-life of Galactose increases during liver diseases.

IV. Tests based on the detoxifying function:

Liver is the major detoxifying organ in the body. This property is used in LFI'.
Hippuric acid test:

Benzoic Acid is detoxified into Benzoyl glycine (Hippuric Acid) in the liver.
When a dose of 6g of benzoic acid is given, the liver detoxifies it into hippuric acid,
which is excreted in the urine.
1n liver diseases, the detoxifying function is hampered and excretion of hippuric acid
in urine is reduced.
V. Tests based on the measurement serum enzymes:

There are two types of enzymes used in LFT's.
i) Enzymes Indicating liver damage (Hepatic jaundice);
AST and ALT, which are rich in liver. Normal levels of these enzymes are,
a) ALT (SGPT) • up to 35 U / liter of serum
b) AST (SGOT) • up to 40 U / liter of serum
These levels increase in liver damages (i.e. Hepatic jaundice).
ii) Enzymes indicating Obstructive diseases (Obstructive jaundice):
a) Alkaline phosphatase (ALP) - Normal serum level 3 - 13 KA units/ dl).
b) GGT (y-glutamyl transferase) - Normal serum level is 7 - 50 U / L
c) 5' - Nudeotidase - Normal serum level is 2- 17 U / L
These enzyme levels increase during posthepatic jaundice (or obstructive jaundice).
Significance of liver function tests:

1. Differential d iagnosis of Hepatic, Prehepatic and Posthepatic diseases.
2. To assess the severity of d iseases and also prognosis of these diseases.

Clinical Biochemi stry 579
van den Bergh reacti on :

Serum Bilirubin level is measured by van den Bergh Reaction.
In this test diazotize d sulfanilic acid reacts with bilirubin to form a purple colored
complex, azobiliru bin.
Normal serum does not give a positive van den Bergh reaction. In jaundice, there are
three types of responses.
i) Direct response: Conjugat ed bilirubin gives purple color immedia tely on mixing
with the reagent. The response is said to be direct positive Van den Bergh response.
Direct response is seen during posthepa tic jaundice.
ii) Indirect response: Unconjug ated bilirubin gives purple color only when alcohol
is added. (Indirect positive Van den Bergh response).
Indirect response is seen during prehepati c jaundice.
iii) Biphasic respons e: If both conjugate d and unconjug a ted bilirubin s are present,
a purple color is obtained immedia tely, and the color intensifies on adding alcohol.
(Biphasic van den Bergh response).
Biphasic response is seen during hepatic jaundice.
A: G ratio:
Normal total serum proteins level is 6 to 8 g/ dl.
Normal serum albumin level is 3.5 to 5.5 g/ dl.
ormal A : G ratio is generally is 1.5: 1 to 2:1.
Significa nce:
The A/G ratio is lowered either due to decrease in albumin or increase in globulins,
as found in the following conditions.
l. Decreased synthesis of albumin by liver usually found in liver diseases and severe
protein malnutrition.
2. Excretion of albumin into urine in kidney damage.
3. Increased productio n of globulins associated with chronic infections etc.
Alkalin e phosph atase (ALP):

Reference range 3-13 KA units/ 100 ml serum.
Alkaline phospha tase enzyme is moderat ely increased in hepatoce llular d isease
(hepatic jaundice ), but markedl y increase d in extra-he pa tic and intra hepa tic
cho lestasis (Obstructive jaundice).
The alkaline phosphat ase level also increases in bone diseases (like rickets and paget's
disease). So the ALP values should be carefully interpretated before coming to conclusion.

Renal Function Tests (RFT)or Kidney Function Tests:

Kidney is the major excretory organ of the body. It has various functions.
Major functions of kidney:
1) Urine formation and excretion of waste products of metabolism.
2) Regulation of water (fluid) and electrolyte balance.
3) Regulation of Acid base balance.
4) Hormonal function: Kidney is involved in synthesis of calcitriol, rennin and
erythropoietin.
Renal Function Tests (RFT)or Kidney Function Tests:

Renal functional tests are the tests to assess the normal functioning of a kidney.
There are two types of tests:

1) Test for glomerular function
2) Test for tubular function
I. Test for glomerular function:
Clearance tests :
Definition:
The clearance of a substance is defined as the volume of blood or plasma completely
cleared off that substance per minute.
Clearance tests measure the amount of a substance in urine as compared to the
concentration of the same substance in blood.
Clearance of a substance is given by formula,
lc;u;v I
Where,
U = Concentration of substance in urine
V = ml.of volume of urine excreted in a minute
P = Concentration of substance in plasma
Clearance value is expressed in ml/min.

3 types of clearance value can be obtained based on the nature of substance used;
1) When the substances are filtered by the glomerulus and is neither reabsorbed
nor secreted by the tubules:
Clearance of these substances give the GFR (Glomerular filtration rate) value.
E.g. lnulin and creatinine.
Values: Inulin and creatinine clearance = 120 ml/ min
Significance: These values decrease during glomerular dysfunction.
2) When the substance are filtered, reabsorbed but not secreted by the tubules:
Clearance of these substances w ill be less than GFR.
E.g.: Urea
Values: Urea clearan ce is 7Sml/ min as about 40% of urea reabsorbed by tubules.
Significance: Urea clearance is decreased d u ring renal damage (can be both
glornerulus dysfw1etion and tubular dysfunction).
3) When the substances are filtered and secreted but not reabsorbed:
Clearan ce of these substances w ill be more than GFR. Th ese values give the renal
plasma flow (RPF).
E.g.: PAH (Para amino Hippuric acid ).
Values: PAH (Para amino H ipp uric acid). Clearance value is about 700 ml/ min.
Significance: P AH clearance is decreased d u ring renal damage.
Comparison
Subst.inces Filtration Reabsorbed Secretion Oearance Value CV)
• lnulin
• Creatinine v X X
CV=GFR
(120 ml / mt)
• Urea v v ><
CV< GFR
(CV = 75 ml/ mt)
• PAH v X v CV>GFR
(CV= 700 ml/mt)
Note :
1) Urea clearance test is not as sensitive as creatinine clearance test as it is influenced by the protein
content ofdiet.
2) Clearance tests may indicate both glomeru/ar and tubular functions. But, generally, clearance tests are
performed to assess the glomerularftmction.

II) Test for tubular function:
A) Measurement of specific gravity (or Urine concentration test):

The simplest test for tubular function is the measurement of specific gravity of urine.
Normal range 1.012 to 1.024.
Significance: Specific gravity of urine decreases during tubular dysfunction because
water reabsorption by tubules is decreased.
B) Measurement of volume of urine :

Normal excretion is about 1.5 liters per day.
Significance: Volume of urine increases during tubular dysfunction.
Note:
• Increased volume of urine and increased specific gravity is also seen diabetes mellius
• Increased volume of urine and decreased specific gravity is seen in diabetes insipidus
Miscellaneous tests :
1) Check for abnormal constituents of urine:
Presence of some substances such as protein, blood etc. in urine indicates a possible
kidney disorders. These are,
a) Protein (albumin): The presence of detectable amount of albumin/protein
(albuminuria or proteinuria) is characteristic of kidney diseases (nephrotic syndrome &
some types of nephritis), where albumin is abnormally filtered at glomerulus and
excreted in urine.
b) Blood: (Hematuria): Presence of RBC in urine occurs during glomerulonephritis
and bleeding of urinary tract.
2) Estimation of constituents of blood/serum and urine :

Serum urea, creatine and uric acid levels are increased in kidney diseases.
1) Normal blood urea level is 12 - 36 mg/ dL.
2) Normal serum uric acid 2 - 6 mg/ dL.
3) Normal serum creatine level is 0.7 - 1.5 mg/ dL.
3) Renogram (using gamma camera) :

Renogram is done to test the extent of functionality of kidneys. Radio-pharmaceutical
substances specific to kidney is injected into the patient and its circulation (progress)
in the kidney is viewed and imaged. In this way one can check for obstructions or
damages in the nephron or kidney cells.

Creatinine clearance test:

Definition: Creatinine clearance test is defined as the volume (ml) of plasma that is completely
cleared off creatinine per minute.
Creatinine is metabolic waste product formed from muscle creatine phosphate. Its production
depends on total muscle mass of the body.
Creatinine is freely filtered by glomeruli and only marginally reabsorbed by tubules. So, creatinine
clear~ce value is close to GFR. lts values are constant and not influenced by body metabolism,
age, exercise or dietary factors. It depends only on renal functions, making it an ideal substance
for kidney function tests.
Test: Volume of 24 hour urine, creatine concentration of 24 hour urine & plasma are measured.
Creatinine clearance is measured by formula,
where, U = Concentration of creatinine in urine

C=UxV V = Volume of urine excreted per minute in ml
p P = Concentration of creatinine in plasma
Reference values: About 120 ml/ min (Close to GFR).
Significance: Creahnine clearance value decreases during glomerular dysfunction.
Urea clearance test:

Definition: Urea clearance test is defined as the volume (ml) of plasma that is completely
cleared off urea per minute.
Urea is the metabolic end p roduct of amino acid / protein metabolism. It is freely filtered by
glomeruli and about 40 % of it is reabsorbed in tubules. So, Urea clearance value is less than GFR.
Test: At 9 AM subject is given a cu p of water and asked to empty the bladder. This u rine is
discarded. After one hour bladder is completely emptied and volume is measured. Measure the
concentration of blood and urinary urea.
The urea clearance values are expressed in 2 ways, depending on the volume of urine excreted.
a) Maximum urea clearance: If the volume of urine is more than 2 ml/minute

Maximum urea clearance is measured by formula,
where, U = Concentration of urea in uri ne (in mg/ ml)
Cm=Ux V V = Volume of urine excreted per minute in ml
p P = Concentration of urea in p lasma (in mg/ ml)
Reference values: 75 ml/min
b) Standard urea clearance: If the volume of urine is less than 2 ml/ minute.
Standard urea clear ance is measured by formula,
where, U = Concentration of ureain urine (in mg/ ml)

Cs= U V = Volume of urine excreted per minute in ml
P = Concentration of urea in plasma (in mg/ ml)
p
Reference values: 54 ml/ min
Significance: Urea clearance va lue is a useful indicator of renal function. It decreases during
renal damage.

Thyroid Hormones:
Thyroid produces two thyroid hormones, namely, thyroxine (T4) and tri iodothyronine
(T3). T3 is functionally more active than T4 •
Functions of thyroid hormones:

• Thyroid hormones are required for the proper functioning of almost all the cells.
They are necessary for general development, tissue differentiation, genetic
expression, cellular metabolism of every ceJl of the body.
• Thyroid hormones increase the basal metabolic rate.
• Calorigenic effect or thermogenesis is the main effect of thyroid hormones. This
thermogen.ic effect is due to the uncoupling property of thyroid hormones in oxidative
phosphorylation. This heat energy is used in maintaining body temperature.
• Thyroid hormones are hyperglycemic hormones; they increase the blood glucose
level by facilitating the glucose absorption in the intestine.
Abnormalities of thyroid hormones:

Hyperthyroidism & hypothyroidism are 2 abnormal conditions of thyroid hormones.
1. Hyperthyroidism (thyrotoxicosis): Increased production of th yroid hormones.

Characteristics: Increased metabolic rate (High BMR), weight loss, nervousness, tachycardia,
irritability, anxiety and sensitivity to heat & often protrusion of eyeballs (exopthalmos) etc.
Types: Hyperthyroidism is of two types; primary and secondary.
i) Primary hyperthyroidism (Due to primary thyroid diseases):
• Grave's disease: (Elevated long acting thyroid stimulators or LATS, the thyroid stimulating
IgGs. LATS activates TSH, which stimulate thyroid hormone synthesis.)
• Toxic goiter
• Increased intake of thyroid hormones
ii) Secondary hyperthyroidism (Olher organ defects causing excess thyroid hormones):
• Due to increased in TSH due to TSH secreting tumors in pituitary glands.
2. Hypothyroidism: Associated with decreased production of thyroid hormones.

Characteristics: Decreased metabolic rate (Low BMR), weight gain, constipation, bradycardia,
sluggishness and sensitivity to cold, puffiness in the neck etc.
• Hypothyroidism in children causes cretinism.
• Hypothyroidism in adults causes myxocdema.
Types and causes: Hypothyroidism is of two types; primary and secondary.
i) Primary hypothyroidism (Due to primary thyroid diseases}:
• Dietary deficiency of iodine
• Hashimoto syndrome: An autoimmune condition affecting thyroid gland.
• Surgical resections of thyroid, 13 I ablation during treatment are other causes.
ii) Secondary hypothyroidism (Other organ defects causing decreased thyroid hormones):
• Due lo decreased TSH secretion by various diseases affecting pituitary gland.

Thyroid Function Tests (TFT)

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

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

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

2) Thyroid uptake studies:

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

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

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

and anti nuclear antibodies are seen in circulation.

5) Miscellaneous tests:
There are some tests that are not used in TFfs nowadays. These have historical importance.
a) TRH response test:

Patient is administered with TRH and T3 and T4 levels in serum are measured.
Normal response: TRH administration will stimula te the production of TSH and if
hypothalamus-pituitary axis is normal T3 and T4 production will be increased.
Significance:
• Hyperthyroidism: Negative feedback effects of T4 overrule the stimu lator effect of TRH.
So there will not be any further increase in thyroid hormone production.
• Hypothyroidism: In primary hypothyroidism, T3 and T4 level will increase after TRH
administration, but in secondary hypothyroidism (due to hypopituitarism), no response
is seen when TRH is given
b) T3 resin uptake test:

• T3 Resin uptake test measures the free binding site on thyroid binding globulin.
• Nowadays, free hormones can be easily measured by ELISA method, hence, this test not
used routinely. This test has only historical importance.
Method: Radioactive Iodine labeled T3 is added to the patient's serum taken in a test tube.
The labeled T3 will occupy the free binding site on TBG. Then a resin is added to the test tube
which absorbs the excess of labeled T3 • The amount of labeled T3 absorbed by the resin (T3
resin uptake) is inversely proportional to the free binding site on TBG and directly proportional
to the amount of thyroid hormones (T3 and T 4}.
Normal values: 25 to 30%.
Significance:
• Hyperthyroidism: Free binding site on TBG will be low.
• Hypothyroidism: Free binding site on TBG will be high.
Free T4 index (FTI):

Free T4 index (or FTI) can be calculated from the Total T4 & T3 resin uptake values.
Free T4 index or (ITI) = Total T4 X T3 resin uptake value.
Significance: FTI is a useful ind icator of thyroid functions. FTI increases in hyperthyroidism
and decreases in hypothyroidism.
c) Serum cholesterol level:

Normal serum cholesterol level is 150 - 220 mg/ di. Hypercholesterolemia is seen in
Hypothyroidism . This test does n ot have much of diagnostic importance as
hypercholesterolemia is also seen in diabetes, hypertension, obstructive jaw1dice etc. But,
this test has prognostic value.
d) Measurement of BMR and serum CPK:

Measurement of BMR & serum CPK are some of the other tests performed in TFTs. But these
are not used in TFfs nowadays. However, these have prognostic va lues.

Gastric Function
Gastric contents are mainly gastric secretion (gastric juice) and sometimes also food.
Gastric Secretion
Daily secretion of gastric juice by stomach in normal subjects is about one liter when
fasting and 2 - 3 liters when taking normal diet.
Composition and functions of gastric juice:
The gastric mucosa has various types of cells,
• The parietal or oxyntic cells which secrete HCI

• The chief cells or peptic cells tha t secrete enzymes
• The m ucous secreting surface epithelial cells
Constituent Secreted by Function
1. Provides optimum pH (1 - 2) for the

action of pepsin.
2. Activates pepsinogen to pepsin.
Parietal cells
HCl 3. Helps in the digestion of proteins by
(Oxyntic cells)
denaturating them.
4. Kms pathogenic microorganisms present
in the food.
Pepsinogen is converted to pepsin in the

stomach. Once formed, this pepsin
Pepsinogen Chief cells catalyses the activation of other
pepsinogen to pepsin (autocatalysis).
Pepsin is a protein digesting enzyme.
Castle's Intrinsic It is a glycoprotein that is required for the

Parietal cells
factor absorption of vitamin 812.
Gastric Protects gastric epithelium from the
Gastric mucus actions of HCl and pepsin.
epithelial cells
Rennin - Helps in the digestion of casein in infants.

Gastric Function Tests:

Gastric function tests involve chemical analysis of gastric contents for diagnosis and
assessment of diseases of stomach and duodenum.
Indications for gastric function tests:

• Diagnosis and assessment of diseases like- gastric ulcer, duodenal ulcer, pernicious
anemia and Zollinger-Ellison syndrome (gastrinoma of pancreas) etc.
• It is also useful in checking the completeness of surgical vagotomy.
Collection of contents:
Gastric contents are collected after introducing a tube (usually Ryle's tube) into the
stomach by nasogastric route (by swallowing) and removing the contents by aspiration.
Principle and Method
There are two kinds of gastric samples that are analyzed for gastric contents -
1) Gastric contents during resting period
2) Gastric content after stimulation.
There are two types of analysis done with both the kinds of gastric sample -
1) Physical analysis
2) Chemical analysis {Again, chemical analysis are of 2 types, qualitative and
quantitative).
Analysis of Gastric contents
f
During resting period After stimulation
I
{ \ / \
Phy~ical analysis Che mical analysis Physical analysis Chemical analysis
Qualitative Quantitative
i
Qualitative
\
Quantitative

I. Analysis of Resting Content:
Resting contents is the gastric contents that are collected in the morning after overnight
fast, therefore, it is also referred to as fasting contents.
Both physical and chemical analysis are done on the sample.
1. Physical Analysis:
This includes noting the volume, color and consistency of the gastric contents.
• Volume: Normal volume of the resting contents is 20 to 50 ml.
• Color: Most often the resting content is clear or colorless. (Sometimes it may be
slightly yellow or greenish due to regurgitation of bile from duodenum.)
• Consis tency: The normal resting gastric juice is fluid in consistency.
2. Chemical Analysis:
This includes both qualitative and quantitative analysis of the gastric contents.
• Qualitative analysis includes tests for blood, bile and starch (food).
Normal gastric juice does not contain any blood, bile or food.
• Quantitative analysis includes determination of acidity-free and total acid ity.
Acidity of the Gastric Juice - Free Acid, Combined Acid and Total Acid
The gastric juice contains free acid and combined acid. The sum of free and combined
acid is referred to as the total acid.
a) Free acid: HCl present in the gastric juice contributes to the free acid.
b) Combined acid: The combined acid is the weak acids present in the gastric juice.
Organic acids (such as lactic acid and butyric acid) and protein hydrochloride contribute
to the combined acid.
c) Total acid = Free acid + Combined acid
Normal Values:
• Free acid: 0 to 30 m Eq/L
• Total acid : 10 to 40 mEq/ L (about 10 mEq/ L higher than free acid).

II Analysis of Gastric Juice after Stimulation:
There are several types of stimulations employed, some of them are,
• Fractional test meal (FTM) employs food as stimulant for gastric secretion.
• Augmented histamine test employs histamine as the stimulant.
• Pentagastrin test employs Pentagastrin as the stimulant.
Fractional Test Meal (FfM):
Procedure:
This test is done in the morning on the subject after an overnight fasting of 12 hours.
1. Collection of fasting (resting) gastric contents and its analysis : The contents
of the stomach are wi thdrawn completely using Ryle's tube and the volume is measured.
This sample is known as fasting gastric juice ((As discussed before.)
2. Ingestion of test meal: 500 ml of test meal is given & the time is noted. (Commonly
used test meal is oatmeal prepared by adding 2 tablespoons of oatmeal in water.)
3. Collection of post-meal samples : After the ingestion of the test meal about 10
ml of gastric contents are removed by means of syringe a ttached to the RyJe's tube
every 15 minutes for 2½ hours.
4. Analysis of the samples : Each sample is analyzed both physically and chemically.
That is, it is analyzed for volume, color, consistency, blood, bile and food.
The free and total acidity of the samples are also estimated in the same way as done
with the resting gastric contents (see above under analysis of the resting gastric contents).
A graph is plotted with acidity levels against time.
Interpretation of FfM
Normal response:
• Normal samples do not contain any b lood, bile or food.
• Maximum acidity is reached at about 15 minutes to 30 minutes after the meal.
• The maximum free acid ranges from 15 to 45 mEq/ liter and the maximum total
acidity ranges from 25 to 55 mEq/liter.

Abnormal responses
There are 3 types of abnormal responses-hyperacidity (hyperchlorhydria), hypoacidity
(hypochlorhydria) and achlorhydria.
1. Hyperchlorhydria
It is the condition in which the maximum free acidity exceed 45 mEq/ 1.
Causes:
Hyperchlorhydriajs found in duodenal ulcer, in about 50% of gastric ulcers, gastric
carcinoma, gastroenterostomy, gastric necrosis, pyloric stenosis etc.
2. Hypochlorhydria
Tt is the condition in which the maximum free acidity is less than 15 mEq/ 1.
Cau ses:
Hypochlorhydria is seen in gastric ulcer, gastric carcinoma, and chronic gastritis and in
old age.
3. Achlorhydria
It is the condition in which there is no secretion of HCl.
Causes:
Achlorhydria seen in gastric carcinoma, chronic gastritis, partial gastrectomy, pernicious
anemia and sub-acute combined degeneration of spinal cord etc.
In pernicious anemia and sub-acute combined degeneration of spinal cord, there is
complete absence of both HCl and pepsin indicating complete absence of gastric secretion
(achylia gastrica).
True achlorhydria (seen in pernicious anemia and sub-acute combined degeneration of
spinal cord) can be differentiated fromhypochlorhydria by stimulation test with histamine
(augmented histamine test).
Interpretation of Physical and Qualitative Tests,
• Volume of the gastric contents increases in pyloric obstruction.

• Food particles may be seen in pyloric obstruction.
• Blood may be present in gastric ulcer and gastric carcinoma.
• Food (test for starch) may be present in cases of pyloric obstruction.
• Bile is found in duodenal obstruction.

Augmented Histamine Test

Histamine is the most powerful stimulant for HCl secretion in the stomach. In the augmented
test, a higher dose of histamine is given compared to that given in the normal histamine test.
Procedure:
1. After an overnight fast, the fasting gastric content is aspirated completely and analyzed.
2. Gastric contents are further collected for next 1 hour (pre-histamine sample) & free acidity
is measured. (Halfvvay through this period an intramuscular injection of an antihistamine
drug like mepyramine meleate (50 - 100 mg) is given to prevent any side effect of histamine).
3. Histamine is then administered subcutaneously (0.004 mg histamine/ Kg body weight).
4. Gastric contents are aspirated for 1 hr (post-histamine sample) & free acidity is measured.
Normal levels: 1n normal conditions, the acid content in pre-histamine sample is upto 10
rnEq / hour and the post-histamine sample contains 10 to 25 mEq/hour.
Significance: Useful in the diagnosis of hyperchlorhydria, hypochlorhydria & achlorhydria.
• True achlorhydria (seen in pernicious anemia and sub-acute combined degeneration of spinal
cord) can be differentiated from hypochlorhydria by augmented histamine test. In pernicious
anemia and sub acute combined degeneration of spinal cord, there is a lack of response to
histamine, that is, there is no increase in acid secretion in the post-histamine sample.
• The test is a guideline to the surgeon to limit acid secretion by vagotomy.
Pentagastrin stimulation test:

Pentagastrin stimulation test employs pentagastrin as the stimulant of gastric secretion.
The gastric juice is aspirated through a Ryle's tube in the fasting condition (Residual juice).
The gastric juice secreted for the next one hour is collected as a single sample (Basal secretion).
The gastric secretion is now stimulated by giving pentagastri.n. Pentagastrin is given in a
dose of 6 mg/ kg body weight. Gastric secretion is collected every 15 minutes for next 1 hour.
Normal Values and significance:
Basal acid output (BAO) is the acid output in milli.mol per hour, in the basal secretion (in the
absence of all intentional and avoidable stimulation).
Normal level: < 10 millimol / hour for men; < 5.5 millimol / hour for women.
Maximal acid output (MAO) is the acid output in millimol per hour, given by the sum of the
acid output of the four 15- minute samples after the stimulation.
Normal level: 7 - 45 millimol / hour for men; 5 - 30 millimol / hour for women.
Peak acid output (PAO) is the acid output in millimol per hour, given by the sum of acid
output of the 2 consecutive 15- minute samples having the highest acid content. The value is
multiplied by 2 to get the value for one hour.
Normal level: 12 -60 millimol / hour for men; 8 - 40 millimol / hour for women.
Significance:
1. Zollinger-Ellison syndrome: It results from a gastrin secreting tumor (gastrinoma of the
pancreas). There is no feedback regulation of gastrin secretion. There is very high level of
gastric acid output along with elevated serum gastrin levels. In this condition, BAO is > 15
mmol/hour (sometimes as high as 150 mmol/hour) and BAO/ PAO ratio is 0. 6 or higher.
2. Chronic duodenal ulcer: BAO, MAO, PAO are considerably increased. BAO may vary from
4-6 mmol/hr & BAO/PAO ratio of more than 0.3 indicates increased basal secretory drive.

Question Bank on Clinical Biochemistry
Essay Questions (10 marks):

1) Discuss in detail various liver function tests
2) Discuss in detail various kidney function tests
Short Essay Questions (5 marks):

I) Discuss in detail various thyroid function tests
2) Discuss in detail various gastric functi.on tests
3) Differential diagnosis of jaundice
4) Clearance tests
Short Answers (2-3 marks):

1) ormal level of any of the serum constituent
2) Creatinine clearance test / Urea clearance test / Inulin clearance test
3) A:G Ratio
4) van den bergh test

1) ormal GFR is
a) 150m1/min b) 120ml/min c) 220ml/min d) 75ml/ min
1) Normal urea clearance value is
a) 75ml / min b) 125ml/min c) ll0ml/min d) 120ml/ min
3) Cause of hemolytic jaundice
a) Malaria b) Cholera c) Viral hepatitis d) Carbon tetra chloride toxicity
4) Serum AST, ALT and ALP remain normal in
a) Hemolytic jaundice b) Paget's disease c) Obstructive jaundice d) Hepatic jaundice
5) Enzyme that increases in serum due to cholestasis
a) AST b) ALT c) ALP d) ACP
6) The following are the salient features of hyperthyroidism
a) Constipation b) Lethargy c) weight loss d) weight gain
7) Normal specific gravity of urine is

a) 1.000-1.1010 b) 1.012-1.1024 c) 1.025-1035 d) 1.035-1.1045
8) Normal serum creatinine level

a) 0.2-0.4 mg% b) 0.3-0.6 mg % · c) 0.7-1.4 mg% d) 1.4-2.8 mg %
9) Jndications of gastric juice analysis are all EXCEPT

a) Duodenal ulcer b) Alcoholism c) Zollinger Ellison syndrome d) completeness of vagotomy
10) Specific gravity of urine will be decreased in
a) Diabetes mellitus b) ephrosis c) Diabetes insipidus d) CRF
Answ ers for MCQ: 1) b 2) a 3) a 4) a 5) c 6) c 7) b 8) c 9) b 10) a
595
Case histor ies and data interpretation
• Knowled ge of biochem istry is essential to understa nd health and diseases. It

should be noted that all diseases have biochem ical basis. Applica tion of
Biochem istry in medicin e often involves biochem ical approac h to understa nd
the basis of these diseases in order to diagnose and effective treatmen t (therapy).
• The results of biochem ical investig ations carried out in clinical laborato ry
will help the clinicians in detectio n of diseases (diagnos is) and monitor ing the
treahnen t (prognosis). Its importa nt to know the reference values to understa nd
any laborato ry data.
• Reference range: Reference range is a set of values of biochemical analytes

that are used as standard values to interpre t the results obtained during various
clinical biochem ical investig ations. Values of specific analytes change in
particula r diseases.
Refer Page 573 for reference range.
• Some case histories with laboratory data are given here with answer keys

75
596
1) A patient h as flatulence and diarrhea following the ingestion of milk. What

is the most probable enzyme deficiency? Write the reaction catalyzed by the
deficient enzyme and the biochemical basis for the symptoms and management
of the patient.
Diagnosis: Lactose intolerance
Refer page 161 for detailed explanation
2) A 45 year male factory worker, known case of diabetes was brought to the
h ospital in comatose state. On examination, he had a ketotic breadth. What
could be the probable cause?
Explain the mechanism in detail.
Diagnosis: Diabetic Ketoacidosis
Refer page 206-208, 231 for detailed explanation.
3) A 40 year man who was on a hunger strike was brought to the hospital in
unconscious state. Blood sugar = 44 mg/ dl, blood pH= 7.21, serum bicarbonate
level = 15 mEq/ L Urine test for sugar was negative and ketone bodies were
positive.
What is the probable diagnosis?
Diagnosis: Starvation Ketoacidosis
Refer page 208, 231 for detailed explanation.
4) A 10 year old boy was brought to the hospital in a comatose state after a
bout of vomiting. Laboratory investigation revealed that is blood sugar was
300mg/ dl, blood pH was 7.2 and serum HCO3• was 20 mEq/ L. his urine showed
the presence of sugar and ketone bodies.
A. What is your probable diagnosis?
B. What are the type's causes of the above disease?
C. Describe the symptoms and the biochemical basis for the various symptoms
of the disease.
D. Name the biochemical test for long term monitoring and management of
this case. What is the normal value of the same?
Diagnosis: Diabetic Ketoacidosis
Refer page 206-208, 231 for detailed explanation.

597
5) Blood report of a 55 year old male obese patient who was being treated at
the OPD for an abscess, showed a random blood glucose level of 355 mg% &
glycated Hb of 10-18%
A) What is your probable diagnosis?
B) What is the significance of glycated Hb
Diagnosis: Diabetes mellitus

Refer page 206-208, 210 for detailed explanatio n.
6) A 2 year old child was brought to the hospital with vomiting and grossly
enlarged abdomen . She had a history of frequent episodes of weakness ,
sweating and pallor that subsided on eating. Lab investigat ion report revealed
low blood glucose, low blood pH, high lactate and Ketonuria . Liver biopsy
revealed large amount of glycogen.
B) What is the biochemical defect?
C) Why does glycogen accumulat e?
Diagnosis: von Gierke's disease

Refer page 190 for detailed explanatio n.
7) A 40 year old business man presented to out- patient departme nt with a

non-healin g wound in the right foot. He also complaine d of frequent urination
and excessive thirst and weight loss of one month's duration.
B) Enumerat e the functions of the hormone implicated in this disorder.
Diagnosis: Diabetes mellitus

Refer page 206-208, 210 for detailed explanatio n.
8) A 4 year old male patient presented with cutaneous xanthoma s on the exterior
surface of his arms, knee & elbows. Lab investiga tions showed a serum
cholesterol level greater than 600 mg/ dl and triglycerid e level of 170 mg/ dl.
Magnetic resonance angiogram (MRA) revealed narrowing of descendin g aorta.
The patient's family history was remarkab le in that both parents had high
cholesterol levels.
For queries and suggestions , contact the author at prasad_text@yahoo.co m or 9986449575

598

B) Name the lipoprotein that transports cholesterol from liver to extrahepatic
tissues & discuss its metabolism
C) Indicate the rate limiting enzyme in cholesterol biosynthesis and its
regulation
Diagnosis: Familial Hypercholesterolemia and atherosclerosis
Refer page 241-242 for detailed explanation.
9) A 60 year old women was referred to a hospital with history of chest pain.
She was noted to have hypertension and her plasma cholesterol level was 410
mg/ dl with an increase in the concentration of LDL. Angiogram demonstrated
a narrowing of the right coronary artery
B) What is the normal serum cholesterol level?
C) Write a note on cholesterol synthesis and name of two hypocholesterolemic
drugs.
D) Discuss the metabolism of the lipoprotein which has a protective role against
this disorder.
D iagnosis: Hypercholesterolemia, atherosclerosis with probable myocardial
infarction
10) A 10 year old child was hospitalized with complaints of abdominal pain.
Laboratory reports indicated hematuria. Ultrasonography reports showed
bilateral calculi. Stone analysis report indicated the presence of cystine. Give
your probable diagnosis. Indicate the biochemical defect.
D iagnosis: Cystinuria
Refer page 266 for detailed explanation.
11) A full term baby, born after normal pregnancy developed severe bouts of
vomiting, grunting respiration and lethargy. He was unresponsive to stimuli.
Blood test details
Urea= 11 mg% (Reference range= 12-36 mg % ).
Ammonia= 468µg% (Reference range= 20-63 µg%).

599
The plasma levels of Citrulline and glutamine were grossly elevated and that
of argininosu ccinate level was decreased .
A) What is the provisiona l diagnosis?
B) What is the biochemi cal basis for increased Ci trulline, glutamine and
decreased argininosu ccinate?
Diagnosi s: Acquired Hyperam monemia and ammonia toxicity due to

citrullinem ia (a urea cycle disorder due to the deficiency of arginosuc cinate
synthetase enzyme
Refer page 254, 256-257 for detailed explanatio n.
12) A 50 year old man was referred to the casualty in a disoriente d state, with
4 week history of increasing abdomina l distension and jaundice of 2 weeks.
Following were the laboratory findings in his blood sample
Parameter Findings ormal values
AST 450 ill / L (3-35 ill /L)
ALT 500 ill/ L (4-40 ill/ L)
Urea 2.Smg/ dL (12-36mg/ dl)
Ammonia 150µg / dL (< 50 µg / dl)
i) Mention the cause for increased level of ammonia, AST and ALT in the blood.
ii) Name the transport form of ammonia in the blood.
iii) Write the reactions of transdeam ination.
iv) How is ammonia detoxified in the brain and liver?
v) Name the regulatory enzyme of urea cycle.
Diagnosis: Acquired Hyperamm onemia & ammonia toxicity due to liver failure
Refer page 254, 256-257 for detailed explanatio n.
13) A 20 year old man came to the emergenc y room with sever lumbar pain.
Subseque nt examinati on and evaluation indicated a kidney stone and increased
excretion of cysteine, arginine and lysine in the urine.
i) What is the probable diagnosis?
ii) What is the cause for kidney stone?
iii) How is cysteine synthesize d from methionin e?
Diagnosis: Cystinuria
Refer page 266 for detailed explanatio n.

600
14) A 3 month old male infant was brought to casualty with history of vomiting .
The baby's mother gave history of lethargy and irritabil ity in the baby.
Biochemical investig ation revealed markedl y elevated plasma ammoni a levels.
A. name the probable disorder
B. name the transpor t form of ammoni a
C. Discuss urea cycle
D. Name any TWO disorder s of urea cycle.
Diagnos is: Ammon ia toxicity due to urea cycle disorder

Refer page 254, 256-257 for detailed explanat ion.
15) A 4 year old boy presente d with diarrhea and dermatit is to pediatric OPD.
Child was lethargi c and showed edema of face and pot belly. His Serum
Albumin level was l.8g/ dl.
A. Identify the nutrition al disorder .
B. Give reasons for the edema formation.
C. What is the dietary supplem entation required for this disorder ?
Diagnosis: Kwashior kor

Refer page 326-327 for detailed explanat ion.
16) A 6 year old boy was taken to the hospital with complai nts of decrease d
vision in the night. The doctor suspecte d a possible nutrient deficiency. Which
nutrient is that? Describe in detail the RDA, absorpti on and functions of the
deficien t nutrient .
Diagnosis: Vitamin A deficiency causing night blindnes s

Refer page 335-338 for detailed explanat ion.
17) A 5 year old boy was brought to the outpatie nt departm ent with bow legs
and pigeon chest. The biochemical findings are given below
Blood urea 16mg%
Serum creatinin e 0.6mg%
Serum calcium 7.0rng%
Serum inorgani c phospho rus l.8mg%
Serum alkaline phospha tase 720U / L (up to 462 U / L)
75
601
i) What is your probab le diagnosis?

ii) ame the causati ve factor and describe the synthes is of its active form
Diagnosis: Rickets
Refer page 339-341 for detaile d explanation.
18) A 58 year old female has a fractured toe that has not healed comple tely
even after three months . On examin ation hemorr hagic patches were seen on
her shins and gingiva e were swollen. She also had poor healing sore on her
body. Histological examin ation of sores reveale d extensive granula tion with
few collagen fibers.
A. What is your probab le diagnosis?
B. Give biochemical explana tions for the sympto ms
C. Add a note on the require ment, dietary sources and functions of the involve d
dietary compon ent.
Diagnosis: Vitami n C deficiency causing Scurvy

Refer page 344-345 for detaile d explanation.
19) A 53 year old grossly overwe ight women present ed with compla ints of
cramps and spasms of both hands. She had positive trousse au's and Chvost ek's
sign. Past medica l history reveale d thyroid ectomy for grave's disease . Her
laborat ory results were as follows
ormal levels
Serum calcium : 7.0 mg% 9-llmg %
Serum Phosph orous : 5.9 mg% 2.5-4.5mg%
Serum ALP : 60 IU /L 40-125 IU/L
i) Comm ent on the laborat ory results and give your probab le diagnosis.
ii) What is the role of PIH In calcium homeostasis?
Diagnosis: accidental removal of parathy roid gland during thyroidectomy. This

causes PTH deficiency and decreas ed serum calcium levels.
20) A 4 year old boy present ed with develop mental delay, irritability and self-
mutilat ing behavior. His serum uric acid level was 10.0mg / dL
or 9986449 575
602
i) What is the likely diagnos is?

ii) What is the biochem ical defect in this conditi on?
iii) Name the drug for treatme nt of hyperu ricemia and its mechan ism of action.
Diagnosis: Lesch Nyhan syndro me

Refer page 415 for detaile d explana tion.
21) A 15 year old boy compla ined of swellin g and pain in the distal phalang eal
joints. Blood investig ations showed the following results:
Fasting blood glucose : 75 mg%
Blood urea : 15 mg %
Blood uric acid : 60 mg %
On diagno sing the patholo gy, the physici an decide d to treat patient with
Allopu rinol
i) What is your probab le diagno sis in the above patient ?
ii) What other blood investig ations would you suggest ?
iii) Comm ent on serum uric acid level and explain the cause of pain in joints.
Diagno sis: Gout

Refer page 413-415 for detailed explana tion.
22) A 12 year old boy present ed at the dermat ology clinic with dry, pigmen ted
skin and fungati ng ulcer. He had always avoided exposu re to sunligh t. A sample
of skin was taken to a radiobi ology laborat ory, they found that tumor cell DNA
contain s excessive amoun t of thymin e dimers and it was still increas ing on
exposu re to sun light. What is you probab le diagno sis and comme nt on the
biochem ical defect involve d?
ii) Discuss DNA Repair mechan ism
D iagnosi s: Xerode rma pigmen tosa

23) Serum of a 56 year old woman showed extra band when subject ed to
electro phoresi s
i) What is your diagnos is?
ii) Name the bands in the electop horetog rarn.
or 9986449 575
For queries and suggestions, contact the author at prasad_t ext@yah oo.com
603
iii) Give the norma l levels of serum total protein , album in and globulins.
iv) Name the protei n that may be excreted in urine in the above condit ion
Diagnosis: Multip le myelo ma

Refer page 538, 543 for detaile d explanation.
24) The following are the results of arterial blood gas analysis:
pH: 7.32, HCQ3-: 15mm ol/L, pCO : 35 mm of Hg
2
A) What is the nature of acid base balance? Explain.
B) What are the causes of the above acid base disturb ance?
C) Name the major buffer system s of plasma .
D) Explain the role of lungs and kidney in buffering.
Diagn osis: Metabolic acidosis

Refer page 553-555 for detaile d explan ation.
25) A 12 year old boy presen ted to the hospit al with anemi a, jaundi ce and
recurr ent bone pain. Periph eral smear showe d numer ous sickled erythr ocytes
i) What is the probab le diagnosis? Explain the biochemical basis for cells and
anemi a
ii) Explain a test available to prove your diagno sis
Diagn osis: Sickle cell anemi a

Refer page 562, 97, 448-449 for detaile d explan ation.
26) A child compl aints of chills and high fever tested positiv e for malari
al
parasi te. The labora tory findin gs are given below.
Serum bilirub in 4mg%
Conju gated bilirub in 0.2mg%
Serum AST 25 U /L
Serum ALT 20 U / L
Bile salts and bile pigme nts are absent in the urine
A) Comm ent on the report
B) Name the test used to estimate serum bilirub in
Diagnosis: Hemo lytic jaundi ce

Refer page 569-570, 575-577 for detaile d explanation.
For queries and suggestions, contact the author at prasad_ text@y

ahoo.co m or 9986449575
604
26) A nine year old baby was admitte d to the hospita l with jaundice. On
admiss ion serum total bilirubi n was 16 mg/ dl. Baby was given phototh erapy
2A) Explain formati on and metabo lism of bilirub in
Diagno sis: Neona tal physio logic jaundic e where unconj ugated bilirub in
increases.
28) A patien t has come to the outpat ient depart ment with yellow ish
discolo ration of the skin and sclera. The physici an suspect s the patient has
viral hepatit is. What are the bioche mical investi gation s that would be
perform ed in this patient and what change s do you expect if it is viral hepatiti s?
Diagnosis: Hepatic jaundic e

Refer page 569-570, 575-577 for detaile d explana tion.
29) Blood report of a new born baby was as follows

Serum bilirubin: 14mg%, Serum direct Bilirub in: 0.5 mg%
A. Indicat e type of jaundic e
B. What is Kemict erus?
C. Discuss the formati on and fate of bilirub in
Diagno sis: eonata l physio logic jaundic e where unconj ugated bilirub in
increas es in neonates.
Refer page 569 for detaile d explanation.
30) The biochem ical investi gations in an adult patient with jaundic e are as
follows. Total bilirubi n -17 mg / dL, direct bilirubin-13 mg/dL , AST-6 6 U/L,
ALT-80 U/L, ALP-458 U/L
A. Indicat e the type of jaundice.
B. List the causes in the type of jaundic e.
C. How is bilirub in formed , transpo rted and excrete d?
0. Discuss van den Bergh's test.
Diagnosis: Obstru ctive jaundic e

Refer page 569-570, 575-577 for detaile d explana tion.
or 9986449575
For queries and suggestions, contact the author at prasad_t ext@yah oo.com
605
31) Liver function test of a 5 days old baby as follows

Total bilirubin: 7 mg% direct bilirubin: 0.4mg% indirect bilirubin: 6.6 mg%
A) What is your diagnosis?
B) Give the biochemical explanation for above condition?
C) Mention the treatment given for above condition and why?
Diagnosis: Neonatal physiologic jaundice where unconjugated bilirubin

increases in neonates.
32) A six year old girl with periorbital edema attended a pediatric OPD.
Laboratory investigation showed proteinuria, hypoproteinemia and
hyperlipidemia. What could be the diagnosis? What are the other causes of
proteinuria?
Diagnosis: Nephrotic syndrome

33) A 36-year-old man admitted to the hospital following episodes of nausea,

vomiting and malaise. On physical examination, his liver was found to be
enlarged.
Serum Findings:
Total bilirubin 9 mg/dl
Direct 3 rng/dl
Indirect 6 mg/dl
Total protein 6.5 gm/dl
Albumin 3.5 gm/dl
Globulins 3.0 gm/dl
ALT 1500 U/L
AST 400U/L
Alkaline phosphatase 19 KA units
Urine finding:
Bile salts Present (+)
Bile pigments Present (+)
Urobilinogen Normal
Diagnosis: Hepatic jaundice

Refer page 569-570, 575-577 for detailed explanation.

University Question papers with answer keys 606
University question papers

with
Answer keys
Dear students / teachers,

This section contains the previous University question papers from the new
revised syllabus (RS 2 Scheme and RS 3 scheme). This is probably the only
textbook which gives the answers close to all the questions from University
papers. Readers can corroborate this.
Answer keys for questions are also included to help the students to save their
precious time. Hope students would find this very useful during exams. This
section can be used as a last minute reading material during University
examinations.

Universit y Question papers with answer keys 607
M.B.B.S. PHASE- I Degree Examin ation- June 2014

Time: 3 Hours BIOCHEM ISTRY (RS-2 & RS -3) [M ax. Marks: 100]
PAPER I (Max. Marks: 50)
Long Essays 1Xl0=10 Marks
1. Classify amino acids based on their structure giving examples (Page 84-86)
Short Essays 5 X 5 = 25 Marks

2. Wha t is an allosteric en zym e? Describe with two exa mples (Page 139-140)
3. Discuss the Embden-m eyerhof pathway that occurs in RBC. Add a not on its energetics.
(Page 174-177)
4. Wha t are the lipoprotei ns? Name the lipoprotei n give their functions (Page 237-238)
5. What is biotransfo rmation? Discuss the different phases of biotransfo rmation with an
example (Page 516-519)
6. Describe the componen ts of ETC (Page 294-296)
Short Answers 5 X3 = 15 Marks

7. Pyruvate deh ydrogenas e com plex(Page 143, 179)
8. GABA, PUPA and SAM and their importanc e (Page 264, 283, 284)
9. Importanc e of iodine (Page 383)
10. FIGLU (Page 281)
11. Epime rism and Anome rism (Page 40-42)
PAPER II (Max. Marks: 50)

1. Define translation and describe steps of translation . ame four inhibitors and their action
(Page 437-442)

2. H ow is bilirubin formed and excreted ? Name the abnormal ities associated w ith bilirubin
metabolis m (Page 569-571)
3. Write the liver function tests based on syn thetic and excretory role (Page 575-577)
4. Write the Henderson-Hasselbalch equation an d its significan ce in bicarbona te buffer system.
(Page 547, 549)
5. Discuss the biochemical changes during starvation (Page 288)
6. What are nucleotid es? What are the sources of C and N atoms o f p urine n ucleotide? Mention
the metabolic disorders associated w ith purine meta bo lism (Page 406-407, 413-415)

7. Monoclon al antibodies. (Page 465-466)
8. PCR and its applicatio ns (Page 457-458)
9. Sou thern blotting technique s (Page 460-461)
10. "Gilberts' syndrom e". (Page 571)
11. Acute intermitte nt Porphyria (Page 567)
For queries and suggestions, contact the author at prasad_te xt@yahoo.com or 99864495
75
Univers ity Questio n papers with answer keys 608
M.B.B.S. PHAS E- I Degre e Exam inatio n- Dec 2013

T im e: 3 H ours BIOCH EMISTRY (RS-2 & RS -3) [Max. Marks: 100]
1X10= 10 Marks
Long Essays
ics and a m phibolic
1. Describ e Citric acid Cycle. H ow it is regu lated? Write about the energet
nature (Page 180-183)
5 X 5 = 25 Marks
Short Essays
2. Describe the fa te an d function s of Methion ine? (Page 263-264)
of this pathwa y
3. Wha t are the reaction s of HMP shunt pathwa y? What is the signific ance
(Page 191-194 )
a nd liver fun ction
4. Clinica l importa nce of the enzyme s in assessm ent of cardiac d isease
(Page 147-148, 578)
aving a
5. utlin e the d e nova synthes is of fatty acid. What are the advanta ges of h
O
multifu nction al emym e comple x? (Page 218-224, 143)
significance o f U rea
6. H ow is urea synthesi.led in the bod y? G ive the reactio ns . What is the
cycle (Page 255-257)
5 X3 = 15 Marks
Short A nswers
7. Phosph olipids (Page 73)
8. Coen zym es (Page 121)
9. Second a ry s tructure of protein s (Page 97-98)
10. Pro~tag landins (Page 67-68)
11. Transam i nation (Page 250-251)
PAPE R II (Max. Marks : 50)

Long Essays 1X10=10 Marks
1. Describ e the synthes is and breakdo wn of hemogl obin. Write a note
on Hemog lobinop athies
(Page 563-564, 568, 561-562)
5 X 5 = 25 Marks
Short Essays
(Page 113)
2. Explain the structu re of tR. A with diagram and mention its function
that effect BMR (Page 308-309)
3. Define BMR. How do you calculat e BMR? Discuss 4 factors
461-471 )
4. Describ e with an exampl e regulati on of gene express ion (Page
e (Page 486)
5. Wh al do you m~an by gene therapy ? Discuss its applicc1tion in medicin
a note on gout 412-414)
6. Describ e the cataboh sm of purme nucleot ide. Add
(Page
Short Answer s
5 X3 = 15 Marks
7. Antican cer agents (Page 410, 418)
8. Plasmid s and oncoge nes (Page 480, 450)
9. Obstruc tive jaundic e and diagnos is (Page 569 -570, )
10. Abnorm al constitu ents of urine (Page 574)
11. Radio isotope s (Page 500-501)
m or 9986449575
For queries and sugges tions, contact the author at prasad_ text@y ahoo.co
M.B.B.S. PHASE- I Degree Examina tion- June 2013

Time: 3 Hours BIOCHEMISTRY (RS-2 & RS -3) [Max. Marks: 100]

1. What are enzymes? Classify enzymes with one example each. Explain any four factors that
affect enzyme activity (Page 118, 123-124, 127-130))

2. Define gluconeogenesis. H ow is alanine converted to glucose? (Page 195-198)
3. What is subs trate level phosphoryl ation? Give 2 e.g.s w ith complete reaction (Page 184)
4. Classify lipoproteins and write the functions of each lipoprotein (Page 237-238)
5. What are tra nsamination reactions? Giving two examples discuss the importance of these
reactions (Page 250-251)
6. Classify proteins based on their function giving an example for each class (Page 95)
Short An swers 5 X3 = 15 Marks

7. High energy com pounds (Page 302)
8. Glycogen storage disorders (Page 189)
9. En zymes of d iagnostic imp ortance (Page 146)
10. FIGLU excretion test (Page 281)
11. Mitoch ondrial sh uttle systems (Page 300-201)
Long Essays 1.XlO=lO Marks

l. What are sources of C and atoms of purine? Describe the biosynthesis of p urine and add
a note on its regula tion (Page 407-410)

2. am e the different types of RNA. Write their salient feature, their function (Page 113-114)
3. What are buffers? Discuss any two buffer system of the body (Page 546-547, 549-550))
4. G ive the biochemical functions of niacin with examples an d manifestatio n of its deficiency
(Page 351-352)
5. What is restriction endonuclea se? Explain their role in recombinan t D A technology (Page
4 77-479)
6. Give an acco unt of phosphorous metabolism (Page 370-371)
S hort Answ e rs 5 X3 ::: 15 Marks

7. Post translationa l modifications. (Page 442)
8. Base pairing rule and Wobble hypothesis (Page 112, 115)
9. Biochemical defect in Thalassernia (Page 563)
10. Cause of Scurvy and Beri Bcri (Page 345,348)
11. Creatinine clearance test (Page 583)
Univers ity Questio n papers with answer keys 61 o
M.B.B.S. PHASE -I Degree Examin ation- Decem ber 2012

Time: 3 Hours BIOCHE MISTRY (RS-2& RS -3) [Max. Marks: 100]

Hpids.
1. Define Lipids. Classify lipids with an example for each . Mention the function s of the
(Page 59-61, 76)

2. Discuss the metaboli c changes during Diabetes Mellitus (Page 206-207)
3. Absorpti on and storage of Iron (Page 373-377)
4. ls Tyrosine an essential amino acid? Justify. Write the reaction s of three importan
t biological
products formed from tyrosine. (Page 267-270) ·
506-7)
5. Give a diagram matic represen tation of mechani sm of steroid hormone action (Page
(Page 18, 27-30)
6. Drawing a diagram of a cell & its organell es, explain the function s of each.

7. Glutathi one (Page 92)
8. Serotoni n (Page 278)
9. Give three enzymes and their therapeu tic uses (Page 148)
10. Ionopho res (Page 298)
11. Digestio n and absorption of disaccharides (Page 157-159)

1. Describe the synthesi s of pyrimidi ne and its regulatio n (Page 416-419)

. List
2. What is normal serum protein level? Mention three importan t function s of albumin
conditions w hen A/G ratio is altered. (Page 538, 540, 579)
3. Discuss the metabolic function of sodium and potassiu m (Page 398-401)
4. Clinical importan ce of AFP and PSA. Give their normal values (Page 453)
5. Give the key reaction of ren al mechani sm in acid base balance. (Page 551-552)
6. What is Go u t? G ive its clinical manifest a tions and line of treatmen t (Page
413-415)

7. Tumor Marker. (Page 453)
8. Four enzymes with copper as integral compon ent. (Page 378)
9. lmmuno globulins. (Page 542-543)
10. Cause for Xeroderm a pigment osa (Page 446)
11. SDA and BMR (Page 308-310)
575
For queries and suggest ions, contact the author at prasad_ text@yahoo.com or 9986449
University Questio n papers with answer keys 61 1
M.B.B.S. PHASE -I Degree Examin ation- June\ July 2012

Time: 3 Hours BIOCHE MISTRY (RS-2 & RS-3) [Max. Marks: lOOJ

1. Give an account of metaboli sm of phenylal anine. Add a note on disorder associate
d with
its metaboli sm (Page 267-272)

2. Cori cycle (Page 198)
3. What is Km? Mention its significa nce (Page 131)
4. What is meant by amphibo lic nature of citric acid cycle? (Page 182-183)
5. Explain enzyme specifici ties with example s (Page 125-126)
6. Transme thylation (Page 263-264)

7. Free radicals (Page 522-523)
8. Mitocho ndria (Page 29)
9. Steatorrhea (Page 166)
10. Hartnup s disease (Page 279)
11. Derivati ve of tryptoph an. (Page 277-278)

l. What is the normal pH of blood? Explain the mechani sm of how it is regulate
d?
(Page 548-550)
S ho rt Essays
5 X 5 = 25 Marks
2. Standard urea clearanc e test (Page 583)
3. Salvage pathway (Page 411)
4. Briefly outline the steps of De nova synthesi s of purine (Page 408-410)
5. Mention the sources, RDA and factors affecting calcium absorpti on (Page 365-369)
6. Deficien cy manifest ation of Vitamin A (Page 338)
Short Answers
5 X3 = 15 Marks
7. Menkes disease (Page 379)
8. Specific Dynamic action of food stuff (Page 310)
9. Types and causes of beri-beri (Page 348)
10. Function s of vitamin K (Page 343)
11. Function s of copper (Page 378)
or 9986449 575
Universit y Question papers w ith answer keys 612
M.B.B.S. PHASE- I Degree Examina tion- Dec 2011 I Jan 2012

Time: 3 Hours BIOCHEMISTRY (RS-2 & RS-3) [Max. Marks: 100)
1. Describe the reactions of Urea cycle. Discuss the interrelati on of Urea cycle and citric acid
cycle. What is the reference range of serum Urea (Page 255-257, 573)

2. Classify transport mechanism s across cell membrane s. Define uniport, symport and antiport.
Give an example of each . (Page 21-24 )
3. Define primary, secondary , Tertiary and Quaterna ry structure of protein. What are the
non-coval ent forces which preserve the secondary structure (Page 96-100)
4. Explain the mechanism of action of Allosteric Enzyme? Name the Allosteric Inhibitor and
A llosteric Activator for Phosphofr uctokinas e and Acetyl CoA-carboxylase. (Page 139)
5. Outline the steps for synthesis of cholesterol. Discuss the rate limiting step and regula tion
of synthesis of cholesterol (Page 234)
6. Describe the reactions of Citric Acid cycle. (Page 180-183)

7. Oncogene s (Page 450)
8. Thyroid function tests-Routine and anti-TPO (Page 586)
9. Cytochrom es (Page 299)
10. GIT Graph for Renal Glycosuri a (Page 210-211)
11. Anti-oxid ants (Page 523)

1. What is gluconeog enesis? Describe the pathway in detail and add a note on its significance
(Pag e 195-197)

2. Briefly outline the steps of De novo synthesis of purine (Page 408-410)
3. Hormona l regulation of Fluid and Electrolyte (Page 396-397)
4. Briefly explain the renal mech anism involved in maintenan ce of pH of blood (Page 551-2).
5. Sources, biochemical role and dietary requireme nt of vitamin A (Page 335-337)
6. Protein Calorie malnutriti on (Page 326-327)
Sh ort Answers 5 X3 = 15 Marks

7. Operon concept (Page 470)
8. Bence Jones proteins (Page 543)
9. What is Porphyria ? Mention the defect and signs and symptom s of acute intermitte nt
Porphyria (Page 566-568)
10. List 3 functions of liver & 3 tests with reference ranges to assess them (Page 575-6).
11. What is a Chimeric DNA? Give applicatio ns of recombin ant technolog y (Page 475,484).
For queries and suggestio ns, contact the author at prasad_ text@yah oo.com or 9986449575
M.B.B.S. PHASE- I Degree Examination- July 2011

Time: 3 Hours BIOCHEMISTRY (RS-2 & RS -3) [Max. Marks: 100]

1. Discuss the formation and fate of ketone bodies (Page 229-231)

2. Glycogenolysis (Page 187-188)
3. Decarboxylation of amino acids (Page 284)
4. Diagnostic uses of enzymes (Page 146)
5. Fatty Acid synthase comp lex (Page 219-224)
6. Detoxification by oxida tion (Page 517)

7. Free radicals and d isease (Page 522-523)
8. Active transport (Page 22-23)
9. Essential amino acids (Page 86)
10. Any three enzymes unique to gluconeogenesis (Page 195-196)
11. Uncouplers of oxidative phosphorylation (Page 298)

1. What is gen etic code? Describe the process of eukaryotic translation (Page 114, 437-442)

2. itrogen bal ance (Page 249)
3. Functions of albumin (Page 540)
4. Biochemical functions and deficiency m anifes tations of vitamin D (Page 340-341)
5. Functions of iodine (Page 382-383)
6. Structure of mRNA (Page 114)

7. Types and causes of beri beri (Page 348)
8. Causes of sickle cell anemia (Page 563, 449)
9. Immunoglobulin A (IgA) and immunoglobulin M (IgM) (Page 542-543)
10. Describe an y three renal function tests (Page 580-582)
11. Deficiency features of orotic acid uria. Explain biochemical basis of manifestation (Page

University Question papers with answer keys 61 4
M.B.B.S. PHASE-I Degree Examination- December 2010

Time: 3 Hours BIOCHEMISTRY (RS-2) [Max. Marks: 100]

1. Describe the metabolism of phenylalanine, tyrosine. Add a note tyrosinemia (Page 267-75)

2. Proto-oncogenes and oncogenes (Page 450-451)
3. Explain substrate level Phosphorylation (Page 184)
4. Rapaport leubering cycle (Page 178)
5. Define Km (Michaelis constant) of an enzyme. Write about its importance w ith a suitable
example (Page 131)
6. Compounds derived from cholesterol (Page 76)

7. Enumerate reactive oxygen species and their characteristics (Page 522-523)
8. What are Xenobiotics? What is the role of Glutathione in detoxification (Page 516, 92)
9. List metabolic functions and clinical significance of Lysosomes (Page 30)
10. Role of dietary fiber in Health and disease (Page 319)
11. What is the role of cytochror.1e P450 in detoxification (Page 517)

1. Explain the steps of activation, elongation, termination of protein synthesis (Page 437-42)

2. Explain the catabolism of purine (Page 412-413)
3. What is Anion gap? Explain normal anion gap acidosis and high anion gap acidosis with
examples (Page 554)
4. Radioactive isotopes of Iodine and their clinical application (Page 501)
5. What is the normal range of serum potassium and w rite about Hypokalemia (Page 401)
6. Mention five biochemical functions of pyridoxine in the body with examples (Page 354)

7. What is genetic code? Explain (Page 115)
8. Renal threshold for glucose and its significance (Page 203)
9. List Biochemical changes in protein - Energy Malnutrition (Page 326-327)
10. Absorption of Iron in the body (Page 373-377)
11. Explain the compensatory mechanisms in Metabolic acidosis (Page 553)

M.B.B.S. PHASE-I Degree Examination- June\July 2010

Time: 3 Hours BIOCHEMISTRY (RS-2 & RS-3) [Max. Marks: 100]

1. Discus in detail oxidative phosphorylation and enumerate its inhibitors (Page 293-298)

2. Glycogenolysis (Page 187-188)
3. Transmethylation reactions (Page 264)
4. Metabolism of chylomicrons (Page 237-238)
5. N on-com petitive enzyme inh ibition (Page 135-136)
6. Rapap ort leubering cycle (Page 178)

7. Nutri tional classification of amino acids (Page 86)
8. Significance of serum Amylase (Page 146)
9. Endoplasmic reticulum (Page 27-28)
10. Niemann p ick disease (Page 228)
11. Den aturation (Page 103-104)

1. Discuss the structure and replication of DNA (Page 111, 423-430)

2. Deficiency manifestations of Vitamin A (Page 338)
3. Protein energy malnutrition (Page 326-327)
4. Extracellular buffers (Page 348-549)
5. Functions of selenium (Page 384)
6. Acute phase proteins (Page 541)

7. Methemoglobin (Page 561)
8. Dietary fiber (Page 320)
9. Respiratory quotien t (Page 307)
10. Renal glycosuria (Page 203)
11. Lesch Nyhan syndrome (Page 419)
For queries and suggestions, contact the author at prasad_tex:t@yahoo.com or 9986449575

M.B.B.S. PHASE-I Degree Examination- December 2009

Time: 3 Hours BIOCHEMISTRY (RS-2 & RS-3) [Max. Marks: 100]

1. What is the normal fasting blood glucose level? Why does it need to be regulated? Describe
the various mechanisms of its regulation (Page 204-205)

2. Composition and function of any two phospholipids (Page 73-74)
3. Cell membrane (Page 19-20)
4. Essential amino acids (Page 86)
5. Enumerate ketone bodies. How they are formed? (Page 229-231)
6. Phenylketonuria (Page 271-272)

7. Formation of ammonia and its toxicity in brain (Page 250-254)
8. What is Zymogen? Give examples of zymogen (Page 141)
9. Diagrammatic representation of mitochondrial electron transport chain and location of
ATP formation sites (Page 294-297)
10. Mechanism of carcinogenesis (Page 447)
11. Give two examples of detoxification by oxidation and reduction (Page 517-518)
Long Essays lXl0=lO Marks

1. Describe the sources, functions, deficiency, manifestations and daily requirement (RDA)
of vitamin A (Page 335-338)

2. Regulation of blood calcium level (Page 365-369)
3. Catabolism of purines and related disorders (Page 412-416)
4. Post-transcriptional modifications (Page 436)
5. Acute intermittent Porphyria (Page 567)
6. Role of kidney in regulation of blood pH (Page 551-552)

7. What is complete protein? (Page 324)
8. Enumerate sources of atoms of purine ring by a diagrammatic representation (Page 407)
9. Give four characteristic feature of genetic code (Page 115)
10. What is recombinant DNA? (Page 475-476)
11. Clinical inte rpretation of TSH level (Thyroid Stimulating Hormone) in Blood (Page 585)

M.B.B.S. PHASE-I Degree Examination- July 2009

Time: 3 Hours BIOCHEMISTRY (RS-2) [Max. Marks: 100]

1. Trace the pathway of gluconeogenesis starting from alanine. Mention the key enzymes and
how they are regulated (Page 195-197)

2. What are the biologically important compounds derived from cholesterol? (Page 76)
3. Prostaglandins (Page 67-68)
4. Give four examples of transmethylation reactions (Page 264)
5. Maple syrup urine disease (Page 282)
6. Energy releasing steps of citric acid cycle (Page 181)

7. Name the two endopeptidases with their specifications (Page 167)
8. What are functions of apolipoproteins? (Page 239)
9. Give the significance of uronic acid pathway (Page 199)
10. Clinical .importance of transamination (Page 251)
11. Detoxification of alcohol (Page 517)

1. Give a detailed account of transcription process. How is it regulated? Name the inhibitors
of transcription (Page 431-436)

2. What are the sources, functions and daily requirement of vitamin A? (Page 335-338)
3. Transport proteins of blood (Page 538-540)
4. Formation and fate of bilirubin in the body (Page 569)
5. Basal metabolic rate (Page 308)
6. Bicarbonate buffer system of blood (Page 549)

7. Polymerase chain reaction (Page 457-458)
8. What is the daily requirement of Thiamine, Niacin an d pyridoxine? (Page 346, 351, 354)
9. Give enzyme defect in the following conditions (Page 194, 571)
a) Drug induced hemolytic anemia b) Crigler- ajjar syndrome
10. Creatinine clearance test (Page 583)
11. Give the normal blood level of the following (Page 573)
a) Fasting blood glucose b) total protein c) Urea d) Bicarbonate e) Sodium f) Potassium

M.B.B.S. PHASE-I Degree Examinat ion- January 2009

Time: 3 Hours BIOCHEMI STRY (RS-2) [Max. Marks: 100]

1. Explain in detail the ~-oxidation of palmitic acid with its energetics (Page 214-216)

2. Functions of carbohydra tes (Page 35)
3. Competitive inhibition and its importance in medicine (Page 132-134)
4. Metabolic changes in diabetes mellitus (Page 206-207)
5. Fatty liver and lipotropic factors (Page 245-246)
6. Disorders of sulphur containing amino acids (Page 266)

7. Mention two isotopes and mention their application in medicine (Page 501)
8. Mention four tumor markers with their significance (Page 453)
9. Role of cytochrome P 450 in detoxification reaction (Page 517)
10. Biologically important compounds derived from tyrosine (Page 268-270)
11. What is reactive oxygen species (ROS)? How are they formed? (Page 522-523,524)
Long Essays lXlO=lO Marks

1. Describe in detail biosynthesis of protein and discuss its regulation (Page 437-442)

2. Name 5 Heme proteins and their functions (Page 376)
3. Unconjugate d hyperbilirub inemia (Page 569-570)
4. Lesch - yhan syndrome (Page 415)
5. Tests based on metabolic and excretory function of liver (Page 575,577)
6. Application of recombinan t D A technology (Page 479)
7. What is the difference between endonuclea se and restriction endonuclea se? Give two
examples of restriction endonuclea sc (Page 477)
8. Deficiency manifestatio n of Vitamin A (Page 338)
9. Role of dietary fiber in the body (Page 320)
10. Biochemical role of pyridoxine (Page 354)
11. Name the trace element . Explain the biochemical role of any 2 trace elements (Page 363)
M.B.B.S. PHASE-I Degree Examination- July 2008

Time: 3 Hours BIOCHEMISTRY (RS-2) [Max. Marks: 100)

1. Describe TCA Cycle. Discuss in detail its energetics, regulation and its role (Page 180-183)

2. Secondary structure of proteins (Page 97)
3. Glycogenesis (Page 185-186)
4. Serotonin (Page 278)
5. Antioxidants (Page 523)
6. General mechanism of action of steroid hormones (Page 506-507)

7. PSA (Page 453)
8. Uncouplers of oxidative phosphorylation (Page 296-297)
9. Refsu m's disease (Page 217)
10. Significance of HMP path way (Page 193)
11. Rancidity (Page 71)
Long Essays lXlO=lO Marks

1. Discuss in detail recombinant DNA technology and its clinical application (Page 477-484)

2. Chloride shift (Page 550)
3. Functions and deficiency manifestations of vitamin C (Page 344-345)
4. Metabolic Acidosis (Page 553)
5. Degradation of pyrimidines (Page 419)
6. Salient features of genetic code (Page 115)

7. Carboxy hemoglobin (Page 561)
8. Fluorosis (Page 380)
9. Imm une electrophoresis (Page 497)
l 0. Anticoagulants (Page 55)
11. Limiting amino acid (Page 325)

M.B.B.S. PHASE-I Degree Examination- January 2008

Time: 3 Hours BIOCHEMISTRY (Revised Scheme II) [Max. Marks: 100]
PAPER I (Max. Marks: SO)

l. Define Isoenzymes. Mention the principles used for separation of lsoenzymes. Write about
the clinical importance of Isoenzymes (Page 144-145)

2. List the important products formed from Tyrosine and write the metabolic pathways leading
to the formation of any two of them (Page 268-270)
3. Mechanisms of action of Glucagon (Page 508)
4. Single electron carrier components of respiratory chain (Page 295)
5. Pyruvate dehydrogenase enzyme action and its biochemical importance (Page 179)
6. List various types of fatty acid oxidation. Write about activation of fatty acids (Page 213-7)

7. Functions of plasma membrane (Page 20)
8. Lipid peroxidation- clinical importance (Page 69)
9. Role of growth factors in carcinogenesis (Page 454,447)
10. Glucose 6 phosphate dehydrogenase deficiency (Page194)
11. Functional classification of proteins (Page 95)
PAPER II (Max. Marks: SO)

l. What is the importance of maintaining acid- base balance in the body? Write in detail ~ow
kidney helps in maintaining acid- base balance (Page 546-548,551-552)

2. Replication of lagging of lagging strand (Page 425,429)
3. List metabolic functions of Ascorbic acid. How do you detect its deficiency? What is the
daily requirement? (Page 344-345)
4. BMR ( Basal Metabolic rate) (Page 308)
5. Degradation of Heme (Page 569)
6. Gene therapy (Page 486)

7. Iodine metabolism (Page 382)
8. Importance of base pairing (Page 112)
9. Molecular defect in and consequences of sickle cell disease (Page 449,563)
10. Sources and beneficial effects of dietary fiber (Page 320)
11. What is reference range? (Page 572)

M.B.B.S. PHASE-I Degree Examination- October 2007

I. LONG ESSAY 2 X 10 = 20 Marks

1. Define glycogenesis and glycogenolysis. Write the reactions of glycogenesis and
glycogenolysis in liver. How are these two pathway reciprocally regulated? (Page 185-189)
2. What are nucleotides? Explain the catabolism of purine nucleotides. Write briefly on
metabolic disorders associated with purine metabolism. (Page 409-415)
II. SHORT ESSAY 10 X 5 = 50 Marks

3. Explain competitive and non competitive inhibition. Mention significance of competitive
inhibition. (Page 133-135)
4. What is fatty liver? Explain its causes (Page 245)
5. Give an account on organization of Electron Transport Chain(ETC) (Page 293-298)
6. Tumor markers (Page 453)
7. Explain the steps of urea cycle and mention the name of its order (Page 255-257)
8. How is bilirubin formed and detoxified in the body? (Page 561)
9. Explain metabolic role of calcitriol (Page 339-341)
10. What is plasmid? What is its application in recombinant DNA technology? (Page 480,484)
11. Give an account of storage and transport of iron (Page 373-377)
12. Explain metabolic and respiratory acidosis (Page 553,555)
III. SHORT ANSWERS 10 X 3 = 30 Marks

13. Explain enzyme profile in myocardial infarction (Page 147)
14. What is normal serum level of cholesterol? Name its four biological func;tions (Page 76,
232-234)
15. What are antioxidants? Name chain breaking and preventive antioxidants (Page 523)
16. Give metabolic functions of endoplasmic reticulum and mitochondria (Page 29)
17. Explain detoxification by conjugation with two examples (Page 519)
18. Which are inhibitors of protein synthesis? (Page 442)
19. Protein calorie malnutrition (Page 326-327)
20. Significance of non-protein nitrogenous substances (Page 574-578)
21. Functions of pyridoxal phosphate (Page 354)
22. Types of RNA and their function (Page 123-114)

M.B.B.S. PHASE-I Degree Examination- February 2007

Time: 3 Hours BIOCHEMISTRY (Revised Scheme II) Max. Marks: 100)

l. Explain the influence of various factors on enzyme activity (Page 127-130)

2. Urea formation in liver (Page 255-257)
3. Glycogen s torage diseases (Page 189)
4. Name any five products formed from Glycine (Page 258-259)
5. Fatty liver and lipotropic factors (Page 245-246)
6. Purine salvage pathways (Page 411)

7. Mechanism of oxidative phosphorylation (Page 296-298)
8. Functions of prostaglandins (Page 67)
9. Oncogenes (Page 450,451)
10. Mutarotation (Page 42)
11. Antioxidants (Page 523)

1. Write in detail about the distribution of calcium in the body, its functions and regulation of
serum levels (Page 365-369)

2. Chemistry, sources and daily requirements of folic acid (Page 356-357)
3. Renal mechanisms in acid base balance (Page 551-552)
4. Transcription (Page 431-436)
5. Genetic code (Page 115)
6. Catabolism of purines (Page 412)

7. Clearance tests (Page 583)
8. Functions of albumin (Page 540)
9. Catabolism of heme (Page 569)
10. Trace elements (Page 363)
11. Mention the normal levels of serum proteins, Urea and Creatinine (Page 524)

M.B.B.S. PHASE-I Degree Examination- October 2006


1. Discu ss the significance of multienzyme complexes w ith respect to carbohydrate and lipid
metabolism. (Page 179, 183, 219-220)
2. Name the coen zym e forms of vitamin involved in amino acid metabolism. Discuss the
biochemical functions and deficiency manifestation of these vitamins (Page 349-352,254)
II. SHORT ESSAY 10 X 5 = 50 M arks

3. How is ammonia detoxified in the body (Page 254-256)
4. Mechanism of oxid ative phosphorylation (Page 296-297)
5. Ketogenesis (Page 229)
6. Gluconeogenesis (Page 195-197)
7. Conjugation in detoxification (Page 519)
8. Clearance tests (Page 583)
9. Blood b uffers and their role in Acid- Base balance (Page 548-550)
10. Biological role of zinc in the body (Page 385)
11. PCR (Page 457-458)
12. BMR (Page 308)

13. Carnitine cycle (Page 216)
14. Biologically active peptides (Page 91)
15. Glycosaminoglycans (Page 54-55)
16. Vitamin-E (Page 342)
17. Methanol poisoning can be alleviated by ethanol, ju stify (Page 134)
18. 2,3, BPG (Page 178)
19. Cyclic Nucleotides (Page 110)
20. Insulin receptors (Page 512)
21. H ow bilirubin is made water soluble? (Page 561)
22. Bence- Jones proteins (Page 543)

M.B.B.S. PHASE-I Degree Examination- April 2006


1. Enumerate the compounds formed from glycine, giving their biological importance. Why
glycine is nutritionally non essential? (Page 258-261)
2. Give an account of the chemistry, sources, daily requirement of vitamin C. Enumerate its
biochemical functions and deficiency manifestations (Page 344-345)

3. Write steps of a-oxidation of fatly acids with energetic (Page 214-216)
4. Give outline of gluconeogenesis and mention it significance (Page 195-197)
5. What are isoenzyme and give their clinical importance citing two examples?
(Page 144-145)
6. What is P: 0 ratio? Which are inhibitors of electron Transport chain? (Page 299,296)
7. Write detail with examples reactions involved in detoxification (Page 516-519)
8. What is normal serum calcium level? Explain its regulation (Page 365-369)
9. Name the clearance tests used to assess kidney functions. Explain any one of the
Clearance tests and mentions its significance. (Page 580-581, 583)
10. Name the buffer system of body fluids, explain any one of them (Page 547-549)
11. Give an account of salvage pathway (Page 411)

12. Hemoglobinopathies (Page 562)
13. Mention the significance of HMP short pathway (Page 193)
14. Name essential fatty acids and mention their importance (Page 66)
15. What is Km value? Explain its importance (Page 131)
16. Name three important growth factors and mention their functions (Page 454)
17. Explain Troponin as a marker for myocardial infarction (Page 147)
18. What is Bence Jones protein? Mention its significance (Page 543)
19. Name bacterial DNA polymerase. Mention its significance (Page 427)
20. Distinguish between RNA and DNA (Page 111)
21. What is gene therapy? Name vectors used for gene therapy (Page 486)
22. Nutritional deficiency anemias (Page 329)
For queries and suggestions, contactthe author at prasad_text@yahoo .com or 9986449575

Universit y Question papers with answer keys 625
M.B.B.S. PHASE-I Degree Examina tion- April 2005

Time: 3 Hours BIOCHEM ISTRY (Revised Scheme II) [Max. Marks: 100)

1. Name the ketone bodies. Give 2 conditions characteri zed by excessive productio n of ketone
bodies. Explain the metabolic derangem ents and consequen ces of ketosis (Page 229-231)
2. Explain the steps of activation, initiation and elongatio n and terminati on of protein
biosynthe sis (Page 437-442)

3. Explain the role of hypoglycemic hormones (Page 204)
4. Metabolic functions of Cysteine (Page 263,265-266)
5. What is oxidative phosphory lation? Explain Chemiosm otic theory (Page 296-297)
6. Classify lipoprotei ns. Mention their functions (Page 237-238)
7. Therapeut ic uses of enzymes (Page 148)
8. Explain the metabolic role of thiamine and in deficiency manifesta tion (Page 4346-348)
9. What is anion gap? Explain normal anion gap acidosis and high anion acidosis with example
(Page 554)
10. Lesch- yhan Syndrome and orotic aciduria (Page 415,419)
11. What is m utation? Give an account on point mutation (Page 448-449)
12. What is nitrogen balance? Explain factors affecting Nitrogen balance (Page 249)
III. SHORT ANSWER S 10 X 3 = 30 Marks

13. Define oncogene. Give examples of oncogene (Page 450-452)
14. Name Mucopoly saccharide s, Mention their functions (Page 55)
15. Give brief account on quaternar y structure of protein (Page 100-101)
16. Define free radicals. How free radicals are generated in the body? (Page 522-523,524)
17. ame sub-cellular organelles . Which are metabolic functions of cytosol? (Page 27)
18. Degradati on of heme (Page 361)
19. Gout (Page 317-319)569
20. What is polymera se chain reaction? Mention it applicatio n (Page 5457-458)
21. What ate metabolic roles of zinc and selenium? (Page 385-384)
22. Which are biological ly important nucleotides? Mention their functions (Page 109-110)

75
Glossary 626
Index
A Aminopter in 134
Acetoaceta te 229-230 Ammonia Transport 254
Acetone 229-230 Amphipath ic lipids 77-78
Acetyl CoA 152,179-182 Amylase 156-157
Acetyl CoA carboxylas e 124,219 Amylopec tin 51-52
Acids, bases 546 Amylose 51-52
Acid Base balance Anapleros is 183
• Acids, bases, buffers 546 Anomerism 41
• Buffer mechanism of body 547-548 Anti diuretic hormone 393,397
• Disorders 553-557 Antioxidan ts 523
• Henderson Hasselbalc h Equation Anti-sense therapy
• Role of lungs 550 Apolipopro teins 239
• Role of kidneys 551 Arachidon ic acid 63,66
Actin 534 Arginine 84-83
Activators 121 Arginosucc inate lyase 254,255
Active transport 22-23 Arginosucc inate synthetase 254,255
Acute phase proteins 541 Ascorbic acid, Vitamin C, 344,345
Adenine 108 Aspartate transamina se 250-251
Adenosine 109 Aspartic acid 84,85
Adenylate cyclase 508 Atheroscle rosis and Treatment 243
Adrenaline , see epinephrin e ATP 109
A/G ratio 579
Alanine 84-85 B
Alanine transamina se 251 2,3 -BPG 178
Albumin 540 Balanced diet 315,316
Aldosteron e 393-397 Basal metabolic rate 308
Alfa fetoprotein (AFP) 453 Base pairing rule 112
Allopurino l 133,415 Bence Jones Proteins 543
Allosteric regulation 139 Benedict's test
AU-trans retinal 336-337 Bicarbonat e buffer 549
Alkaptonu ria 273 Bile salts 76,235-236
Amino acid Bimolecula r leaflet 78
• Classification 85-87 Biological importance 95
• Essential amino acids 86 Biogenic amines 284
• Amino acid pool 249 Biological value of proteins 322
• Amino acid Catabolism 250-254 Blood glucose regulation 204,205
Amino acid pool 249 Buffers 547
Buffer mechanism of body 547-549

Glossary 627
C D
Calcitoni n 368,453 Debranch ing enzyme 187
Calcitriol 339,368 Decarbox ylation 284
Calcium 365-369 Denatura tion 103
Calmodu lin 367 Derived proteins 94
Calorific value 306 Deoxysu gars 44
Cancer 450 Dermata n sulphate 56
Carbohyd rates Chemistr y Detoxification
• Absorpti on, 160 • Definition 516
• Classification 33 • Types 517-520
• Definition 33 Dextran 53
• Digestion, 156-159 Dextrin 54
Carboxy peptidase 167,169 Diabetes mellitus 206-208
Cardiolip in 74 Diagnost ic enzymes 146
Cathepsi n Diastereo isomerism 40
CEA (Carcino embryon ic antigen) 453 Dietary carbohyd rates 318-320
Cell structure 17 Dietary Fibres 319
Cell membran e function 20 Dietary lipids 321
Cell membran e Structure 19 Digestion and Absorpti on
Cephalin • Carbohy dra tes
Chemios motic Theory 297 - Digestion 156-159
Chloride 402 - Absorpti on 160-161
Choleste rol 76 • Lipids
Choleste rol metaboli sm 234 - Digestion 162-163
Citric acid cycle 180-183 - Absorpti on 164-165
CK-MB 145 - Malabsor ption syndrom e 166
Coenzym e 121 • Proteins
Cofactors 121 - Digestion 167-169
Collagen 530-531 - Absorpti on 170
Competi ve enzyme inhibition 132-134 Disaccharides 47-48
Compou nd lipids 60 D A: Structure and Function 111-
Conjugat ed proteins 94 112
Connecti ve tissues 530 Dopamin e 269
Contracti le proteins 534
Copper 378
Cori cycle (Lactate metabolis m) 198 E
C-reactiv e protein 541 Eicosanoids 67-68
Creatine 260,535 Elastin 532
Creatine phosphat e 535 Electrolyte balance
Cysteine 265-266 • Distribut ion of electrolyt es 395-396
• Electrolyte balance 396-397

75
Glossary 628
Essential fatty acids 66

Electron Transport Chain & Exocytosis 26
Oxidative phosphorylation Evaluation of glycemic status
• ETC Organization 293-295
• Chemiosmotic Theory 297
• Shuttle mechanisms 300-301 F
• Inhibitors 296
Facilitated diffusion 22
• Uncouplers 298
Fat soluble vitamins
Electrophoresis 490-491
• Vitamins A 335-338
ELISA 495-496
• Vitamin D 339-341
Enantiomerism 39
• Vitamin E 342
Endocytosis 26
• Vitamin K 343
Enzymes
Fats 69-70
• Activators 121
Fatty acids 62-6
• Active site 119
Fatty acid oxidation 214-216
• Allosteric regulation 139-140
Fatty acid synthase 219-220
• Classification 123-124
Fatty liver 245
• Coenzyme 121
Feedback regulation 142
• Cofactors 121
Feedback inhibition 142
• Competive enzyme inhibition 132-134,136
Feedback repression 142
• Covalent modification 141
Ferritin 375
• Definition 118 Fluid mosaic model of cell membrane
• Diagnostic enzymes 146
19-20
• Effect of enzyme concentration 127
Fluoride 380-381
• Effect of pH 129
Free radicals 522-523,524
• Effect of substrates 130
Fructosamine
• Effect of temperature 128
Fructose 33,37
• Feedback regulation 142
Fructose metabolism 201
• Induction/ repression 139
• Isoenzymes 144-145
• Km value and its importance 131 G
• NonCompletive enzyme inhibition 135 G6PDdeficiency 194
• Mechanism of Enzyme action 48 Galactose metabolism 202
• Proenzyrnes 141 Gene therapy 486
• Specificity 125-126 Globular and fibrous 94
• Therapeutic enzymes 148 Glucagon 204
• Uncompetitive enzyme inhibition 136 Gluconeogenesis 195-197
• Zyrnogens 141 Glucose tolerance test 209
Epimerism 40 Glucose 33,37
Epinephrine 269 Glucose transporters 160
Essential amino acids 86 Glycated haemoglobin 210
Glycerophospholipids 73,74

Glossary 629
Glycine 258-261 Hormones

Glycogen storage diseases 189 • Classification 504
Glycogenesis 185 • Mechanism of hormone action 506-513
Glycogenolysis 187 Hyaluronic acid 56
Glycolysis J 74 Hybridization
Glycoprote ins 532 Hybridoma technology
Glycosuria 203 Hyperchol esterolemi a 242
Genetic code 115
Globulins 541 I
Glutamate 84
Imino acid 84-85
Glutamine 283,254
Immunogl obulins 542-543
Glucagon - Function 204
Inducer 139
Glucagon - Action 508
Intracellular Organelles 27-30
Glycine 258-261
Insulin - Function 508
Glycogen 52
Insulin - Mechanism of action 512
Gout 413-415
Insulin - Structure 93
Growth factors 454
Iodine number 71
GTI 209-210
Ion channels 21
Gyrase 428
Iron 373-377
Isoelectric pH 105
H Isoenzyme s 144-145
Hartnup's disease 79 Isoelectric precipitati on 105
HDL 237 Isomerism 38-43
Heme 559
Heme degradatio n 569
Heme synthesis 564 J
Jaundice 570-571
Hemochro matosis 377
Juvenile onset diabetes 206
Hemoglobin:
• Structure and functions 559
• Abnormal Hemoglob ins 562 K
Henderson Hasselbalc h Equation 547 Keratan sulphate 56
Heparin 55 Kerasin 75
Heteropoly saccharide 54-55 Ketone body
Hexokinas e 175 • Ketogenesis 229
High energy compound s 302 • Ketolysis 230
Hippuric acid 519 • Ketosis 231
Histidine • Rotheras Test 231
HMP shunt pathway 191-193 Km and its importanc e 131
Homopoly saccharide 51-54 Koshland's induced fit theory 120
Hormonal control of glucose 204-205 Kreb's cycle 180-182
Kwashiork or 326

Glossary 630
Minerals (continued)
L • Calcium 365-369
Lac Operon 470 • Chloride 402,
Lactate dehydroge nase 175,177 • Copper 378
Lactose • Fluoride 380-381
Lactose intolerance 166 • Iodine 382-383
Lactase 164 • Iron 373-377
Lactic acidosis 184 • Magnesium 372
LDL243-244 • Manganese 387
Leuci ne 84-85 • Molybden um 386
Limiting amino acids 325 • Phosphoro us 370-371
Linoleic acid 66 • Potassium 401
Linolenic acid 66 • Selenium 384
Lipids • Sodium 399-400
• Absorption 164 • Sulphur 403
• Amphipath ic lipids 77-78 • Zinc 385-386
• Classification 59-61 Mitochond ria 29
• Definition 59 Molisch test 41
• Digestion 162 Molybden um 458
Lipogenesi s 218-224 Monoclonal antibodies
Lipolysis 213 Monosacch arides 19
Lipoproteins 237-239 Multiple myeloma 372
Lipotropic factors 246 Mutarotati on 25
Liver function test 575-579 Mutation 343-346
Lohmann's reaction 535
Lyase 124 N
Lysine 84-85 NAD 360
Lysosomes 30 NADP 360
et protein utilization 322
M Neurotran smitters 513
Magnesium 372 Nitrogen Balance 249
Malabsorp tion syndrome 166 Non-Comp letive enzyme inhibition 135
Ma nganese 387 Nor epinephrin e 269
MAO 268,279 Normal levels of analytes 573
Marasmus 326 ucleic Acids
Mechanism of Enzyme action • DNA structure 111
Metabolic acidosis 553 • RNA structure 113
Metabolic alkalosis 555 Nucleotide s 109-110
Methionin e & Cysteine 263-266 Nucleotide Synthesis 406-411
Methyl cobalamin ucleotide degradatio n 412-414
Micelle 77 Nucleolus 27
Minerals Nucleus 27
• Classification, 363-364 N yctalopia 338

Glossary 631
0 Phosphate buffer 549

Obesity 328-329 Phosphoro us 370-371
Obstructiv e Jaundice Phospholip ids 226-228
Oil 69 Polysaccha rides 51-55
Oligopep tide 81 Porphyria 566-568
Oligosacch arides 50 Post-transl ational modification436
Oncogenes 450 Post-transc riptional modification442
Oncogenic viruses 447 Potassium 401
Optical isomerism 39 Probes 459
Orni thine 82 Proline 84,85
Osmosis 21 Proteins
Osteogene sis imperfecta • Biological importance 91
Osteomala cia 341 • Charge properties 104
Oxidative deaminatio n 252 • Classificati on 94-95
Oxidative decarboxy lation 184 • Conjugated Proteins 94
Oxynervon 60 • Digestion 167-168
Oxytocin 91 • Globular and fibrous 94
• Protein conformati on 96-102
p • Protein quality 324
Palmi tic acid 62 • Protein calorie malnutritio n 326-327
Palmitoleic acid 63 • Protein turnover 249
Pantotheni c acid 353 • Simple Proteins 94
PAPS (Phosphoadenosylphosphosulphate) 110 Prostaglan dins 67
Parathyroi d hormone {PTH) 368 Prostate specific Antigen (PSA)453
Para thormone 368 Protein calorie malnutritio n 326-327
PCR 457-458 Protein turnover 249
Pellagra 352 Proteoglyc ans 532-533
Pentose phosphate pathway 191 Proto-onco genes 450
Pepsin 168 Pyruvate kinase 175,177
Peptide bond 90 Pyruvate oxidation 179
Peptide Chemistry 91-93
Peroxisom e 29 Q
Plasma membrane Quaternar y structure of Proteins 100-
• Structure 19 101
• Functions 20 Q (Ubiquinon e, CoQ) 295
Plasma proteins
• Types 538 R
• Functions 539 Radioisoto pes 500-501
Phenylalan ine 84,85 Rancidity 71
Phenylalan ine metabolism 267-269 Rapaport Leubering cycle 178
Phenyl Ketonuria (PKU) 271-272 RDA (Recomme nded Dietary
Allowance ) 313
J
Glossary 632
Recombinant DNA Technology Sphingomyelin 474,60

• Gene therapy 486 Sphingophospholipids 60,73
• PCR 457-458 Spliceosomes 436
• Restriction enzymes 477-479 Starch 51-52
• Vectors 480 Starvation 288
Refsum's disease 217 Stearic acid 62
Regulation Gene Expression Stereoisomerism 38
• Regulated and constituted genes 469 Steroid hormone action 506-507
• Lac Operon 470 Substrate level phosphorylation 184
Renal function test 580-583 Sugar derivatives 43-46
Renal rickets 341 Suicide inhibition 137
Replication 423-430 Sulphur 263
Respiratory acidosis 555 Synthesis of fatty acids 218-223
Respira tory alkalosis 555
Restriction enzymes 477-479 T
Reverse transcriptase 452 TCA cycle 180-183
Rickets 341 Terpenes 78
RIA 494,496 Tertiary structure of proteins 100
RNA: Therapeutic enzymes 148
• Types 113-114 Threonine 84-85
• Structure 113 Thyroid function test 584-587
• Function 113 Transamination 250-251
RQ 307 Transcription 431-436
Transferases
s Translation 437-442
SAM (S-adenosyl Methionine) 110,264 Transport across the membrane 21-24
SDA (Specific dynamic action) 310-311 Triacylglycerols
Second messenger 508 • Structure and composition 70
Secondary s tructure of proteins 97-99 • Functions 70
Selenium 384 • Synthesis 225
Serine 262 Triple helix (Collagen helix)
Serotonin 278 Troponin 534
Sickle cell anemia 97 Tropomyosin 534
Simple diffusion 21 Trypsin 167-168
Simple proteins 96 Trypsinogen 167-168
Sitosterol 244 Tryptophan 276-279
Sodium 398-400 Tumor suppressor genes 451
Sodium pump 25 Tumor markers 453
Southern Blotting 460 Tyrosine 272-278

Glossary 633
u
Ubiquin one 295 z
Uncoup lers 298 Zinc 385-386
Uronic acid pathwa y 199 Zwitter ions 105
Urea Cycle 255-257 Zymoge ns 141
Urea 255-257
Uric acid 574
Urobilin ogen 576
V
Va line 84-85
Van der Waal's forces 101
Van den Bergh reaction s 579
Vectors 480
Vitamins:
• Antivita mins 332
• B-cornplex vitamin s 246-360
• Definiti on, Classifi cation, 332-334
• Vitamin s A 335-338
• Vitamin D 339-341
• Vitamin C 344-345
• Vitamin E 342
• Vitamin K 342
Von Gierke's disease 189
w
Water balance
• Functio ns of water 390
• Distribu tion of water in body 391
• Regulat ion of water balance 393
Waxes 59
Wernick e korsako ff syndrom e 348
Western blotting 461
X
Xanthin e 108
Xanthin e oxidase 386,412
Xenobiotics 516
Xeroph thalrnia 338
Xylulose 33

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Referen es 634
REFERENCE BOO KS
subject
Studen ts should develo p the habit of reading multip le books to understand the
in differe nt perspectives. There are several good books in the subject of
Biochemistry
for reference. Some of them are listed here.
1. Harper 's Bioche mistry
2. Lippin cott's Review of Bioche mistry
3. Textbo ok of Bioche mistry by D.M Vasud evan
4. Bioche mistry by U. Satyan arayan a
5. Bioche mistry by Debjyo ti Das
6. Textbo ok of Bioche mistry by Dinesh Puri
7. Bioche mistry by Albert Lehnin ger
8. Bioche mistry by Lubert Styer
9. Bioche mistry by Thoma s Devlin
10. Bioche mistry by MN Chatte rjea and Rana Shinde
11. Bioche mistry by Pankaj a Naik
12. Bioche mistry b y Voet and Voet
13. Bioche mistry by AR Aroor
14. Bioche mistry by Seetha ramaia h Chittip rol
15. Princip les of Bioche mistry by T.N. Pattab iraman
16. Bioche mistry by Rafi
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Revised F urth Edition 2016

Textbook of Biochemistry for Medical Students

Published by RM publications, Mangalore, India

For orders Call 09986449575, 9141949575

This book is also available Online@ www.parasredkart.com

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PREFACE TO THE FOURTH EDITION - 20 14

What's new in this edition?

This book is dedicated to

All the students who are willing to learn .. .

Prof. C V Rag uveer. MD, DCP, FICPath,

Co-Authors and Contributors

Author would like to acknowledge the role of various contributors in the

1. Dr. Urban D'Souza, Professor of Physiology, KVG Medical College,

2. Dr. Shankar Bhat, Professor & Head, Department of Physiology,

3. Dr. Reshma Kumarchandra, Faculty, Department of Biochemistry,

4. Dr. Shivanand Baliga, Faculty, Department of Biochemistry, Kasturba

I NOTE TO THE STUDENTS !

Format of the book:

1) Contents of the chapter

3) Question Bank including Multiple Choice Questions (MCQ)

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Cell, Chemistry of Biomolecules and Enzymes :

Nutrition, Vitamins, Minerals and Water:

Molecular Biology, Genetics, Recombinanat DNA, Techniques, Cancer:

Applied and Clinical Biochemistry):

I. Introduction and Scope of Biochemistry

II. Cell and Sub Cellular Structures (3 hours)

IV. Isotopes and their applications in Medicine (1 hou r)

V. Chemistry of Carbohydrates (4 hours)

VI. Chemistry of Proteins, Peptides and Amino acids (6 Hours)

VII. Chemistry of Lipids (4 Hours)

VIII. Chemistry of Nucleic Acids (4 Hours)

IX. Enzymes & Clinical Enzymology (7 Hours)

XI. Biological Oxidation (3 Hours)

XII. Digestion & Absorption (3 Hours)

XIII. Carbohydrates Metabolism (9 Hours)

XIV. Lipid Metabolism (10 Hours)

XV. Protein and amino acid metabolism (10 hours)

XVI. Purine and pyrimidine metabolism (3 hours)

XVII. Intermediary metabolism (2 hours)

XVIII. Minerals (4 hours)

XIX. Molecular genetics & proteins biosynthesis (lOhours)

XXI. Liver functions & kidney functions (3 Hours)

XXII. Nutrition and energy metabolism (5 Hours)

XXIII. Biochemistry of cancer (2 Hours)

XXIV. Endocrine function: (3 hrs)

XXV. Biochemical tests for atherosclerosis & myocardial infarction

XXVI. SI units, Quality control and standardization (1 hour)

• Scope of Biochemistry and Nutrition in Medicine

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Importance and Scope of Biochemistry and Nutrition in Medicine:

• Nutrition, one of the integral part of health care, is a branch of Biochemistry:

• Biotechnology, which is a branch of Biochemistry, has created wonders in the field of

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• Cell structure: Structural features and general structure of a typical cell

• Cytoplasm and subcellular organelles:

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Prokaryotic and Eukaryotic cell organization:

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Structural or morphological features of a cell:

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