(Eğitim Tanrısı) Anil Gupta - Comprehensive Biochemistry For Dentistry - Textbook For Dental Students-Springer Singapore (2019)

Anil
Gupta
Comprehensive
Biochemistry
for Dentistry
Textbook for Dental Students
Comprehensive Biochemistry for Dentistry
Anil Gupta
Comprehensive
Biochemistry for Dentistry
Textbook for Dental Students
Anil Gupta
Department of Physiology and Biochemistry
Eklavya Dental College and Hospital
Kotputli
Rajasthan
India
ISBN 978-981-13-1034-8 ISBN 978-981-13-1035-5 (eBook)

https://doi.org/10.1007/978-981-13-1035-5
Library of Congress Control Number: 2018950138
© Springer Nature Singapore Pte Ltd. 2019

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Singapore
I dedicate my book to Almighty
Shirdi Sai Baba.
Foreword
It gives me a great satisfaction to write a brief note on Comprehensive Biochemistry

for Dentistry by Dr. Anil Gupta. In this book, the latest information has been incor-
porated to create a comprehensive text that is suitable for beginners.
Every effort has been made to update the text with the possible latest informa-
tion. The information is clearly presented, logically arranged, and well-illustrated.
References have been included to guide readers to the classical and current litera-
ture. I am confident that this textbook will prove to be a useful compendium for all
students at the graduate and postgraduate levels, igniting their interest and enabling
a greater involvement in the growing dental field.
O. P. Jangir
Director Academics
FELM, IASE (Deemed to be) University
Gandhi Vidya Mandir
Sardarshahr
Rajasthan, India
vii
Foreword
It is my privilege to review and comment on the book Comprehensive Biochemistry

for Dentistry. The 24 chapters of this book begin with cells and organelles—the
fundamental units of life. The next two sections contain information on structural
and molecular biology components and intermediary metabolism. The fourth sec-
tion is dedicated to medical biochemistry, explaining the importance of acid–base
balance, nutrition, and diagnostic enzymes. The final section focuses on dental bio-
chemistry, which is of more clinical interest. This unique section examines the role
of various molecules and macromolecules in the normal functioning of oral tissues
and in the instigation of dental diseases. The index is quite good and easily steers
the reader toward references of interest. Most chapters present information on
applied biochemistry, including the biochemistry and treatment of specific diseases,
making the book useful for both physicians and students. It also serves as a refresher
on the fundamentals of biochemistry for students and physicians. The author pres-
ents up-to-date information on biochemistry and emphasizes relevant physiologic
and pathophysiologic biochemical concepts. The book uses simple and clear lan-
guage that can be easily understood by students, physicians, and researchers. I am
sure this book will be welcomed by many undergraduate and postgraduate students,
physicians, and researchers.
Rajesh Dabur
Professor and Head
Department of Biochemistry
M. D. University
Rohtak, India
Foreword
I am glad to know about the publication of Comprehensive Biochemistry for

Dentistry. This book covers the complete syllabus for undergraduate courses, as
well as being a source of knowledge for professionals. I wish the author great suc-
cess with this book.
S. P. S. Sodhi
Principal
Dashmesh Institute of Research and Dental Sciences
Faridkot, India
Executive Member
Dental Council of India
New Delhi, India
Member–Academic Council
Member–Board of Studies
Member–Planning Board
Baba Farid University of Health Sciences
Faridkot, India
ix
Foreword
It is a pleasure to write a foreword for Comprehensive Biochemistry for Dentistry,

which was written by my younger brother, Dr. Anil Gupta. What impressed me most
was his deep insight on the subject and his method of presenting even the most
complex concepts in a simple, easily understandable, and convincing manner. With
each chapter, he provides the details of biochemistry as applied to the understanding
of dental disorders. The book is organized in a manner to help students. It is a book
to be opened with expectation and closed with profit.
I have reviewed this book and appreciate the simple language, straightforward
style, and clear presentation. This book will definitely prove to be very useful for all
dental students and practitioners in India.
S. K. Singhal
MD, LLB
Medico Legal Consultant and Author of Seven Books
Medical Director
Professor, and Head of the Department of Forensic Medicine and Toxicology
A.C.P.M. Medical College
Dhule, India
Foreword
Biochemistry is a vast and challenging field for dental students. Comprehensive

Biochemistry for Dentistry is a complete and student-friendly book. It offers the
users an excellent opportunity to understand the basic fundamentals of biochemistry
and its applications to the medical field. A dedicated effort has been made by Prof.
(Dr.) Anil Gupta to systematically compile the book into six units covering the
entire syllabus. I am sure that the textbook will be an informative read for under-
graduate students, as well as clinicians. The author deserves appreciation and recog-
nition for his efforts to carry out this commendable and arduous task.
Sanjay Bansal, MDS
Principal
EDCH
Kotputli, India
xi
Foreword
I wish great success and my heartiest congratulations to my senior colleague, Dr.

Anil Gupta, for publishing Comprehensive Biochemistry for Dentistry. This book
fills a great need of students, especially those studying dentistry. I have known Dr.
Anil for years; he is deeply loved and respected by our students for his skills in the
subject, his teaching methodology, and his constant pursuit for excellence. He is a
great academician, a great person, and above all, a kind human being. Dr. Anil has
been continuously working on this title for years, trying to create the best possible
textbook on biochemistry with up-to-date content and simple language, allowing its
readers to understand the complexities of the biochemicals in the human body. It is
a great fortune of mine to be associated with Dr. Anil Gupta. I believe this title will
be a superb choice for students of biochemistry thanks to its comprehensive content,
simplicity, and ease of understanding.
Nitul Jain, MDS
Professor and Head Department of Oral Pathology
Eklavya Dental College and Hospital
Kotputli, Rajasthan, India
Foreword
It gives me great pleasure to unveil yet another noteworthy work penned by the
author.
Dr. Anil Gupta is a prolific writer and no stranger to the world of books. His
exceptional style of writing, creativity, and groundbreaking ideas have only
improved with time.
Biochemistry is one of the most important basic sciences in the preclinical years
of medical and dental schools. For me personally, it was a daunting subject.
This book complements the classical textbooks of biochemistry. It blends the
fundamental and dynamic concepts of biochemistry and provides exhaustive knowl-
edge for dental students in a single, clearly presented volume. The chapters are
skillfully written and easy to understand.
I recommend this book with enthusiasm and give my best wishes to Dr. Anil
Gupta for undertaking this endeavor.
Prithvi Raj Singh
MD Pathology
Reader, Department of Pathology
EDCH, Kotputli, India
xiii
Foreword
Dr. Anil Gupta, the author of this book, deserves to be congratulated on presenting
the speciality of biochemistry to medical and dental students at different levels of
education, as well as to professionals of different specialists. This book provides a
comprehensive and clear account of the current principles of biochemistry knowl-
edge. The author has done this by drawing on his rich experience as a teacher of
biochemistry for more than 10 years. This book defines all topics in a narrative
format so that the information is palatable to students and professionals. In this
book, Dr. Gupta has adopted a conventional approach to characterize food constitu-
ents, describing their formulae, properties, and place in human biological and
chemical reactions. The language is simple and easily understandable. The chapters
on the metabolism of carbohydrates, lipids, proteins, and nucleic acids are exhaus-
tive and naturally presented. Students will benefit greatly from the descriptive
account of vitamins, hormones, and enzymes, with special emphasis on dental top-
ics. This book will be welcomed by teachers of biochemistry, dental students, medi-
cal students, and professionals for revising and refreshing one’s knowledge in
biochemistry. I am confident that this book will go through many editions in the
coming years and the author will add more details on the topics herein.
P. K. Garg, MBBS, MD (Pathology)

Professor, Pathology
Venkateshwara Institute of Medical Sciecnes
Gajraula, UP, India
Foreword
This book, Comprehensive Biochemistry for Dentistry, is a good idea and great
effort by the author. The book is very educative and informative for graduates.
I wish Dr. Gupta good luck for this great endeavour.
Naveen Bansal
Head, Department of Orthodontics
Genesis Institute of Dental Sciences and Research
Ferozepur, India
xv
Foreword
I sincerely thank the author for allowing me to be part of his precious book. The
chapters within are unique and contain a plethora of information, not only for under-
graduate studies but also for postgraduate entrance examinations. The genetics sec-
tion may be especially helpful for this purpose. As a faculty member at a reputed
coaching centre for postgraduate entrance examinations, I find that most under-
graduate students lack a clear understanding of the concepts of biochemistry.
Therefore, I think this book is very helpful for clarifying the concepts. It includes a
section on dental biochemistry that is an additional benefit for students. To con-
clude, I want to quote Francis Bacon: “Some books are to be tasted, others swal-
lowed, and some few to be chewed and digested.” The content of this book should
be chewed and absorbed properly for undergraduate and postgraduate entrance
examinations.
Puneet Garg, MBBS, MD (Pathology)

PCMS-I, Govt. Medical College and Rajindra Hospital
Patiala, India
Foreword
I congratulate Dr. Anil Gupta for his extensive teaching and research experience,
which were utilized to write this book, Comprehensive Biochemistry for Dentistry.
I am absolutely sure that students of dentistry will find this book highly useful.
Hasan Kamal, PhD (Biochemistry)

Associate Professor
Department of Biochemistry
IDST, Modinagar
Ghaziabad, India
xvii
Preface
Biochemistry is an essential subject in the medical, dental, nursing, paramedical,

physiotherapy and homeopathy streams in various universities across the world.
Knowledge of the fundamentals of biochemistry is necessary for the study of normal
structure and functioning of organs and organ systems, as well as the pathogenesis of
diseases.
A number of biochemistry books are available in the market. This book is
unique in its approach to the vital concepts of biochemistry. It blends the funda-
mentals and dynamic concepts of biochemistry with the theory of dental sciences
to provide valuable, coherent, and exhaustive knowledge for dental students and
professionals.
Comprehensive Biochemistry for Dentistry is composed of 24 chapters on the
structure, function, and metabolism of biomolecules. The information is presented
in a simplified approach and enriched with diagrams, flow charts, tables,, and
graphs. Important concepts, definitions, and clinical significance are provided in a
summary format for easy understanding and recapitulation. Topics such as cell and
organelles, vitamins, hormones, nucleic acids, biological oxidation, metabolism,
nutrition, and serum enzymes have been prepared in such a way to provide exhaus-
tive knowledge to students using clear and straightforward language. In particular,
the information on dental biochemistry is unique to this book. Proteins, lipids, car-
bohydrates, vitamins, and hormones are discussed in relation to the normal structure
and functioning of oral tissues, as well as their roles in dental diseases.
This book is a unique attempt to integrate the fundamentals of biochemistry with
the science of dentistry for the purpose of imparting knowledge to dental students
and professionals. I hope it will disseminate the knowledge of biochemistry to
undergraduate students and serve as a reference book for professionals and acade-
micians. I have made sincere and honest efforts to prepare and present a factually
accurate book. However, I welcome criticisms, comments, and suggestions for
improvements to forthcoming editions of this book.
Kotputli, India Anil Gupta

27 March 2018
xix
Acknowledgements
The Almighty Shri Shirdi Sai Baba bestowed upon me the knowledge and persever-
ance for writing the book. My father, Shri Ved Parkash Gupta, always inspires me to
undertake great endeavours. I am highly indebted to my father for instilling the
habit of learning in me since my school days.My wife persistently motivates me to
achieve my goals and she stands by me in odd hours.
I owe my gratitude to Dr. Bhavik Sawhney, Associate Editor–Biomedicine,
Springer (India) Pvt. Ltd, for his support, cooperation, and understanding, which he
offered to me to complete the writing of this book. I am also thankful to Ms. Saanthi
Shankhararaman, Project Coordinator (Books) and her production team for creating
an attractive and appealing book design.
xxi
Contents
1 Introduction to Biochemistry�� 1

1.1 Definition�� 1
1.2 Historical Development of Biochemistry �� 1
Part I Cellular Biochemistry
2 Cell and Organelles�� 5

2.1 Cell�� 5
2.1.1 Definition�� 5
2.2 Landmark Discoveries�� 5
2.3 Cell Theory Postulates�� 5
2.4 Modern Concept of Cell�� 6
2.5 Prokaryotic Cell�� 6
2.6 Eukaryotic Cell �� 6
2.7 Plasma Membrane�� 8
2.7.2 History�� 8
2.7.3 Chemical Composition �� 8
2.8 Models of Plasma Membrane Structure�� 10
2.8.1 Lipid Bilayer Model�� 10
2.8.2 Sandwich Model �� 11
2.8.3 Unit Membrane Model �� 11
2.8.4 Fluid Mosaic Model�� 12
2.8.5 Functions of Plasma Membrane�� 13
2.9 Cytoplasmic Organelles�� 14
2.9.1 Mitochondria�� 14
2.9.2 Ribosomes�� 18
2.10 Golgi Body�� 21
2.10.1 Functions of Golgi Body�� 24
2.11 Endoplasmic Reticulum�� 25
2.12 Nucleus �� 28
2.13 Nucleolus�� 32
Suggested Readings �� 32
xxiii
xxiv Contents
Part II Structural Biochemistry
3 Protein and Amino Acids�� 35

3.1 Historical Facts �� 35
3.3 Classification of Proteins�� 35
3.3.1 Classification Based upon Size and Shape�� 36
3.3.2 Classification Based upon Biological Functions�� 36
3.3.3 Classification Based upon Chemical Composition
and Physical Properties �� 37
3.3.4 Classification Based upon Quality
and Nutritional Value�� 41
3.4 Structural Organization of Proteins�� 42
3.4.1 Primary Structure�� 43
3.4.2 Insulin Structure�� 46
3.4.3 Secondary Structure�� 47
3.4.4 Tertiary Structure�� 51
3.4.5 Quaternary Structure of Protein�� 52
3.5 Amino Acids Definition�� 54
3.6 Number of Amino Acids �� 55
3.7 Structure of Amino Acid �� 55
3.8 Important Characteristics of Amino Acids�� 55
3.9 Classification of Amino Acids�� 56
3.9.1 Classification Depending on Position of Amino Group�� 56
3.9.2 Classification Depending on Proteinogenic Property �� 57
3.9.3 Classification Depending on Polarity of Side Chain�� 58
3.9.4 Classification Depending on Nutritional Value�� 58
3.9.5 Classification Depending on Chemical Structure
of Side Chain�� 59
3.9.6 Classification Depending on Chemical Property�� 63
3.10 Applied Biochemistry �� 64
3.10.1 Arginine�� 64
3.10.2 Casein Phosphopeptides (CPP)�� 64
4 Plasma Proteins�� 67
4.1 Classification of Plasma Proteins�� 67
4.2 Plasma Proteins�� 67
4.3 Albumin�� 67
4.4 Globulins�� 69
4.5 α1-Globulins�� 69
4.5.1 Alpha-1: Acid Glycoprotein�� 69
4.5.2 Alpha-1 Fetoglobulin�� 69
4.5.3 Alpha-1 Antitrypsin�� 70
4.6 α2-Globulins�� 71
4.6.1 Ceruloplasmin �� 71
4.6.2 Haptoglobin�� 71
Contents xxv
4.7 β-Globulins�� 72

4.7.1 Transferrin�� 72
4.7.2 C-Reactive Protein�� 72
4.7.3 Hemopexin�� 73
4.8 Other Important Plasma Proteins�� 73
4.8.1 Bence-Jones Protein�� 73
4.8.2 Fibrinogen �� 74
4.9 Biological Functions of Plasma Proteins�� 74
5 Hemoglobin�� 77
5.2 Characteristics of Hemoglobin �� 77
5.3 Structure of Hemoglobin�� 78
5.3.1 Heme �� 78
5.4 Two-State Model of Hemoglobin �� 80
5.5 Functions of Hemoglobin �� 82
5.6 Variants of Hemoglobin�� 82
5.6.1 Adult Hemoglobin (HbA) �� 83
5.6.2 Fetal Hemoglobin (HbF)�� 83
5.6.3 Glycosylated Hemoglobin (HbA1C) �� 83
5.7 Hemoglobinopathies �� 84
5.7.1 Sickle-Cell Hemoglobin (HbS)�� 84
5.7.2 Sickle-Cell Trait�� 86
5.7.3 Hemoglobin C (HbC)�� 87
5.7.4 Hemoglobin D (HbD) �� 87
5.7.5 Hemoglobin E (HbE)�� 87
5.7.6 Thalassemia�� 87
5.8 Derivatives of Hemoglobin �� 89
5.8.1 Oxyhemoglobin�� 89
5.8.2 Carboxyhemoglobin�� 89
5.8.3 Carbamino-Hemoglobin �� 90
5.8.4 Sulfhemoglobin�� 90
5.8.5 Methemoglobin �� 90
5.9 Biodegradation of Hemoglobin�� 91
5.10 Biosynthesis of Hemoglobin�� 94
5.10.1 Biosynthesis of Heme �� 94
5.10.2 Biosynthesis of Globin�� 97
5.10.3 Regulation of Heme Biosynthesis�� 99
6 Carbohydrates�� 101
6.2 Classification�� 101
6.2.1 Monosaccharides�� 101
6.2.2 Disaccharides�� 102
6.2.3 Oligosaccharides �� 102
6.2.4 Polysaccharides�� 103
xxvi Contents
6.3 Characteristics of Monosaccharides �� 103

6.3.1 Asymmetric Carbon Atom�� 103
6.3.2 Vant Hoff’s Rule of “n” for Number of Isomers�� 103
6.3.3 Isomerism in Monosaccharides�� 104
6.3.4 Optical Isomerism�� 106
6.3.5 Racemic Mixture�� 107
6.4 Biologically Important Carbohydrates�� 107
6.4.1 Monosaccharides�� 107
6.4.2 Disaccharides�� 108
6.4.3 Polysaccharides (Glycans)�� 111
6.5 Classification of Mucopolysaccharides�� 116
6.5.1 Acidic and Non-sulfated Mucopolysaccharides �� 116
6.5.2 Acidic and Sulfated Mucopolysaccharides�� 117
6.5.3 Neutral Mucopolysaccharides�� 119
7 Lipids�� 123
7.2 Classification of Lipids �� 123
7.2.1 Simple Lipids�� 123
7.2.2 Compound Lipids �� 124
7.2.3 Derived Lipids�� 125
7.3 Derived Lipids�� 125
7.3.1 Fatty Acids�� 125
7.3.2 Types of Fatty Acids �� 126
7.4 Essential Fatty Acids (EFA)�� 128
7.4.1 History�� 128
7.4.2 Types of Essential Fatty Acids�� 128
7.4.3 Functions of EFA�� 129
7.4.4 Clinical Significance of EFA�� 130
7.5 Cholesterol�� 130
7.5.1 Occurrence�� 130
7.5.2 Dietary Sources�� 131
7.5.3 Properties�� 131
7.5.4 Chemical Structure�� 131
7.5.5 Functions�� 132
7.6 Compound Lipids�� 132
7.6.1 Phospholipids�� 132
7.6.2 Glycolipids�� 139
7.6.3 Sulfolipids�� 142
7.7 Simple Lipids�� 142
7.8.1 Dietary Omega: Three Fatty Acids and Dental Diseases�� 144
Contents xxvii
8 Nucleic Acids�� 145

8.1 Historical Background�� 145
8.2 Occurrence�� 146
8.3 Chemical Composition of DNA�� 146
8.4 Types of Deoxyribonucleotides in DNA�� 149
8.5 Structure of DNA Strand�� 149
8.6 Watson and Crick DNA Model �� 152
8.7 Functions of DNA�� 156
8.8 Types of DNA �� 156
8.9 Replication of DNA�� 157
8.9.2 Site of Occurrence�� 158
8.9.3 Models of Replication of DNA�� 158
8.9.4 Semiconservative Model of DNA Replication�� 158
8.9.5 Mechanism of Semiconservative Model�� 159
8.10 Transcription �� 165
8.10.2 Prerequisites of Transcription �� 165
8.10.3 Mechanism of Transcription �� 168
8.11 Ribonucleic Acid (RNA)�� 172
8.11.1 Structure�� 172
8.11.2 Types of RNA �� 172
8.12 Translation (Protein Synthesis)�� 177
8.12.2 Prerequisite of Translation�� 177
8.12.3 Mechanism of Translation�� 178
8.12.4 Post-translational Modification of Polypeptide Chain�� 184
9 Enzymes�� 185
9.2 Characteristics of Enzymes�� 185
9.3 Nomenclature and Classification of Enzymes�� 187
9.4 Enzyme Specificity �� 188
9.5 Mechanism of Enzyme Action�� 189
9.5.1 Theory of Activation Energy�� 189
9.5.2 Enzyme-Substrate Complex Theory �� 191
9.5.3 Lock and Key Model of Enzyme Action�� 191
9.5.4 Induced-Fit Model�� 192
9.6 Factors Regulating Enzyme Action�� 194
9.6.1 Effect of Enzyme Concentration�� 194
9.6.2 Effect of Substrate Concentration �� 194
9.6.3 Effect of Product Concentration �� 195
9.6.4 Effect of Temperature �� 195
xxviii Contents
9.6.5 Effect of pH�� 195

9.6.6 Effect of Coenzymes and Cofactors�� 196
9.6.7 Effect of Inhibitors�� 196
9.7 Enzyme Inhibition�� 197
9.7.2 Competitive Enzyme Inhibition Features �� 197
9.7.3 Noncompetitive Enzyme Inhibition Features �� 198
9.7.4 Uncompetitive Enzyme Inhibition Features �� 198
9.7.5 Suicide Inhibition Features �� 199
9.8 Isoenzymes or Isozymes �� 201
9.8.2 Occurrence�� 201
9.8.3 Structure of LDH Isoenzymes�� 201
9.8.4 Function of LDH�� 202
9.8.5 Clinical Importance�� 203
9.9 Ribozymes�� 203
9.10 Lysozyme�� 204
10 Hormones�� 207
10.1 Hormones�� 207
10.2 Comparison and Contrast�� 207
10.3 Classification of Hormones�� 208
10.3.1 Depending Upon Chemical Structure �� 208
10.3.2 Depending Upon Nature of Site of Action�� 210
10.4 Mechanism of Action of Hormones�� 211
10.4.1 Intracellular Receptor-Based Mechanism
of Hormonal Action�� 211
10.4.2 Cell-Surface Receptor-Based Mechanism
of Hormonal Action�� 215
10.5 Hormones from Hypothalamus�� 223
10.6 Hormones from Pituitary Gland�� 224
10.6.1 Prolactin�� 224
10.6.2 Thyroid-Stimulating Hormone�� 225
10.6.3 Adrenocorticotropic Hormone�� 226
10.6.4 Follicle-Stimulating Hormone (FSH) �� 227
10.6.5 Luteinizing Hormone (LH) �� 228
10.6.6 Growth Hormone�� 228
10.7 Hormone from Posterior Pituitary Gland�� 230
10.7.1 Antidiuretic Hormone (ADH) �� 230
10.7.2 Oxytocin�� 233
10.8 Parathormone (Parathyroid Hormone)�� 235
10.9 Insulin �� 238
10.10 Glucagon �� 241
Contents xxix
10.11 Thyroid Hormones�� 243

10.11.1 Biosynthesis�� 244
10.12 Calcitonin�� 251
10.13 Somatostatin�� 252
10.14 Adrenal Cortical Hormones (Corticosteroids)�� 253
10.14.1 Glucocorticoids �� 256
10.14.2 Mineralocorticoids�� 261
10.14.3 Adrenal Medulla Hormones�� 264
10.15 Hormones of Gonads�� 266
10.15.1 Androgens �� 267
10.15.2 Female Sex Hormones�� 270
11 Vitamins�� 279
11.2 Important Facts About Vitamins �� 279
11.3 Classification of Vitamins �� 279
11.4 Fat Soluble Vitamins �� 280
11.4.1 Vitamin A�� 280
11.4.2 Vitamin D�� 292
11.4.3 Vitamin E�� 301
11.4.4 Vitamin K�� 307
11.5 Water Soluble Vitamins�� 312
11.5.1 Vitamin C�� 312
11.5.2 Vitamin B Complex�� 319
11.5.3 Vitamin B1 (Thiamine) �� 319
11.5.4 Vitamin B2 �� 326
11.5.5 Nicotinic Acid �� 331
11.5.6 Pantothenic Acid �� 335
11.5.7 Vitamin B6 �� 339
11.5.8 Biotin�� 344
11.5.9 Folic Acid�� 346
11.5.10 Vitamin B12�� 355
Part III Metabolism
12 Digestion and Absorption of Proteins�� 367

12.1 Dietary Sources�� 367
12.2 Digestion of Proteins�� 368
12.2.1 Digestion in Oral Cavity �� 368
12.2.2 Digestion in Stomach�� 368
12.2.3 Digestion in the Small Intestine�� 369
12.3 Absorption of Amino Acids�� 372
xxx Contents
12.4 Mechanism of Absorption�� 372

12.4.1 Passive Diffusion of Amino Acids�� 372
12.4.2 Active Transport of Amino Acids�� 372
12.4.3 Influence of Meister Cycle (Gamma-Glutamyl Cycle)
in Amino Acid Absorption�� 373
12.5 Clinical Significance �� 373
13 Metabolism of Proteins and Amino Acids�� 377
13.1 Amino Acid Pool�� 377
13.1.1 Sources Providing Free Amino Acids
into Amino Acid Pool�� 378
13.1.2 Sources Utilizing Free Amino Acids
from Amino Acid Pool�� 379
13.2 Transamination�� 380
13.3 Deamination�� 383
13.3.1 Types of Deamination �� 383
13.4 Transdeamination�� 387
13.5 Urea Cycle�� 388
13.5.1 Steps in Urea Cycle�� 389
13.5.2 Biological Significance �� 392
13.5.3 Regulation of Urea Cycle�� 392
13.5.4 Clinical Significance �� 392
14 Digestion and Absorption of Carbohydrates �� 395
14.1 Definition of Digestion �� 395
14.3 Digestion of Carbohydrates�� 396
14.3.1 Digestion in Oral Cavity �� 396
14.3.2 Digestion in the Stomach�� 396
14.3.3 Digestion in the Duodenum�� 396
14.3.4 Digestion in the Small Intestine�� 397
14.4 Absorption of Carbohydrates�� 397
14.4.2 Rate of Absorption of Monosaccharides �� 397
14.4.3 Mechanism of Monosaccharide Absorption�� 398
15 Metabolism of Carbohydrates�� 403
15.1 Introduction�� 403
15.2 Glycolysis �� 403
15.3 Pyruvate Metabolism�� 412
15.4 Citric Acid Cycle�� 414
15.5 Shuttle System�� 418
Contents xxxi
15.6 Hexose Monophosphate Shunt (HMP) �� 420

15.7 Gluconeogenesis �� 423
15.8 Gluconeogenesis from Lactic Acid (Cori’s Cycle) �� 424
15.9 Glycogenolysis�� 426
15.10 Glycogenesis �� 428
15.11 Inherited Disorders of Glycogen Metabolism�� 433
15.12 Disorders of Carbohydrate Metabolism�� 435
15.12.1 Diabetes Mellitus�� 435
15.12.2 Types of Diabetes Mellitus�� 435
15.12.3 Laboratory Investigation of Diabetes Mellitus�� 437
16 Digestion and Absorption of Lipids�� 441
16.1.1 Digestion of Triglycerides�� 441
16.1.2 Digestion of Cholesteryl Ester�� 444
16.1.3 Digestion of Phospholipids �� 444
16.2 Absorption of Lipids �� 444
16.2.1 Lipolytic Theory�� 445
16.2.2 Frazer’s Partition Theory�� 445
16.2.3 Bergstrom Theory �� 445
17 Metabolism of Lipids �� 451
17.2 Oxidation of Fatty Acids�� 452
17.2.1 Types of Fatty Acids Oxidation�� 452
17.3 Biosynthesis of Cholesterol�� 459
17.3.1 Steps in Biosynthesis�� 460
17.3.2 Regulation of Cholesterol Biosynthesis�� 463
17.4 Biodegradation of Cholesterol�� 464
17.5 Ketogenesis �� 465
17.5.1 Steps in Ketogenesis�� 466
17.5.2 Biological Significance of Ketone Bodies�� 467
17.6 Ketosis�� 468
17.7 Lipoproteins�� 469
17.7.1 Classification of Lipoproteins �� 469
18 Metabolism of Minerals�� 473
18.1 Calcium �� 473
18.1.1 Functions of Calcium�� 476
18.1.2 Regulation of Plasma Calcium Level�� 477
18.1.3 Disorders of Calcium Metabolism�� 479
18.2 Phosphorus�� 481
xxxii Contents
18.3 Selenium �� 482

18.4 Copper�� 483
18.5 Zinc �� 484
18.6 Fluorine �� 485
18.6.1 Fluoride Toxicity �� 486
18.7 Iron Metabolism�� 488
18.7.1 Clinical Significance �� 491
19 Biological Oxidation�� 495
19.2 Methods of Biological Oxidation�� 495
19.3 Standard Reduction Potential (Redox Potential)�� 497
19.4 Oxidoreductase Enzymes�� 498
19.5 Electron Transport System (ETS) �� 500
19.5.1 Structural Components of Electron Transport
System (ETS)�� 500
19.5.2 Electron Carriers (Acceptors) �� 500
19.5.3 Enzyme Complexes in Electron Transport System �� 504
19.5.4 Physiology of Electron Transport System�� 504
19.6 Oxidative Phosphorylation�� 508
19.6.1 Theories of Oxidative Phosphorylation�� 509
19.6.2 Inhibitors of Electron Transport System and Oxidative
Phosphorylation�� 511
Part IV Medical Biochemistry
20 Acid-Base Balance�� 517

20.2 Sources of Acids in Body�� 517
20.3 Sources of Bases in Body �� 519
20.4 Acid-Base Homeostasis�� 520
20.4.1 Buffer Systems�� 520
20.4.2 Role of Buffer System in Acid-Base Homeostasis�� 520
20.4.3 Types of Buffer Systems �� 520
20.4.4 Role of Respiratory System in Acid-Base Homeostasis �� 525
20.4.5 Role of Renal System in Acid-Base Homeostasis�� 526
20.5 Disorders of Acid-Base Balance �� 529
20.5.1 Acidosis�� 529
20.5.2 Alkalosis�� 530
21 Nutrition�� 533
21.2 Food�� 533
21.3 Definition of Calorific Value of Food�� 533
Contents xxxiii
21.4 Measurement of Calorific Value�� 534

21.5 Respiratory Quotient of Foods (RQ)�� 535
21.6 Basal Metabolic Rate (BMR)�� 536
21.6.1 Estimation of Basal Metabolic Rate�� 536
21.6.2 Factors Regulating BMR�� 538
21.6.3 Clinical Significance of BMR �� 540
21.7 Specific Dynamic Action (SDA)�� 540
21.8 Balance Diet�� 541
21.8.1 Basic Food Groups�� 542
21.8.2 Types of Basic Food Groups �� 542
21.8.3 Types of Balanced Diet �� 544
21.9 Nutritional Value of Dietary Carbohydrates �� 547
21.10 Nutritional Value of Dietary Proteins�� 548
21.10.1 Quality of Dietary Proteins �� 548
21.10.2 Quantity of Dietary Proteins �� 552
21.11 Nutritional Value of Dietary Lipids�� 552
21.12 Protein Energy Malnutrition �� 553
21.12.1 Kwashiorkor�� 554
21.12.2 Marasmus�� 555
22 Serum Enzymes and Organ Function Tests�� 557
22.2 Types of Plasma Enzymes�� 557
22.3 Significance of Clinical Enzymology �� 558
22.4 Serum Enzymes in Heart Diseases �� 559
22.4.1 Creatine Kinase (CK or CPK)�� 559
22.4.2 Serum Glutamate Oxaloacetate Transaminase (SGOT)�� 560
22.4.3 Lactate Dehydrogenase (LDH) �� 561
22.5 Serum Enzymes in Liver Diseases�� 561
22.5.1 Serum Transaminases�� 562
22.6 Serum Enzymes in Gastrointestinal Tract Diseases�� 564
22.6.1 Serum Amylase �� 564
22.6.2 Serum Lipase�� 564
22.7 Serum Enzymes in Bone Diseases�� 565
22.8 Liver Function Tests�� 565
22.8.1 Classification of Liver Function Tests�� 566
22.8.2 Group I (Tests for Excretory Function of the Liver) �� 567
22.8.3 Group II (Tests for Enzymes of the Liver)�� 570
22.8.4 Group III (Tests for Synthesizing Function of the Liver) �� 572
22.8.5 Group IV (Test for Carbohydrate Metabolism)�� 573
22.8.6 Types of Glucose Tolerance Test�� 573
22.8.7 Group V (Test for Lipid Metabolism) �� 574
22.8.8 Group VI (Test for Amino Acid Metabolism) �� 575
22.8.9 Group VII (Test for Detoxification Function) �� 575
22.9 Renal Function Tests (RFT)�� 576
xxxiv Contents
Part V Immunochemistry
23 Immunoglobulins �� 585

23.2 Structure of Immunoglobulin�� 586
23.2.1 Edelman-Gally Model of Immunoglobulin�� 586
23.3 Characteristics of Individual Immunoglobulins�� 588
23.3.1 Immunoglobulin A (IgA)�� 588
23.3.2 Immunoglobulin G (IgG)�� 588
23.3.3 Immunoglobulin M (IgM)�� 589
23.3.4 Immunoglobulin D (IgD)�� 589
23.3.5 Immunoglobulin E (IgE) �� 590
Part VI Dental Biochemistry

24 Dental Biochemistry�� 595
24.2 Enamel�� 595
24.2.1 Chemical Composition of Enamel�� 596
24.3 Dentine�� 598
24.3.1 Chemical Composition of Dentine�� 598
24.4 Cementum �� 599
24.4.1 Chemical Composition of Cementum�� 599
24.5 Biochemical Basis of Dental Caries �� 600
24.5.1 Dental Caries�� 600
24.5.2 Role of Biochemical Compounds Involved
in Dental Caries�� 600
24.5.3 Fluoride as Anticaries Agent �� 601
24.5.4 Salivary Proteins in Dental Caries�� 601
24.6 Chemical Composition of Saliva�� 602
24.7 Periodontal Diseases and Immunity�� 604
About the Author
Anil Gupta is currently working as Professor and Head of the Department of

Physiology and Biochemistry at Eklavya Dental College and Hospital, Kotputli
(affiliated to the Rajasthan University of Health Sciences, Jaipur, India). He gradu-
ated in biosciences from Punjab University in 1989. He obtained his Bachelors in
Dental Surgery from the University of Poona in 1984. Later, he completed his
Masters in Biochemistry in 2009 and PhD in Biochemistry in 2012 from SJJT
University, Rajasthan. Over the past 6 years, he has been independently, pursuing
postdoctoral research on the nutritional status of children aged 2–5 years. He has
presented research papers at reputed universities, including Thaper University,
Patiala; Birla Institute of Technology and Sciences, Pilani; Punjabi University,
Patiala; M.D. University, Rohtak; and Arya P.G. College of Krukshetra University.
Dr. Gupta has had more than 30 research papers accepted and published in high-
impact, peer-reviewed, and indexed journals. During his academic period, he was
accorded with merit certificates, merit scholarships, and medals.Dr. Gupta has
9 years of teaching experience, 6 years of postdoctoral experience, and 4 years of
PhD supervisor experience, with more than 21 years of clinical experience. He is a
guide to PhD scholars at universities and supervises research scholars in distinctive
fields such as heavy metal contamination of water, quality analysis of drinking
water, and the predisposition of blood types to diabetes mellitus and dyslipidemia).
One research scholar has been awarded PhD degree under his supervision. He
serves as adjunct faculty, teaching research methodology to research scholars at
universities. Additionally, he serves as a reviewer and editorial board member of
national and international journals. His research interests are focused on human
physiology, nutrition, and associated pathophysiology.
xxxv
Introduction to Biochemistry
1
Biochemistry is the study of chemical compounds that are structural and functional
components of living organisms. These chemical compounds exist in organic and
inorganic forms in the body of living organisms. Life is the highly organized form
of biochemicals that exhibit proliferation, growth, development, irritation, and
reproduction.
Biochemistry deals with study of basic structure of these biochemicals and pro-
portions in which biochemicals aggregate to form a living cell. Study of biochemis-
try also emphasizes upon the transformation of biochemicals within the body in
health and diseases. The subject of biochemistry also deals with molecular changes
that are inherently associated with pathogenesis of diseases. Field of biochemistry
is highly extensive. It also describes the role of biomolecules as biomarkers in the
diagnosis and prognosis of diseases.
1.1 Definition
Biochemistry is a branch of life science (medical science) which deals with

study of the structure, functions, and transformation of biomolecules within
living organisms together with their role in diagnosis and pathogenesis of
diseases.
The word biochemistry is derived from “bio” which means “living” and
“chemistry” which means the study of chemicals. Biochemistry deals with the
study of chemicals that are essential part of living matter.
1.2 Historical Development of Biochemistry
• Roots of modern biochemistry are traceable to the science of Indian medicine

called as Ayurveda. Its origin coincides with the development of mankind.
Ayurvedic science focuses on seven tissues of the body called as DHATUS. They
© Springer Nature Singapore Pte Ltd. 2019 1

A. Gupta, Comprehensive Biochemistry for Dentistry,
https://doi.org/10.1007/978-981-13-1035-5_1
2 1 Introduction to Biochemistry
are rasa, rakta, mamsa (muscles), meda (fats), asthi (skeletal system), majja
(bone marrow), and shukra (semen). Ayurvedic principles strongly emphasize
upon the positive interrelationship among AHARA (quality of diet), VIHARA
(lifestyle), and DHATUS.
• Charaka was an Ayurvedic physician which dates back to 300 BC. He compiled
a compendium of Ayurveda called as Charak Samhita. It describes types of
foods, quality of food, and role of nutrition in health and diseases.
• Modern medical science relies on biochemical assaying of blood, urine, and
cerebrospinal fluid to diagnose diseases.
• Paracelsus (1493–1541) introduced the chemicals in the field of medicine.
• Carl Wilhelm Scheele (1742–1786) studied organic compounds like tartaric
acid, citric acid, and lactic acid.
• Antoine Lavoisier (1743–1794) was recognized as father of modern chemistry.
He was pioneer in the study of oxidation of foods inside body cells.
• J. von Liebig (1803–1973) contributed to biological chemistry. In 1842, he
wrote a book of organic chemistry with applications to physiology and
pathology.
• JJ Berzelius (1779–1848) is considered as one of the founders of modern bio-
chemistry. He coined the term protein. He wrote a book named as animal
chemistry.
• Friedrich Miescher discovered nucleic acid in 1869.
• Kuhne used the term enzyme in 1877.
• Emil Fischer proposed lock and key theory to explain mechanism of enzyme
action in 1998.
• Carl Neuberg was a German scientist. He is credited with title of Father of
Modern Biochemistry. The term biochemistry was proposed by Carl Neuberg
in 1903.
• Embden-Meyerhof-Parnas provided understanding about oxidation of
glucose.
• A. Szent-Gyorgyi contributed to discovery of fumaric acid in 1930.
• Kendell was credited with isolation of thyroxine in 1914.
• Hans A Krebs described TCA cycle in 1937.
• Watson and Crick lead to pioneering research in molecular biology. They dis-
covered double helical model of DNA in 1953.
• Har Gobind Khorana (1922–2011) led to remarkable research. He demon-
strated significance of ribonucleotides (genetic codes) (UCUCUCU) in coding
serine and leucine. He synthesized polyribonucleotides.
• Herbert Boyer in 1937 synthesized recombinant DNA by transducing DNA
fragment into plasmid. Human insulin has been synthesized by DNA recombi-
nant technology.
• Today, modern medicine is heavily relied on biochemistry in the diagnosis of
diseases and in designing drugs.
• In the future, mapping of human genome will lead to gene therapy.
Part I
Cellular Biochemistry
Cell and Organelles
2
2.1 Cell
2.1.1 Definition
Cell can be defined as fundamental structural and functional unit of life bound
by plasma membrane that can reproduce independently.
All living organisms are composed of basic structural and functional fundamen-
tal units, which are called as Cells. The body of living organisms like bacteria,
protozoans and blue-green algae is unicellular. Single cell in these organisms per-
forms all the functions. Plants, fungi, animals are multicellular organisms and their
bodies are made up of millions of cells. The body of man is composed of nearly one
trillion cells. The cell are highly specialized in their structure and functions in mul-
ticellular organisms.
2.2 Landmark Discoveries
In 1665, Robert Hooke investigated a piece of cork under microscope. He found

that cork was made up of small compartments; he called them as cells.
In 1672, Leeuwenhoek was the first who observed sperms, bacteria, and red
blood cells under microscope.
In 1831, Robert Brown postulated that all cells had a nucleus in the center.
In 1839, Schleiden and Schwann postulated cell theory.
2.3 Cell Theory Postulates
• Schleiden (1838) and Schwann (1839) together proposed Cell theory.

All living organisms are made up of one or more than one cells.
• Cell is the fundamental unit of life.

https://doi.org/10.1007/978-981-13-1035-5_2
6 2 Cell and Organelles
• Later on, Rudolph Virchow in 1885 described that “Omnis Cellula e Cellula”.
The modified cell theory is as follows:
1. The body of all living organisms is made up of cells.
2. The cell is the basic structural and functional unit of life.
3. All cells arise from preexisting cells (Omnis Cellula e Cellula).
Exception to Cell theory.
i. Viruses
ii. Viriods
iii. Prions
2.4 Modern Concept of Cell
• All living organisms are made up of one or more cells.

• All living cells arise from preexisting cells by division.
• The cell is the basic unit of structure and function among all living organisms.
• Biochemical reactions involving catabolism and anabolism in organism occur
inside cells.
• Cells contain genetic material which is transferred from one cell to other cell.
2.5 Prokaryotic Cell
Pro- means “primitive,” and karyon means “nucleus.”
Features
• Prokaryotic cell has primitive nucleus. It is not enclosed by nuclear membrane.

• Cell has a single double-stranded circular DNA molecule in cytoplasm. It is
called as nucleoid as in Fig. 2.1.
• Cell membrane is covered by cell wall.
• Cytoplasm lacks membrane-bound cytoplasmic organelles.
• Cell contains 70S ribosomes freely scattered in cytoplasm.
• Cell lacks internal cytoskeleton.
• Cell lacks mitochondria. Enzymes for respiration in prokaryotes are found within
the infoldings of cell membrane and these infoldings are called as mesosomes.
• Cell division occurs by fission.
2.6 Eukaryotic Cell
The word Eu means “true” and karyon means “nucleus.”
Features
• Eukaryotic cells have properly defined nucleus. It is surrounded by nuclear

membrane.
2.6 Eukaryotic Cell 7
Circular
DNA [nucleoid]
Ribosome
Cytoplasm
Plasma
membrane
Cell wall
Capsule
Pilli
Fig. 2.1 Diagram of prokaryotic cell
Cell Membrane
Lysosomes
Ribosomes Rough
endoplasmic
reticulum
Golgi body Nuclear pore
Chromatin network
Nucleolus
Smooth
endoplasmic
reticulum Nucleus
Cytosol Mitochondria
Ribosome
Fig. 2.2 Diagram of eukaryotic cell
• Cell contains single double-stranded helically coiled DNA which associates with
histone proteins to form chromosomes as in Fig. 2.2.
• Cell membrane has phospholipid bilayer structure.
• Cytoplasm has membrane-bound organelles.
• Cell contains internal cytoskeleton.
• Cell contains 80S ribosomes attached to endoplasmic reticulum.

• Cell contains mitochondria containing enzymes for oxidative phosphorylation
and energy production.
• Cells divide by mitosis.
2.7 Plasma Membrane
2.7.1 Definition
Plasma membrane is a lipoprotein aceous semipermeable structure that sur-

rounds and supports protoplasm.
2.7.2 History
• In 1855, Nageli and Cramer proposed the term “cell membrane” for membrane
surrounding protoplasm.
• In 1895, Charles Ernest Overton described lipid nature of plasma membrane.
• In 1917, Irving Langmuir proposed an orientation of hydrophilic heads and
hydrophobic tails of phospholipid molecules in plasma membrane.
• In 1925, Gorter and Grendel described lipid bilayer model of plasma mem-
brane without protein.
• In 1935, Danielli and Davson proposed sandwich model (protein-lipid-protein).
• In 1950, Robertson proposed a unit membrane model of plasma membrane.
• In 1972, Singer and Nicholson proposed fluid mosaic model of plasma membrane.
2.7.3 Chemical Composition
Plasma membrane is essentially composed of carbohydrates, lipids, and proteins in

varying proportions. The relative proportion of macromolecules present in plasma
membrane is dependent on type of cells.
In general, plasma membrane contains the following components:
• Example: Lipid contents vary between 20% and 75%

• [24% lipids in inner membrane of mitochondria and 74% in myelin sheath].
• Protein contents vary between 20% and 76%
• Example: [20% lipids in myelin sheath and 77% lipids in inner membrane of
mitochondria].
• Carbohydrate contents vary between 1% and 10%
• Example: [1% carbohydrates in endoplasmic reticulum and 7% in plasma
membrane].
Lipids in Plasma Membrane

Lipids constitute the essential component of plasma membrane. Each lipid mole-
cule is amphipathic in nature. It is composed of a hydrophilic (polar) head and
2.7 Plasma Membrane 9
hydrophobic (nonpolar) tail. Hydrophilic head is oriented outward towards the

aquous medium and hydrophobic tail is directed inwardly.
Types of Lipids
• Fatty acids
Fatty acids are the building blocks of phospholipids and glycolipids in plasma
membrane. Nearly 50% of the total fatty acids in membranes are the saturated
fatty acids like palmitic acid and steric acid. Remaining 50% of fatty acids are
unsaturated fatty acids like are oleic acids, arachidonic acid, linoleic acid and
linolenic acids. The number of fatty acids in lipids is variable.
• Phospholipids
Phospholipids are the principal structural components of membrane lipids.
Glycerophospholipids are important part of lipids in biomembranes. Lecithin
and cephalin are common glycerophospholipids in biomembranes of animals.
• Sphingolipids
Sphingolipids are important constituents of biomembranes of neurons in animals.
Cerebrosides, gangliosides, and sphingomyelin are common sphingolipids.
• Cholesterol
Cholesterol is an additional component of biological membranes in animals.
Cholesterol is a sterol that is exclusively found in the biological membranes of
animals. It is not found in plant tissues. Cholesterol forms about 20% of the total
lipids of cell membrane.
Proteins in Plasma Membrane

Proteins constitute another essential structural part of biological
membranes.
Types of Proteins
• Integral membrane protein

They are deeply implanted in biological membrane. Integral membrane proteins
are permanently attached to hydrophobic region of lipid layer by van der Waals
forces. They are also called as intrinsic proteins.
• Peripheral membrane proteins
They are weakly attached to hydrophilic regions of phospholipid layer by ionic
bonds. These proteins are present on surface of membrane. They are also called
as extrinsic proteins. They can be easily separated from membrane by denatur-
ation and detergents.
• Transmembrane proteins
Transmembrane proteins span across the membrane extending from its outer sur-
face to inner surface. They are intrinsic proteins. They are linked by hydrophobic
amino acid residues to nonpolar region of phospholipids. Transmembrane pro-
teins serve as receptors for vast variety of drugs. These proteins also serve as ion
channels for the transport of ions, solutes across the plasma membrane.
Carbohydrates in Plasma Membrane

Carbohydrates constitute a small fraction of biological membranes. Carbohydrates
are covalently linked to membrane proteins. This process is called as glycosylation
of proteins. Common monosaccharides are D-galactose, D-mannose,
N-acetylglucosamine, and N-acetylneuraminic acid.
Glycosylation can be achieved through two types of glycosidic bonding as:
• N-glycosidic linkage
It is a bonding between glycans and nitrogen of either asparagine or arginine
residue. It occurs in lumen of endoplasmic reticulum in eukaryotic cells.
• O-glycosidic linkage
It is a linkage between glycans to oxygen of hydroxyl group on serine,
Hydroxyproline, hydroxylysine, tyrosine and threonine residues. It occurs in
lumen of Golgi bodies in eukaryotic cells.
2.8 Models of Plasma Membrane Structure
2.8.1 Lipid Bilayer Model
Characteristics
• Lipid bilayer model of plasma membrane was proposed by Gorter and Grendel
in 1925.
• Two workers studied the plasma membrane of RBCs.
• Plasma membrane is composed of two layers of lipid molecules.
• Each lipid molecule has hydrophilic head and hydrophobic tail. Heads of all lipid
molecules are oriented toward aqueous medium, while tails of molecules are
oriented inwardly as in Fig. 2.3.
Polar head
Outer Phospho
phospholipid Non-polar tail lipid molecule
layer
Inner
phospholipid
layer
Fig. 2.3 Diagram Showing Lipid Bilayer Model

2.8 Models of Plasma Membrane Structure 11
Outer protein layer

Polar head
Phospho
Non-polar tail lipid molecule
Inner protein layer
Fig. 2.4 Diagram of Sandwich Model
2.8.2 Sandwich Model
Characteristics
• Sandwich model was proposed by Danielli and Davson in 1935.

• Plasma membrane is made up of a phospholipid bilayer and two layers of pro-
teins to form a protein-lipid-protein structure of plasma membrane.
• Lipid bilayer in Danielli and Davson model has the same structure as was pro-
posed in lipid bilayer model.
• Lipid bilayer is surrounded (sandwiched) on either side by sheets of beta proteins
as in Fig. 2.4.
2.8.3 Unit Membrane Model
Characteristics
• Unit membrane model was proposed by Robertson in 1950.

• All biological membranes have unit membrane structure.
• Unit membrane is made up of a central phospholipid bilayer, and it is sandwiched
between two sheets of proteins. This arrangement of lipoprotein layers results in
formation of a trilaminar structure of plasma membrane as in Fig. 2.5. The trila-
mella together constitute as a unit to surround and support cell and organelles.
• Thickness of unit membrane is 75 Å.
–– Thickness of lipid bilayer is 35 Å.
–– Thickness of each layer of protein is 20 Å.
Outer protein layer [20A°]
Unit
Phospholipid bilayer [35A°] membrane
[75A°]
Inner protein layer [20A°]
Fig. 2.5 Diagram of Unit Membrane Model
2.8.4 Fluid Mosaic Model
Features
• It is a highly accepted model of plasma membrane.

• Fluid mosaic model of plasma membrane structure was proposed by Singer and
Nicolson in 1972. It is also called as Singer and Nicolson model.
Characteristics of Fluid Mosaic Model
• Plasma membrane has a thickness of 75 Å.

• Plasma membrane is a dynamic structure. It has a feature of expansion, con-
traction, invagination, evagination, and repair.
• Plasma membrane has a quasi-fluid nature. It is partly solid as it surrounds and
protects cell and organelles. It is partly liquid as it allows passage of substances
through it.
• Quasi-fluid nature of membrane allows lateral movement of protein mole-
cules through lipid bilayer as in Fig. 2.6.
• Plasma membrane structure represents a mosaic of lipids and proteins. The
protein molecules like icebergs float in a sea of lipid molecules.
• On outer surface of plasma membrane, oligosaccharides are linked to extrinsic
proteins and phospholipid molecules to form glycoproteins and glycolipids.
Nature of Lipids in Fluid Mosaic Model
• Lipid molecules have amphipathic nature. Lipid molecules are arranged into two
layers forming a lipid bilayer.
• Each lipid molecule has a polar head (hydrophilic) and a nonpolar tail (hydro-
phobic). Polar heads of all lipid molecules are directed outward toward aqueous
2.8 Models of Plasma Membrane Structure 13
Extrinsic protein
Intrinsic protein
Hydrophilic
head
Phospho Cholesterol
lipid
Hydrophobic
tail
Fig. 2.6 Structure of cell membrane (Fluid Mosaic Model)
medium. Nonpolar tails of lipid molecules are oriented inward in such a way that
tails of one lipid bilayer face tails of another lipid bilayer.
• Lipid bilayer in membrane is quasi-fluid in nature.
Nature of Proteins in Fluid Mosaic Model
• Integral membrane protein

They are deeply implanted in biological membrane. Integral membrane proteins
are permanently attached to hydrophobic region of lipid layer by Van der Waals
forces.
• Peripheral membrane proteins
They are weakly attached to hydrophilic regions of phospholipid layer by ionic
bonds. These proteins are present on surface of membrane.
A few extrinsic protein molecules are covalently attached to carbohydrate moi-
eties to form glycoproteins.
Protein molecules can translocate laterally due to quasi-fluid nature of lipid
bilayer. Proteins provide structural and functional specificity to membrane.
2.8.5 Functions of Plasma Membrane
• Plasma membrane surrounds and protects cell.

• Plasma membrane permits exchange of substances between cytoplasm and extra-
cellular compartment.
• Plasma membranes form different types of junctions that help communication
among cells.
• Plasma membrane allows movement of selected molecules to pass through it

(selective permeable), while other molecules cannot pass through membrane.
• Plasma membrane contains receptors for drugs and hormones.
• Plasma membrane contains carrier protein molecules for active transport of
molecules.
• Plasma membrane of mitochondria carries ATPase for synthesis of ATP.
• Plasma membrane of enterocytes has fingerlike outgrowths and microvilli that
increase absorptive area.
• Plasma membrane of neurons helps in nerve impulse generation and conduction.
Plasma membrane of bacteria contains respiratory enzymes.
2.9 Cytoplasmic Organelles
2.9.1 Mitochondria
Definition
Mitochondria are filamentous, self-replicating, double-walled organelles
located in cytoplasm of eukaryotic cells.
Mitochondria are termed as powerhouse of cell.
The term mitochondrion is derived from Greek words mitos which means
thread and chondros which means granule (owing to appearance of mitochon-
dria in spermatogenesis).
History
• In 1880, Kolliker discovered mitochondria in insect flight muscles.

• In 1890, Altman described mitochondria in cells and called them as bioblasts
(living organelles inside cell).
• In 1898, Benda coined the term mitochondria.
• In 1941, Claude separated mitochondria from other fractions of cell.
Occurrence
Mitochondria are located in cytoplasm of eukaryotic cells.
Color
Mitochondria have brownish red color.
Size
Size of mitochondria is variable. Mitochondria may have 0.5–3 μm length and
0.1–0.5 μm diameter.
Shape
Mitochondria have variable shapes. Under simple microscope, mitochondria appear
as minute threadlike structures. They may have sausage, spherical, or filament-
like shape.
2.9 Cytoplasmic Organelles 15
Number
The number of mitochondria in a cell is dependent on metabolic activity.
• Minimum number is one mitochondrion per cell. Example: green algae

belonging to genera Micromonas, Trypanosoma, and Chlorella, yeast
• The highest number is 50,000 mitochondria/cell. Example: insect flight
muscles
• Absence of mitochondria. Example: human red blood cells
Generally, the number of mitochondria varies between 100 and 2000 mitochon-
dria per cell.
Structure
Mitochondria structure can be described in three separate headings as:
Mitochondrial Membranes
Mitochondria are bounded by two successive membranes. Each one has unique bio-
logical functions.
Outer Mitochondrial Membrane
• Outer membrane has a thickness of about 60 Å.

• Its chemical composition is similar to that of plasma membrane of eukaryotic
cell. It is composed of phospholipids and proteins which are present in a 1:1
ratio.
• Outer membrane proteins
Outer membrane contains integral membrane proteins (transmembrane pro-
tein). These proteins act as channels and hence proteins are called as porins.
–– Porin proteins allow transport of molecules (7000 dalton mw) across the outer
mitochondrial membrane.
Inner Mitochondrial Membrane
• Inner mitochondrial membrane has high protein contents than phospholipids.

The ratio of proteins to phospholipids is 3:1.
• Inner membrane phospholipid
Inner membrane contains a prominent phospholipid called as cardiolipin (exclu-
sively found in inner membrane in eukaryotes, in bacterial cell wall).
• Inner membrane proteins
Inner membrane contains more than 100 polypeptides. They act as ATP synthase
enzyme, carrier proteins, and proteins for mitochondrial fission and fusion.
• Inner membrane is impermeable to ionic and polar compounds. These
molecules require carrier proteins to pass across inner membrane as in
Fig. 2.7.
• Cristae
Inner membrane gives rise to a number of inward folding which are called as
cristae. They increase the surface area of inner membrane for oxidative
phosphorylation.
Cristae divides mitochondrial matrix into compartments. Cristae are arranged at
right angle to longitudinal plane of mitochondria (parallel in neurons and skeletal
muscles) as in Fig. 2.8.
–– Oxysomes
They are tiny knob-like particles which are attached to M-face of inner mem-
brane and cristae. They are also called as elementary particles or Parson’s
particle or Fernandez-Moran particle, F0F1-particles.
Circular DNA
Outer
mitochondrial
membrane
Ribosome Inter
membrane
space
Oxysome Inner
membrane
Crista
Fig. 2.7 Mitochondrian
Outer
membrane
Inter
membrane
space
Base Crista
Oxysome Stalk
Head
Inner
membrane
Fig. 2.8 Structure of Crista

Fig. 2.9 Structure of Oxysome

Head
100A˚
Stalk
35A˚
45A˚ Base
About 10,000 to 100,000 oxysomes can be found in a mitochondrion.

Each oxysome has head piece, stalk, and base. Oxysome contains enzymes for
electron transport chain as in Fig. 2.9.
Intermembrane Space (Perimitochondrial Space)

It is a space between two mitochondrial membranes. It has a width of 6 nm. It con-
tains water and organic and inorganic solids.
Mitochondrial Matrix
• It is the viscous granular fluid which is present inner to inner membrane.

• It is composed of water and organic and inorganic solids.
• Matrix contains all enzymes for TCA cycle except succinate dehydrogenase
(attached to inner membrane). It also contains enzymes for urea cycle, trans-
amination, and lipid metabolism.
• Matrix contains 70S ribosome. It is called as mitoribosome.
• Matrix contains circular naked DNA. It is called as mt-DNA. There are about ten
copies of DNA in each mitochondrion. The mt-DNA can replicate and form
mRNA for synthesis of riboproteins. Each mitochondria contains more than
1000 proteins. Only 1% of mitochondrial proteins are riboproteins, while the
remaining 99% of proteins are synthesized in cytoplasmic ribosomes.
Functions of Mitochondria
Synthesis of ATP
NADH2 and FADH2 are oxidized with the help of coenzymes located in cristae.
Oxidation results in formation of ATP molecules.
Role in TCA Cycle
In presence of molecular O2, pyruvate undergoes oxidative decarboxyaltion to form

acetyl CoA. It further undergoes series of enzymatic conversions to form CO2 and
water and NADH2 and FADH2.
Role in Urea Cycle
Mitochondria contain carbamoyl phosphate synthetase-I enzyme and ornithine

transcarbamylase enzyme. These enzymes are necessary for urea formation.
Thermiogenesis
In mitochondria, proton leak causes heat production. It is not stored in ATP and the
process is called as thermiogenesis.
Role in Cytosolic Calcium Homeostasis
Mitochondria can store a good amount of cellular calcium in matrix. Calcium enter
matrix via calcium uniporter molecule. Calcium can be released into cytosol. In this
way, mitochondria act as cytosolic calcium regulator.
2.9.2 Ribosomes
Definition
Ribosomes are highly organized non-membrane-bound organelles located in
cytoplasm that are involved in synthesis of protein.
History
• In 1943, Claude isolated ribosomes from cytoplasm.
Ribosomes are microscopic ribonucleoprotein particles either attached to

rough endoplasmic reticulum or lying free in cytoplasm.
The word ribosome is etymologically derived from Ribo (ribonucleic acid)
and Soma (body).
Occurrence
• Ribosomes are found in both prokaryotic cells and eukaryotic cells.

• In prokaryotes, ribosomes occur free in cytoplasm.
• In eukaryotes, ribosomes are attached to RER and occur in free state in
cytoplasm.
• In mitochondria (mitoribosomes) and chloroplasts (plastidoribosomes),
ribosomes are present in matrix.
• In mature sperm and RBC, ribosomes are absent.
Number
The number of ribosomes varies depending on cellular content of ribonucleic acid.
Cells actively involved in protein synthesis have higher number of ribosomes.
Example: plasma cells, secretory cells.
Types of Ribosomes
Ribosomes are of two types depending on sedimentation coefficient (represents
ability of a biological particle to sediment (settle) during centrifugation). It is
expressed in Svedberg unit (S).
• 70S Ribosomes
–– They occur in prokaryotic cells, mitochondria, and chloroplasts.
–– Sedimentation coefficient is 70S.
• 80S Ribosomes
–– They occur in eukaryotic cells.
–– Sedimentation coefficient is 80S.
Structure of Ribosomes
• Each ribosome is composed of two unequal subunits, larger subunit and smaller
subunit. These subunits remain separated in cytoplasm. During protein synthesis,
subunits join together.
• Larger subunit
It is dome shaped. It is attached to membrane of RER through a ribophorin
glycoprotein. Larger subunit contains three binding sites for tRNA as:
–– A site (aminoacyl-tRNA binding): This is the first binding site. It attaches to
aminoacyl-tRNA which contains activated amino acid that is to be incorpo-
rated into growing polypeptide chain.
–– P site (peptidyl-tRNA binding site): It is the second binding site. It attaches
to tRNA holding elongating polypeptide chain.
–– E site (deacylated tRNA site or Exit Site): It is the third binding site. It holds
deacylated tRNA that leaves the ribosome.
• Smaller subunit
It is oval shaped. It attaches to flat side of larger subunit as a cap. It has mRNA
binding site.
• In 70S ribosome, large subunit is 50S and small subunit is 30S. Its molecular
weight is 9 × 106 daltons.
• In 80S ribosome, large subunit is 60S and small subunit is 40S. Its molecular
weight is 1.8 × 106 daltons as in Fig. 2.10.
Chemical Composition
Chemically, ribosomes are composed of rRNA and protein.
In 70S Ribosome
• It has 60% rRNA and 40% proteins in a ratio of (1.5:1).

• The rRNA belongs to three types as:
Fig. 2.10 Ribosome 80s (Eukaryotic cell)
Large subunit
60 S
ribosome
40 S Small
subunit
ribosome
–– 23S and 5S types of rRNA are present in 50S larger subunit.

–– 16S type of rRNA is present in 30S smaller subunit.
• 70S ribosome contains 55 types of core proteins.
In 80S Ribosome
• It has 45% rRNA and 55% proteins in a ratio of (0.8:1).

• The rRNA belongs to four types as:
–– 28S, 5S, and 5.8S types of rRNA are present in 60S larger subunit.
–– 18S type of rRNA is present in 40S smaller subunit.
• 80S ribosome contains 70 types of core proteins.
Polyribosomes
It is the cluster of many ribosomes on the strand of mRNA. It is also called as poly-
somes. A number of ribosomes in a polysome depend on length of mRNA strand.
Generally, 8–10 ribosomes assemble on mRNA strand. It occurs in the presence of
Mg++ ions.
Functions of Ribosomes
• Protein Synthesis
Free ribosomes are the site of synthesis of cellular proteins. Ribosomes are called
protein factory of cell.
• Synthesis of Enzymes
RER-bound ribosomes synthesize secretory proteins like enzymes. These are
translocated and act. Example: pancreatic enzymes.
• Synthesis of Hormones
RER-bound ribosomes synthesize hormones. These are located and act. Example:
peptide hormones.
2.10 Golgi Body 21
2.10 Golgi Body
Definition
Golgi body is the highly organized complex of interconnected flattened sacs
and vesicles that function as secretory and intracellular transport
organelle.
History
In 1898, Camillo Golgi (Italian scientist) described a complicated internal network
inside nerve cells of barn owl and called as internal reticular apparatus. Later on,
it was named as Golgi bodies.
It is also called as Golgi complex or Golgi apparatus.
Occurrence
• Golgi body is present in all eukaryotic cells except mature RBC.

• It is absent in prokaryotic cells.
Size and Number

Size and number of Golgi complex are variable and depend on metabolic activity of
cells. In secretory cells, their size is large in comparison with nonsecretory cells.
Liver cell may contain 30–50 Golgi bodies near the nucleus.
Position
Golgi complex is single and its location is permanent. It is located near the nucleus
and above centrioles. In liver cells and nerve cells, multiple units of Golgi bodies are
found scattered in cytoplasm.
Structure
Depending on electron microscope study, Golgi body is composed of three
structural elements as:
Cisternae
• Cisterna is a flattened double-walled saclike structure. Cisternae are compactly

arranged parallel to each other to form a stack. About 4–6 cisternae are placed in
a stack. Adjacent cisternae are separated by a distance of 100–300 Å. It is called
as intercisternal space which contains fluid and protein fibrils. Ends of each cis-
tern are swollen and called as Golgian vacuoles.
• Cisterna is surrounded by double-layered membrane. Internally, a cistern has
lumen of 90 Å. It contains a fluid.
• Cisternae curved and exhibit polarity. Each cistern has two distinct sides as:
–– Forming Face: It is convex in shape and is positioned toward the nucleus.
It is also called as F-face or cis-face. Thickness of F-face membrane is
40 Å. The cis-face of cisternae together constitutes cis-Golgi network
(CGN).
–– Maturing Face: It is concave in shape and positioned toward plasma mem-
brane. It is also called as M-face or trans-face. Thickness of M-face mem-
brane is 80 Å. The trans-face of cisternae represents trans-Golgi network
(TGN) as in Fig. 2.11.
• Transitional vesicles contain secretory proteins (enzymes, hormones, antibodies
synthesized by ribosomes and delivered to lumen of RER and packaged into tran-
sitional vesicles). Transitional vesicles are pinched off from RER. They are coated
with COAP-I (coat protein complex). They transport secretory proteins from RER
to cis-Golgi network (anterograde transport). Transitional vesicles fuse with
membrane of cis-face (F-face) of cisternae. Secretory proteins are processed within
lumen of cisternae and packaged into secretory vesicles. They are transported to
different locations including fusion with plasma membrane as in Fig. 2.11.
Tubules
• A network of thin tubules is located at periphery of cisternae. Tubules have diam-

eter of 300 Å. They help to interlink adjacent cisternae.
Vacuoles
Maturing face
Inter cisternal space
Cisterna
Forming face
Transition vesicles
Fig. 2.11 Golgi complex

2.10 Golgi Body 23
Vesicles
• Vesicles are membrane-bound saclike structures. They have diameter between

200 and 800 Å. Vesicles are oriented toward tubular Golgi network (TGN).
They contain processed substances and are pinched off from M-face of Golgi
body. These vesicles may also be called as secretory vesicles. They are two types
as:
–– Smooth Vesicles
These vesicles have smooth surface. They contain secretory proteins.
–– Coated Vesicles
These vesicles have rough surface which is coated with coat protein (e.g.,
clathrin protein). Clathrin coat vesicles fulfill intracellular vesicular traffick-
ing function.
Golgian Vacuoles
• They are dilated ends of cisternae. They are modified and contain granular sub-
stances. They are released from trans-face of Golgi network. They may function
as lysosomes.
Formation of Vesicles
Ribosomes synthesize proteins and deliver them into lumen of RER. These large
molecules are unable to pass through plasma membrane of cytoplasmic organelles.
Within RER, large molecules are packaged into tiny, membrane-bound, saclike
structures called as vesicles. Plasma membrane of ER bulges out and pinches off
transport vesicles. They are transported to cis-face cisternae and fuse with its mem-
brane. In this way, transport vesicles deliver proteins from ER to Golgi body with-
out moving through plasma membrane. These proteins are called as cargo
proteins.
Within lumen of cisterna, proteins undergo processing. Carbohydrate and lipid
moieties are added to proteins to form glycoproteins, lipoproteins, and proteogly-
cans. It is called as post-transcriptional modifications.
Proteins are transported from cis-face to trans-face of Golgi body through shuttle
vesicles.
At trans-Golgi network, TGN (M-face), proteins are packaged into vesicles
that are transported to different intracellular locations and even extracellular sites.
Types of Secretions in Vesicles
1. Vesicles may contain antibodies secreted by plasma cells. These vesicles are
pinched off from M-face and transported toward plasma membrane. They fuse
with membrane to release their contents to extracellular space.
2. Vesicles may contain peptide hormones and stored in cell. After a signal trans-
duction, vesicles release hormones in extracellular space.
3. Vesicles may contain hydrolytic enzymes. These are transferred to lysosomes.
Model of Intracellular Vesicular Trafficking
• Anterograde Vesicular Transport

Cargo proteins move in COP-II-coated vesicles (coat protein complex-II)
from RER to cis-face of cisternae.
• Retrograde Vesicular Transport
Proteins also move in COP-I-coated vesicles from older cisternae (M-face) to
young cisternae (F-face).
2.10.1 Functions of Golgi Body
Synthesis of Secretory Proteins
• Secretory proteins (hormones, enzymes, antibodies) are synthesized in RER and

transported to inside of endoplasmic reticulum. Proteins are translocated in coat
vesicles (coated with COPII) to cisternae. Proteins undergo modification and
packaged into secretory vesicles. Membrane of vesicle fuses with plasma mem-
brane and imports secretory proteins in extracellular space (exocytosis).
Example: Golgi body synthesizes enzymes in the pancreas, thyroxine in the thy-
roid gland, and antibodies in plasma cells.
Synthesis of Glycoproteins and Glycolipids

Glycolipids
• Gangliosides (glycolipids in the brain) are synthesized in Golgi body. Its lipid
component (ceramide) is synthesized in smooth endoplasmic reticulum.
Ceramide is glycosylated with moieties glucosyl UDP and galactosyl
UDP. Reactions are catalyzed by enzymes glucosyltransferase and galactosyl-
transferase along with other transferases located in Golgi body.
Glycoproteins
• Cell membrane proteins and secretory proteins are glycosylated with carbohy-
drate moieties. It can occur by two types of glycosylations as:
–– N-linked Glycosylation
Oligosaccharide moiety is attached to N-atom of asparagine residue in protein
molecule.
–– O-linked Glycosylation
Oligosaccharide moiety is attached to O-atom of either serine or threonine
residue in protein molecule (e.g., addition of glycosaminoglycans to proteins
forms proteoglycans).
2.11 Endoplasmic Reticulum 25
GLYCOGENIN is GLYCOSYLATED TYROSINE.

It is a primer to initiate synthesis of glycogen. It is a glycoprotein and acts a
glycosyltransferase enzyme.
Synthesis of Proteoglycans
• Proteins are glycosylated with mucopolysaccharides like hyaluronic acid, chon-

droitin sulfate, and keratin sulfate in Golgi body and form proteoglycans. They
serve as important structural constituents of ground substance of extracellular
matrix.
• The term ground substance refers to amorphous gelatinous substance which
is transparent, viscous, and colorless.
• It fills interstitial spaces between fibers and cells in connective tissues.
Synthesis of Acrosome
• Acrosome contains hydrolases to digest layers of egg. It is synthesized from

Golgi body.
Synthesis of Lysosomes
• Proteins are glycosylated at asparagine residues by addition of mannose-6-

phosphate moiety within Golgi body. Mannose-6-phosphate is a signaling mol-
ecule for protein to serve as acid hydrolase. These proteins are packaged into
clathrin-
coated vesicles and move to acidic vesicles called as endosomes
(membrane-bound intermediate cellular organelle that helps in sorting of materi-
als). Mature endosomes fuse with lysosomes.
2.11 Endoplasmic Reticulum
Definition
Endoplasmic reticulum is a network of interconnected structures that serve as
cytoskeletal of cell.
History
• In 1897, Garnier discovered endoplasmic reticulum.

• In 1945, structure of ER was described by Claude, Porter, and Fullam.
• The term endoplasmic reticulum was coined by Porter.
Occurrence
• Endoplasmic reticulum is found in the cytoplasm of eukaryotic cells. Exception:

mature RBC, germinal cells.
• In prokaryotes, ER is absent.
Types
• Smooth endoplasmic reticulum (SER)

It does not contain ribosomes. It is also called as agranular endoplasmic
reticulum.
• Rough endoplasmic reticulum (RER)
It contains ribosomes. It is also called as granular endoplasmic reticulum.
Structure
Endoplasmic reticulum is a cytoskeleton with well-defined structures namely
cisternae, tubules, and vesicles which are described as follows:
Cisternae
• They are cylindrical double-walled structures of ER. Cisternae are placed one

above the other to form an interlinked compact structure.
• Diameter of a cisterna is about 40 μm.
• In cells with high protein synthesis, cisternae contain ribosomes.
• Cisternae are located nearer to the nucleus as in Fig. 2.12.
Ribosomes
Rough
endoplasmic
reticulum pore
Smooth
endroplasmic
reticulum
Vesicles
Tubules
Fig. 2.12 Components of endoplasmic reticulum

2.11 Endoplasmic Reticulum 27
Tubules
• Tubules are wide-branched double-walled structures. Each tubule has a diameter

100 μm.
• Tubules are devoid of ribosomes.
• Tubules are located close to plasma membrane.
Vesicles
• Vesicles are double-walled oval structures. Each vesicle has a diameter 500 μm.
• Vesicles contain ribosomes.
Characteristics of SER
• SER is located near plasma membrane.

• It lacks ribosomes.
• It is mainly found in tissues concerned with synthesis of lipids.
Example: Adipose cells, liver, muscle fibers, adrenal cortical cells.
Characteristics of RER
• RER is located close to the nucleus.

• It contains ribosomes.
• It is found in protein-synthesizing cells.
Examples: Islets of Langerhans, pancreatic acini, Nissl’s granules, plasma

cells.
Functions
• Endoplasmic reticulum acts as cytoskeleton. It gives mechanical support to cell.

It helps to maintain shape of cell.
• It helps in import and export of substances.
• Endoplasmic reticulum is involved in formation of lysosomes.
• Rough ER is essential for synthesis of nuclear envelope.
• RER is essential for synthesis of regulatory proteins and membrane
proteins.
• RER is converted into SER through loss of ribosomes.
• ER is helpful in glycogen storage.
• SER is essential for synthesis of triglycerides in adipose cells.
• SER is essential in synthesis of steroidal hormones.
• SER is essential in glycogenolysis in the liver.
• SER in skeletal muscles is helpful in action potential across the muscle fiber.
• SER in muscle fiber (sarcoplasmic reticulum) is essential in cytosolic release
of calcium ions which in turn promotes muscle contraction.
2.12 Nucleus
Definition
Nucleus is defined as a double membrane-bound cytoplasmic organelle in
eukaryotic cells containing genetic material for inheritance of information and
regulation of cellular functions.
Nucleus is derived from the Latin word nucleus which means kernel or seed.
History
• During 1632–1723, Antonie van Leeuwenhoek (microscopist) was the first who
described presence of a lumen (nucleus) in the RBC of salmon.
• In 1931, Robert Brown (botanist) described nucleus in root cells of orchid plant.
• In 1953, J Hammerling described nucleus a storehouse of genetic material in his
work on Acetabularia (unicellular alga).
Facts
• Nucleus is a characteristic feature of eukaryotic cells.

• Nucleus is absent in prokaryotic cells.
• In humans, nucleus is absent in mature erythrocytes. It is present in erythrocytes
in maturation stage.
Number
• Anucleate
Mature RBCs are without nucleus.
• Uninucelate
A cell containing single nucleus is termed as uninucleate cell.
Example: adipose cells, fibroblasts, osteoblasts, plasma cells, monocytes,
lymphocytes.
• Multinucleate
A cell containing multiple nuclei is termed as multinucleate cell.
Example: Osteoclasts (four to five nuclei), skeletal muscle fibers,
proerythroblasts.
• Giant cell
Giant cell is multinucleated cell which is formed by fusion of macrophages or
monocytes in pathological conditions.
Example: Langerhans giant cell (granulomatous diseases), foreign body
giant cell.
• Syncytium
It is fusion of uninucleate cells by plasma membrane to appear as a multinucleate
single mass.
2.12 Nucleus 29
Example: Cardia muscle fibers are uninucleate cells. They are intercon-
nected by intercalated discs and function as a single mass of multinucleate
cells called as syncytium.
Shape
• Shape of nucleus is highly variable depending on metabolic activity of cells.

• Generally, nuclei have round or oval shapes.
• Neutrophils have trilobed nucleus which becomes hypersegmented (>3 lobes) in
megaloblastic anemia.
Size
• Size of nucleus is highly variable and depends on metabolic activity of cell.

• Nuclear size is directly proportional to volume of cytoplasm and it is termed as
nucleoplasmic ratio.
• Nucleoplasmic ratio is altered in megaloblastic anemia and malignancy.
Position
• Generally, nucleus is positioned in the center of cell which is based on metabolic

activity of cell.
• Nucleus occupies area of maximum activity inside cytoplasm.
• However, in plasma cells, nucleus is eccentric in positon:
In adipose cells, nucleus is confined to periphery of cytoplasm due to accumu-
lation of fats.
In glandular cells, nucleus assumes basal position.
Ultrastructure
Nucleus during interphase has a diameter between 5 and 20 μm. It is composed of
five parts as follows:
• Nuclear membrane
–– Nuclear membrane is also called as karyotheca. It separates nucleus from
cytoplasm.
–– It is a double-layered structure. Its outer layer is called outer nuclear mem-
brane, and inner layer is called inner nuclear membrane. These layers are
separated by a distance of 100–500 Å. The space between outer and inner
nuclear membranes is called perinuclear space.
–– Each membrane has unit membrane structure. Each membrane is composed
of phospholipid bilayer surrounded by two protein layers as in Fig. 2.13. Each
membrane has a width of 70–90 Å. The outer membrane is rough and bears
ribosomes. It is connected with endoplasmic reticulum. The inner membrane
is smooth and is in contact with nucleoplasm.
Chromatin
threads
Heterochromatin
Peri nuclear space
Karyo lymph
Nuclear pore
Outer nuclear membrane
Inner nuclear membrane
Nucleolus
Fig. 2.13 Structure of Nucleus
–– Nuclear membrane is perforated by presence of nuclear pores. Both layers of

nuclear membrane are in contact with each other at nuclear pore.
–– Nuclear pores
Nuclear pores are channel exchange of water-soluble substances between
cytoplasm and nucleus. Each nuclear pore has a diameter of 1200 Å.
Nuclear pores belong to family of transmembrane protein complexes which
are located in nuclear membrane as in Fig. 2.13.
They are called as nuclear pore complex or nucleoporins. There are about
30 proteins that form nucleoporins.
Nucleoplasm
• It is a viscous, transparent, and granular fluid that fills up space inside the nucleus.
It is a type of protoplasm that is bound by nuclear membrane. It is also called as
nuclear sap or karyoplasm
• Nucleoplasm contains chromatin and nucleolus.
• Nucleoplasm contains organic and inorganic compounds like nucleoside phos-
phates, proteins, and enzymes and minerals.
Chromatin
• Chromatin is a DNA-protein complex (nucleoprotein) that has affinity to basic

dyes.
• Chromatin is in the form of highly fine, coiled filament-like structure present
in nucleoplasm. It is seen during interphase.
2.12 Nucleus 31
Intra nuclear
chromatin
Fibrils
Granules
Matrix
Perinuclear
chromatin
Fig. 2.14 Structure of Nucleolus
• During cell division, chromatin appears thick and ribbon shaped and called as
chromosome (color-stained bodies). Chromosomes are observed during cell
division (M phase).
• A chromosome is composed of helically coiled DNA which is coated with his-
tone protein (nucleoprotein).
• Chromatin is of two types as:
–– Euchromatin
Euchromatin is a light-stained diffusely condensed chromatin network. It has
a width of about 100 Å. It forms 95% part of chromatin. Euchromatin is
actively involved in transcription.
–– Heterochromatin
Heterochromatin is a dark-stained highly condensed chromatin. It has width
of 1000 Å. The DNA in heterochromatin is inert. Heterochromatin is not
involved in transcription of mRNA.
Heterochromatin is of two types as:
Constitutive heterochromatin
Constitutive heterochromatin is highly condensed chromatin. It is made up of
multiple tandem repeats (when nucleotides are repeated as ATTCG
ATTCG ATTCG). DNA with tandem repeats is called satellite or repetitive
DNA. It is not transcribed. Constitutive heterochromatin is present in centro-
meres and telomers. It is necessary for pairing of homologous chromosomes
in meiosis.
Facultative heterochromatin
Facultative heterochromatin can be condensed or uncondensed and can act as
euchromatin or heterochromatin. It has the ability of gene expression.
Example: Bar body
2.13 Nucleolus
• It is a round or irregular structure which is attached to nucleolar organizer region

of chromosome.
• Nucleolus is not covered by membrane as in Fig. 2.14. It is composed of four
components as:
–– Nucleolar matrix
It is amorphous portion of nucleolus. It is in the form of semisolid viscous
fluid. It contains proteins.
–– Granular part
Granular part contains ribosomal subunits which are in formative stages.
–– Fibrillar part
Fibrillar part contains small fibrils of 50 Å length. Fibrils are made up of pro-
teins and rRNA molecules. These fibrils are precursors to ribosomes.
–– Chromatin
Chromatin is associated with peripheral portions of nucleolus and is called as
perinucleolar chromatin. It grows inwardly into nucleolus as ingrowths and
called as intranucleolar chromatin.
Nucleolus is the main site for synthesis of rRNA.
Functions of Nucleus
• Nucleus is an essential part of eukaryotic cell that contains genetic material in

the form of DNA.
• DNA in nucleus controls growth, proliferation, differentiation, metabolism, and
apoptosis of cells.
• Nucleus regulates cell metabolism by transcription of mRNA.
• DNA in nucleus is the site of crossing over and variations.
• DNA in nucleus is the site for mutation.
• Nucleus is responsible for speciation.
• Nucleus regulates cell division.
• Nucleolus in cell is the site for synthesis of rRNA.
Suggested Readings
Berg JM (2007) Biochemistry. Freeman WH, New York
Campbell NA, Reece JB (2005) Biology, 7th edn. Pearson Benjamin Cummings, San Francisco,
CA
Karp G (2007) Cell and molecular biology, 5th edn. Wiley, Hoboken
Part II
Structural Biochemistry
Protein and Amino Acids
3
3.1 Historical Facts
• In 1835, Dutch chemist, Mulder GJ, started chemical analysis of albuminous

substances from egg white, milk, gluten, and serum. Mulder concluded that a
“common fundamental radical” was present in all those substances. This funda-
mental radical is combined with sulfur and phosphorous in varying proportions
to yield albuminous nature of substances.
• In 1838, Swedish scientist, Berzelius JJ, proposed the word “protein” for the
common fundamental radical, suggested by Mulder.
• The word protein has its root in the Greek word “proteios” which stands for
“primary.” It signifies prime importance of proteins in the anatomy and physiol-
ogy of acellular to multicellular organisms.
3.2 Definition
Protein is defined as “polymer of amino acids that constitutes as basic struc-

tural and functional units of body tissues of living organisms.”
Proteins are made up of carbon, hydrogen, and oxygen as the constituent ele-
ments. Nitrogen is an essential element in proteins, and its proportion is nearly 16%
of total molecular weight of protein. Other elements like sulfur and phosphorus are
also present in small proportion in some proteins. Proteins may have molecular
weight ranging from 5000 to millions.
3.3 Classification of Proteins
Proteins can be classified by four criteria as follows:
1. Classification based upon size and shape

https://doi.org/10.1007/978-981-13-1035-5_3
36 3 Protein and Amino Acids
2 . Classification based upon functions

3. Classification based upon chemical composition and physical properties
4. Classification based upon quality and nutritional value
3.3.1 Classification Based upon Size and Shape
Proteins can be classified into two groups based on size and shape as:
• Fibrous Proteins
–– These proteins have axial ratio more than 10.
–– They have an elongated shape which may be either threadlike or resemble a
sheet.
–– Proteins are water insoluble.
–– They provide mechanical strength and protection to body tissues.
Example: Collagen fibers, elastin fibers, alpha-keratin in hairs and nails,
beta-keratin in silk
• Globular Proteins
–– They have an axial ratio less than 10.
–– They are also called corpuscular proteins.
–– These proteins have spherical or ovoid shape.
–– They form colloidal solution with water.
–– They may have a role as enzyme, hormone, or antibody.
Example: Hemoglobin, myoglobin, immunoglobulins, ribonuclease
3.3.2 Classification Based upon Biological Functions
Proteins perform multiple functions. They can be categorized into several groups.
• Structural Proteins
–– They are the essential structural components of various tissues of the body.
Example: Collagen protein in bone, elastin protein in cartilage, alpha-
keratin in skin and nails
• Enzymes
–– They catalyze metabolic reactions in body tissues.
Example: Amylase, lipase, protease
• Hormones
–– They are the secretions from endocrine glands. They regulate metabolic reac-
tions in body tissues.
Example: Insulin, thyroxin
• Transport Proteins
–– They help in the gaseous transportation from one type of tissues to another tis-
sues. Hemoglobin helps in transport of oxygen from pulmonary alveoli to body
tissues and carbon diaoxide from tissues to pulmonary alveoli. Myoglobin helps
in transport of oxygen from erythrocytes to mitochondria within muscles.
Example: Hemoglobin, myoglobin
3.3 Classification of Proteins 37
• Protective Proteins
–– They provide immunity against the pathogens.
Example: IgA, IgG, IgM, IgD, IgE
• Contractile Proteins
–– They are the functional and contractile units of skeletal muscles and are
responsible for muscle contraction.
Example: Actin, myosin
3.3.3 C
lassification Based upon Chemical Composition
and Physical Properties
The classification is widely accepted by academicians and scientists. It was pro-

posed by the British Physiological Society and American Physiological Society in
1907–1908. This system classifies proteins on the basis of complexity in chemical
composition of protein molecules.
Proteins are classified into three groups as:
(a) Simple proteins

(b) Conjugated proteins
(c) Derived proteins
(a) Simple Proteins

Simple proteins are the linear polypeptides of amino acids which upon
hydrolysis yield constituents amino acids.
They are further subclassified on the basis of coagulability and solubility
into seven types of proteins.
1. Albumins
• These proteins are heat coagulable.
• They are soluble in water and dilute salt solutions.
• They have low isoelectric pH (4.7).
• They behave as acidic at pH 7.4.
• Albumins are precipitated by saturated ammonium sulfate solution.
• They are deficient in glycine amino acid.
Example: Ovalbumin of egg, lactalbumin of milk, albumin in plasma,
legumelin in legumes, leucosine of cereals
2. Globulins
• They are heat coagulable.
• They are water insoluble but soluble in dilute salt solutions.
• They are precipitated by half saturated ammonium sulfate solution or full
saturated sodium chloride solution.
Example: Plasma globulin, ovoglobulin, glycinin in soybeans, edestin
in hemp seeds, legumin in peas
3. Gliadins
• They are plant proteins and also called prolamines.
• They are insoluble in water and absolute alcohol.
• They are soluble in 60–80% ethyl alcohol.
• Gliadins are rich in proline amino acid but poor in lysine.
Example: Hordein in barley, gliadin in wheat, zein in maize
4. Glutelins
• They are plant proteins.
• They are insoluble in water, alcohol, and salt solution.
• They are soluble in dilute solutions of acids and alkalies.
• They are coagulated by heat.
• Glutelins are rich in glutamic amino acid.
Example: Glutenin in wheat, glutelin in maize, oryzenin in rice
5. Protamines
• They are basic proteins and mostly found in animals.
• They are soluble in water and dilute solutions of acids and alkalies.
• They are heat non-coagulable.
• They are rich in arginine amino acid.
• They are deficient in cysteine, tyrosine, and tryptophan amino acids.
• They have high isoelectric pH (7.4).
• They link to nucleic acids and form nucleoproteins.
Example: Salmine in sperm of salmon, cyprinine in carp, clupeine in
sperm of herring
6. Histones
• They are basic proteins and mostly found in animals.
• They are soluble in water and dilute solutions of acids and alkalies.
• They are heat non-coagulable.
• They are rich in arginine and histidine amino acids.
• They have high isoelectric pH (7.4).
• They link to nucleic acids and form nucleoproteins.
• They are deficient in cysteine, tyrosine, and tryptophan amino acids.
Example: Globin in hemoglobin, nucleoproteins
7. Scleroproteins
• They are also called as albuminoids and occur as supporting tissues in animals.
• They are insoluble in water, solutions of acid and alkalies, and 60–80% alcohol.
• They are soluble in concentrated solution of acids.
Scleroproteins are characterized into three types of proteins based upon loca-
tion and function.
• Keratins
Alpha-keratin
–– They are insoluble in water, alcohol, acids, or alkali.
–– Alpha-keratin is rich in sulfur-containing amino acid, cysteine.
–– Alpha-keratin contains a right-handed, α helix.
–– Hard alpha-keratin contains higher number of disulfide bonds in compari-
son with soft or pseudo keratin.
Example: Proteins in human hairs, nails, outermost layer of skin, and

mammalian tissues like wool, horns, claws, and hooves
Beta-keratin
–– It is rich in amino acids glycine and alanine and poor in cysteine.
–– Beta-keratin has β-pleated sheet structure.
–– It is comparatively harder than α keratin.
Example: Proteins in silk fibroin, scales of reptiles, feathers, beaks,
and claws of birds
• Elastins
–– They are rich in amino acids like valine, leucine, and proline.
–– They are deficient in cysteine, methionine, and histidine.
–– They provide elasticity to tissues.
Example: Proteins in elastic fibers in ligaments, cartilages, and loose
areolar connective tissues
• Collagen
–– They are long fibrous proteins.
–– They provide mechanical strength to tissues.
Example: Proteins in bones, muscles, skin, tendons
(b) Conjugated Proteins
These proteins are linked to non-protein components, called as prosthetic
groups which can be either a metal or an organic compound.
Simple protein + Prosthetic group ------ Conjugated protein
(Apo-protein) (Holoprotein)
Type of prosthetic group determines subclass of conjugated protein as follows:

• Mucoproteins
–– Mucoproteins are composed of simple proteins linked to mucopolysac-
charides as prosthetic group.
–– In mucoproteins, mucopolysaccharide concentration is >4%.
Example: Mucoproteins in blood group antigens, umbilical cord, beta
ovomucoid in egg white, vitreous humor
–– Mucins are mucoproteins containing nearly 70–80% of carbohydrates con-
sisting of N-acetylglucosamines, N-acetylgalactosamines, sialic acid, and
fucose.
Glycophorin is a mucoprotein in the membrane of erythrocytes. It
contains high content of carbohydrates nearly 60%.
• Glycoproteins
–– Glycoproteins are composed of simple proteins attached to mucopolysac-
charides as prosthetic group.
–– In glycoproteins, mucopolysaccharide concentration is <4%.
Example: Serum albumin, serum globulin, immunoglobulins
• Nucleoproteins
–– Nucleoproteins are composed of simple proteins of protamine and histones
conjugated with nucleic acids as prosthetic group.
Example: Deoxyribonucleoproteins are present in nuclei of cells, chlo-
roplasts, and mitochondria.
Ribonucleoproteins are present in nucleoli within eukaryotic nuclei.
• Chromoproteins
–– Chromoproteins are composed of simple proteins with pigmented com-
pounds as prosthetic group.
Example:
1. Hemoglobin, myoglobin, cytochromes, and enzyme catalase are
hemoproteins containing heme as prosthetic group.
2. Iodopsin, cyanopsin, porphyropsin, and rhodopsin are photosensi-
tive pigments in cones and rods in the retina of eyes.
3.
Flavin adenine dinucleotide (FAD) and flavin mononucleotide
(FMN) are coenzymes containing riboflavin as prosthetic group.
• Lipoprotein
–– Lipoproteins are composed of simple proteins attached to lipids as pros-
thetic group.
Example: Serum lipoproteins
• Phosphoproteins
–– Phosphoproteins are composed of proteins linked to phosphoric acid as
prosthetic group.
Example: Casein protein in milk, vitellin protein in egg yolk
• Metalloproteins
–– Metalloproteins are composed of simple proteins and metallic ions as
prosthetic group.
Example:
Carbonic anhydrase contains zinc (Zn++).
Hemoglobin contains iron (Fe++).
Ceruloplasmin contains copper (Cu++).
(c) Derived Proteins
Derived proteins are obtained from the simple and conjugated proteins.
Derived proteins are produced due to activity of physical factors like X-rays, UV
rays, and heat and chemical factors like acids, alkali, and enzymes upon simple
and conjugated proteins.
Derived proteins can be subclassified into two groups of proteins as follows:
• Primary Derived Proteins
–– Primary derived proteins are denatured native proteins.
–– They have molecular weight similar to native proteins.
–– They differ from native proteins in terms of solubility and crystallization
properties.
–– In primary derived proteins, all secondary forces like ionic bonds, hydro-
phobic bonds, van der Waals forces, and hydrogen bonds are disrupted.
–– Peptide bonds remain intact.
Types
1. Coagulated proteins: They are insoluble in water.
Example: Coagulated egg and cooked meat
2. Proteans: They are insoluble in water.
Example: Myosan derived from myosin, fibrin derived from fibrinogen
3. Metaproteins: They are insoluble in water but soluble in acid or alkali.
Example: Acid metaproteins, alkali metaproteins
• Secondary Derived Proteins
–– Secondary derived proteins are obtained by hydrolysis of native
proteins.
–– They have molecular weight lesser than native protein.
–– In secondary derived proteins, hydrolysis occurs at peptide bonds.
Types
–– According to the level of hydrolysis, they are of three types as proteoses,
peptones, and peptides:
1. Proteoses: They are produced by hydrolysis of proteins by acid or
enzyme. They are water soluble. They are heat coagulable.
2. Peptones: They are produced by hydrolysis of proteoses by acid or
enzyme. They are soluble in water. They are non-coagulable by heat.
3. Peptides: They are made up of few amino acids linked by peptide
bonds. They can be dipeptides or tripeptides. They are water soluble
and not coagulated by heat (Fig. 3.1).
3.3.4 Classification Based upon Quality and Nutritional Value
Quality of proteins is determined by the amount of essential amino acids in proteins.

The higher the amount of essential amino acids, the greater will be its nutritional
value. Proteins can be subclassified into three groups on the basis of quality and
nutritional value.
• Complete Proteins
–– These proteins contain all the essential amino acids in proportional
composition.
–– They are also called as first-class proteins.
–– These proteins can promote growth of children.
–– These proteins are generally obtained from animals.
Example: Egg, meat, fish, milk
• Incomplete Proteins
–– These proteins are deficient of one essential amino acid.
–– These proteins are generally obtained from plants.
–– These proteins are inadequate for normal growth of children.
–– These proteins can maintain normal metabolism in adults.
Example: Pulses are deficient of amino acid methionine. Cereals are defi-
cient of amino acid lysine
Fig. 3.1 Complete
hydrolysis of protein
molecule Proteins
Proteans
Metaproteins
Proteoses Peptones Peptides
Amino acid + Amino acid+ - - - - - - - - -+ Amino acid + Amino acids
• Poor Proteins
–– These proteins are deficient of more than one amino acid.
Example:
Legumes are deficient of amino acids tryptophan and methionine.
Maize is deficient of amino acids lysine and tryptophan.
3.4 Structural Organization of Proteins
Proteins are biopolymers of α-amino acids. These are linked together through pep-
tide bonds. A condensation of two amino acids forms a dipeptide.
Example: Carnosine, anserine, pseudoproline
Similarly, condensation of more amino acids results into formation of tripep-
tides, tetrapeptides, and oligopeptides and proteins.
A polypeptide is formed by linking of 10–50 amino acids, and a protein molecule
is composed of more than 50 amino acids. Natural proteins are composed of 20
α-amino acids.
3.4 Structural Organization of Proteins 43
For example, a tetrapeptide contains only four amino acids. These can be any 4
out of total of 20 amino acids. These four amino acids can have (204 = 160,000)
types of arrangements. Therefore, proteins are synthesized in the body by altering
the sequence of amino acids.
Except glycine, all amino acids contain chiral carbon and are L stereoisomers.
Proteins exhibit wide structural complexity. Proteins have four levels of struc-
tural organization as:
Primary structure
Secondary structure
Tertiary structure
Quaternary structure
3.4.1 Primary Structure
Definition
Primary structure of protein is defined as “linear arrangement of amino acids
in a definite sequence in a polypeptide chain.”
haracteristics of Primary Structure

C
• Primary structure of protein is formed by condensation of amino acids in a linear
arrangement in a polypeptide chain.
• Each polypeptide chain has definite sequence of amino acid residues.
• Example: In a tetrapeptide, four amino acids are condensed to form differ-
ent types of tetrapeptides as in Fig. 3.2.
• Polypeptide chain shows polarity.
–– Amino terminal (N-terminal)
On the left hand side of a chain, the first amino acid is present. It has a free
amino group. This end of a polypeptide chain is called “amino terminal” or
(N-terminal).
–– Carboxy terminal (C-terminal)
Opposite end of polypeptide chain contains last amino acid. This amino acid
has a free carboxylic group. This end of chain is called carboxy terminal or
C-terminal.
By convention, numbering and sequence of amino acids are determined
from N-terminal as in Fig. 3.3.
Fig. 3.2 Linear structure Ala – Thr – Val - Leu

of tripeptide 1st tetrapeptide
Ala – Leu – Thr – Val
2nd tetrapeptide
Ala – Val – Thr – leu
3rd tetrapeptide
H H H H
H2N – C – CONH – C – CONH – C – CONH – C – COOH
Amino (N)
Terminal R1 R2 R3 R4
Carboxlic
C-Terminal
Peptide Bonds
Fig. 3.3 Polarity in peptide chain
Fig. 3.4 Formation of peptide H H

bond
H2N C COOH + H2N C COOH
R1 R2
Condensation
H H
H2N C CONH C COOH + H2O
R1 R2
• In a polypeptide chain, amino acids are linked together by formation of covalent

bonds called as peptide bonds (–C–N–) as in Fig. 3.4. It has the following
features:
–– Peptide bond is formed by condensation of carboxylic group (COOH) of one
amino acid with the amino group (NH2) of successive amino acid with the loss
water molecule. The (CO–NH) groups in a peptide are called peptide or
amide group.
–– The peptide bond is a covalent bond. The four atoms, namely, carbonyl car-
bon, carbonyl oxygen, amide nitrogen, and hydrogen in peptide group (CO–
NH), are located in the same plane. Peptide bond has a planar geometry.
–– Peptide bond is a partial double bond. The length of a single covalent C–N
bond in amines is around 1.49 Å, while the length of double (C〓N) bond in
imines is around 1.27 Å. However, the length of C–N bond in peptide bond is
1.32 Å. This indicates that the peptide bond has a length lesser than single
bond but greater than a double bond. Partial double bond nature of peptide
bond is due to resonance in peptide bond. The nitrogen atom donates its
valency electrons to carbonyl carbon. The valency electrons shift to oxygen
and make it negatively charged ions, and nitrogen assumes positive charge.
This delocalization of electrons is called resonance as in Fig. 3.5.
Fig. 3.5 Resonance in peptide bond O
O– O
C C
N+ N
H H
–– Peptide bond is a rigid bond. There is absence of free rotation around the
C–N bond in peptide group. Therefore, peptide group can assume two
isomeric conformations, namely, cis and trans. In cis form, two α-carbons
lie on the same side of peptide bond, while in trans form, two α-carbons lie on
the opposite side of peptide bond.
However, in nature, most of peptide bonds in proteins belong to
trans-conformation.
–– Two single bonds are present on either side of peptide bond. One single bond is
formed by alpha carbon and carbonyl carbon (Cα–CI) of first amino acid, while
the other bond is between amide nitrogen and alpha carbon (NI–Cα) of second
amino acid. These single bonds can rotate freely around peptide bond and can
assume a number of positions. This free rotation is responsible for coiling and
recoiling of polypeptide chains. It determines shape of protein molecule.
• The amount of rotation of single bonds is determined by torsion angle or dihedral
angle. The angle formed by (Cα–CI) bond with the peptide bond (C–N) is called
Psi (Ψ) torsion angle, whereas the angle between (NI–Cα) and peptide bond
(C–N) is called Phi (Φ) torsion angle. Pioneering research over the (Ψ, Φ) tor-
sion angles of amino acid resides in small polypeptide chain was performed
by Dr. G N Ramachandran during 1960-1963.
A diagrammatic representation of torsion angles is called Ramachandran plot.
Example:
Dystrophin is a large-sized primary protein containing 3685 amino acid resi-
dues weighing about 427,000 dalton. It is present in skeletal, cardiac, and
smooth muscle fibers.
• In a primary structure of protein, a linear polypeptide may be linked to another
polypeptide with the help of disulfide bonds. These bonds can be within the same
polypeptide chain (intra-chain) or between two adjacent polypeptide chains
(inter-chain). For example, insulin represents a primary structure of protein.
3.4.2 Insulin Structure
It was described by Sanger in 1955.
• Insulin is composed of two polypeptide chains as:

–– Chain A (glycine chain): It contains 21 amino acid residues.
–– Chain B (phenylalanine chain): It has 30 amino acid residues.
–– Chain A contains one intra-chain disulfide bridge between 6th cysteine resi-
due and 11th cysteine residue.
• Insulin molecule contains two inter-chain disulfide bridges:
–– First inter-chain disulfide bridge is formed between seventh cysteine resi-
dues of chain A and chain B.
–– Second inter-chain disulfide bridge is formed between 20th cysteine residue
of chain A and 19th cysteine residue of chain B as in Fig. 3.6.
• In chain A, amino acid sequence at eighth, ninth, and tenth positions under-
goes variation among different species.
–– In humans, sequence (Thr-Ser-Ile) is present.
–– In bovine, sequence (Ala-Ser-Val) is present.
–– In pig, sequence (Thr-Ser-Ile) is present.
–– In sheep, sequence (Ala-Gly-val) is present.
• Structure of insulin among human, pig, and bovine is almost identical except the
difference of amino acid at 30th position in chain B. It is amino acid threonine in
humans, whereas it is alanine in pig and bovine.
3.4.3 Secondary Structure
Secondary structure is defined as “steric relationship between the closely

placed amino acid resides in a polypeptide chain.”
A linear polypeptide chain undergoes coiling or folding to attain a three-
dimensional shape of a helix or a sheet or an intermediate shape. Non-covalent force
like hydrogen bond is responsible for folding of polypeptide chain in secondary
structure.
Intra chain disulfide bridge
S S
A 6 7 11 20
Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn
S S
Inter chain disulfide bridge
S S
Phe Val Asn Glu Ile Leu Cys Gly Ser His Leu Val Glu Ala Lys Tyr Leu Val Cys Gly Glu Aeg Gly Phe Phe Tyr Thr Ala Pro Lys Thr
7 19
Fig. 3.6 Structure of Insulin

• Hydrogen bonding
–– It is a non-covalent and weak electrostatic force of attraction. Hydrogen bond
is formed between a hydrogen atom covalently linked to an electronegative
atom like fluorine, oxygen, or nitrogen in one molecule and an electronegative
atom in another molecule.
–– In secondary protein structure, hydrogen bond is formed between the oxygen
atom of carbonyl (–C〓O) group in one amino acid and the hydrogen atom of
amide (–NH) group in another amino acid. Hydrogen bonding stabilizes the
secondary structure of proteins.
econdary Structures in Proteins

S
1. Alpha helix
2. Beta-pleated sheet
3. Beta-bends (reverse turns)
4. Triple helix
1. Alpha Helix (Historical Facts)

• In 1930, William Astbury was a molecular biologist who did pioneering
work in X-ray diffraction studies on hair and wool. On the basis of diffraction
patterns, he concluded that fibrous proteins have folded structure and unfolded
or stretched structure. He used nomenclature as alpha for folded form and
beta for stretched form of secondary structures.
• In 1951, it was Pauling, Corey, and Branson who described alpha helical
model of secondary structure of protein. The research was published in the
proceedings of the National Academy of Sciences.
• Possibly, they proposed the structure of helix earlier than sheet structure. So
nomenclature of alpha and beta are used.
• Alpha helix is the most common type of secondary structure of proteins.
Alpha helix Characteristics
• It is a helically coiled rodlike structure. It is formed by tight folding of poly-
peptide chain around an imaginary long axis. The carbonyl group (–C〓O)
and amide group (–NH) form the backbone of helix. Peptides and hydrogen
bonds are placed parallel to long axis of helix. The side chains of amino acid
residues are directed outwardly and are placed perpendicular to helical axis.
• The alpha helix is stabilized by hydrogen bonds.
• One complete turn of helix contains 3.6 amino acid residues. The distance
between two amino acid residues is 1.5 Å (translation) along the helical axis,
and each one has 100° of rotation. The vertical distance between two suc-
cessive turns is called pitch, and it is the product of amino acid residues (3.6)
and translation (1.5 Å) and is equal to 5.4 Å in alpha helix.
• The folding in alpha helix can be either right handed or left handed. Right-
handed alpha helix offers more stable conformation than left-handed helix.
• In left-handed folding, side chains exhibit steric interferences on the (–C–N–)

backbone. Therefore, left-handed alpha helix is unstable and rarely exists. In
nature, all alpha helices found in proteins are right-handed structures.
• Proline contains a secondary amino group (–NH), and it cannot furnish hydro-
gen atom for hydrogen bonding. So it does not favor formation of alpha helix.
Glycine has the shortest side chain and it has highly flexible steric conforma-
tion. Proline and glycine are present in beta-bends in proteins.
• Pauling and Corey α-helix is called as (3.613–helix). It indicates 3.6 amino
acid residues in 1 complete turn of helix which is formed by 13 atoms includ-
ing hydrogen bonds as in Fig. 3.7.
Example: Alpha-keratin, ferritin
2 . Beta-Pleated Sheet Structure
• It was William Astbury, a molecular biologist who initially proposed beta
sheet structure in 1930. Later on, Pauling and Corey elucidated the beta sheet
structure and called it as beta sheet. The beta sheet structure of protein is dis-
tinctly different from alpha helical rod structure.
• A beta sheet is formed by polypeptide chains which are called beta
strands. There can be two or more beta strands in a beta sheet as in
Figs. 3.8 and 3.9.
• The beta strands have almost completely extended (unfolded) form. It con-
tains nearly four to ten amino acid residues. Adjacent beta strands are held
together by two or more hydrogen bonds which gives the sheet a pleated
Fig. 3.7 Alpha Helix
C R
O C H
C Intra chain
H hydrogen bond
R
O
R C C N C
C O H H O
H N
C R 5.4A˚ pitch
H
R C
R H O
C H
H
C
N H N
H H 3.6 Amino acids coil
C
N
O
Fig. 3.8 Beta-pleated parallel R R

sheets
C N C NH2 Amino Terminal
H R
O O
O
NH R R
C NH
NH2 Amino Terminal
R
Fig. 3.9 Beta-pleated anti- R

parallel sheets
H
C N C Amino Terminal
H R NH
O O
O
R
HN C HN
COOH Carboxy Terminal
R
appearance. The distance between two amino acid residues is 3.5 Å in the
beta strand. The peptide bonds in adjacent beta strands are placed adjacent to
each other. The side chains in adjacent strands are outwardly directed.
• A beta sheet can have adjacent beta strands either placed in the same
direction (parallel) or in the opposite direction (antiparallel) or follow
mixed pattern. Generally, beta sheet contains four to ten beta strands.
• In antiparallel arrangement of beta sheet, the N-terminus of one strand
faces C-terminus of adjacent strand as in Fig. 3.9. The hydrogen bonds are
placed perpendicular to strand and are planar. The space between a pair of
hydrogen bonds is less. Antiparallel beta sheet structure is highly stable.
Example: Superoxide dismutase enzyme, fibroin in silk
• In parallel arrangement of beta sheet, all the N-terminals of all strands
are placed in the same direction as in Fig. 3.8. The hydrogen bonds are non-
planar and the hydrogen bond pairs are greatly spaced. This arrangement is
comparatively less stable. Example: Flavodoxin
• In mixed pattern of arrangement in sheet, a few strands run in parallel
direction, while others are directed in antiparallel direction. Example:
Carbonic anhydrase enzyme
3 . Beta-Bends (Reverse Turns)
Beta-turns are the frequent structural motifs in protein molecule. It was
described by Venkatachalam in 1968.
• A beta-turn is made up of four amino acid residues. These are designated
as (i, i+1, i+2, i+3).These residues do not form alpha helix. The alpha carbon
of residue (i) and alpha carbon of residue (i+3) are located at a distance less
than 7 Å. The amino acid residues are held by intra-chain hydrogen bonding.
Fig. 3.10 Beta bends Beta bend
-C-Terminal
-N-Terminal
Polypeptide chain
Polypeptide chain
-N-Terminal
-C-Terminal
Beta-turn is the region in polypeptide chain where it reverses its direction by

180° and coils upon itself. These conformations are also called as reverse
turns as in Fig. 3.10.
• Beta-turns are of two types as type I and type II. These types differ from
each other by psi and phi angles at amino acid residues i+1 and i+2.
• Beta-turn determines the shape of protein molecule. It helps the polypeptide
chain to assume a globular shape. Generally, the turns are present on the outer
surfaces of proteins. So it may help in the recognition of an antigen. Also, it
may provide surface for the attachment of an antigen.
4 . Triple Helix
• Collagen protein forms a triple helix structure. It is called tropocollagen.
• It is made up of three polypeptide chains which run in parallel direction.
Each chain is composed of 1000 amino acid residues. The glycine, proline,
and hydroxyproline are the constituent amino acids as in Fig. 3.11.
• Each polypeptide chain has a repeating proline residue, and the chain is called
as poly-proline polypeptide chain. Within the chain, alpha carbon atoms on
(–CO) and (–NH) groups are located on the opposite side of peptide bond.
Thus chain is called as left-handed poly-proline II helix. Each chain under-
goes right-handed coiling around each other to form a triple helix.
• The polypeptide chains are linked by hydrogen bonds. The hydrogen is
donated by (–NH) group of glycine and oxygen of carbonyl group (–CO) on
the proline. Three poly-proline helices are tightly packed in triple helix. There
are 3.3 amino acid residues in triple helix. Every third residue in triple helix
is glycine. There is a repeating sequence of glycine-AA2-AA3, where AA2
and AA3 can be proline and hydroxyproline.
Collagen polypeptide chains [precursors]
Triple helix procollagen
Fig. 3.11 Triple helix collagen
3.4.4 Tertiary Structure
Tertiary structure can be defined as “steric relationship of distantly placed

amino acid residues in a polypeptide chain.”
Characteristics
• Tertiary structure is the three-dimensional conformation of the entire polypep-
tide chain. It is composed of a single polypeptide chain. The peptide groups form
the backbone of tertiary structure.
• The side chains of distant amino acid residues undergo various bond formations
between each other. These interactions produce several folds and super folds in
polypeptide chain. The distant amino acid residues come closer to each other in
the chain as in Fig. 3.12.
• The three-dimensional conformation is a physiologically active protein and
is called native protein.
• Tertiary structure is stabilized by five types of non-covalent bonds as:
–– Hydrogen bonds: These are non-covalent and weak electrostatic force of
attraction. Hydrogen bonds are formed between the polar side chains in amino
acid residues.
Example: Arginine, histidine, glutamate, aspartate, lysine, serine, tyrosine
–– Ionic bonds (electrostatic bonds): These are non-covalent bonds which are
formed between oppositely charged side chains in amino acid residues.
Example: Arginine, histidine, glutamate, aspartate, lysine
Fig. 3.12 Tertiary Protein
SS H
S S H
S
S
H S S
NH2
COOH Amino terminal
Carboxy terminal
–– Hydrophobic interactions: These are force of attraction between non-polar

side chains of amino acid residues. These are main forces that stabilize ter-
tiary structure of proteins.
Example: Alanine, leucine, isoleucine, phenylalanine
–– Van der Waals forces: These are very weak forces that occur between non-
polar side chains.
–– Disulfide bonds: These are formed by oxidation of sulfhydryl groups in cys-
teine amino acid residues in side chains.
Example of tertiary structure: Myoglobin
3.4.5 Quaternary Structure of Protein
Quaternary structure is the three-dimensional arrangement of two or more

folded polypeptide chains.
The folded protein subunits are positioned relative to each other in the qua-
ternary structure. These subunits are held by non-covalent forces.
• The complex aggregate of protein subunits is called as oligomer. It is biologi-

cally functional.
• Constituent polypeptide chains are called as monomers as in Figs. 3.13 and
3.14.
Monomer
Monomer
NH2
NH2
COOH COOH
NH2
NH2
COOH
COOH
Fig. 3.13 Quaternary Protein
Based on the number of monomeric units, quaternary structure is called as:
• Dimer (two monomers), for example, creatine phosphokinase is dimer

• Trimer (three monomers), for example, collagen, porins
• Tetramer (four monomers), for example, hemoglobin, immunoglobulin
• Pentamer (five monomers), for example, GABAA receptor
• Hexamer (six monomers), for example, viral capsides
Similarity or Dissimilarity of Monomers
• In a quaternary protein, monomers can be similar or dissimilar.

• Proteins with similar monomers are called as homodimer or homoteramer.
Example: Apoferritin is a protein. It is made up of 24 homo-monomers.
• Proteins with dissimilar monomers are called as heterodimer or heterotetramer.
Example: Hemoglobin is a heterotetramer. It is made up of two alpha and

two beta polypeptide chains.
Immunoglobulin is a heterotetramer. It is made up of two heavy chains and
two light chains.
Fig. 3.14 Proteins
Primary structure
Organization
Secondary structure
Tertiary structure
Quaternary structure
3.5 Amino Acids Definition
Amino acids are the organic compounds containing amino and carboxylic
groups.
These are fundamental structural components of proteins. Amino acids have
essential nutritive value for living organisms.
3.8 Important Characteristics of Amino Acids 55
3.6 Number of Amino Acids
More than 300 amino acids have been discovered and isolated. There are only 20
amino acids in nature which are necessary for biosynthesis of all the proteins. It is
the definite and unique sequence of amino acids in a polypeptide chain that enable
the synthesis of large number of proteins.
3.7 Structure of Amino Acid
It is an amino-carboxylic acid that is made of five components.
• Asymmetric carbon atom (C)

–– It is the central carbon in amino acid. It linked to four different groups. So it
is a chiral carbon. All amino acids except glycine contain chiral carbon. This
carbon atom is placed immediately next to carbon of carboxylic group and is
also called as α-carbon, and amino acid is called as α-amino acid. All 20
amino acids are alpha in nature.
–– The amino acids have (d) and (l) configurations. In (d) amino acid, the amino
group is positioned on the right-hand side of alpha carbon. In (l) amino acid, it
is positioned on the left-hand side of alpha carbon in the molecule. In nature, all
alpha amino acids have (l) configuration except glycine. Amino acid serine has
both (d and l)) configurations. d-serine is found in the cell wall of bacteria.
• Amino group (–NH2)
• Carboxylic group (–COOH)
• Hydrogen atom (H)
• Hydrocarbon side chain (R)
–– Hydrocarbon side chains provide identity to amino acids. Side chain may
contain acidic or basic groups. It may contain hydrophilic or hydrophobic
groups. These are necessary for the steric conformation of proteins. They
determine the structure and properties of proteins as in Fig. 3.15a.
3.8 Important Characteristics of Amino Acids
1. Stereoisomerism
Amino acids carry chiral carbon atom except glycine. So amino acids have
two isomers. The (d) amino acids carry amino group on the right-hand side,
while (l) amino acids have amino group on the left-hand side of the chiral car-
bon as in Fig. 3.15b.
2. Optical Isomerism
All amino acids except glycine rotate the plane polarized light into right- (d)
and left-hand side (l) as in Fig. 3.15.
a structure of amino acid b stereoisomerism in amino acid

Hydrogen Atom H H
H
H2N C COOH HOOC C NH2
H2N C COOH
R R
Amino group Carboxylic
R Group L - Amino acid D - Amino acid
Side chain
c H H H
H3N+ C COOH H3 N+ C COO– H2N C COO–
R R R
Acidic PH Isoelectric PH Alkaline PH
Cation Zwitterion Anion
Fig. 3.15 Zwitterion
3. Zwitterions
Amino acids possess (COOH) and (NH2) groups. The carboxylic group
donates proton and acts as acid. The amino group accepts proton and acts as
base. This is amphoteric property. Amino acids with amphoteric property are
called as ampholytes as in Fig. 3.15c.
So the carboxylic group becomes negatively charged, while amino group
becomes positively charged. At a particular pH, amount of negative and positive
charge equals each other, and the net charge on amino acid is zero. Amino acid
behaves as dipole ion and is called zwitterion. It is a neutral dipolar ion.
The pH at which amino acid carries no charge and it does not migrate toward
any electrode in an electric field is called as isoelectric pH.
3.9 Classification of Amino Acids
Depending on various criteria, amino acids can be classified in different ways:
• Classification depending on position of amino group

• Classification depending on proteinogenic property
• Classification depending on polarity of side chain
• Classification depending on chemical structure of side chain
• Classification depending on nutritional value
• Classification depending on chemical property
3.9.1 Classification Depending on Position of Amino Group
Amino acids can be subclassified into three groups depending upon position of
amino group.
3.9 Classification of Amino Acids 57
Table 3.1 Showing name and nature of 20 amino acids

Serial number Name of amino acid Abbreviation Nature of amino acid
1 Methionine Met Essential
2 Valine Val Essential
3 Isoleucine Ile Essential
4 Leucine Leu Essential
5 Phenylalanine Phe Essential
6 Threonine Thr Essential
7 Tryptophan Trp Essential
8 Lysine Lys Essential
9 Histidine His Semi-essential
10 Arginine Arg Semi-essential
11 Alanine Ala Nonessential
12 Asparagine Asn Nonessential
13 Aspartic acid Asp Nonessential
14 Cysteine Cys Nonessential
15 Glutamic acid Glu Nonessential
16 Glutamine Gln Nonessential
17 Proline Pro Nonessential
18 Serine Ser Nonessential
19 Tyrosine Tyr Nonessential
20 Glycine Gly Nonessential
• Alpha amino acids

–– Amino group is attached to a carbon atom immediately next to carbox-
ylic group and is called as alpha amino group. Carbon atom is called as (C1)
or alpha (α) carbon.
Example: All 20 amino acids have alpha amino groups in Table 3.1
• Beta amino acids
–– Amino group is attached to a carbon atom next to alpha carbon and is called
beta (β) amino group, and carbon is called as (C2 ) or beta (β) carbon.
• Gamma amino acids
–– Amino group is attached to a carbon atom next to beta carbon and is called
gamma amino group, and carbon is called as (C3 ) or gamma (γ) carbon.
3.9.2 Classification Depending on Proteinogenic Property
According to proteinogenic property, amino acids can be subclassified into two

groups:
1. Proteinogenic amino acids

The amino acids that can be utilized for synthesis of proteins in living organ-
isms are called as called proteinogenic amino acids. These amino acids are coded
by DNA through triplet codons and also called as standard amino acids.
Example: 20 amino acids as in Table 3.1
Out of 20 amino acids, 9 proteinogenic amino acids are essential as they cannot
be synthesized by body tissues and have to be supplemented with diet, while 1
proteinogenic amino acid is semi-essential as its physiological need is raised dur-
ing periods of pregnancy and active growth of children. The remaining ten pro-
teinogenic amino acids are synthesized in the body and called as nonessential.
Example: Essential and nonessential amino acids are proteinogenic as in
Table 3.1.
2 . Non-proteinogenic amino acids
The amino acids that cannot be incorporated into structure of proteins during
their biosynthesis in body tissues are called non-proteinogenic amino acids.
They exist in cytoplasm either as metabolites or structural components of non-
protein compounds. They are also called as nonstandard amino acids.
Example: Gama amino butyric acid, hydroxyproline, ornithine, citrul-
line, carnitine, glycine
3.9.3 Classification Depending on Polarity of Side Chain
Amino acids are subclassified into two groups depending on affinity of side chain to
water molecules:
1. Hydrophobic amino acids

The hydrocarbon side chains do not react with water molecules and are
termed as non-polar side chains. Such amino acids are called as hydrophobic or
non-polar amino acids.
Example: valine, alanine, tyrosine, phenylalanine, proline, tryptophan as
in Table 3.1
2. Hydrophilic amino acids
The hydrocarbon side chains have high affinity for water molecules and are
termed as polar side chains. Such amino acids are called as hydrophilic or polar
amino acids. The side chains in these amino acids can be either positively charged
or negatively charged.
Example: serine, asparagine, threonine, glutamine, histidine, tyrosine in
Table 3.1
3.9.4 Classification Depending on Nutritional Value
Amino acids are necessary nutritional components tissues. Depending on their

nutritional value, amino acids can be subclassified into three groups:
1. Essential amino acids

These amino acids are not synthesized by body tissues and have to be supple-
mented with diet to fulfill their physiological demand. They are also called as
indispensable amino acids.
Example: as listed in Table 3.1
2. Semi-essential amino acid

It is also called as conditionally essential amino acid as its essentiality in the
body is determined by developmental stage of children and pregnancy and lacta-
tion among women. The amino acid has to be supplemented with diet during
these conditions.
3. Non-essential amino acids
These amino acids are synthesized in the body and do not have to be supple-
mented with diet.
3.9.5 C
lassification Depending on Chemical Structure of Side
Chain
Amino acids can be subclassified into different groups depending on chemical

structure of hydrocarbon side chains.
1. Simple amino acid

The side chain has the simplest structure in these amino acids as in Fig. 3.16.
Example: Glycine, alanine
2. Branched chain amino acids
The side chains are short and branched as in Fig. 3.17. Example: Valine,
isoleucine, leucine
Fig. 3.16 Simple Protein

amino acids
H H
H2N – C – COOH H2N – C – COOH
H CH3
Glycine Alanine
Fig. 3.17 Branched H H H
chain amino acids
H2N – C – COOH H2N – C – COOH H2N – C – COOH
CH – CH3 CH2 CH – CH3
CH3 HC – CH3 CH2
CH3 CH3
Valine Leucine Isoleucine
Fig. 3.18 Hydroxy amino H H

acids
∝
CH2 – OH CH2 – OH
Serine CH3
Threonine
Hydroxy amino acids
Fig. 3.19 Sulfur-containing H H
amino acids
CH2 CH2
SH CH2
Cysteine S CH3
Methionine
Sulfur containing A. Acids
Fig. 3.20 Amide-containing H H
amino acids
CO – NH2 CH2
Asparagine CH2
CO – NH2
Glutamine
Amide Group containing A. Acids
3. Hydroxy amino acids

The amino acids have hydroxyl groups in side chains as in Fig. 3.18.
Example: Serine, threonine
4. Sulfur-containing amino acids
The amino acids contain sulfur atoms in the groups attached in side chains
as in Fig. 3.19. Example: Cysteine, methionine
5. Amide groups containing amino acids
The amino acids possess carboxy-amide groups (R–CO–NH2–R) in the side
chains as in Fig. 3.20. Example: Asparagine, glutamine
Abovementioned groups of amino acids have an open chain structure and
possess one amino and one carboxylic group. So they are called as aliphatic
monoamino-monocarboxylic acids. These amino acids are neutral in
reaction.
6. Monoamino-dicarboxylic acids
The amino acids contain an additional carboxylic group in the side chain and
are called as monoamino-dicarboxylic acids as in Fig. 3.21. These are acidic
amino acids. Example: Aspartic acid, glutamic acid
7. Diamino monocarboxylic acids
The amino acids contain additional amino group in the side chain and are
called as diamino monocarboxylic acids as in Fig. 3.22. These are basic amino
acids. Example: Arginine, lysine, and a derived amino acid called as
hydroxylysine
Above two groups of amino acids have open chain structure and called as
aliphatic amino acids. The amino acid histidine is dibasic monocyclic carbox-
ylic acid but it has a heterocyclic structure.
8. Diamino dicarboxylic acids
These amino acids contain carboxy-amide group (–RCO–NH2–) in the side
chain as in Fig. 3.20. Amino acids have two amino and two carboxylic groups.
These amino acids are neutral. Example: Asparagine, glutamine
Fig. 3.21 Monoamino-dicarboxylic H H
acid
∝ ∝
β CH2 β CH
2
COOH γ CH
2
Asparatic Acid COOH

Glutamic Acid
Mono amino dicarboxylic Amino acids
Fig. 3.22 Diamino-monocarboxylic H H
acids
NH2 C COOH HN2 C COOH
∝ ∝
β CH2 β CH2
γ CH2 γ CH2
δ CH δ CH
2 2
NH ε CH2
C = NH+ NH3+
NH2 Lysine
Arginine
Diabasic amino acids
(Diamino monocarbaxolic acids)
9. Aromatic amino acids

The amino acids contain aromatic rings in the side chains as in Fig. 3.23.
They are neutral. Example: Phenylalanine, tyrosine
10. Heterocyclic amino acids
The amino acids possess heterocyclic rings in the side chains as in Fig. 3.24.
It contains dissimilar atoms like nitrogen, sulfur, or oxygen in the ring struc-
ture. Example: Tryptophan, histidine. The amino acid tryptophan is neu-
tral and histidine is basic in nature.
11. Amino acids containing amino group in side chain
The amino acids contain amino group (–NH) in side chains as in Fig. 3.25.
These amino acids are basic in nature. Example: Proline, hydroxyproline.
The nitrogen is a constituent atom of five-membered ring in these amino acids.
It is free to form peptide bond.
However, these amino acids lack a free amino group (–NH2). Hydroxyproline
is a non-proteinogenic amino acid. It is derived by hydroxylation of proline
during posttranslational modifications. It contains a hydroxyl group attached to
gamma carbon.
Benzene H2 Phenol H2
Ring Group
C HO C
CH NH2 CH NH2
COOH COOH
Phenyl alanine Tyrosine

Aromatic amino acids
Fig. 3.23 Aromatic amino acids
Fig. 3.24 Heterocyclic CH2

Indole
amino acids group
CH NH2
N
COOH
Tryptophan
Imidazole CH2
group
HN N CH NH2
COOH
Histidine
Fig. 3.25 Proline Pyrrolidine group
COOH
NH
Proline
3.9.6 Classification Depending on Chemical Property
Amino acids can be subclassified into three groups based on the reaction in aqueous
medium.
• Acidic amino acids

–– These amino acids have one amino and two carboxylic groups. These are
acidic in nature. Example: Monoamino dicarboxylic acids
• Basic amino acids
–– These amino acids have two amino and one carboxylic group. These are basic
in nature. Example: Arginine, histidine, lysine, and a derived amino acid
named as hydroxylysine
• Neutral amino acids
–– These amino acids have one amino and one carboxylic group. These are neu-
tral in reaction. Example: Aliphatic amino acids, hydroxy amino acids,
aromatic amino acids, heterocyclic amino acids, sulfur-containing amino
acids, amide-containing amino acids (dibasic dicarboxylic acids)
Essential amino acids can be memorized as “TT named PVM is ILL

today,” where TT stands for threonine and tryptophan, while PVM stands for
phenylalanine, valine, and methionine, whereas ILL means isoleucine, leucine,
and lysine.
• Selenocysteine is a rare amino acid and is considered the 21st amino acid.
In it, cysteine contains selenium instead of sulfur. It is coded by termina-
tion codon named as UGA among prokaryotes, eukaryotes, and humans. It
contains trace element, selenium having an antioxidant property. Example:
Glutathione peroxidase enzyme contains selenocysteine.
• Pyrrolysine is another rare amino acid and considered as 22nd in number.
It is coded by UAG codon. It is present in methane-producing archaebac-
teria. Pyrrolysine amino acid is absent in the body of humans.
• Tyrosine amino acid is necessary for biosynthesis of catecholamines like
thyroxin, adrenaline, and nor-adrenaline. It is necessary for melanin pig-
ment synthesis.
• Methionine is coded by AUG codon and it starts the translation. Activated
methionine, S-adenosyl methionine (SAM), is a methyl donor to lipids,
proteins, and nucleic acids.
• Histidine undergoes decarboxylation to form histamine. It initiates allergy
and immune reaction.
• Trytophan is necessary for synthesis of nicotinic acid and serotonin.
• Glycine is necessary for synthesis of heme.
3.10 Applied Biochemistry
3.10.1 Arginine
• It is an essential amino acid. Its oral health benefit has been tried. Kleinberg
(2002) proved that topical preparation containing calcium carbonate and argi-
nine bicarbonate gets deposited over the open dentinal tubules. The arginine
molecule helps to block open dentinal tubules and minimizes dental
hypersensitivity.
3.10.2 Casein Phosphopeptides (CPP)
• It has been demonstrated that milk phosphor-protein, casein has a role in the remin-
eralization of dental enamel. Casein phosphopeptides (CPP) are extracted from milk
casein by tryptic digestion. Reynolds (1997) observed that topically administered
CPP complexes are absorbed into the dental plaque. It has affinity for calcium and
phosphate from saliva and results into an increase in localized concentration of cal-
cium and phosphate over tooth surfaces.
• Casein phosphopeptides (CPP) possess sequences of (-Pse-Pse-Pse-Glu-Glu-),
where Pse indicates “phosphoseryl” residue. These residues stabilize calcium
and phosphate ions in oral cavity. These ions are precipitated into amorphous
calcium phosphate (ACP) complex. The CPP molecule combines with ACP to
form complexes, CPP-ACP.
• The ACP molecules over tooth surfaces can be converted into octacalcium phos-
phates. It is an intermediate of hydroxyapatite crystals in dental enamel.
• The CPP-ACP nanoclusters are formed at pH between 5 and 9.
• The CPP-ACP complexes can inhibit growth of Streptococcus mutans in dental
plaque.
• The complexes raise oral pH which can prevent dental demineralization.
• The ACP nano-complexes can be transformed into hydroxyapatite crystals and
help to remineralize the incipient dental carious lesion.
• Configuration of a molecule means the arrangement of atoms or groups

within a molecule in three-dimensional space. The isomers are called as
configurational isomers. For example: d-amino acid and l-amino acid
• Conformation of a molecule means the rotation of atoms or groups about a
bond within a molecule in three-dimensional space. The isomers are called
as conformational isomers or rotamers. For example, peptide bond has cis
and trans conformers in polypeptide chain.
• Carnitine is an amino acid found in skeletal muscles, heart muscles, liver,
and kidneys. It is synthesized in the liver from methylation of lysine. It is
chemically, β-hydroxy-γ-N-trimethylaminobutyric acid
Suggested Readings 65
• Carnosine is a dipeptide made up of alanine and histidine. It is found in

skeletal muscles and brain tissues.
• A tripeptide, creatine, is synthesized by glycine, arginine, and
methionine.
• A tripeptide and antioxidant, glutathione, is synthesized by glycine, glu-
tamic acid, and cysteine.
• Bradykinin is a peptide of nine amino acids. It is a vasodilator.
• Angiotensin I is a peptide of ten amino acids. It is a potent vasoconstrictor.
It is converted into angiotensins II and III by cleavage of two and three
amino acids from angiotensin I, respectively.
Suggested Readings
Kleinberg I (2002) SensiStat. A new saliva-based composition for simple and effective treatment
of dentinal sensitivity pain. Dent Today 21:42–47
Reynolds EC (1997) Remineralization of enamel subsurface lesions by casein phosphopeptide-
stabilized calcium phosphate solutions. J Dent Res 76:1587–1595
Branden C, Tooze J (1999) Introduction to protein structure. Garland, New York
Murray RF, Harper HW, Granner DK, Mayes PA, Rodwell VW (2006) Harper’s illustrated bio-
chemistry. Lange Medical Books/McGraw-Hill, New York
Van Holde KE, Mathews CK (1996) Biochemistry. Benjamin/Cummings, Menlo Park, CA
Plasma Proteins
4
Plasma proteins constitute important organic component in plasma. They are

also called as serum proteins. Plasma proteins are comprised of simple as well as
conjugated proteins. The average concentration of plasma proteins is 7.4 g%, and it
varies between 6.5 g% and 8.4 g% under normal condition of health.
Amount of plasma proteins and relative proportion of individual proteins in
plasma are affected by diseases. Therefore, plasma proteins have diagnostic and
prognostic significance.
4.1 Classification of Plasma Proteins
A large number of plasma proteins have been identified and separated from plasma.
Various methods have been utilized to separate plasma proteins. The concentration of dif-
ferent proteins in plasma is highly variable. Today, around 100 plasma proteins have been
identified. Majority of proteins exist in plasma in trace amount and belong to subclasses of
major plasma proteins. It is difficult to classify plasma proteins. A list of plasma proteins
is given based on their fractions in electrophoretic method of separation (Table 4.1).
4.2 Plasma Proteins
Individual plasma proteins are described in the following portion as below:
4.3 Albumin
• Characteristics
–– Albumin is a globular protein. It is protein made up of single polypeptide
chain. It has 610 amino acids.

https://doi.org/10.1007/978-981-13-1035-5_4
68 4 Plasma Proteins
Table 4.1 Showing different types of plasma proteins

Type of plasma protein Concentration of plasma protein
1. Albumin 3.5–5.5 g/dl
2. Globulins 2.5–3.5 g/dl
(α1)-Globulins 0.1–0.4 g/dl
(α1)-Acid glycoprotein (orosomucoid) 60–140 mg/dl
(α1)-Fetoprotein <1 μg/dl
(α1)-Antitrypsin 200–400 mg/dl
(α2)-Globulins 0.4–0.8 g/dl
Ceruloplasmin 30 mg/dl
Haptoglobin 30–200 mg/dl
(β)-Globulins 0.5–1.2 g/dl
Transferrin 200–350 mg/dl
Hemopexin 50–100 mg/dl
C-reactive protein <1 mg/dl
(γ)-Globulins 0.7–1.5 g/dl
IgA 150–250 mg/dl
IgG 600–1600 mg/dl
IgM 60–179 mg/dl
IgD 3 mg/dl
IgE 10–70 μg/dl
Other proteins
Fibrinogen 200–400 mg/dl
Prothrombin 5–10 mg/dl
Transthyretin (pre-albumin) 10–25 mg/dl
Transcortin 7 mg/dl
Thyroxine-binding globulin 1 mg/dl
Transcobalamin I 1–20 μg/l
–– Its molecular weight is 69,000.

–– Albumin constitutes nearly 50–70% of the total plasma proteins.
–– Its normal serum concentration varies between 3.5 and 5.5 g% in adults.
–– Its isoelectric pH is 4.7.
–– Albumin can be precipitated with full saturation of ammonium sulfate.
–– It is synthesized in the liver.
• Functions of Albumin
–– It exerts colloidal osmotic pressure inside plasma in vessels. Albumin is a
macromolecular organic compound. It cannot egress from capillary endothe-
lium. So albumin maintains 70–80% of the total colloidal osmotic pressure in
vessels. In a capillary, COP ranges from 25 to 30 mm Hg, and albumin con-
tributes to COP about 22 mm Hg.
–– Colloidal osmotic pressure or oncotic pressure is the force that draws the
water inside the plasma from interstitial fluid. It helps to maintain water con-
tent of plasma.
4.5 α1-Globulins 69
–– Albumin helps in the transportation of calcium ions, unconjugated bilirubin,

nonesterified fatty acids, and thyroid hormones.
–– Albumin is a transporter of acidic and neutral drugs like warfarin sodium,
penicillin, diazepam, acetyl salicylic acid, and furosemide.
• Clinical Significance
–– A decrease in the concentration of serum albumin is found in liver disease,
protein energy malnutrition, and glomerulonephritis. It results into movement
of fluid from vascular compartment into interstitial spaces. This leads to
edema formation in the body. Generally, it occurs when the serum albumin
concentration falls below 2.5 g%.
4.4 Globulins
• Characteristics
–– Globulins are globular group of proteins. They are insoluble in water.
–– They have molecular weight ranging from 90,000 to 1,300,000.
–– Globulins can be differentiated α-globulins, β-globulins, and γ-globulin frac-
tions with the help of electrophoresis.
4.5 α1-Globulins
4.5.1 Alpha-1: Acid Glycoprotein
• Characteristics
–– It is also called as orosomucoid protein.
–– Its normal serum concentration is 60–140 mg/100 ml.
–– It is a carrier of basic drugs like quinidine, propranolol, and morphine.
–– It transports steroidal hormone like progesterone.
–– It is a biomarker for acute inflammation in the body, so it is called as acute-
phase protein.
4.5.2 Alpha-1 Fetoglobulin
• Characteristics
–– It is present in fetal blood circulation in pregnancy.
–– In adults, its normal concentration is <1 μg/100 ml.
–– This protein is a tumor marker for hepatocellular carcinoma.
4.5.3 Alpha-1 Antitrypsin
• Characteristics
–– It is a serum trypsin inhibitor, and it inhibits the activity of proteases.
–– Its normal concentration is between 200 and 400 mg/100 ml in adults.
–– It is an important acute-phase reactant protein. Its concentration increases in
inflammation and infection as acute injury, liver cirrhosis, hepatocellular car-
cinoma, malignancy, and burns.
–– It protects the tissues from lytic activity of elastase enzyme. It is secreted by
neutrophils, and the enzyme degrades elastin protein in lung and liver tissues.
1. Lung disease
• Alpha-1 antitrypsin deficiency is a genetic disorder. It is due to presence of
defective alleles such as PiM, PiZ, and PiF. The alleles PiZ in homozygous
state (ZZ) is responsible for severe deficiency of alpha-1 antitrypsin in
blood. The persons with genotypes (PiMM) and (PiZZ) have normal and defi-
cient alpha-1 antitrypsin levels.
• Persons with ZZ genotype have higher susceptibility to chronic
obstructive pulmonary disease and liver cirrhosis.
• Cigarette smoking in ZZ genotype persons predisposes to emphysema and
COPD. Smoking oxidizes methionine 358 residue in alpha-1 antitryp-
sin and renders it inactive.
• Therefore, elastase disrupts lung tissues in the absence of alpha-1 antitryp-
sin as in Fig. 4.1.
2. Liver cirrhosis
• Deficiency of alpha-1 antitrypsin leads to juvenile liver cirrhosis.
3. Diagnostic Tool
• Alpha-1 antitrypsin has a diagnostic role as tumor marker in malignancy of
gonads.
Fig. 4.1 Flow chart

showing proteolysis of
ZZ genotype
lung tissues
Absent or low alpha-1 Antitrypsin level
Inhibition of formation of elastase-alpha-1 Antitrypsin complex
Proteolysis of lung tissues by active elastase enzyme

4.6 α2-Globulins 71
4.6 α2-Globulins
4.6.1 Ceruloplasmin
• Characteristics
–– It is a glycoprotein containing eight copper atoms as cofactor.
–– Its normal serum concentration is 30 mg/100 ml in adults.
–– Ceruloplasmin contains about 90% of the total serum copper.
–– It is an important ferroxidase enzyme. It helps in conversion of ferrous ion
into ferric ion.
–– Its serum concentration is decreased in liver disease and mineral deficiency.
–– In Wilson’s disease, copper accumulates in tissues of the liver and brain. It is
an autosomal recessive trait. It is characterized by edema of the legs and abdo-
men, yellow discoloration of the skin, anxiety, emotional disturbance, and
behavior change.
–– In Menkes disorder, deficiency of copper occurs in the body. It is an X-linked
recessive trait. It is characterized by brittle hairs, muscular weakness, growth
deficiency and damage to brain development, and seizures. The disease starts
early in infancy, and generally, baby dies by the age of 2–3 years.
4.6.2 Haptoglobin
• Characteristics
–– Haptoglobin is composed of two light chains (alpha) and two heavy chains
(beta) linked covalently with each other by disulfide bridges.
–– Its normal serum concentration is between 30 and 200 mg/100 ml in adults.
–– In normal healthy persons, breakdown of RBCs in blood vessels is a common
event. About 10% of total erythrocytes can undergo hemolysis.
Hemoglobin is released from ruptured erythrocytes within blood vessels.
–– Free heme is highly toxic to tissues of blood vessels. Ferrous iron (Fe++) in
heme undergoes Fenton reaction to produce reactive oxygen species (ROS) in
blood vessels. These can damage lipid layer, protein, and DNA.
–– Haptoglobin binds with free hemoglobin through its alpha chain and
forms a haptoglobin-hemoglobin complex. This complex cannot cross
through glomerular filtration.
–– Haptoglobin prevents loss of free hemoglobin in urine.
–– Haptoglobin-hemoglobin complex is captured by macrophages and spleen.
Free hemoglobin undergoes biodegradation.
–– Haptoglobin has antioxidant and cytoprotective functions.
–– Haptoglobin is an acute-phase protein. Its serum level increases in acute

inflammation and infection.
–– Haptoglobin helps in diagnosis of hemolytic anemia. Its serum concen-
tration decreases in hemolytic anemia.
4.7 β-Globulins
4.7.1 Transferrin
• Characteristics
–– Transferrin is an iron-containing glycoprotein in plasma.
–– Its normal serum concentration is 200–350 mg/100 ml in adults.
–– Transferrin is a carrier of iron in plasma.
–– Transferrin is made up of a single polypeptide chain called as “apo-
transferrin” which binds with two ferric ions and is called as transferrin.
–– Transferrin helps in the distribution of iron in ferric state. It delivers iron
to bone marrow for biosynthesis of hemoglobin.
–– Transferrin has a positive role in providing innate immunity.
–– Serum transferrin concentration is increased in iron deficiency anemia.
–– Serum transferrin concentration is decreased in liver cirrhosis, glomerulone-
phritis, protein-energy malnutrition, acute infection, and burns.
4.7.2 C-Reactive Protein
• Characteristics
–– It is a beta globulin protein present in plasma.
–– It is a pentameric protein. It is made up of five polypeptide chains.
–– Its normal concentration is less than 1 mg/100 ml in adults. Aging, pregnancy,
inflammation, and burns increase serum level of C-reactive protein.
–– This protein has a capability to react with antigenic polysaccharide of group
C present in pneumococci, so-called C-reactive protein.
–– C-reactive protein has the ability to bind with phosphocholine in the plasma
membrane of dead cells and bacteria. C-reactive protein-phosphocholine
complex activates complement system and helps in the activation of T lym-
phocytes and macrophages.
–– It is an acute-phase reactive protein. Its serum concentration increases in
acute inflammation and infection. So it is a non-specific biomarker for inflam-
mation and infection. It is helpful in the prognosis of a disease.
–– C-reactive protein is a better indicator of inflammation than erythrocyte sedi-
mentation rate.
4.8 Other Important Plasma Proteins 73
4.7.3 Hemopexin
• Characteristics
–– It is a beta globulin. It is made up of a single polypeptide chain of 439 amino
acids.
–– Hemopexin has the strongest affinity for binding with “heme.” This affinity is
the highest among known proteins. Hemopexin binds to heme in 1:1 ratio.
–– Its serum concentration is between 50 and 100 mg/100 ml in adults. In infancy,
its serum concentration is low, and it comes up to adult serum level within first
year of life.
–– Hemopexin has the cytoprotective and antioxidant properties similar to
haptoglobin.
• Clinical significance
–– Serum hemopexin level is decreased in hemolytic anemia. So it has a diagnos-
tic value for assessing hemoglobin level and RBC count.
4.8 Other Important Plasma Proteins
4.8.1 Bence-Jones Protein
• Characteristics
–– It is a paraprotein (an antibody that arises from rapid proliferation of
monoclonal plasma cells during malignancy).
–– It is an abnormal immunoglobulin.
–– Its molecular weight is 45,000.
–– It is made up of immunoglobulin light chain. So it is a subunit of a normal
immunoglobulin.
–– Light chain can be either kappa (κ) chain or lambda (λ) chain.
–– Light chain is made up of around 217 amino acids.
–– Bence-Jones protein appears in blood and urine in persons suffering from
multiple myeloma. It is a tumor of plasma cells. Normally, plasma cells pro-
duce thousands of antibodies which are grouped into five categories, such as
IgA, IgG, IgM, IgD, and IgE. Tumor plasma cell produces a particular type of
antibody in large excess.
–– Serum paraprotein concentration more than 3 mg/100 ml is a diagnostic of
multiple myeloma.
–– This protein is identified by heating a sample of urine to 60 °C temperature.
The protein gets precipitated. Further heating of urine dissolves the precipi-
tate. If the urine sample is cooled under tap water, the precipitate reappears.
4.8.2 Fibrinogen
• Characteristics
–– It is a soluble plasma protein. It is called clotting factor I.
–– Fibrinogen is the precursor to fibrin, which is necessary for blood clotting.
–– Its normal serum concentration is 200–400 mg/100 ml in adults.
–– It is a hexameric protein. It is made up of two Aα chains, two Bβ chains, and
two γ polypeptide chains. These chains are interlinked by disulfide bridges.
–– Its molecular weight is between 350,000 and 450,000.
–– Fibrinogen molecule has a highly elongated structure with an axial ratio of
20:1.
–– Fibrinogen is necessary for blood clotting.
–– Its serum concentration is decreased in liver diseases.
–– Its low serum concentration is correlated to higher bleeding tendency.
–– Fibrinogen is an acute-phase reactant protein. It is a biomarker of coronary
heart disease.
4.9 Biological Functions of Plasma Proteins
• Acid-base Regulation
–– Plasma proteins are amphoteric in nature. So they can buffer an acid or base
in blood. It helps to maintain acid-base balance of blood and body fluid.
• Colloidal Osmotic Pressure (Oncotic Pressure)
–– Plasma proteins offer colloidal osmotic pressure. It is normally about 25 mm
of Hg. Albumin exerts maximum COP due its larger concentration in plasma.
Colloidal osmotic pressure is necessary for normal distribution of body water
in blood vessels and interstitial spaces.
• Role in Blood Clotting
–– Plasma contains fibrinogen, prothrombin, and other blood clotting factors in
inactive form. At the time of injury, these components are activated in a cas-
cade fashion and helps in blood clotting.
• Role in Immunity
–– Plasma contains immunoglobulins. These are synthesized by B lymphocytes.
They protect the body against pathogens.
• Role in Transport of Substances
–– Albumin and globulins transport large substance from blood to tissues. Drugs,
hormones, and enzymes are transported by plasma proteins.
• Nutritive Role
–– Plasma proteins are simple proteins. They are source of amino acids. So they
have play a nutritive role in the body.
• Blood Viscosity maintenance

–– Plasma proteins make the blood viscous. It is due to presence of globulins and
fibrinogen in plasma. Blood viscosity is necessary for maintenance of normal
blood pressure.
• Storage of Enzymes
–– Plasma is a store house of several enzymes like, lipase, amylase, and trans-
aminases in minute amounts. The variation in the amount of plasma enzymes
is helpful in the diagnosis and prognosis of diseases.
• Act as Reserve Proteins
–– Under fasting condition, plasma proteins are broken down to amino acids.
These are carried to tissues by blood circulation. They are utilized by tissues
for synthesis of tissue proteins.
Suggested Readings
Alberti KGMN (ed) (1978) Recent advances in clinical biochemistry. Churchill Livingstone,
London
Baron DN (1982) A short textbook of chemical pathology, 4th edn. Wiley, New York
Conn EE, Stump PK (1969) Outline of biochemistry, 2nd edn. Wiley, New Delhi
Hemoglobin
5
5.1 Definition
Hemoglobin
• Hemoglobin refers to a red colored conjugated protein consisting of heme as

prosthetic group and globin as an apoprotein.
Nature of Hemoglobin
• Hemoglobin is a metalloprotein owing to presence of ferrous ions as prosthetic

moiety.
Chromoprotein
• Hemoglobin is a chromoprotein owing to presence of a pigmented prosthetic

moiety.
5.2 Characteristics of Hemoglobin
• Hemoglobin constitutes about 95% of dry weight and 35% of wet weight of
erythrocytes.
• Normal Concentration of Hemoglobin
–– At birth: 23 g%
–– After infancy: 12.5 g%
–– Adult males: 14–18 g%

https://doi.org/10.1007/978-981-13-1035-5_5
78 5 Hemoglobin
–– Adult females: 12–16 g%

–– Clinical value: 14.7 g% regardless of gender variation
• Hemoglobin is an essentiality for life of aerobic organisms including
humans.
5.3 Structure of Hemoglobin
• Hemoglobin is composed of two components as:

–– Apoprotein: It is called globin.
–– Prosthetic group: It is called heme.
• Globin
It is a globular protein. It is composed of four polypeptide chains. Depending
on number and sequence of amino acid residues, chains are designated as:
–– α chains
Globin contains two α chains. Each one is composed of 141 amino acid
residues.
–– β chains
–– Globin contains two β chains. Each one is composed of 146 amino acid
residues.
• Hydrophobic amino acids are located interiorly, and hydrophilic amino acids are
placed externally in hemoglobin polypeptide chains. This feature makes hemo-
globin water soluble. Cluster of hydrophobic amino acid residues constitutes
hydrophobic or heme pocket in each polypeptide chain.
• Each heme pocket contains one heme moiety. Therefore, one hemoglobin mole-
cule contains four heme moieties.
Hemoglobin is heterotetramer composed of two α chains and two β chains.
5.3.1 Heme
Heme is iron-protoporphyrin IX complex. Its structure is described as

follows:
Structure of Protoporphyrin IX
It is a common type of porphyrin. It is a planar molecule. It has tetrapyrrole struc-
ture which is composed of four pyrrole rings fused together.
• Pyrrole ring: It is a five-membered ring made of four carbon atoms and one
nitrogen atom as in Fig. 5.1. Pyrrole rings are numbered from I to IV. They are
interlinked with methyne bridges (〓CH–).These bridges are labeled as α, β, γ,
and δ, respectively. Inner carbon atoms of pyrrole rings are linked to methyne
bridges.
5.3 Structure of Hemoglobin 79
Fig. 5.1 Pyrrole ring HC CH
HC CH
NH
Fig. 5.2 Structure of Methyl group

Vinyl
hemoglobin group
H3C – C1 C2 – CH= CH2 H 3C – C 3 C4 – CH= CH2
I II
C C CH C C
IV IV
Fe++
δ CH β CH
N N
γ
C CH C
IV III
H 26
H3C – C8 C7 HOOC – CH2 – C C5 – CH3
CH2
CH2
N - atom of imidazole
Propionic acid COOH ring of histidine
]Globin
• Outer carbon atoms of pyrrole rings are free and numbered from 1 to 8. Hydrogen
atoms from positions 1 to 8 are substituted by different groups.
• Hydrogen atoms at positions 1, 3, 5, and 8 are substituted by methyl groups (–
CH3), while at positions 2 and 4 are substituted by vinyl groups (–CH〓CH2),
and positions 6 and 7 are replaced with propionic acid groups (–CH2.CH2.
COOH) as in Fig. 5.2.
Valencies of Iron
• Iron in heme exists in ferrous state. Ferrous iron has valence 6 and has 6
coordinate positions.
• Iron is positioned at the center of tetrapyrrole. Ferrous iron is attached to
four nitrogen atoms (N) of pyrrole rings by four coordinative bonds.
80 5 Hemoglobin
• Ferrous iron is also linked to nitrogen (N) atom of imidazole ring of histidine
amino acid residue in polypeptide chain through its fifth coordinative position.
• In oxygenated state, ferrous iron is linked reversibly to one molecule of oxygen
through sixth coordinative bond.
• In deoxygenated state, sixth coordinative position of iron is occupied by H2O
molecule.
A hemoglobin molecule:
• Contains four polypeptide chains

• Contains four heme moieties
• Contains four ferrous atoms
• Carries four oxygen molecules
Binding of hemoglobin with oxygen molecule is called as oxygenation. This

process is not called oxidation because iron remains in ferrous state in
oxyhemoglobin.
5.4 Two-State Model of Hemoglobin
It is called as Monod-Wyman-Changeux model of hemoglobin.
Basis of Model
• An allosteric protein (a protein that can have many sites for binding with multi-
ple ligands) is made up of multiple subunits.
• Each subunit has ability to exist in two conformational states as tense state (T)
and relaxed state (R).
• These two conformers (T and R) are in equilibrium with each other.
Hemoglobin molecule is a tetramer with four subunits which are arranged

in two identical dimers.
Each dimer has one α-subunit and one β-subunit and are designated as
(α1β1) and (α2β2).
They are held together by non-covalent forces.
Depending on relative position of dimmers and affinity toward ligand, hemoglo-
bin molecule exhibits two thermodynamic forms as follows:
“T” Form of Hemoglobin
• T represents tense form of hemoglobin. Non-covalent forces restrict conforma-

tional change both within dimer and between two dimmers. Hemoglobin has less
affinity to oxygen molecule in T form. This form is deoxy form of hemoglobin.
5.4 Two-State Model of Hemoglobin 81
The oxygen binding coordinate site of ferrous iron is vacant. It is occupied by a

water molecule. Low pO2, high pCO2, and low pH always favor T form of hemo-
globin as in Fig. 5.3.
• R represents relaxed form of hemoglobin. In a heme residue, binding of oxygen
to Fe++ iron brings about rearrangement of its electrons. Ferrous iron undergoes
a change in its position and comes to lie in plane of protoporphyrin ring as in
Fig. 5.4.
Fig. 5.3 T form of N N

hemoglobin
Fe++ Iron is
N N Domed
N
H
Deoxy haemoglobin
[ T- Form]
Fig. 5.4 R form of H

hemoglobin Hydogen bonding
O O
N N
Iron is
planar Fe++
N N
N
H
Oxy haemoglobin
[R- Form]
82 5 Hemoglobin
• Ferrous iron is attached to proximal histidine in polypeptide chain; therefore,

shift in position of iron induces a change in histidine position. Finally, one poly-
peptide chain undergoes conformational shift.
• Conclusively, relative interactions between one polypeptide chain and contigu-
ous polypeptide chains undergo complete shift. One dimer undergoes rotation of
15 degrees relative to position of adjacent dimer in hemoglobin molecule.
• The conformational change enhances affinity of heme residues to oxygen.
Therefore, every additional oxygen molecule binds with higher affinity in
comparison with earlier oxygen molecule, and this property is called coop-
erative binding.
• R form oxy form of hemoglobin.
• High pO2, low pCO2, and high pH always favor R form of hemoglobin.
• T form is deoxy form of hemoglobin.
• High pCO2, low pO2, and low pH always favor T form of hemoglobin.
• Rotation of one dimer relative to other dimer is responsible for interconver-
sion of T form into R form and vice versa.
5.5 Functions of Hemoglobin
1 . It helps in transportation of oxygen from pulmonary alveoli to body tissues.

2. It transports carbon dioxide from body tissues to pulmonary alveoli.
3. It also acts as protein buffer which is an important buffer of plasma.
5.6 Variants of Hemoglobin
Various types of hemoglobin are found in population. Hemoglobin variants are due
to different globin genes which control synthesis of globin chains as follows:
• Chromosome 16
• Two α-globin genes are located on each chromosome 16
• Chromosome 11
• This chromosome contains three types of genes as:
• Single β-globin gene on each chromosome 11
• Two γ-globin genes and one δ-globin gene
These variants are expressed due to gene expression. These variants differ in
their structure of globin fraction of hemoglobin molecule. Throughout all variants,
heme moiety remains unaltered.
Normal hemoglobin variants neither exhibit any biochemical changes nor
clinical manifestation in the individuals.
5.6 Variants of Hemoglobin 83
5.6.1 Adult Hemoglobin (HbA)
It is normal hemoglobin in adult population. It exists into two forms as:
• Hemoglobin A
• Hemoglobin A2
Hemoglobin A (HbA)
It is composed of 2α chains and 2β chains. It is designated as (α2 β2). This hemo-
globin is found in 90–95% of adult population.
Hemoglobin A2 (HbA2)
It is composed of 2α chains and 2δ chains. It is designated as (α2 δ2). Beta chains
are substituted by delta chains. Each delta chain contains 146 amino acids. Delta
chain has ten amino acid residues different from beta chain. This Hb is found in
around 2.5% of adult population.
5.6.2 Fetal Hemoglobin (HbF)
• It is composed of 2α chains and 2γ chains. It is designated as (α2 γ2). Beta

chains are substituted by gamma chains. Each gamma chain has 146 amino acid
residues. It has 37 amino acids different from beta chains.
• Fetal Hb is the predominant hemoglobin during 20th-week period of intra-
uterine life.
• HbF has much higher affinity for oxygen. At pO2 of 20 mm of Hg, oxygen
saturation of HbA is 35% and HbF is 70%.
It can transport greater volume of oxygen at low partial pressure of oxygen. This
characteristic is helpful in oxygen transport from maternal circulation to fetus.
• At birth, fetal hemoglobin represents 80% of the total Hb of the body of infant.
It is replaced by adult Hb after 6–12 months.
5.6.3 Glycosylated Hemoglobin (HbA1C)
It is a form of adult hemoglobin. A small fraction of hemoglobin undergoes glyco-

sylation with glucose moiety. It is designated as HbA1c. It represents nearly 2% of
total adult hemoglobin. However, individuals suffering from diabetes mellitus pos-
sess higher proportion of glycosylated Hb (5–10%).
Its amino acid number and sequence are similar to adult hemoglobin. It differs
from HbA in having a sugar residue linked to valine amino acid residue by
glycosylation.
84 5 Hemoglobin
5.7 Hemoglobinopathies
Hemoglobinopathies refer to group of genetic disorders producing abnormal-

ity in structure of one of the globin chains and is characterized by altered bio-
chemical reactions and clinical manifestation in the body of individuals.
Variants of Abnormal Hemoglobin

Globin synthesis is regulated by genes. A pair of α-globin genes and a pair of
β-globin genes are located on chromosomes 16 and 11. They control translation of
two α and β polypeptide chains. Mutation in globin genes is responsible for altered
structure of globin chains.
Gene mutation manifests into mutant hemoglobin which in turn expresses into
clinical signs and symptoms.
Therefore, abnormal hemoglobin is mutant hemoglobin. There are more than
1000 variants of abnormal hemoglobin which have been detected. They can be cat-
egorized into two groups depending on type of gene mutation as:
Mutation in Structural Gene
• This mutation is responsible for substitution of an amino acid residue in adult

hemoglobin by another amino acid residue. Sequence of amino acid residues is
affected. Synthesis of polypeptide chains remains unaffected.
Example: HbS, HbD
Mutation in Regulatory Gene
• This mutation affects synthesis of polypeptide chains. This condition manifests

either affected synthesis of alpha chain or beta chain, and disorder is termed as
thalassemia.
5.7.1 Sickle-Cell Hemoglobin (HbS)
It is the hemoglobin that is contained in sickle-shaped (crescent-shaped) erythro-

cytes. It is a highly frequent variant of abnormal hemoglobin in population.
Occurrence
This disorder was mentioned by James B Herrick, an American physician in 1930.
Sickle-cell anemia is more common in African countries. Black population is
more prone to occurrence of disease. According to an estimate, more than 80% of
sickle-cell anemia sufferers are confined to Southern Africa.
Characteristics of Sickle-Cell Hemoglobin
• It is an abnormal hemoglobin with deviated structure. It has two alpha

chains which are normal. They have amino acid sequence similar to that of
adult hemoglobin.
5.7 Hemoglobinopathies 85
• Two beta chains have altered sequence of amino acid residues.

• Amino acid glutamic acid at sixth position in each beta chain is substituted by
valine amino acid.
Pathogenesis
• Defect is caused by a mutant β-globin gene located on chromosome 11.

• Glutamic acid is a polar amino acid, and it is replaced with non-polar, valine
amino acid. It results into formation of a sticky patch on the outer surface of beta
chains.
• Sticky patch is found in oxygenated state of hemoglobin-S (R form) as well as
deoxygenated state of hemoglobin-S (T form). Sticky patch is absent in oxygen-
ated state of HbA.
• Deoxygenate state of Hb-S also contains a site complementary to sticky patch
and is called as complementary site.
• Complementary site on oxygenated state of Hb-S is masked.
During deoxygenation of Hb-S, sticky patch of one molecule can bind with comple-
mentary site of another molecule. It results into polymerization of deoxygenated
Hb-S molecules. They are manifested as long fibrous polymers that extend across
the interior of erythrocytes. These fibers bring about distortion in shape of
erythrocytes.
During oxygenation of Hb-S, sticky patch of one molecule cannot bind with
complementary site of another molecule as complementary site is masked.
Distortion in shape of erythrocytes can be minimized by either of two fac-
tors as:
• Deoxygenation of Hb-S should be minimized.

• Oxygenation of Hb-S should be prolonged.
Effects of Sickle-Cell Anemia

Hemolytic Anemia
• Erythrocytes become fragile.

• They have increased tendency to rupture.
• It results into repeated and excessive hemolysis.
• Its manifestation is hemolytic anemia.
Damage of Body Tissues
• Sickle-shaped erythrocytes have tendency to become lodged into capillaries.

• It results into low perfusion of body organs.
• Low blood perfusion into organs causes hypoxia and necrosis of tissues.
86 5 Hemoglobin
Predisposition to Infection
• Hemolytic anemia is accompanied by poor nutrition and weak immunity.

• Affected individuals develop high disposition to infections.
Short Life Span
• Life span of homozygous suffers is limited.

• They die before age of 20 years.
5.7.2 Sickle-Cell Trait
It is a disorder characterized by heterozygous condition of β-globin gene.

Sickle-cell anemia is caused by mutant β-globin gene.
Mutant genes exhibit two conditions as:
Homozygous Genes
In this condition, both genes are mutant. One gene is inherited from the father and
other one from the mother. Both beta chains are structurally abnormal. This condi-
tion is homozygous.
Individuals with heterozygous genes suffer from sickle-cell anemia.
Heterozygous Genes
In this condition, one inherited gene is mutant, while the other gene is normal. One
beta chain is structurally abnormal, while the other one is normal. This condition is
heterozygous.
Individuals with heterozygous genes are carriers of sickle-cell anemia. They do
not suffer from clinical manifestations of disorder. They have a normal life span.
Clinical Significance of Sickle-Cell Trait

Plasmodium falciparum is a malarial parasite. Its incidence is highest in tropical
areas of the world. It is associated with high rate of mortality and morbidity.
Coincidentally, sickle-cell trait is higher in tropical areas of the world.
It has been found that sickle-cell trait offers resistance against malaria. The
fact is based on following assumptions as:
• High tendency for hemolysis in sickle-cell anemia.

• Rupture of erythrocytes disrupts the life cycle of parasite.
• Infestation of malarial parasites inside erythrocytes decreases pH of erythro-
cytes. This condition further aggravates distortion in shape of erythrocytes
(>sickling).
• Increasing rate of RBC sickling helps to interrupt plasmodium life cycle.
Sickle-cell trait has become an adaptation among humans to malaria for

survival.
5.7 Hemoglobinopathies 87
5.7.3 Hemoglobin C (HbC)
The alpha chains are two in number. Each chain has the same sequence of amino
acid residues as in adult Hb. The beta chains undergo change in sequence of amino
acid residues. In each beta chain, glutamic acid is replaced by lysine amino acid at
6th position.
The condition is called as Cooley’s hemoglobinemia. It is characterized by
hemolytic anemia in affected individuals.
5.7.4 Hemoglobin D (HbD)
This hemoglobin has normal alpha chains. In each beta chain, glutamic acid is
replaced by glutamine at 12th position. This hemoglobin has several varieties which
have been identified from different regions. Example: HbD (Punjab)
5.7.5 Hemoglobin E (HbE)
This variant of hemoglobin is common in population. It has been estimated that

nearly 15% of Southeast Asian population is suffering from HbE disorder.
The two alpha chains have the same sequence of amino acid residues as found in
adult hemoglobin. In each beta chain, glutamic acid is substituted by lysine at 26th
position. The affected individuals exhibit no clinical manifestation.
5.7.6 Thalassemia
Definition
Thalassemias are hereditary disorders characterized by impaired synthesis of
polypeptide chains of hemoglobin.
Occurrence
Thalassemia is derived from the Greek word thalassa which means sea. Earlier it
was named as Mediterranean anemia.
• It is more frequently found in population around Mediterranean Sea. It is also

prevalent in India, Central Africa, and West Asia.
• Its highest rate of incidence has been found in Maldives population.
Molecular Changes in Hemoglobin
• Globin molecule has two alpha chains and two beta chains. The alpha chains are
synthesized by two alpha globin genes located on chromosome 16. These have
four alleles which encode alpha globin chains.
88 5 Hemoglobin
• The beta chains are synthesized by one beta globin gene on chromosome 11.
These two alleles encode beta globin chains.
• Mutation in genes is responsible for defective synthesis of globin chains.
Types of Thalassemia
α-Thalassemia
• α-Thalassemia is characterized by partial or complete absence of synthesis

of alpha chains.
• Synthesis of beta chains in adults and gamma chains in new born babies increases
as a compensatory mechanism.
• Increased synthesis of beta chains or gamma chains results into formation of
defective tetrameric structure of hemoglobin. Ultimately, its oxygen carrying
capacity is affected negatively.
β-Thalassemia
• β-Thalassemia is characterized by partial or complete absence of synthesis

of beta chains in hemoglobin.
• As a compensatory mechanism, excessive synthesis of alpha chains occurs. This
forms an alpha chain tetramer which has compromised oxygen-carrying capacity.
Types of β-Thalassemia
β-Thalassemia Minor
• One allele out of a pair is mutant, while allele is normal. It is a heterozygous

condition. Small fraction of beta chain is synthesized.
• Persons are carrier of thalassemia. They remain asymptomatic throughout
life.
β-Thalassemia Major
• Both alleles are mutant. It is a homozygous condition.

• Synthesis of beta chains is completely absent.
• Persons are suffering from severe symptoms. They are inflicted with severe ane-
mia. Their life span is shorter and may die at age of 2 years.
Clinical Manifestations of Thalassemia

Hemolytic Anemia
• There is frequent hemolysis in thalassemia which causes severe anemia. It is

more common in β-thalassemia major.
5.8 Derivatives of Hemoglobin 89
Splenomegaly
• Damaged erythrocytes are removed by the spleen. Its hyperactivity results into
enlargement in size of the spleen.
Infections
• Individuals have compromised immunity. It leads to higher prevalence of bacte-

rial and viral infections among thalassemia patients.
Iron Overload
• Patients need frequent blood transfusions to compensate for anemia. It results

into iron overload. Iron is stored in the liver, skeletal muscles, and heart. It
becomes fatal to life.
5.8 Derivatives of Hemoglobin
Heme moiety interacts with different ligands and forms hemoglobin derivatives.
A few important derivatives of hemoglobin are discussed as follows:
5.8.1 Oxyhemoglobin
• It is binding hemoglobin with oxygen molecule. The binding is reversible and

loose. Hemoglobin can rapidly bind with oxygen at high pressure, and it can
liberate oxygen at low pressure.
• High partial pressure of oxygen favors oxygenation of hemoglobin, and high
partial pressure of CO2 favors deoxygenation of hemoglobin.
• This property of hemoglobin makes it an effective conjugated protein for trans-
port of gases from the lungs to tissues and vice versa.
5.8.2 Carboxyhemoglobin
It is an association of hemoglobin with carbon monoxide. It is also called as

carbonyl-hemoglobin.
• CO is a colorless gas. It is released by incomplete burning of carbonaceous

substances.
• Hb binds with CO as it binds with molecular oxygen. Hemoglobin has 200 times
higher affinity to CO than oxygen molecule. It binds with CO irreversibly and
strongly. CO is fatal to body tissues. It can inhibit cytochrome oxidase enzyme
in electron transport chain.
90 5 Hemoglobin
5.8.3 Carbamino-Hemoglobin
• It is an association between amino group of globin protein and CO2.
Hb.NH2 +CO2 Hb.NHCOOH

(Carbaminohemoglobin)
This is a reversible binding. This property of Hb helps in transport of nearly 10%

of CO2 from tissues to the lungs.
5.8.4 Sulfhemoglobin
• It is an association between hemoglobin and hydrogen sulfide gas. It is a greenish

pigment.
• Excessive accumulation of sulfhemoglobin in blood circulation is described as
sulfhemoglobinemia.
• It results into cyanosis of the skin and mucous membrane. It is caused due to
exposure to sulfur compounds and administration of sulfonamide drugs.
5.8.5 Methemoglobin
• It is an oxidized form of hemoglobin in which ferrous iron is oxidized into ferric

iron.
• Oxidation of hemoglobin can be caused by exposure to poisons like nitrities,
nitrates, nitrobenzene, antipyrin, aniline dyes, and sulfonamide drugs.
• In healthy individuals, a fraction of methemoglobin (0.3 g/dl) which represents
around 1.5% of total hemoglobin is found.
• Methemoglobin in normal persons is converted into hemoglobin by methemo-
globin reductase enzyme. A deficiency of enzyme is accompanied by accumula-
tion of methemoglobin in blood. This inherited disorder is called as familial
methemoglobinemia.
Excessive amount of methemoglobin in blood circulation is described as

methemoglobinemia.
At 10% level of methemoglobin:
• Dyspnea, cyanosis, and change in behavior
At 20–30% level:
• Anxiety, loss of concentration, and headache
At 50% level of methemoglobin:

5.9 Biodegradation of Hemoglobin 91
• Seizures, coma, and death may occur.
Methemoglobin unlike hemoglobin is unable to bind with oxygen.
5.9 Biodegradation of Hemoglobin
Erythrophagocytosis
• The erythrocytes have 120 days life span in the human body. Senile erythrocytes are
removed from the circulation by macrophages of the spleen which is called as grave-
yard of senile erythrocytes. The physiological process of sequestration of senile
erythrocytes by macrophages from circulation is called as erythrophagocytosis.
• The spleen is the main site of erythrophagocytosis. But the liver and bone mar-
row also act as scavenging organs for mature erythrocytes.
• Under normal health condition, turnover (rate of sequestration of senile RBCs
from circulation and rate of release of fresh RBCs into circulation) of erythro-
cytes is around 2 × 106 per second.
Proteolysis of Hb
• Senile erythrocytes have fragile cell membranes. They pass through sinusoidal
spaces of the spleen, and cell membranes are deformed and rupture. Hemoglobin
is released from mature RBCs. Macrophages in the spleen phagocytose hemo-
globin and catalyze proteolysis of hemoglobin into free globin and heme moiety
as in Figs. 5.5 and 5.6.
Fate of Globin
• Globin enters blood circulation, and it is reutilized for synthesis of hemoglobin

in bone marrow.
• Globin is hydrolyzed into constituent amino acids which enter amino acid pool
of the body for reutilization.
Fate of Heme
• Heme is iron-protoporphyrin ring. Heme is acted upon by heme oxygenase

enzyme. The enzyme splits alpha-methene bridge in porphyrin nucleus. Reaction
occurs in presence of molecular oxygen and NADPH2.
• Porphyrin ring opens up at alpha-methene bridge position between pyrrole ring I
and pyrrole ring II. The Fe++ is released and enters iron store of body for reuse.
• Porphyrin is converted into green-colored pigment called as biliverdin with
release of CO and H2O. The coenzyme is oxidized into NADP+.
92 5 Hemoglobin
SENILE RBCS
sequesterated
in SPLEEN, Born Marrow
HEMOLYSIS
Rupture of cell membrane
Hemoglobin liberated
Hemoglobin
within SPLEEN, Born Marrow
Hydrolysis
Globin Release of HEME

Proteolysis Globin O2
Acted upon by
NADPH2
Heme – ∝ – Metheyl oxygenase
NADP+
( Found in R.E. Cells)
Reutilization
of Oxidative splitting
Release Globin in erythropoiesis of Iron - Porphyrin ring
of
Constitutent A+ ∝ Methyne Bridge [ Between
Amino [ Pyrrole
Co [ ring I & II
acids
Opening of Ring
Enter
Amino acid Formation of
R.E.Cells
Pool Biliverdin
Fe++ Iron
Reused Released bilirubin NADPH2
reductase
NADP+
Bilirubin
Fig. 5.5 Biodegradation of hemoglobin
Reduction of Biliverdin
• Within macrophages, biliverdin is reduced into yellow-colored pigment called as

bilirubin. Reaction is catalyzed by bilirubin reductase enzyme in the presence of
coenzyme NADPH2 as in Figs. 5.5 and 5.6.
5.9 Biodegradation of Hemoglobin 93
Fate of Bilirubin
Formation of bilirubin
in spleen, bone marrow
Blood
Bl d
circulation
Bilirubin
Lipid soluble
Binds with albumin to form
Bilirubin-albumin
complex
E t
Enters li
liver
Enters
systemic
Biliru
Bilirubin
Bil
Bi irubin
r bin
rubiin
bi Glucuronyl circulation
ttran
ran
ran
ansf
sffe
erra
e
transferase as
se
Indirect bilirubin
Glucuronic
Glucu
Glu
G lu
ucu
curon
oni
oni
nic
acid
acid
ac
aci
Urobilinogen enters
Bilirubin (5%) systemic circulation
diglucuronide
(direct bilirubin)
Kidney
Enterohepatic
Urobilinogen circulation
Bilirubin
diglucuronide
Urobilinogen
Excreted
Bilirubin
diglucuronide
Oxidized into
Colon Bacteria
Urobilin
Deconjugates Reabsorbed
bilirubin from ileum
(70-80%)
Diglucuronide
Bilirubin
Urobilinogen Reduced into stercobilinogen

(20%) oxidized
Stercobilin
Stool (50-100mg/day)
Fig. 5.6 Fate of bilirubin

94 5 Hemoglobin
Fate of Bilirubin
• Bilirubin from the spleen enters blood circulation. This bilirubin is lipid soluble
and is called as indirect bilirubin. It combines with albumin, and it is distributed
as bilirubin-albumin complex.
• Within the liver, bilirubin-albumin complex enters liver sinusoidal spaces. The
membranes of hepatocytes have bilirubin receptors, and bilirubin from complex
attaches to receptors. Albumin is released into circulation.
–– Within smooth endoplasmic reticulum of hepatocytes, bilirubin under-
goes conjugation with two molecules of glucuronic acids. Reaction is cata-
lyzed by glucuronyl transferase enzyme. There is transfer of glucuronic acid
moiety from active nucleotide (UDP-Ga) to carboxylic group of propionic
acid in bilirubin to form glucuronide. Bilirubin diglucuronide is formed
which is water soluble and is called as direct bilirubin or conjugated bilirubin
as in Fig. 5.6.
• Bilirubin is secreted in the bile. It enters the duodenum and reaches the colon.
Bacteria in the colon secrete beta-glucuronidase enzyme which deconjugates
bilirubin diglucuronide. Free bilirubin is released in the colon.
• Bilirubin undergoes a series of reduction reactions to form dihydrobiliru-
bin → meso-bilirubin → l-stercobilinogen (colorless compound) as in
Fig. 5.6.
• About 25% of l-stercobilinogen is excreted in stool. It undergoes auto-oxidation
in air and is converted into yellowish brown pigment called as stercobilin.
Another 60–70% of l-stercobilinogen is reabsorbed from the intestine and enters
hepatic portal vein. It reaches the liver for secretion into the bile. It is called as
enterohepatic circulation. A small fraction of l-stercobilinogen (5%) escapes
enterohepatic circulation and enters systemic circulation and reaches kidneys. It
is called as urobilinogen and excreted in urine. On exposure to air, it is oxidized
into l-urobilin which imparts yellow color to urine.
5.10 Biosynthesis of Hemoglobin
It can be described under two stages as follows:
• Biosynthesis of globin
• Biosynthesis of heme
5.10.1 Biosynthesis of Heme
Heme is iron-containing protoporphyrin IX compound. Heme is synthesized in

normoblasts.
5.10 Biosynthesis of Hemoglobin 95
The different steps explaining heme synthesis are described as follows:
Synthesis of δ-Amino Levulinic Acid (ALA)
This step occurs in mitochondria:

• A molecule of succinyl CoA undergoes condensation with glycine to form
δ-amino levulinic acid.
• Reaction is catalyzed by δ-amino levulinic acid synthase enzyme in presence of
pyridoxal phosphate as coenzyme. Reaction occurs in two steps as follows:
–– In the first step, there is formation of alpha-ketoadipic acid and release of
CoA-SH.
–– In the second step, there is formation of delta ALA with release of a molecule
of CO2.
–– Both steps are catalyzed by ALA synthase enzyme with pyridoxal phosphate
as coenzyme.
–– Deficiency of pyridoxal P results into failure of ALA formation and interferes
in heme synthesis. It is cause of anemia.
• ALA synthase is mitochondrial enzyme. It is a rate-limiting step of heme synthe-
sis as in Fig. 5.7.
Synthesis of Porphobilinogen (PBG)
This step occurs in cytoplasm:

• Two ALA molecules undergo condensation to form porphobilinogen with
removal of a molecule of water.
• Reaction is catalyzed by ALA dehydratase enzyme. Enzyme requires zinc ions
as cofactor.
Synthesis of Uroporphyrinogen (UPG)

• Four molecules of porphobilinogen condense together to form uroporphyrinogen
with loss of four molecules of ammonia.
• Reaction is catalyzed by PBG deaminase or uroporphyrin I synthase.
• Uroporphyrinogen is a linear tetrapyrrole and called as hydroxy methyl
bilane (HMB). The HMB undergoes spontaneous cyclization to form uropor-
phyrinogen I.
• Uroporphyrinogen I is changed into uroporphyrinogen III by enzyme uroporphy-
rinogen III synthase enzyme as in Fig. 5.7.
96 5 Hemoglobin
ALA Synthase
Pyridoxal P Mitochondria
Succinyl CoA + Glycine alpha-keto Adipic acid
CoA.SH
ALA Synthase/Pyridoxal P Mitochondria
alpha-keto Adipic acid delta-amino Levulinic acid (ALA)
CO2
ALA Dehydratase Cytosol
(2 mole) Delta-ALA Porphobilinogen (PBG)
2 H2O
PBG deaminase Cytosol
(4 mole) Porphobilinogen Uroporphyrinogen III (UPG)
(4 mole) NH3
UPG Decarboxylase Cytosol
Uroporphyrinogen III Coproporphyrinogen III (CPG)
(4 mol) CO4
CPG Oxidase Mitochondria
Coproporphyrinogen III Protoporphyrinogen III (PPG)
CO2 NADP+ NADPH2
PPG Oxidase Mitochondria
Protoporphyrinogen III Protoporphyrin III
4 hydrogen ions
Heme Synthase Mitochondria

Protoporphyrin III Heme
Fig. 5.7 Showing synthesis of HEME
Synthesis of Coproporphyrinogen (CPG)

• Uroporphyrinogen III undergoes decarboxylation to form coproporphyrinogen
with loss of four molecules of CO4.
• Reaction is catalyzed by uroporphyrinogen decarboxylase enzyme.

• Acetate groups in uroporphyrinogen are decarboxylated into methyl groups as in
Fig. 5.7.
Synthesis of Protoporphyrinogen III (PPG)

• Coproporphyrinogen undergoes oxidation in mitochondria to form protoporphy-
rinogen III.
• Reaction is catalyzed by coproporphyrinogen oxidase (enzyme exhibits series III
specificity).
• Propionic acid side chains decarboxylated in presence of molecular oxygen to
form vinyl groups as in Fig. 5.7.
Synthesis of Protoporphyrin (PP)

• Protoporphyrinogen III undergoes oxidation to form protoporphyrin.
• Reaction is catalyzed by protoporphyrinogen oxidase.
• Methylene bridges (-CH2) converted into methenyl bridges (–CH〓).
• This formation of protoporphyrin IX as in Fig. 5.7.
Synthesis of Heme

• In the last stage, ferrous iron is attached to protoporphyrin IX in mitochondria.
• Reaction is catalyzed by heme synthase (ferrochelatase) enzyme. It is located in
mitochondria.
• Ferrous form (Fe++) of iron in heme imparts red color to heme moiety.
However, presence of ferric iron (Fe+++) in heme converts it into hematin
moiety. It imparts brown color to hematin moiety.
• This property is in utilized in Sahli’s hemoglobinometer for assaying hemo-
globin concentration.
5.10.2 Biosynthesis of Globin
Globin molecule is made up of two alpha chains and two beta chains. Their synthe-
sis is regulated by globin genes located on chromosome 16 and chromosome 11,
respectively. The expression of alpha genes and beta genes is well controlled.
98 5 Hemoglobin
5’
ZETA 2
ZETA 1
MU
CHROMOSOM 16
Psudo
Alpha - 1 T
R
TRANSCRIPTION A ALPHA
Alpha 2 mRNA N CHAINS
S
L
Alpha 1 A
T
I
THETA O
N
3’
ALPHA - GLOBIN
GENE CLUSTURE
Fig. 5.8 Alpha globin genes
Alpha Globin Genes
• Single chromosome 16 contains two alpha globin genes. Overall, two chromo-
somes 16 have 4 alpha globin genes.
• Four alpha globin genes regulate synthesis of two alpha polypeptide chains.
• Alpha globin gene has seven gene loci. They are named as zeta1, zeta 2, mu,
pseudo alpha 1, alpha 2, alpha 1, and theta in 5′ → 3′ direction as in Fig. 5.8
• Alpha gene cluster transcribe mRNA which directs synthesis of alpha globin
chains.
Beta Globin Genes
• Each chromosome 11 contains single beta globin gene.

• Two beta globin genes in two chromosomes 11 regulate synthesis of beta poly-
peptide chains.
• Beta gene has five gene loci and is called as beta gene cluster. They are named
as epsilon, gamma G, gamma A, delta, and beta in 5′ → 3′ direction as in
Fig. 5.9
• Beta gene transcribes mRNA which controls synthesis of beta chains in adults.
5’
EPSILON
GAMMA G
CHROMOSOM 11
GAMMA A
T
R
A
DELTA N
S BETA
L
A CHAINS
TRANSCRIPTION
mRNA T
BETA I
O
N
3’
SYNTHESIS OF β-GLOBIN
BETA - GLOBIN
GENE CLUSTURE
Fig. 5.9 Beta-globin genes
5.10.3 Regulation of Heme Biosynthesis
ALA Synthase
• It is a chief regulatory enzyme in heme biosynthesis. It is a rate-limiting enzyme.

Its activity is influenced by following compounds as:
–– Hematin is formed in the body from surplus ferrous iron. It is oxidized into
ferric state to form hematin. It acts as allosteric inhibitor to ALA synthase.
–– High concentration of glucose in cells inhibits synthesis of enzyme ALS
synthase.
–– Heme prevents synthesis of enzyme ALA synthase through negative feedback
mechanism.
Lead
• Lead inhibits ALA dehydratase enzyme and regulates heme synthesis.
Isonicotinic Acid Hydrazide (INH)
• Prolonged intake of antitubercular drug, INH, leads to deficiency of pyridoxal

phosphate. It is necessary for synthesis of δ-ALA.
100 5 Hemoglobin
Barbiturates
• Drugs like barbiturates are metabolized by heme-containing cytochrome P450

enzyme in the liver. They induce synthesis of heme.
Covalent Bond
• A bond formed by sharing of electrons between two atoms.
Coordinate Bond
• A bond formed by donating a pair of electrons by one atom called as donor atom.
Suggested Readings
Adamson JW, Finch CA (1975) Hemoglobin function, oxygen affinity and erythropoietin. Annu
Rev Physiol 37:351
Bunn HF (1981) Hemoglobin: structure and function. In: Beck WS (ed) Hematology. MIT Press,
Cambridge
Ganong WF (2003) Review of medical physiology, 21st edn. Lange Medical Book, New York
Wallerstein RO (1987) Laboratory evaluation of anemia. West J Med 146:443
Carbohydrates
6
6.1 Definition
Carbohydrates are polyhydroxy aldehydes or ketones or compounds which yield poly-
hydroxy aldehydes and ketones upon hydrolysis.
Carbohydrates are organic biomolecules abundantly distributed in animals and

plants. In humans, glucose and glycogen forms of carbohydrates serve as an instant
and important source of energy for physiological activities. Highly specific carbo-
hydrates like ribose, lactose, and galactose serve as structural components of nucleic
acid, lipids, and breast milk.
6.2 Classification
Carbohydrates are classified into four main categories as follows:
1. Monosaccharides
2. Disaccharides
3. Oligosaccharides
4. Polysaccharides
6.2.1 Monosaccharides
• The word “saccharide” is derived from the Greek word “sakkharon” which
means “sugar” or “sweet.”
• Monosaccharides are simple sugars. They are the carbohydrates which can-
not be hydrolyzed into more simple sugars.
• Their molecular formula is [CnH2nOn).
Examples: Glucose, fructose, galactose, ribose, erythrose

https://doi.org/10.1007/978-981-13-1035-5_6
102 6 Carbohydrates
Classification of Monosaccharides
Monosaccharides can be further subdivided on the basis of two criteria as
follows:
• On the basis of number of carbon atoms

–– Monosaccharides can be trioses, tetroses, pentoses, hexoses, and heptoses.
• On the basis of functional group
–– Monosaccharides containing aldehyde (–CHO) group are called as
“aldoses” and those containing ketone (–CO) group are called as
“ketoses.”
Number of carbon atom Aldoses Ketoses

Trioses Glyceraldehyde Dihydroxyacetone phosphate
Tetroses Erythrose Erythrulose
Pentoses Ribose, arabinose, xylose Ribulose, xylulose
Hexoses Glucose, galactose, mannose Fructose
Heptoses Glucoheptose, galactoheptose Sedoheptulose
6.2.2 Disaccharides
• They are the carbohydrates which yield two molecules of similar or dissimilar
monosaccharides upon hydrolysis.
• Their molecular formula is [Cn(H2O)n–1].
• Example:
They are the carbohydrates which yield two molecules of similar or dissimilar
monosaccharides upon hydrolysis.
Their molecular formula is [Cn(H2O)n–1]
Example:
Sucrose Glucose + Fructose
Lactose Glucose + Galactose
Lactulose Galactose + Fructose
Maltose Glucose + Glucose

Isomaltose Glucose + Glucose
Trehalose Glucose + Glucose
Cellobiose Glucose + Glucose
6.2.3 Oligosaccharides
• They are the carbohydrates which yield three to ten monosaccharide units upon
hydrolysis.
• Example:
6.3 Characteristics of Monosaccharides 103
They are the carbohydrates which yield 3-10 monosaccharide units upon hydrolysis.
Example:
Trisaccharides
• Raffinose Glucose + Galactose + Fructose
• Maltotriose 3(Glucose)
Tetrasccharides
• Stachyose [Glucose + 2(Galactose) + Fructose]
Pentasaccharides
• Verbascose [Glucose + 3(Galactose) + Fructose]
6.2.4 Polysaccharides
• They are the carbohydrates which yield more than ten monosaccharide units
upon hydrolysis.
• Polysaccharides are also called as glycans.
• Their molecular formula is [C6H10O5]n.
• Depending on nature of hydrolytic products, polysaccharides are of two types as
follows:
–– Homopolysaccharides (Homoglycans)
These polysaccharides yield similar monosaccharide units upon hydrolysis.
Example: Starch, glycogen, inulin, cellulose
–– Heteropolysaccharides (Heteroglycans)
These polysaccharides yield dissimilar monosaccharide units upon hydrolysis.
Example: Hyaluronic acid, heparin, chondroitin sulfate, keratan sulfate
6.3 Characteristics of Monosaccharides
Monosaccharides exhibit characteristic features which are described as follows:
6.3.1 Asymmetric Carbon Atom
• When four dissimilar atoms or groups are attached to a carbon atom, it is called
as asymmetric carbon atom or “chiral carbon.”
• Monosaccharides contain asymmetric carbon atoms.
• Asymmetric carbon is responsible for isomerism in monosaccharides as in Fig. 6.1.
6.3.2 Vant Hoff’s Rule of “n” for Number of Isomers
• According to Vant Hoff’s rule of “n.”

–– The number of isomers in a compound depends on the number of asymmetric
carbon atoms in that compound.
104 6 Carbohydrates
• The “n” represents number of asymmetric carbon atoms.

• Number of isomers in any monosaccharide is given by 2n.
• Aldohexose (glucose) has four asymmetric carbon atoms at positions C2, C3,
C4, and C5. So it has 16 isomers (2x2x2x2=16). Ketohexose (fructose) has three
symmetric carbon atoms at positions C3, C4, and C5. So it has eight isomers
(2x2x2=8). All isomers are not biologically active in body tissues.
6.3.3 Isomerism in Monosaccharides
Definition
Isomerism is defined as a phenomenon in which compounds having similar molecu-
lar formula exhibit difference in their chemical structures.
• The word Isomerism is derived from Greek word and it conveys (‘isos means
equal and meros means parts). Monosaccharides exhibit isomerism as
described below:
1. Stereoisomerism
Stereoisomerism is a type of isomerism in which compounds have similar
molecular formula, but they differ in relative arrangement of atoms or groups
in three-dimensional space.
• Monosaccharides show stereoisomerism.

Example: Glucose exhibit two forms as d-glucose and l-glucose. These ste-
reoisomers differ in arrangement of H and OH groups on C5 atom in glucose
molecule. The C5 atom in glucose molecule is called as “reference carbon atom”
or “penultimate carbon atom” as in Fig. 6.2.
Fig. 6.1 Asymmetric CHO
Carbon atom
H C OH
R
Fig. 6.2 Stereoisomerism O O
in glucose
1C H 1C H
H 2C OH OH 2C H
HO 3C H 3C
PENULTIMATE H OH
H 4C OH CARBON HO 4C H
H 5C OH HO 5C H
6CH 6CH
2OH 2OH
D - GLUCOSE L - GLUCOSE
6.3 Characteristics of Monosaccharides 105
• Reference or penultimate carbon is the carbon atom adjacent to the last pri-
mary alcohol carbon in a compound. It determines “d” or “l” series of com-
pounds. In d-glucose, OH group is located on right-hand side in C5 carbon atom.
In l-glucose, position of OH group is reversed in C5 carbon atom as in Fig. 6.2.
• Aldohexose exhibits 16 stereoisomers. They exist in “d” and “l” series. There
are eight d-aldohexoses as d-allose, d-altrose, d-idose, d-talose, d-gulose, d-
glucose, d-galactose, and d-mannose. There are eight l-aldohexoses as
enantiomers.
2. Enantiomers
The stereoisomers that are nonsuperimposable and mirror image of each other.
Example: d-Glucose and l-glucose are mirror images and nonsuperimpos-
able molecules. So they are called as enantiomers as in Fig. 6.2.
• D series of sugars are predominant in nature. Only D sugars are metabolically

active in the human body. Out of 16 stereoisomers, only d-glucose, d-galactose,
and d-mannose exist commonly in nature.
3. Epimers
Epimers are the isomers which differ from each other in the arrangement of atoms
or groups around a single chiral carbon.
• Example: d-Glucose and d-galactose differ around C4 atom. d-Glucose and

d-mannose differ around C2 atom as in Fig. 6.3.
• The process of conversion of one epimer into another in body tissues is called as
epimerization. In the liver, galactose is converted into glucose by epimerase
enzyme.
4. Anomers
Anomers are defined as the cyclic monosaccharides which differ from each other
in the arrangement of atoms or groups around C1 atom in aldoses and C2 atom in
ketoses.
Fig. 6.3 Epimers CHO CHO CHO

H C OH H C OH HO C2 H
HO C H HO C H HO C H
4C 4
H OH HO C H H C OH
H C OH H C OH H C OH
CH2OH CH2OH CH2OH
D - GLUCOSE D - GALACTOSE D - MANNOSE

106 6 Carbohydrates
• Chiral carbon in anomers is called as “anomeric carbon.”

• Anomers are cyclic epimers as in Fig. 6.4.
Example:
–– d-Glucose exhibits two anomers, namely, α-d-glucose and β-d-glucose. In
α-d-glucose, OH group is below the plane of molecule.
In β-d-glucose, OH group is above the plane of molecule.
6.3.4 Optical Isomerism
Asymmetric carbon atom confers optical activity to a compound. When a plane

polarized light passes through a monosaccharide solution, it rotates the light beam
either to right or left depending. The phenomenon is called as optical isomerism.
Accordingly, a monosaccharide is called as “dextrorotatory” and “levorotatory.”
• The dextrorotatory glucose molecule is represented as (d-glucose) or (+) glu-

cose, and levorotatory glucose is represented as (l-glucose) or (−) glucose.
• The notations “d” and “d” represent stereoisomer and optical isomer.
Example: d-Glucose is (+) or dextrorotatory. It means a glucose molecule
with OH group on the right-hand side on C5 carbon atom can rotate a plane
polarized light in right-hand direction. Another example is d-fructose is (−)
or levorotatory.
CH2OH
C C
H H
H 1C
C
OH H
OH OH
C C CH2OH
5C
H OH OH
H
α-D-GLUCOPYRANOSE H
4C H 1C
OH H
O
CH2OH OH 3C 2C
C C H OH
H OH
GLUCOSE [HAWORTH PROJECTION]
H 1C
C
OH H
OH H
C C
H OH
β-D-GLUCOPYRANOSE
Fig. 6.4 Haworth projection of glucose (anomers)

6.4 Biologically Important Carbohydrates 107
6.3.5 Racemic Mixture
It is a mixture which contains equal amount of dextrorotatory and levorotatory iso-

mers of a compound.
• Racemic mixture is represented with prefix (±)- or (dl). A racemic mixture is

inactive optically. Its net optical rotation is zero.
6.4 Biologically Important Carbohydrates
6.4.1 Monosaccharides
1. Trioses
• Important trioses like d-glyceraldehyde and dihydroxyacetone phosphate are
the important intermediate metabolites in glycolysis cycle. They are con-
verted into glycerol which is a structural constituent of lipids.
2. Tetroses
• Erythrose 4-phosphate is an intermediate metabolite in hexose monophos-
phate shunt. It is a precursor for biosynthesis of tryptophan, phenylalanine,
and tyrosine amino acids.
3. Pentoses
• d-Ribose is a structural residue of RNA, NAD, FAD, and coenzyme A.
• d-2-Deoxyribose is a structural residue of DNA molecule.
• d-Ribulose and d-xylulose are intermediate metabolites in HMP shunt.
• l-Xylulose is a marker in hereditary disorder called as “pentosuria.” It accu-
mulates in the urine of patient.
4. Hexoses
• d-Glucose
–– d-glucose is a colorless and crystalline monosaccharide. It is readily solu-
ble in water.
–– It exists in nature in free state in fruits like grapes. It is found in bound
state in cellulose, maltose, and mucopolysaccharides. It is called as
grape sugar.
–– It is the physiologically active sugar present in the human body. Normal
blood glucose level is between 80 and 120 mg/dl under fasting state.
–– Glucose is the chief source of energy for body tissues. Brain cells and
erythrocytes utilize glucose exclusively for their energy demand.
–– Glucose is stored in the liver and skeletal muscles in the form of
glycogen.
–– d-Glucose is dextrorotatory and also called as “dextrose.”
–– It is a reducing sugar.
108 6 Carbohydrates
–– It forms glucosazone crystals. Their shape resembles cluster of yellow

needles.
• d-Galactose
–– It exists in bound form in nature. Galactose is a structural component of
mucopolysaccharides, glycoproteins, and compound lipids.
–– It is a structural unit of lactose. In mammary glands, glucose is epimerized
into galactose.
–– d-Galactose is dextrorotatory.
–– It forms galactosazone crystals. Their shape resembles rhombic plates.
• d-Fructose
–– It is found mainly in fruits and honey. It is called as fruit sugar.
–– It is a colorless and crystalline ketohexose.
–– It is a structural unit of sucrose.
–– It is sweeter than sucrose.
–– d-Fructose is levorotatory and is also called as “levulose.”
–– d-Fructose exhibits anomerism. The C2 atom in d-fructose is anomeric,
and it forms two anomers, namely, α-d-fructose and β-d-fructose.
needles.
• d-Mannose
–– It is not an essential sugar in the human body. It is biosynthesized from
glucose by epimerization.
–– It was extracted from plant “mannans.”
–– It is a structural unit of glycoproteins in the human body.
needle-shaped crystals.
5 . Heptoses
• Sedoheptulose
–– It is one of the few heptoses existing in nature. It is found in plants of
sedum family.
–– Sedoheptulose 7-phosphate is an intermediate metabolite in HMP shunt.
6.4.2 Disaccharides
Three disaccharides are biologically important. These are as follows:

1. Lactose
• Lactose is called as “milk sugar.”
• It is a reducing disaccharide.
• It is composed of d-glucose and d-galactose. These are linked together by
beta-1,4 glycosidic bond as in Fig. 6.5.
• It forms lactosazone crystals. Their shape resembles cotton ball or hedge-
hog as in Fig. 6.10.
2. Maltose
• It is commonly called as malt sugar.
• It is a colorless and sweet monosaccharide with crystalline structure.
• It is composed of two molecules of d-glucose linked by alpha-1,4 glycosidic
bond as in Fig. 6.6.
• It is a reducing disaccharide.
• In the human body, maltose is produced by enzymatic hydrolysis by amylase
on dietary starch.
Fig. 6.5 Lactose CH2OH CH2OH
HO 5
O H O OH
H H
4 1 O 4
3 2 3 2
H H H
β-D-GaLACTOSE D-GLUCOSE
Fig. 6.6 Maltose CH2OH CH2OH
5
O H H 5
O H
4 1 4 1
3 2 O 3 2
OH
α-D-GLUCOSE α-D-GLUCOSE
110 6 Carbohydrates
• Maltose is hydrolyzed into two glucose moieties by maltase in the gut.

• It forms maltosazone crystals. Their shape resembles sunflower petals as
in Fig. 6.10.
3. Sucrose
• It is commonly called as table sugar or cane sugar as it is present in
sugarcane.
• It is a colorless and crystalline disaccharide. It is readily soluble in water. It is
sweet in taste.
• It is composed of d-glucose and d-fructose linked by alpha-d-glucosyl-beta-
d-fructoside (alpha-1,2) linkage as in Fig. 6.7.
• Aldehyde and ketone functional groups are linked together in sucrose.
It is a nonreducing disaccharide. There is absence of free functional
group.
• It does not form osazone.
• Sucrose does not show mutarotation as both anomeric carbons are linked
together.
• Sucrose exhibits dextrorotation (+62.5°). Its hydrolytic residues are glucose
and fructose. Glucose has dextrorotation (+52.5°), while fructose shows levo-
rotation (−92°). Therefore, the hydrolytic residues invert the optical rotation.
So the mixture has more levorotation than dextrorotation, and it is called as
“invert sugar.” The enzyme which hydrolyzes sucrose is called as invertase or
sucrose.
Example: Honey contains invert sugar and fructose.
Fig. 6.7 Sucrose CH2OH

CH2OH
O H H
5 O
4 1 2 5
3 2 O
3 4 CH2OH
α-D-GLUCOSE α-D-GLUCOSE
6.4.3 Polysaccharides (Glycans)
Homopolysaccharides
Homopolysaccharides are the polymers of the same monosaccharide. They yield the
same monosaccharide units on hydrolysis. They are also called as “homoglycans.”
Starch
Occurrence
Starch is synthesized by green plants as a reserve food material. It is found in differ-
ent parts of plants like leaves, stem, roots, and fruits. Staple foods, namely, potato,
rice, wheat, maize, and cassava, are rich source of starch. Starch is the most com-
mon dietary source of energy for humans.
Characteristics and Chemical Composition
• Starch is a white, odorless, and crystalline polymer of d-glucose.

• It is poorly soluble in cold water. Upon heating, starch powder swells up in water
and forms gel-like mass.
• Starch gives deep blue color with iodine solution. It is not a reducing
carbohydrate.
• Starch is made up of amylose and amylopectin. They are polymers of d-
glucose residues. They have different structures and properties.
• Amylose
Amylose represents about 20% of starch by weight. It has molecular weight
around 60,000. Amylose is soluble in water. It shows deep blue color with iodine
solution. Amylose is a linear polymer of around 300 d-glucose residues. They
are linked by alpha-1 → 4 glycosidic bonds.
• Amylopectin
Amylopectin represents about 80% of starch by weight. It has molecular weight
around 500,000–600,000. It is insoluble in water. It swells up in water and forms
gel-like mass. It shows reddish violet color with iodine solution as in Fig. 6.8.
Amylopectin is a greatly branched polymer of d-glucose residues. Main trunk
and branches of amylopectin have d-glucose residues linked by alpha-1 → 4 gly-
cosidic bonds. A branch of d-glucose residues is linked to the main trunk by
alpha-1 → 6 glycosidic bond. After every 25 d-glucose residues, a branch is pres-
ent, and around 70–80 branches are present in amylopectin molecule.
112 6 Carbohydrates
α-1→6 - BONDING
G G G G
G
G G G G G G G G
α-1→4 - GLYCOSIDIC BOND
Fig. 6.8 Amylopectin
Hydrolysis of Starch
• By Action of Alpha-Amylase
Alpha-amylase is found in saliva and pancreatic juice. Salivary amylase acts at
optimum (pH 6.7), and pancreatic amylase acts at optimum (pH 7.1). The alpha-
amylase cleavages alpha-1 → 4 glycosidic bonds randomly within starch
molecule.
In the initial stage, enzymatic hydrolysis yields “amylodextrin.” It shows violet
color with iodine solution. Further hydrolysis of starch yields “erythrodextrin”
which shows red color with iodine solution. More hydrolysis of starch yields
“achrodextrin” which does not give any color with iodine solution. Finally, enzy-
matic hydrolysis yield maltose.
Alpha-amylase hydrolysis of starch yields a mixture of maltose and a few
residues of dextrin.
• By Action of Beta-Amylase
Beta-amylase is found in sprouted seeds, germinated cereals (called as malt), and
almond. Beta-amylase starts cleavage of alpha-1 → 4 glycosidic bonds from
nonreducing end of starch molecule. Beta-amylase cannot cleavage alpha-1 → 6
glycosidic bonds at branch points in starch. It results into formation of limit
dextrin.
Beta-amylase hydrolysis of starch yields a mixture of maltose and limit dex-
trin. Limit dextrin is a large residual polymer of d-glucose residues which is
produced during beta-amylase hydrolysis of starch. It cannot be further
hydrolyzed.
Functions of Starch
• Starch is the dietary source of energy for humans and higher animals. In the
body, starch is hydrolyzed into maltose and ultimately into glucose.
Glycogen
Occurrence
• Glycogen is the chief reserve food of animal kingdom. Glycogen is stored in the
liver and skeletal muscles in humans and higher animals.
• Glycogen is analogous to starch in plants. So glycogen can be called as “animal
starch.”
• Some fungi, yeast, and bacteria also possess glycogen in their body.
• Glycogen is a white, odorless, and crystalline polymer of d-glucose.

• It is poorly soluble in water and forms an opaque solution.
• Its molecular weight ranges from 1,000,000 to 5,000,000.
• It shows deep red color with iodine solution.
• Glycogen is a highly branched polymer of d-glucose residues. The main trunk of
glycogen molecule is made up of d-glucose residues linked by alpha-1 → 4 gly-
cosidic bonds. Branches of d-glucose residues are linked to the main trunk by
alpha-1 → 6 glycosidic bonds as in Fig. 6.9. After every 15 glucose residues, a
branch is present.
• Molecular structure of glycogen is more complex than amylopectin.
Fig. 6.9 Glycogen Branches
α - 1→ 6 - bonding
Main stem
α - 1→ 4 - bonding
Glucose
moieties
Branched chain glycogen structure

114 6 Carbohydrates
Functions of Glycogen
• Glycogen is the stored food for animals. It is converted into glucose by glycoge-
nolysis in the liver and skeletal muscles in humans. Glucose is utilized by body
tissues for fulfilling energy demand.
Inulin
Occurrence
• Inulin is found in plants like onion, garlic, dahlia, and dandelion. It is a reserve
food of plants.
• It is a white, odorless, and crystalline polymer of d-fructose.

• Its molecular weight is 5000.
• It does not give color with iodine solution.
• Inulin is made up of d-fructose residues linked by beta-(1 → 2) glycosidic bonds.
• Inulin is hydrolyzed by inulinase enzyme present in plant tissues.
Functions of Inulin
• Inulin is not metabolized in the human body. Inulin does not have any nutritional
value for humans.
• Inulin is used to estimate glomerular filtration rate (GFR). It is an indicator of
renal function.
• Inulin is used to estimate proportion of extracellular fluid (ECF).
Needle shaped Sun flower shaped Cotton shaped

crystals crystals crystals
[Glucosazones] [Maltosazone] [Lactosazone]
Fig. 6.10 Osazones
Cellulose
Occurrence
• Cellulose is the abundantly found organic substance on earth.

• It constitutes 95% of cotton and 55% of wood.
• Cellulose is the main structural constituent of cell wall of green plants.
• It is also found in cell wall of a few algae and water molds.
• It is a white, odorless, and crystalline polymer of d-glucose.

• It is hydrophilic but insoluble in water.
• Its molecular weight ranges from 27,000 to 900,000.
• Cellulose is a linear chain polymer composed of d-glucose residues linked by
beta-(1 → 4) glycosidic bonds. The number of d-glucose residues is variable.
Cellulose polymers in cotton and wood are made up of around 10,000 residues
and 2000 d-glucose residues, respectively.
• When cellulose is treated by mineral acids, it results into formation of “cellobi-
ose.” It is a reducing disaccharide. It is made up of two units of d-glucose linked
by of beta-(1 → 4) glycosidic bonds.
Functions of Cellulose
• Humans consume cellulose along with vegetables and fruits. It is not digested in
the human body. It is due to absence of cellulose-digesting enzyme among
humans.
• Cellulose does not have any nutritional value for humans.
• Cellulose has roughage value for humans. Ingestion of cellulose in the form of
vegetables and fruits increases contents of the large intestine. Distension of the
intestine stimulates peristalsis and helps in evacuation of bowels. It relieves
constipation.
• Cellulose is digested by termites and ruminants. Termites possess Trichonympha
in the gut, and ruminants harbor anaerobic bacteria in the large intestine. These
microbes help in digestion of cellulose by action of cellulase enzyme.
Dextran
Occurrence
• Dextran was initially discovered by microbiologist Louis Pasteur in wine. It is

synthesized from sucrose by action of Gram-positive cocci named as Leuconostoc
mesenteroides. These organisms induce polymerization of glucose residues in
sucrose and result into formation of dextran.
116 6 Carbohydrates
• It is a colorless, odorless, and crystalline polymer of glucose.

• It is readily soluble in water.
• Its molecular weight ranges from 1,000,000 to 2,000,000.
• Dextran is a neutral polymer and exhibits high colloidal osmotic pressure.
• Dextran is a highly branched chain polymer of d-glucose residues. There is
alpha-(1 → 6) glycosidic linkage between d-glucose residues in straight chain.
The branch points are attached by alpha-(1 → 3) linkage.
Functions of Dextran
• Dextran is not metabolized in the body.

• Dextran is used as plasma expander. It is infused intravenously and dextran
remains in blood vessels for hours. It helps to increase volume of plasma. It is
useful to manage hypovolemia due to acute blood loss when blood transfusion is
not possible.
• Dextran is a relatively safe intravenous fluid. However, its use is associated with
side effects like anaphylaxis, acute renal failure, and difficulty in blood grouping
and cross matching.
Heteropolysaccharides (Heteroglycans)
Heteropolysaccharides are of polymers of different monosaccharides. They yield
mixture of monosaccharide units on hydrolysis. They are also called as
“heteroglycans.”
This group of glycans is structurally associated with amino sugars and uronic acid.
Therefore, they were described by “Jeanloz” as “glycosaminoglycans” (GAGs).
These heteropolysaccharides are characterized by formation of slimy and vis-
cous solution. So they are called as “mucopolysaccharides.”
6.5 Classification of Mucopolysaccharides
6.5.1 Acidic and Non-sulfated Mucopolysaccharides
Hyaluronic Acid
Occurrence
• It is found in synovial fluid in joints, vitreous humor of the eye, epithelial tissues,
connective tissues, brain tissues, and umbilical cord.
• It is an acidic mucopolysaccharide at body pH.

• Its molecular weight is around 5,000,000.
6.5 Classification of Mucopolysaccharides 117
• Hyaluronic acid exists in free state and in bound state with proteins. It forms a
viscous gel with proteins which is a component of extracellular matrix.
• Hyaluronic acid is a polymer of d-glucuronic acid and N-acetyl glucosamine
(NAG) residues. These residues are linked by beta-(1 → 3) and beta-(1 → 4)
glycosidic linkages.
• Upon hydrolysis, it yields equimolar proportion of d-glucuronic acid, N-acetyl
glucosamine, and acetic acid in solution.
Functions of Hyaluronic Acid
• Hyaluronic acid is an integral structural component of extracellular matrix in

tissues.
• It acts as cementing substance in body tissues.
• It acts as shock absorbant and lubricant in joints.
• It forms a viscous gel that fills the intercellular spaces. So it resists the invasion
of pathogenic bacteria in tissues.
• It is present in high concentration in embryonic tissues. It is helpful in cell migra-
tion and formation of granulation tissues. It is necessary for wound repair in
embryonic tissues.
• It is found in basement membrane of glomerulus. It is helpful in glomerular
filtration.
6.5.2 Acidic and Sulfated Mucopolysaccharides
Keratan Sulfate
Occurrence
• Keratan sulfate was isolated from the bovine cornea. It is found in the cornea,
cartilage, and bone.
• Keratan sulfate is a polymer of disaccharide residues of N-acetyl d-glucosamine

and d-galactose. These residues are linked by beta-(1 → 3) linkages.
• Sulfate residues are present on the C6 of N-acetyl d-glucosamine residues.
• Uronic acid residue in keratin sulfate is absent.
Types of Keratan Sulfate

It is classified into two types on the basis of its occurrence in body tissues:
• Keratan Sulfate-I
It is called corneal keratan sulfate.
118 6 Carbohydrates
• Keratan Sulfate-II
It is also called as non-corneal keratin sulfate. It is found in cartilage and bone.
Functions of Keratan Sulfate
• Corneal keratan sulfate is an important structural constituent of stroma of human

cornea. It is made up of collagen fibers and proteins and glycosaminoglycans. Its
deficiency results into macular corneal dystrophy.
• Non-corneal keratan sulfate is a structural component of cartilages and bone.
Chondroitin Sulfate
Occurrence
• Chondroitin sulfate is found in bone, cartilage, skin, tendons, and heart valves of
humans.
Types and Chemical Composition

Depending on chemical composition, chondroitin sulfate is classified into four
types:
• Chondroitin sulfate A
It is a polymer of disaccharide of N-acetyl d-galactosamine and d-glucuronic
acid. The sulfate residues are present on C4 of N-acetyl d-galactosamine.
It is found in cartilages, cornea, and bones.
• Chondroitin sulfate B
It is a polymer of disaccharide of N-acetyl d-galactosamine and l-iduronic acid.
d-Glucuronic acid undergoes epimerization into l-iduronic acid in chondroitin
sulfate B. The sulfate residues are present on C4 of N-acetyl d-galactosamine.
It is found in human skin, heart valves, blood vessels, lungs, and tendons.
Chondroitin sulfate B is called as “dermatan sulfate” due to its presence in the
skin. It shows weak anticoagulant property and is also called as “beta-heparin.”
• Chondroitin sulfate C
acid. The structure of chondroitin sulfate C and chondroitin sulfate A resembles
each other except the position of sulfate residues. The sulfate residues are present
on C6 of N-acetyl d-galactosamine in chondroitin sulfate C.
It is found in tendons and cartilages.
• Chondroitin sulfate D
acid. The sulfate residues are present on C6 of N-acetyl d-galactosamine and C2
of d-glucuronic acid.
It is found in shark cartilage.
6.5 Classification of Mucopolysaccharides 119
Functions of Chondroitin Sulfate
• Chondroitin sulfate is the prime structural constituent of ground substance of

bone, cartilage, tendon, and skin.
• It also shows weak anticoagulant property.
• It has a role in neuronal growth and neuronal repair due to its presence in extra-
cellular matrix in brain tissues.
• It maintains elasticity and resilience of articulating cartilage in joints.
• Dermatan sulfate has a role in wound repair in skin and blood vessels. It is impli-
cated in cardiovascular disease, carcinogenesis, and infection.
Heparin
Occurrence
• Heparin was isolated from the liver. It is synthesized by mast cells in the liver.
Heparin also occurs in the blood vessels, lungs, spleen, thymus, and skin.
• Heparin is a highly sulfated mucopolysaccharide. It is also called as “alpha

heparin.”
• Its molecular weight varies from 15,000 to 20,000.
• It is a polymer of disaccharide of d-glucosamine and d-glucuronic acid or l-
iduronic acid. These residues are linked by alpha-(1 → 3) linkages.
• In d-glucosamine, sulfate residues are present at C2 and C6.
• The C2 position in d-glucuronic acid or l-iduronic acid is sulfated.
• In initial stage of heparin polymerization, it contains d-glucuronic acid residues.
Later on, about 95% of d-glucuronic acid is replaced by l-iduronic acid through
epimerization.
Functions of Heparin
• Heparin is stored in granules of mast cells. It is released into blood vessels and
act as anticoagulant.
• Heparin may act as anti-inflammatory in bronchial asthma and ulcerative colitis.
It has been proved in clinical trials.
6.5.3 Neutral Mucopolysaccharides
• Blood group substances are glycoprotein. They consist of neutral mucopolysac-

charides covalently linked to peptides. They contain carbohydrate residues like
120 6 Carbohydrates
N-acetylated galactosamines, N-acetylated glucosamine, fucose, galactose, and

sialic acid.
• Ovalbumin contains neutral mucopolysaccharides linked to peptides.
Rhamnose occurs in nature as deoxy sugar. It is chemically described as

either methyl-pentose or 6-deoxy-hexose. Rhamnose exists as l-rhamnose. It
is an exception to other naturally occurring sugars which exist in d-form.
Fucose occurs as hexose deoxy sugar. It is present in insects and mamma-

lian tissues. It is associated with N-acetylated glycans.
Arabinose is an aldopentose and exists in nature predominantly as l-

arabinose. It is a component of hemicellulose and pectin. It was initially isolated
from gum arabic or acacia plant whose sap gets hardened on exposure to air.
Hemicellulose is a heteropolysaccharide present in cell walls of plants

along with cellulose. It is made up of glucose, xylose, mannose, galactose,
rhamnose, and arabinose. It has an amorphous structure unlike cellulose
which has crystalline structure.
Mucoprotein is a conjugated protein made up of oligopeptides covalently

linked to glucose, arabinose, xylose, mannose, and fucose carbohydrates.
Carbohydrate proportion of mucoprotein is more than 4%, and it can
range between 10% and 70%. Orosomucoid contains 50% of carbohy-
drates by weight.
Glycoprotein is a conjugated protein made up of oligopeptides covalently

linked to glucose, arabinose, xylose, mannose, and fucose carbohydrates.
Carbohydrate proportion of glycoprotein is less than 4%. It is found in
plasma membrane of cells. Example: Mucin.
Proteoglycan is a conjugated protein composed of oligopeptides cova-

lently linked to glycosaminoglycan chains. Carbohydrate proportion of pro-
teoglycans is much higher in range between 70% and 90%. Proteoglycans are
negatively charged molecules due to presence of sulfate moieties. They repre-
sent the main constituent of extracellular matrix in connective tissues. They
are of various types depending on the presence of glycosaminoglycans.
Peptidoglycan is also called as “murein.” It is a polymer formed by cross-

linking of carbohydrates and oligopeptides. It is made up of repeated residues
of N-acetyl glucosamine (NAG) and N-acetylmuramic acid (NAM) covalently
linked to oligopeptides. It forms an important constituent of cell wall of
bacteria.
Suggested Readings
Aspinall GO (1985) The polysaccharides. Academic Press, New York
Bailey RW (1964) Oligosaccharides. Pergamon Press, Oxford
Binkley RW (1988) Modern carbohydrate chemistry. Marcel Dekker, San Diego
Florkin M, Stotz E (1963) Comprehensive biochemistry: carbohydrates. Elsevier, New York
Guthrie RD (1974) Introduction to carbohydrate chemistry, 4th edn. Clarendon Press, Oxford
Heath EC (1971) Complex polysaccharides. Annu Rev Biochem 40:29
Jaques LB (1979) Heparin: an old drug with a new paradigm. Science 206:528–533
Lennarz WJ (ed) (1980) The biochemistry of glycoproteins and proteoglycans. Plenum, New York
Lipids
7
7.1 Definition
Lipids are heterogenous organic compounds which are either fatty acids or
linked to fatty acids and are soluble in organic solvents.
7.2 Classification of Lipids
Lipid classification was proposed by Bloor in 1925. He proposed a term “lip-

ides” to a group of fatty substances.
7.2.1 Simple Lipids
Simple lipids are esters of fatty acids with alcohols. They are also called as
“homolipids.”
Simple lipids can be subdivided into two groups based on type of alcohol.
• Neutral Fats
–– They are esters of fatty acids with glycerol.
–– Three fatty acid molecules are esterified with one molecule of glycerol. So
they are also called as triglycerides or triacylglycerol as in Figs. 7.1 and 7.2.
• Waxes
–– They are esters of fatty acids with monohydroxy aliphatic alcohols. These
alcohols have high molecular weight.
–– True waxes are ester of fatty acids with cetyl alcohol (C16 H33 O) or other
higher long-chain alcohols.

https://doi.org/10.1007/978-981-13-1035-5_7
124 7 Lipids
Fig. 7.1 Glycerol 1 CH2OH
2 CHOH
3 CH2OH
[Glycerol]
Fig. 7.2 Triglyceride O
(∝) 1 CH2 – O – C – R1
O
(β) 2 CH – O – C – R2
O
(∝′) 3 CH2 – O – C – R3
[Triglyceride]
Examples:
1. “Beeswax” is an ester of palmitic acid with triacontanol (C30 H62O). It is
also called myricyl alcohol. It is an animal wax.
2. “Spermaceti” is found in the head of sperm whale. It is a wax and is pro-
duced inside spermaceti organ in sperm whale. Spermaceti is also found in
oils of whales. It is an animal wax.
3. “Lanolin” is also called as “wool wax.” It is obtained from sebaceous
glands of sheep.
4. “Cerumen” is also called as “earwax.” It is secreted by ceruminous glands
in external auditory canal in humans.
5. “Carnauba wax” is obtained from palm leaves. It is a diester of
4-hydroxycinnamic acid and ω-hydroxycarboxylic acids with long-chain
alcohols. It is a plant wax.
6. “Paraffin wax” is a synthetic wax obtained as petroleum product. It is made
up of long chain of alkane hydrocarbons.
–– Cholesterol ester is an ester of fatty acids with cholesterol.
–– Vitamin A ester is an ester of palmitic acids with retinol.
–– Vitamin D ester is an ester of palmitic acid with cholecalciferol.
7.2.2 Compound Lipids
Compound lipids are esters of fatty acids and alcohols containing additional groups.
They are also called as “heterolipids.”
Compound lipids can be subdivided based on presence of additional group as
follows:
• Phospholipids
Phospholipids are composed of fatty acids, alcohol, phosphoric acid, and nitrog-
enous base.
7.3 Derived Lipids 125
Example: Lecithin, choline, sphingomyelin

• Glycolipids
Glycolipids are composed of fatty acids, alcohol, and carbohydrate residues.
Example: Cerebrosides and gangliosides.
• Lipoproteins
Lipoproteins are composed of lipids linked to proteins.
Example: HDL (High density lipoprotein), LDL (Low density lipoprotein),
VLDL (Very low density lipoprotein).
• Sulfolipids
Sulfolipids are lipids that possess sulfate residues.
Example: Sulfatide. It is synthesized in endoplasmic reticulum.
7.2.3 Derived Lipids
Derived lipids are obtained by hydrolysis of simple and compound lipids. They can
be the following types:
• Fatty acids
• Partial triglycerides like diglycerides and monoglycerides
• Alcohols: straight-chain alcohols like glycerol and cetyl alcohol and unsatu-
rated alcohols like sphingol and phytol.
• Steroids
–– C27steroids: Cholesterol, cholestanol, coprostanol
–– C28 steroids: Ergosterol
–– C18,19,21 steroids (gonadal and adrenal cortex hormones)
–– C24 steroids (bile acids)
–– Secosteroids: Cholecalciferol
• Terpenes
Squalene is a triterpene. It is found in the liver of shark and mammals and sebum
of humans.
• Carotenoids
7.3 Derived Lipids
7.3.1 Fatty Acids
Fatty acids are organic compounds containing a hydrocarbon chain and a ter-
minal carboxylic group.
126 7 Lipids
7.3.2 Types of Fatty Acids
Fatty acids are various types which are described as follows:
traight-Chain Saturated Fatty Acids

S
• These fatty acids have hydrocarbon straight chain without double bond.
• Their molecular formula is (Cn H2n+1 COOH).
• Examples:
Acetic acid (CH3COOH)
Propionic acid (C2H5COOH)
Butyric acid (C3H7COOH)
Palmitic acid (C15H31COOH)
Stearic acid (C17H35COOH)
• Depending on the number of carbon atoms in hydrocarbon chain, they are
sub-classified into different types.
• Types of straight-chain saturated fatty acids
–– Short-chain fatty acids: Straight-chain fatty acids containing up to eight car-
bon atoms
–– Example: Butyric acid (C4)
–– Medium-chain fatty acids: Straight-chain fatty acids containing up to 12
carbons atoms
–– Example: Capric acid (C10) and lauric acid (C12)
–– Long-chain fatty acids: Straight-chain fatty acids containing more than 12
carbon atoms
–– Example: Palmitic acid (C16), stearic acid (C18)
• Cow milk contains predominantly butyric acid, stearic acid, and palmitic
acid.
traight-Chain Unsaturated Fatty Acids

S
• These fatty acids have hydrocarbon straight chain with double bonds.
• Depending on number of double bonds, they are sub-classified into different types.
• Types of straight-chain unsaturated fatty acids
–– Monounsaturated fatty acids (MUFA): They contain single double bond.
Their molecular formula is (CnH2n-1COOH).
Example:
Oleic acid (18:1;9).It contains C18 atoms with one double bond between C9
and C10 atoms in hydrocarbon chain. Oleic acid is present in milk.
–– Polyunsaturated fatty acids (PUFA): They contain two or more than two
double bonds.
Example:
Linoleic acid (C18), linolenic acid (C18), arachidonic acid (C20). They
have high biological importance for humans.
• Unsaturated fatty acids exhibit isomerism.
–– They have two isomeric forms.
7.3 Derived Lipids 127
–– In “cis” form, two hydrogen atoms around the double bond remain on the
same side of chain.
–– In “trans” form, two hydrogen atoms around the double bond are placed on
the opposite side of chain.
Example:
Oleic acid has cis configuration and elaidic acid has trans configuration.
Both have the same molecular formula.
• Trans fats.
–– Unsaturated fatty acids mostly exist in “cis” form in nature.
–– Trans fats are unsaturated straight-chain fatty acids with trans
configuration.
–– They are produced during partial hydrogenation of vegetable oils. The pro-
cess converts unsaturated vegetable oils into semisolid saturated fats.
Example: margarine. It is synthesized from vegetable oils by
hydrogenation.
–– During the process, a few unsaturated double bonds are converted into trans
form. Thus the vegetable fat contains trans fats.
–– The amount of trans fats in daily food should not be more than 1% of total
fats.
–– Natural trans fats are limited in number.
Example:
Conjugated linoleic acid and vaccenic acid. They are found in milk of cattle.
–– Trans fats have no known benefit to humans.
–– They increase the level of LDL and decrease the level of HDL. Tans fats
are implicated in atherosclerosis and coronary artery disease.
ssential Fatty Acids

E
• Essential fatty acids are those polyunsaturated fatty acids which are essential for
physiological functions of the body. They are supplemented with diet.
Example:
Linoleic acid (C18), linolenic acid (C18), arachidonic acid (C20)
ranched-Chain Fatty Acids (BCFA)

B
• These are basically saturated fatty acids with a branch on methyl group or on the
hydrocarbon chain.
• They are abundantly found in dairy products.
Example:
Phytanic acid
• They are structural component of bacterial cell membrane. Example: Lactobacilli.
yclic Fatty Acids

C
• They have a ring structure internally in the molecule.
Example:
Chaulmoogric acid
• It is derived from seeds of chaulmoogra. It is used in treatment of leprosy.
128 7 Lipids
7.4 Essential Fatty Acids (EFA)
7.4.1 History
• In 1927, Herbert Evans and George Burr observed a deficiency in the growth of
rats. They hypothesized the necessity of a lipid substance (EFA) in diet and
called it “vitamin F.”
• In 1930, it was George Burr and his wife, Mildred Burr, who discovered linoleic
acid and coined the term “essential fatty acids” pertaining to their essentiality for
growth and overall health of experimental albino rats.
• In 1958, a deficiency of EFA was reported among infants who were fed upon a
diet deficient of EFA.
Definition
Essential fatty acids are polyunsaturated fatty acids which are not synthesized in the
body and need to be supplemented with diet to maintain normal health.
EFA are highly essential for physiological functions of the body. They are not
synthesized in the body. They are supplemented with diet.
7.4.2 Types of Essential Fatty Acids
Linoleic acid and linolenic acid are EFA. They are polyunsaturated fatty acids.
Linoleic Acid
• It is a C18 compound. It has two double bonds at C9 and C12 positions. They are
present between C9–C10 carbon atoms and C12–C13 carbon atoms. It is repre-
sented as (18:2;9,12).
• Linoleic acid is found in cottonseed oil, sunflower oil, soya bean oil, and egg
yolk.
• It exists as ester of triglyceride.
• Linoleic acid is a precursor to lipoxins, eicosanoids, and endocannabinoids.
• In fatty acids, carboxylic group is present at one end of molecule. The carbon
atom next to carboxylic carbon is called as α-carbon, and the carbon atom of
methyl group on the opposite end is called as ω-carbon (omega).
• Linoleic acid is called as “ω-6 fatty acid.” It has first double bond located at
ω-6 carbon atom.
Linolenic Acid
• It is a C18 compound. It has three double bonds at C9, C12, and C15 positions.
They are present between C9–C10, C12–C13, and C15–C16 atoms in hydrocar-
bon chain, respectively. It is represented as (18:3;9,12,15).
7.4 Essential Fatty Acids (EFA) 129
• Linolenic acid is found in rapeseed oil, sunflower oil, linseed oil, cod liver oil,
sea algae, and phytoplankton.
• Linolenic acid is called as “ω-3 fatty acid.” It has first double bond located
at ω-3 carbon atom.
• Three omega-3 fatty acids are biologically important for humans. They are
α-linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid
(DHA).
• EFA cannot be synthesized in the body of mammals and humans. They lack
fatty acid desaturase enzyme which is necessary for biosynthesis of EFA.
Arachidonic Acid
• It is a C20 compound. It has four double bonds. They are present between C5–
C6, C8–C9, C11–C12, and C14–C15 carbon atoms. It is represented as
(20:4;5,8,11,14).
• Arachidonic acid is found in peanut oil, cod liver oil.
• It is synthesized from linoleic acid in the human body through chain elongation
and desaturation. It has high biological importance.
• Arachidonic acid is a precursor to prostaglandins, leukotrienes, and
thromboxane.
Conditionally Essential Fatty Acids
• These are polyunsaturated fatty acids that become essential in physiological

and/or pathological condition and need supplementation with diet.
Example: γ-Linolenic acid, DHA (docosahexaenoic acid)
7.4.3 Functions of EFA
• Essential fatty acids are vital for growth and normal health of individual.
• They are structural components of body tissues.
• Essential fatty acids are components of lipids in mitochondrial membrane. In
deficiency of EFA, oxidative phosphorylation is reduced.
• EFA are integral components of lipids in plasma membranes. Arachidonic acid
constitutes about 15% of the lipids in cell membranes.
• EFA are necessary for biosynthesis of prostaglandins from arachidonic acid
by cyclooxygenase enzyme activity.
• EFA are necessary for biosynthesis of leukotrienes through lipoxygenase
activity.
• Diet rich in EFA helps to lower low-density lipoprotein.
• Essential fatty acids help to minimize fatty liver condition.
• Docosahexaenoic acid (DHA) is abundantly found in the retina. It is biosyn-
thesized from dietary alpha linolenic acid. So EFA are necessary for normal
vision.
130 7 Lipids
• Docosahexaenoic acid (DHA) is the essential component of the plasma mem-

brane of brain tissues. DHA is essential in development of the brain and cog-
nitive functions among preschool children.
• Essential fatty acids have cis configuration. They provide fluidity to plasma
membranes.
7.4.4 Clinical Significance of EFA
• Deficiency of EFA is rarely observed with normal diet.

• Essential fatty acids bring about esterification of cholesterol. They are helpful in
excretion of cholesterol.
• Deficiency of EFA may result into fatty liver.
• Deficiency of EFA results into excessive thickness of the stratum corneum
(hyperkeratosis) and epithelial hyperplasia of Malpighian layer with hyperpig-
mentation of the skin (acanthosis).
• Deficiency of EFA may be associated with abnormality in growth of body
tissues.
• EFA deficiency leads to Higher chances for retinitis pigmentosa disorder. It is a
hereditary degenerative disorder characterized by loss of retinal cells and
blindness.
• Diet with higher levels of trans fatty acids replaces EFA and predisposes to coro-
nary artery disease.
7.5 Cholesterol
Cholesterol is a biologically important sterol in the human body.

The word is derived from Greek words “chole” which means bile, “stereos”
which means solid, and “ol” which means alcohol.
It was first discovered by Francois Poulletier de la Salle from gallstones.
7.5.1 Occurrence
• Cholesterol is distributed widely in body tissues. Healthy human body contains

about 2 g of cholesterol per kg of body weight. A person of 70 kg of body weight
has 140 g of cholesterol in the body.
• Normal serum total cholesterol level is 200–240 mg/dl.
• Brain tissues contain comparatively higher amount of cholesterol. It is around
25% of total cholesterol of the body.
• Adipose tissues contain around 30 g of cholesterol.
• The liver contains around 15 g of cholesterol.
• Almost all cells of the body can synthesize cholesterol.
7.5 Cholesterol 131
7.5.2 Dietary Sources
• Milk, cream, cheese, butter, eggs, meat, and shellfish are rich in cholesterol.
7.5.3 Properties
• It is a white to light yellowish, odorless crystalline solid.

• It is insoluble in water but soluble in alcohol, chloroform, ether, and other organic
solvents.
• It is an important animal sterol.
• It is absent in prokaryotes and plants.
• In animal tissues, cholesterol exists in “free state” as well as “esterified state.”
Brain tissues mainly contain free state of cholesterol, whereas the adrenal cortex
chiefly contains cholesterol ester.
7.5.4 Chemical Structure
• Molecular formula of cholesterol is (C27H45OH).

• It is a solid organic alcohol.
• Cholesterol has “cyclo-pentano-perhydro-phenanthrene nucleus.” It is also
called as “sterane ring” as in Fig. 7.3.
• The nucleus system is composed of four rings which are nonlinearly fused. Three
rings are designated as A, B, and C which are cyclohexane. Fourth ring is desig-
nated as D which is cyclopentane. The three cyclohexane rings together consti-
tute “phenanthrene” (polycyclic aromatic organic compound).
• Cholesterol has one hydroxyl group (OH) present at C3 position.
• It has one double bond between C5–C6 atoms.
• It possesses methyl groups at C10 and C13 atoms.
• It possesses a chain of eight carbon atoms linked to C17 atom as in Fig. 7.4.
Fig. 7.3 Structure of 18

Cholesterol 12 17
11 13 16
1 9 C D
2 14 15
10 8
A B
3 7
5
4 6
Cyclopentanoper hydro Phenanthrene
nucleus
[Sterane nucleus]
132 7 Lipids
Fig. 7.4 Cholesterol CH3

CH3
CH2– (CH2)3 – CH
CH3 CH3
17
Aliphatic side
13 chain
CH3
1 15
2 10
3 5
4 Cyclopentane
OH
6 Ring
Phenanthrene nucleus
[ Cholesterol ]
Fig. 7.5 7-Dehydro cholesterol CH2 CH2
CH2
CH2
CH2
1
2
3 5
4 7
OH
6
7-DEHYDRO CHOLESTROL
7.5.5 Functions
• Cholesterol is a structural component of plasma membrane.

• It helps to maintain fluidity of plasma membrane.
• It is necessary for biosynthesis of bile acids and bile salts.
• It is vital for biosynthesis of steroidal hormones like glucocorticoids, testoster-
one, and estrogen.
• Synthesis of calcitriol (vitamin D3) requires 7-dehydrocholesterol in the skin. It
is a precursor to vitamin D3 (Fig. 7.5).
7.6 Compound Lipids
7.6.1 Phospholipids
Phospholipids are compound lipids which contain nitrogenous base/additional sub-

stituents attached to phosphate residue in addition to fatty acids and alcohol.
7.6 Compound Lipids 133
They are also called as “phosphatides.”
Classification of Phospholipids
Celmer and Carter classified phospholipids on the basis of type of alcohol pres-
ent in phospholipids.
Phospholipids are classified into three groups as:
1. Glycerophosphatides
2. Phosphoinositides
3. Phosphosphingosides
Glycerophosphatides
Composition
• They contain fatty acid + glycerol + phosphoric acid + nitrogenous base.
• Two fatty acid molecules are linked to glycerol at C1 and C2 positions. These
ester bonds are hydrophobic in nature and form the hydrophobic tail of
phospholipid.
• The phosphoric acid is attached to C3 of glycerol forming a hydrophilic head of
phospholipid. Glycerophosphatides have “amphipathic character.”
• Fatty acid, glycerol, and phosphate together form “phosphatidic acid.”
• Nitrogenous base is attached to phosphate residue through ester linkage.
Types
Depending on type of nitrogenous base, phospholipids are the following types:
Lecithin/Phosphatidylcholine
Occurrence
• Lecithin is the chief phospholipid in biological membranes of animals and

plants.
• It is abundantly found in the brain, liver, sperm, and egg yolk.
• It is present in sprouts and seeds. Important plant seeds with high lecithin content
are peanuts, flaxseeds, sunflower seeds, sesame seeds, and cottonseeds.
Composition
• It is composed of fatty acids + glycerol + phosphoric acid + choline as in

Fig. 7.6.
• Saturated fatty acid and unsaturated fatty acid are linked to C1 and C2 positions
on glycerol through ester linkage.
134 7 Lipids
O
Glycerol ∝ CH2 – O – C – R1
moiety
O Fatty acids
β CH – O – C – R2
O CH3
∝¢ CH2 – O – P – O – CH2 – CH2 – N+ CH3
OH CH3
Phosphoric acid Polar choline
Fig. 7.6 Lecithin
• Phosphoric acid is linked to C3 position on glycerol through ester linkage. This

molecule is called as “phosphatidic acid.”
• Choline is linked to phosphate moiety through ester bond.
Hydrolysis of Lecithin
Lecithin is hydrolyzed by various phospholipases found in animal tissues.
Action of Phospholipases A1 and A2
• Phospholipases A1 andA2 are present in mammals and humans. Phospholipase

A1acts at SN-1 position of phospholipid (lecithin) and liberates fatty acid from
C1 of glycerol.
Phospholipase A1
Lecithin Lysolecithin + Fatty acid
• Phospholipase A2 is found in humans. It acts at SN-2 position of phospholipids

and liberates fatty acid from C2 of glycerol.
Phospholipase A 2
Lecithin Lysolecithin + Arachidonic acid
Action of Phospholipase B
• This enzyme is found in humans.

• It can act at SN-1 and SN-2 positions of phospholipid. Its substrate is
lysophospholipid.
• It is also called as lysophospholipase.

Phospholipase B
Lysolecithin Glyceryl phosphoryl choline + Fatty acid
Action of Phospholipase C
• It is found in humans, plants, and Clostridium welchii.

• It cleavages phosphate ester bond and releases 1,2-diacylglycerol and phospho-
ryl choline.
Phospholipase C
Lecithin 1,2Diacyl glycerol + Phosphoryl choline
Action of Phospholipase D
• This enzyme is found in plants, bacteria, and viruses.

• It cleavages phospholipid into phosphatidic acid and choline.
Phospholipase D
Lecithin Choline + Phosphatidic acid
Cephalin/Phosphatidylethanolamine
Occurrence
• Cephalin is the chief phospholipid of bacteria.

• It is found in biological membranes of animals and humans. It constitutes around
20% of total phospholipids.
• It is abundantly found in nervous tissues like white matter of the brain, spinal
cord, and nerve fibers. Cephalin constitutes around 50% of total phospholipids of
the brain.
Composition
• Cephalin is composed of fatty acids + glycerol + phosphoric acid + ethanol-

amine as in Fig. 7.7.
Plasmalogen
Plasmalogen is a class of ether phospholipid. It is different from other phospholip-
ids with the presence of ether bond at C1 and ester bond at C2 of glycerol.
136 7 Lipids
Occurrence
• It is found in biological membranes of animals.

• It is rich in brain tissues, mitochondria, and muscles.
• Plasmalogen constitutes about 15% of total phospholipids.
Composition
• Plasmalogen is composed of fatty acids + glycerol + phosphoric acid + etha-

nolamine/choline as in Fig. 7.8.
• In plasmalogen, saturated fatty acid is attached to C1 position of glycerol by ester
linkage, and PUFA is attached to C2 position of glycerol by ether linkage.
Cardiolipin
Occurrence
• Cardiolipin is a main phospholipid in inner mitochondrial membrane of animals

and plants.
• It was extracted from bovine heart. So it derived its name.
• It is also found in bacterial cell membrane.
Fig. 7.7 Chephalin

∝ CH2 – O – CO – R1
Fatty acids
β CH – O – CO – R2
O
∝¢ CH2 – O – P – O – CH2 – CH2 – NH2
OH
Ethanol amine
Phosphoric acid
Fig. 7.8 Plasmalogen CH2 – O – CH = CH – R1

(containing ethanolamine
moiety) CH – O – CO – R2
O
CH2 – O – P – O – CH2 – CH2 – NH2
OH
Ethanolamine
Composition
• It is composed of fatty acids + glycerol + phosphoric acids.

• Cardiolipin has a unique chemical structure. Glycerol molecule forms the back-
bone of the cardiolipin. It has two phosphatidic acid molecules. These are linked
at C1 and C3 positions on central glycerol moiety. So it is also called as “diphos-
phatidylglycerol” or “1,3-bis phosphatidylglycerol.”
• Cardiolipin contains two phosphoric acid molecules. So it has greater affinity to
proteins in inner mitochondrial membrane.
Phosphoinositide
Phosphatidylinositol
Occurrence
• It is found in biological membranes of animals and plants.

• It is more in brain tissues in mammals.
Composition
• It is composed of fatty acids + glycerol + phosphoric acids + inositol as in Fig. 7.9.

• Inositol is a cyclic polyhydroxy alcohol. It is a hexahydroxy cyclohexane. It is
the major structural constituent of phosphatidylinositol.
Phosphosphingosides
Sphingomyelin/Phosphatidyl-Sphingoside
Occurrence
• They are predominantly found in nervous tissues in the brain, spinal cord, and
nerves.
• They are the main phospholipid in myelin sheath of axons of nerves.
Fig. 7.9 Phosphatidyl O

inositol
CH2 – O – C – R1
CH – O – CO – R2
O
CH2 – O – P – O – CH2OH
OH OH
C
H H
C
H OH OH
OH H
138 7 Lipids
Choline
Phosphoric acid moiety
O CH3
=
CH3 – (CH2)12 – CH = CH – CHOH – CH – CH2 – O – P – O – CH2 – CH2 – N+ CH3
NH H CH3
Fatty acid C=O

R
Fig. 7.10 Sphingomyelin
Composition
• They are composed of fatty acid + sphingol + phosphoric acid + choline as in

Fig. 7.10.
• Sphingol is a C18 amino alcohol. It is also called as “sphingosine.”
• Fatty acid molecule is attached to –NH2 group on sphingol. It results into forma-
tion of “ceramide.” It is a waxy lipid and important constituent of cell mem-
branes of animal cells.
• Ceramide is further linked to phosphoric acid to form “ceramide phosphate.”
Choline is attached to phosphate through ester bond to form sphingomyelin.
Functions of Phospholipids
• Phospholipids are structural constituents of biological cell membranes of ani-

mals, plants, and bacteria.
• Phospholipid like cardiolipins is an integral component of inner mitochondrial
membrane. It is associated with normal functioning of mitochondrial enzymes. It
helps in oxidative phosphorylation.
• Phospholipid like sphingomyelins is an integral component of myelin sheath of
myelinated nerve fibers. It forms an insulating layer over axon of nerve cells.
• Phospholipid like plasmalogens protects body tissues against oxidative damage
from reactive oxygen species.
• Phospholipids are necessary for blood coagulation along with blood clotting
factors.
• Phospholipid like lecithin decreases surface tension of water in intestinal lumen.
They are helpful in emulsification of fats.
• Phospholipids are helpful in the formation of chylomicrons within enterocytes.
They help in the transport of dietary triglycerides from the intestine to blood
circulation.
• Phospholipids help in the transport of endogenous triglycerides from the liver to
body tissues.
• Lecithin provides choline. It is a lipotropic factor and prevents fatty liver.

• Phospholipids of cell membranes provide arachidonic acid through enzymatic
action. It is a precursor for prostaglandins and leukotrienes.
• Phospholipids act as cofactor for tissue lipase.
Clinical Significance of Phospholipids
• Dipalmitoyl-Phosphatidyl-Choline (DPC)
–– It is lecithin which acts as “pulmonary surfactant.”
–– It is composed of two molecules of palmitic acids linked to
phosphatidylcholine.
–– It lowers the surface tension in pulmonary alveoli.
–– DPC increases the ability of pulmonary alveoli to expand (pulmonary
compliance).
–– DPC prevents the collapse of pulmonary alveoli after expiration
(atelectasis).
• Niemann-Pick Disease
–– It is a hereditary recessive disorder of lipid storage (lipidoses).
–– It is due to deficiency of sphingomyelinase enzyme.
–– It is characterized by excessive accumulation of sphingomyelin in the brain,
spleen, and liver.
–– Its onset is early in infancy. The affected children have enlargement of the
liver (hepatomegaly), enlargement of the spleen (splenomegaly), and progres-
sive degeneration of brain tissues.
7.6.2 Glycolipids
Glycolipids are compound lipids containing a carbohydrate moiety attached to

lipid molecule by glycosidic bond.
They are non-phosphorylated lipids.
Occurrence
• Glycolipids are found in white matter of the brain, spinal cord, myelin sheath,
spleen, and erythrocytes.
Composition
• They are fatty acids + sphingol + carbohydrates.
Types
Glycolipids are two types:
140 7 Lipids
Cerebrosides/Monoglucosylceramides
Occurrence
• Cerebrosides are abundantly found in white matter of the brain, spinal cord, and
myelin sheath of nerve fibers and dendrites.
Composition
• They are composed of fatty acids + sphingol + carbohydrates (galactose) or

(glucose).
• Cerebrosides are devoid of glycerol, phosphoric acid, and nitrogenous
base.
Types
Based on fatty acid in cerebrosides, they are the following four types:
• Kerasin
–– It contains lignoceric acid. It is a C24 fatty acid.
• Cerebron
–– It contains hydroxy lignoceric acid, called as cerebronic acid. It is a hydroxyl-
ated lignoceric acid.
• Nervon
–– It contains nervonic acid. It is a monosaturated fatty acid with C24 atoms.
• Oxynervon
–– It contains oxygenated nervonic acid.
Function
• Cerebrosides are structural components of white matter of the brain, myelin

sheath, and spinal cord.
• They are helpful in normal brain functioning.
• They modulate signal transduction.
• They are necessary for impulse conduction.
Clinical Significance
• Gaucher’s Disease
–– It is a hereditary autosomal recessive disorder of cerebroside metabolism.
–– The disorder is due to deficiency of beta-glucocerebrosidase enzyme.
–– It is characterized by excessive accumulation of glucocerebrosides (kerasin)
in the brain, liver, reticuloendothelial cells, and bone marrow.
–– Disease affects children and adults. Failure of growth, mental retardation, and
muscle spasticity are characteristic feature in infants and children. In adults,
bone marrow is infiltrated with histiocytes. Bone pain, anemia, and thrombo-
cytopenia are main manifestations in adults.
Ganglioside
Ganglioside is a carbohydrate-enriched glycolipid isolated from ganglionic cells
of bovine brain by Ernst Klenk in 1942.
Occurrence
• Gangliosides are found in the spleen, liver, and brain.

• They are predominantly found in white matter of the brain, spinal cord, gangli-
onic cells, spleen, and dendrites.
Composition
• They are composed of fatty acids + sphingol + carbohydrates (galactose) or

(glucose) + N-acetyl neuraminic acid (NANA) also called as sialic acid.
• Gangliosides contain 1–3 NANA residues.
Types
Based on number of sialic acid (NANA) residues, gangliosides are the following
types:
• One sialic acid residue containing gangliosides is designated as “GM.”

–– GM-1, GM-2, GM-3
• Two sialic acid residues containing gangliosides are designated as “GD.”
–– GD-1a, GD-1b, GD-2, GD-3
• Three sialic acid residues containing gangliosides are designated as “GT.”
–– GT-1b, GT-3
• Four sialic acid residues containing gangliosides are designated as “GQ.”
–– GQ-1
Function
• Gangliosides are structural components of biomembranes.

• They act as receptors for pituitary hormones.
• They modulate signal transduction.
• They have a role in cell recognition.
• They have a role in transportation of amines through plasma membranes.
142 7 Lipids
• Tay-Sachs Disease
–– It is a hereditary recessive disorder of ganglioside metabolism.
–– It is due to deficiency of hexosaminidase enzyme.
–– The disorder is characterized by excessive accumulation of GM-2 ganglioside
in the ganglionic cells and brain tissues.
–– Its onset is early in infancy. Ganglionic cells of the brain and retina are injured.
Infants suffer from blindness, mental retardation, and seizures.
–– Prognosis is extremely poor.
7.6.3 Sulfolipids
Sulfolipids are the compound lipids which possess sulfur groups. The sulfur group
is attached to glycolipid through esterification. Sulfolipids are abundantly found in
biomembranes of nervous tissues.
7.7 Simple Lipids
Neutral Fats
Neutral fats are the tri-esters of glycerol with similar or dissimilar fatty acids.
They are also called as “triacylglycerol” or “triglycerides.” The carbon atoms of
glycerol are designated as C1, C2, and C3. Esterification of glycerol with similar fatty
acids results into formation of simple triglycerides. Esterification with dissimilar
fatty acids results into mixed triglycerides.
Physical Properties
• Triglycerides are colorless, tasteless, and odorless.

• They are insoluble in water.
• Triglycerides containing higher proportion of unsaturated fatty acids are liquid
at room temperature and called as “oil.” Example: Cottonseed oil, sunflower
oil.
• Triglycerides with higher proportion of saturated fatty acids are semisolid at
room temperature and called as “fats.” Example: Butter (butyric acid),
coconut oil (lauric acid).
• Their specific gravity is <1. They float on the surface of water.
• They undergo emulsification in aqueous medium in the presence of bile salts,
soap, and proteins.
• Melting point of neutral fats with short-chain fatty acids is low, for example, but-
ter (MP = 35 °C), and fats with long-chain fatty acids is high, for example, tripal-
mitin (67 °C).
7.7 Simple Lipids 143
O
O CH2 – O – C – C17 H33 CH2 OH
Hydrolysis
H33 C17 – C – O – CH O CH OH
CH2 – O – C – C17 H33 CH2 OH
Glycerol
Triglyceride
[Triolein] +
3 C17 H33 COOH
Oleic Acid
Fig. 7.11 Hydrolysis of triglyceride
Chemical Properties
• Hydrolysis
–– Neutral fats can be hydrolyzed by alkali, acids, or enzyme.
–– Lipase is found in saliva, gastric juice, pancreatic juice, intestinal juice, and
body tissues.
–– Lipase cleavages fats into glycerol and constituent fatty acids as in Fig. 7.11.
• Saponification
–– Saponification is the hydrolysis of neutral fats by alkali. It results into forma-
tion of glycerol and alkali salts of fatty acids. These salts are called as
“soap.”
Tripalmitin + Sodium hydroxide Glycerol + Sodium palmitate
(Soap)
• Rancidity
–– The occurrence of unpleasant smell and bad taste in the neutral fats upon
aging is called rancidity.
–– Rancidity is hydrolytic rancidity and oxidative rancidity.
–– In hydrolytic rancidity, fats are hydrolyzed by lipase in the presence of mois-
ture and warm temperature. It yields glycerol, free fatty acids, and
monoacylglycerol.
–– In oxidative rancidity, unsaturated fatty acids in fats are oxidized to form per-
oxides and aldehydes. They have unpleasant smell and taste.
–– Natural oils possess antioxidants like vitamin E, hydroquinones, and phenols.
They prevent rancidity.
–– Natural oils containing higher quantity of PUFA are more prone to
rancidity.
144 7 Lipids
7.8.1 Dietary Omega: Three Fatty Acids and Dental Diseases
Periodontitis is a chronic and progressive inflammatory disease of periodontium

of teeth. It is a bacterial disease. It is characterized by inflammation of gums,
tooth mobility, pain, and suppuration. Polyunsaturated fatty acids like omega-3
fatty acids and omega-6 fatty acids have anti-inflammatory properties. Topical
application of omega-3 fatty acids on teeth and gums in experimental models has
proved beneficial in protection of tissue damage and bone loss associated with
periodontitis.
Randomized clinical trial in humans suffering from periodontitis had been done.
Systemic administration of eicosapentaenoic acid and gamma-linolenic acid was
followed for 12 weeks. Reduction in periodontal pockets in patients receiving EPA
and GLA was observed.
Suggested Readings
Campan P, Planchand PO, Duran D (1997) Pilot study on n-3 polyunsaturated fatty acids in the
treatment of human experimental gingivitis. J Clin Periodontol 24:907–913
Guan X, Wenk MR (2008) Biochemistry of inositol lipids. Front Biosci 13:3239–3251
Hasturk H, Kantarci A, Ohira T, Arita M, Ebrahimi N, Chiang N, Petasis NA, Levy BD, Serhan
CN, Van Dyke T (2006) RvE1 protects from local inflammation and osteoclast- mediated bone
destruction in periodontitis. FASEB J 20:401–403
Hunt SM, Groff JL, Gropper SA (1995) Advanced nutrition and human metabolism. West,
Belmont, CA
Hunter JE (2006) Dietary trans fatty acids: review of recent human studies and food industry
responses. Lipids 41(11):967–992
Rosenstein ED, Kushner LJ, Kramer N, Kazandjian G (2003) Pilot study of dietary fatty acid
supplementation in the treatment of adult periodontitis. Prostaglandins Leukot Essent Fatty
Acids 68:213–218
Vance JE, Vance DE (2002) Biochemistry of lipids, lipoproteins and membranes. Elsevier,
Amsterdam
Zhao G, Etherton TD, Martin KR, Gillies PJ, West SG, Kris-Etherton PM (2007) Dietary alpha-
linolenic acid inhibits proinflammatory cytokine production by peripheral blood mononuclear
cells in hypercholesterolemic subjects. Am J Clin Nutr 85:385–391
Nucleic Acids
8
8.1 Historical Background
• In 1868, Swiss physician, Friedrich Miescher, isolated nucleic acid from nuclei
of human WBC (pus cells). He called it as nuclein.
• In 1884, it was Oscar Hertwig who emphasized upon the importance of nuclein
as genetic material.
• In 1889, Altman named nuclein as nucleic acid.
• In 1919, Russian biochemist P. Levene proposed polynucleotide model of nucleic
acid of yeast. Later on, he suggested tetranucleotide model comprised of four
nucleotides arranged in the same order as G-C-T-A-G-C-T-A. Levene also
declared sequence (phosphate-sugar-base) of nucleotide constituents.
• In 1944, Oswald Avery postulated that DNA was the core molecule for inheri-
tance of information.
• In 1950, Erwin Chargaff, Austrian biochemist, concluded against Levene that
one nucleotide never replicates in the same sequence. He also noted that compo-
sition of nucleotide differs in different species.
• In 1950, Rosalind Franklin produced X-ray diffraction study of DNA structure.
Her pioneering work paved a way to 3D structure of DNA by Watson and Crick.
She remained unacknowledged.
• In 1953, American biologist, James Watson, and English physicist, Francis Crick,
restructured data regarding DNA and put forward a double-helix model of DNA.
• In 1961, DNA was synthesized by Kornberg.
Definition
Deoxyribonucleic Acid (DNA)
DNA is the largest biopolymer consisting of deoxyribonucleotides.
It constitutes genetic material of organisms. However, certain organisms like
tobacco mosaic virus (TMV) and human immunodeficiency virus (HIV) contain
ribonucleic acid (RNA) as a genetic material.

https://doi.org/10.1007/978-981-13-1035-5_8
146 8 Nucleic Acids
DNA as Genetic Material

In 1902, Sutton and Boveri promulgated the role of chromosomes in transmis-
sion of genetic information from one generation to another.
In 1952, the experiment conducted by Alfred Hershey and Martha Chase on
bacteriophage paved the way to declare DNA as genetic material due to follwo-
ing characteristics such as:
• DNA is present in all cells.

• It can replicate itself as carbon copy.
• It is inherited from parents to offspring.
• DNA can undergo mutations which are essential for adaptation and
variations.
8.2 Occurrence
In Eukaryotic Cells
• DNA is located within the nucleus of cell. It is associated with basic proteins and
forms chromosomes.
This may be called as nuclear DNA.
• DNA is also located within mitochondrial matrix.
This may be called as mitochondrial DNA (mtDNA).
In Prokaryotic Cells
• DNA is located in the cytoplasm of cell (nucleoid).
8.3 Chemical Composition of DNA
• DNA is a biomacromolecule. It is made up of two strands of

deoxyribonucleotides.
• Each deoxyribonucleotide is composed of three units as in Figs. 8.1, 8.2, and 8.3.
–– Nitrogenous bases
–– Deoxyribose sugar
–– Phosphoric acid
• Nitrogenous Bases
These are nitrogen-containing cyclic organic compounds. They are categorized
into two groups depending upon their chemical structure such as:
–– Purines
Purine is a nine-membered heterocyclic organic compound. Purine is com-
posed of pyrimidine ring attached to imidazole ring. Purine has four nitrogen
atoms at 1, 3, 7, and 9 positions as in Fig. 8.4.
Adenine and guanine are two purine bases in DNA.
8.3 Chemical Composition of DNA 147
Fig. 8.1 Ribose Sugar O

HOH2C5 H
C 4 1C
H H H OH
3 2
C C
OH OH
Fig. 8.2 Deoxyribose O
HOC5H2 H
Sugar
C 4 1 C
H H H OH
3 2
C C
OH H
Fig. 8.3 Phosphoric acid H3PO4 OH
HO P O
OH
NH2 O
C C
6 5 N 6 5 N
N 1 C 7 HN 1 C 7
8 CH 8 CH
HC 2
C C 2
C
9 9
3 4 3 4
N N
N H H2N N H
ADENINE GUANINE
PURINES
Fig. 8.4 Purines
–– Pyrimidines
Pyrimidine is a six-membered heterocyclic organic compound. Pyrimidine struc-
ture resembles benzene ring. It has two nitrogen atoms at 1 and 3 positions.
Thymine and cytosine are two pyrimidine bases in DNA.
• Deoxyribose Sugar
148 8 Nucleic Acids
–– It is an aldopentose. It contains five carbon atoms. Deoxyribose sugar

(C5H10O4) has one oxygen atom less than ribose sugar (C5H10O5). In deoxyri-
bose sugar, C2 contains H-C-H group, whereas ribose sugar contains H-C-OH
group.
• Phosphoric Acid
It forms phosphodiester bond with two sugar residues as in Fig. 8.5a.
O NH2
a
C CH3 C
6 6
HN 1 5 C N 1 5 CH
C 2 4 CH C 2 4 CH
3 3
O N O N
H H
THYMINE CYTOSINE
PYRIMIDINES
b
Nitrogen Base + Deoxyribose Sugar
NUCLEOSIDE
Glycosidic Bond
c
NUCLEOSIDE + Phosphoric acid
NUCLEOTIDE
Esterification
Fig. 8.5 (b) Nucleoside structure. (c) Nucleotide structure

8.5 Structure of DNA Strand 149
In a Deoxyribonucleotide
Purines and pyrimidines are attached to deoxyribose sugar by glycosidic bond. The
nitrogen atom at position 9 in purines is linked to C1of deoxyribose sugar, whereas
nitrogen atom at position 1 in pyrimidines is linked to C1 of deoxyribose sugar as in
Fig. 8.5b.
Phosphodiester Bond
Phosphodiester bond is formed between two OH groups of phosphoric acid
and OH groups of sugar at 3′ and 5′ positions.
Polydeoxyribonucleotides in a strand are linked together by phosphodiester
bonding. These sugar-phosphate linkages constitute the backbone of DNA. It is
the structural basis of DNA.
The 3′ and 5′ positions in deoxyribose sugar are available for ester linkage.
Phosphoric acid is attached to OH group at 3′ carbon of deoxyribose sugar in one
nucleotide and OH group at 5′ carbon of deoxyribose sugar in adjacent nucleotide.
Therefore, phosphodiester linkage at 3′ and 5′ positions provides stability to
DNA strand as in Fig. 8.5c.
8.4 Types of Deoxyribonucleotides in DNA
DNA contains four types of deoxyribonucleotides depending on the nature of

nitrogenous base.
1. Deoxyadenosine Monophosphate (dAMP)

It is also called as 5′-deoxyadenylic acid or deoxyadenylate as in Fig. 8.6.
2. Deoxyguanosine Monophosphate (dGMP)
It is also called as 5′-deoxyguanylic acid or deoxyguanylate as in Fig. 8.7.
3. Deoxythymidine Monophosphate (dTMP)
It is also called as 5′-deoxythymidylic acid or deoxythymidylate as in Fig. 8.8.
4. Deoxycytidine Monophosphate (dCMP)
It is also called as 5′-deoxycytidylic acid or deoxycytidylate as in Fig. 8.9.
8.5 Structure of DNA Strand
• It is polymer of deoxyribonucleotides. Adjacent nucleotides are linked together

by phosphodiester bond.
• Each DNA strand has polarity.
–– 3′ End (3 prime)
It is the end of DNA strand where C3 of deoxyribose sugar is not linked and
is free.
–– 5′ End (5 prime)
It is the end of DNA strand where C5 of deoxyribose sugar is not linked and
is free as in Figs. 8.10 and 8.11.
150 8 Nucleic Acids
Fig. 8.6 Adenosine NH2

monophosphate
(Adenylic acid)
6
7
5 1
O
8 ADENINE
O P O 4 2
9
O CH2 N 3
5
O
4 1
SUGAR
3 2
OH
Fig. 8.7 O
Guanosine
monophosphate
(Guanylic acid)
7
5 1
O
8 GUANINE
O P O 6
9
O CH2 N
5 NH2
O
4 1
SUGAR
3 2
OH
One strand of DNA molecule exhibits 3′–5′ polarity, whereas other strand
has 5′–3′ polarity.
Base Pair in DNA

Base pair signifies two nitrogenous bases held together through hydrogen bonding.
Each pair of base consists of purine in one strand and pyrimidine in another strand.
Particular purine always pairs with particular pyrimidine.
8.5 Structure of DNA Strand 151
Fig. 8.8 CH3

Thymidine
monophosphate
(Thymydlic acid) O O
5
O P O 4 6C
O CH2 THYMINE
5
O N
3 1
NH
2
4 1
SUGAR
O
2
Fig. 8.9 Cytidine NH2

monophosphate (Cytidylic
acid)
C
6
5 1N
O CYTOSINE
4 2
O P O C
3
O CH2 N O
5
O
4 1
3 2
This concept of base pair is called as complementary base pairs (base pair is
nonidentical).
Adenine pairs with thymine by two hydrogen bonds.
Guanine pairs with cytosine by three hydrogen bonds.
Justification of Complementary Base Pair
• Width of DNA helix is 20 Å. This space can be properly filled by a purine and a
pyrimidine. Contrarily, pairing between two purines would require space larger
than 20 Å. Furthermore, pairing between two pyrimidines would leave an empty
space, and hydrogen bonding could not be possible. A purine molecule has size
twice as wide as pyrimidine molecule.
• Three-dimensional arrangement of adenine and thymine is perfect to confer
hydrogen bonding between two bases.
152 8 Nucleic Acids
GLYCOSIDIC
BOND A Adenine
C Cytosine
P P
G Guanine
S A T S
T Thymine
P P
PHOSPHO DIESTER
BOND S C G S RIBOSE SUGAR
P P PHOSPHATE GROUP
S T A S
P P
S G C S
P P
S C G S
P P
S A T S
P P
S G A S
P P
HYDROGEN
BONDING
Fig. 8.10 Fragment of DNA molecule
• Similarly, guanine and cytosine alignment can only confer hydrogen bonding
between two bases.
Therefore, two DNA strands show:
• Polarity
Each strand shows polarity either in 3′–5′ direction or 5′–3′ direction.
• Complementary base pairing
A〓T, G≡C
• Antipolarity (antiparallel)
In DNA helix, two strands run in antiparallel direction.
8.6 Watson and Crick DNA Model
In 1953, American biologist, James D. Watson, and British physicist, Francis
H.C. Crick, postulated a 3D model of DNA. They restructured X-ray diffraction
data provided by earlier pioneers, notably Franklin and Wilkins, regarding DNA.
CH3 3′ END
O O
H
H
H RIBOSE SUGAR
O 5′ END N 2 3′
N
O P O O
H
O N
7 N
RIBOSE SUGAR O
8.6 Watson and Crick DNA Model
5′ CH2 THYMINE
5′ CH2
O O
9
H 5′ N O P O
HYDROGEN BONDING
3′ H
H ADENINE O
H
5′
H
N
O N H O H O 5′ CH2
O P O PHOSPHATE
MOIETY
O N O
C N HN G
5′ CH2 RIBOSE SUGAR O P O
O O
5 N
H 1
3′ H 5′ END
CYTOSINE O H N GUANINE
O H
3′ END
153
Fig. 8.11 Segment of DNA

154 8 Nucleic Acids
In 1962, Watson and Crick got Nobel Prize together with Wilkins.
Characteristics of DNA Model
• DNA molecule is made up of two polynucleotide strands that are linked with
each other by hydrogen bonds placed at regular intervals.
• DNA strands are antiparallel. One strand runs in 3′–5′ direction, while other
strand runs in 5′–3′ direction.
• Two polynucleotide strands are spirally coiled around an imaginary common
axis and form a double helix.
• Double helix has a diameter of 20 Å (2 nm). Diameter is uniform throughout the
DNA molecule as in Fig. 8.12.
• Grooves in double helix
–– Double helix contains major grooves and minor grooves that arise owing to
antiparallel nature of strands. Major and minor grooves oppose each other in
helix and are alternately placed along the entire length of DNA molecule.
Fig. 8.12 Watson - Crick

Model of DNA
3’
5’
5’
3’
S T A S 34 A°
P P
3.4 A°
S A T S
P P
S G C S
8.6 Watson and Crick DNA Model 155
–– Width of major groove is 22 Å and minor groove is 12 Å.

–– Within grooves, base pairs are easily accessible to other molecules for interac-
tion. DNA-binding proteins have affinity for major grooves, for example,
transcription factors that attach major grooves and modulate transcription.
Histones, polymerases, and nucleases bind at major grooves.
• Spiral of double helix
–– One complete spiral (turn) of double helix has length of 34 Å (3.4 nm), called
pitch. It contains ten base pairs. A distance of 3.4 Å (0.34 nm) is present
between adjacent base pairs.
–– Nitrogenous bases are oriented to the interior of helix, while sugar-phosphate
backbone is directed outwardly. Purines and pyrimidines are oriented perpen-
dicular to sugar-phosphate backbone.
–– Adenine pairs with thymine through two hydrogen bonds, while guanine pairs
with cytosine through three hydrogen bonds. It is called as complementary
base pairing.
–– Double helix is coiled in right-hand direction. DNA turns are directed
clockwise.
DNA double helix is comparable to coiled ladder. Vertical bars of ladder repre-
sent sugar-phosphate components of DNA molecule. Horizontal steps of ladder rep-
resent nitrogenous bases which are linked to each other by hydrogen bonds, while
they remain attached to sugar-phosphate backbone.
Primarily, DNA double helix is stabilized by two forces such as:
• Hydrogen bonds
Forces of attraction between purines in one polynucleotide chain and pyrimi-
dines in other polynucleotide chain. This interchain hydrogen bonding is elusive
in defining the double helix.
• Phosphodiester bonds (covalent bond)
Present between two OH groups of phosphoric acid and OH groups of deoxyri-
bose sugars at 3′ and 5′ positions
• Nucleobase-stacking interactions
These are non-covalent forces of attraction between aromatic bases since they
contain pie bonds. Nitrogenous bases offer strong π-π stacking interactions.
Therefore, base-stacking interactions constitute other stabilizing forces in helix.
These forces are hydrophobic. These interactions are partially inter-strand and
partially intra-strand in DNA double helix.
Chargaff Rule
Erwin Chargaff states that in DNA of all organisms, the amount of adenine is equal
to the amount of thymine and the amount of guanine is equal to the amount of
cytosine.
The amount of purines should be equal to the amount of pyrimidines (1:1 ratio).
(A + G = T + C OR (A + G/T + C = 1)
156 8 Nucleic Acids
8.7 Functions of DNA
1. DNA has a unique property of duplicating itself into an exact copy. It is essential
for transmission of characters from parents to offspring. Hence, it is a genetic
material of cells.
2. DNA can transcribe mRNA on its strand. The mRNA contains codons for protein
synthesis.
3. DNA regulates synthesis of proteins through codons.
4. DNA controls gene expression. It can regulate cellular functions like secretion,
excretion, proliferation, and apoptosis.
5. DNA can undergo mutations which are essential for origin of species.
6. DNA is essential for crossing over and recombination during meiosis. They are
necessary for variation in same species.
7. It controls differentiation of cells in embryonic development.
8.8 Types of DNA
Linear DNA
• DNA molecule is linear in shape. It is made up of two strands. Each strand has
two free ends.
• Linear DNA is associated with nucleoprotein and is organized into
chromosome.
• It is found in eukaryotes.
Circular DNA
• DNA molecule is circular in shape. Ends of circular DNA are joined together. It
is not associated with protein.
• Circular DNA can be single stranded in viruses like chicken anemia virus
(CAV) and porcine circovirus 2 (PCV2). They are pathogenic organisms.
• Circular DNA can be double stranded in mitochondria, chloroplasts, bacteria,
and viruses.
Double-Stranded DNA
• DNA molecule has two strands which are helically coiled. It is found in
eukaryotes.
A-DNA
• It is a right-handed molecule.
• One turn of helix contains 11 base pairs.
8.9 Replication of DNA 157
B-DNA
• One turn of helix contains ten base pairs.
• Length of the helix is 34 Å.
• It is the Watson and Crick DNA.
C-DNA
• One turn of helix has nine base pairs.
D-DNA
• One turn of helix has eight base pairs.
Z-DNA
• This DNA molecule was observed by Robert Wells, and its structure was deci-
phered by Alexander Rich in 1979.
• It is a left-handed molecule.
• One turn of helix has 12 base pairs.
• Length of helix is 45 Å.
• Sugar-phosphate backbone adopts a zigzag pattern giving its name as Z-DNA.
Palindromic DNA
• A DNA molecule that contains palindromic sequence. Reading of sequence in

one strand in 5′ → 3′ direction exactly matches with sequence in other strand in
5′ → 3′ direction.
• 5′ G-G-A-T-G-T-G 3′
• 3’G-T-G-T-A-G-G 5′
• Palindrome sequence is found in prokaryotes and eukaryotes.
• It was described by Wilson and Thomas in 1974.
8.9 Replication of DNA
8.9.1 Definition
Replication of DNA is an autocatalytic multistep biological process of synthesizing

two DNA molecules from a parental DNA molecule.
158 8 Nucleic Acids
8.9.2 Site of Occurrence
In Eukaryotes
• DNA replication occurs in the nucleus during S-phase of interphase (interval

between two cell divisions).
• DNA replication is semiconservative, bidirectional, and
semi-discontinuous.
In Prokaryotes
• DNA replication occurs in cytoplasm of cell.

• DNA replication is semiconservative, bidirectional, and continuous.
8.9.3 Models of Replication of DNA
Three models of DNA replication have been proposed.
Semiconservative Model
Two parental strands of DNA molecule separate. Each strand serves as template for
synthesis of a new strand of DNA. After replication, each DNA molecule has one
parental strand and other new strand. Parental strands are semiconserved within
two daughter DNA molecules as in Fig. 8.13.
Conservative Model
Parent DNA molecule regulates synthesis of two new strands. After replication, new
strands coil helically to form a daughter DNA molecule. Parental strands are con-
served in one daughter DNA molecule as in Fig. 8.14.
Dispersive Model
Parental DNA double helix is broken into short double-helix segments. Each seg-
ment synthesizes short new double-helix segment. After replication, all segments
reassemble into two DNA molecules. Each daughter DNA molecule consists of
partially parental double helix and partially new double helix. Parental strands are
dispersed randomly in two daughter DNA molecules as in Fig. 8.15.
8.9.4 Semiconservative Model of DNA Replication
Historical Facts
• In 1953, Watson and Crick documented a semiconservative model of DNA rep-
lication in a paper. They commented that original strands of DNA served as tem-
plate in DNA replication.
Fig. 8.13 Semi- Parental DNA

conservative Model of
DNA Replication
Semi conservative model
• In 1957, Taylor described the semiconservative model of DNA replication in

eukaryotes.
• In 1958, Meselson and Stahl provided experimental evidence in favor of
semiconservative model of replication.
8.9.5 Mechanism of Semiconservative Model
Prerequisites of DNA Replication
1. Deoxyribonucleoside Monophosphates (dAMP, dGMP, dTMP, and dCMP)

• Deoxyribonucleoside monophosphates constitute raw material for DNA rep-
lication. They are present in nuclear sap.
2. Enzymes
• DNA helicase
Helicase is a motor protein (molecular motor; able to move in a specific direc-
tion along the substrate; can utilize chemical energy released by hydrolysis of
ATP for mechanical movement).
160 8 Nucleic Acids
Fig. 8.14 Conservative Parental DNA

Model of DNA Replication
Conservative model
Helicase is an essential enzyme in living organisms. Helicase traps chemi-

cal energy released from hydrolysis of ATP. Helicase moves along the
sugar- phosphate backbone and disrupts hydrogen bonds in annealed
nucleotide bases (complementary bases held by hydrogen bonds).
Helicase does function to separate helically coiled DNA strands.
• DNA topoisomerase
–– This is an isomerase enzyme. It regulates geometry (topology) of DNA
molecule. It induces break in DNA strand to relive coiling stress.
• DNA ligase
–– It is a ligase enzyme. It brings about formation of phosphodiester bonds
between adjacent nucleotides and catalyzes polymerization.
–– It is helpful in DNA replication and repair. It helps to join two Okazaki
fragments after removal of RNA primer. It seals DNA segments after cor-
rection of mismatched nucleotide bases.
• Primase
–– It is a type of RNA polymerase. It catalyzes formation of RNA primer on
the DNA template.
–– DNA polymerases
These enzymes are essential for synthesis of DNA utilizing deoxyribonu-
cleoside triphosphates. However, DNA polymerase is unable to initiate
Fig. 8.15 Dispersive Parental DNA

Model of DNA Replication
Dispersive model
DNA synthesis. It can insert a free deoxyribonucleotide to 3′ end of a

newly synthesized strand. It helps to elongate a new strand in 5′–3′
direction.
In eukaryotes, DNA polymerase are the following types:
DNA polymerase α
It helps in the synthesis of RNA primer. It helps in the synthesis of lagging
DNA strand.
DNA polymerase β
It helps in DNA repair.
DNA polymerase γ
It has 3′ → 5′ exonuclease activity. It helps in the synthesis of mitochon-
drial DNA.
DNA polymerase δ
It helps in the synthesis of leading DNA strand. It inserts deoxyribonucleo-
tides after removal of RNA primer.
DNA polymerase ε
It helps to insert correct deoxyribonucleotides in growing DNA chain after
excision of mismatched nucleotide bases.
–– In prokaryotes, DNA polymerase are the following types:
162 8 Nucleic Acids
DNA polymerase I
This polymerase removes RNA primer between Okazaki fragments. It helps
to fill gap through its 5′ → 3′ synthesizing activity. It also has 3′ → 5′ exo-
nuclease activity which is involved in proofreading and DNA repair.
DNA polymerase II
This polymerase has activities similar to that of DNA polymerase I.
DNA polymerase III
It is mainly involved in 5′ → 3′ DNA replication. It promotes elongation of
DNA strand. It also has proofreading property.
• Proteins
Single-stranded DNA-binding protein
–– This protein has been found in prokaryotes and eukaryotes.
–– It binds with single strand of DNA. It prevents recoiling of single DNA
strand into duplex. It helps to stabilize uncoiled DNA strand.
Steps in DNA Replication
1. Phosphorylation of Deoxyribonucleoside Monophosphates

• Deoxyribonucleoside monophosphates constitute raw material for DNA rep-
lication. They are found in the nuclear sap. The four types are as follows:
–– dAMP (deoxyadenosine monophosphate)
–– dGMP (deoxyguanosine monophosphate)
–– dTMP (deoxythymidine monophosphate)
–– dCMP (deoxycytidine monophosphate)
• Deoxyribonucleoside monophosphates undergo phosphorylation to form
deoxyribonucleoside triphosphates as dATP, dGTP, dTTP, and dCTP.
• Reaction is catalyzed by phosphorylase enzyme in the presence of ATP
molecules.
2. Origin of Replication
• Origin of replication is a specific sequence in DNA which leads to initiation
of DNA replication. Origin of replication is also called as Ori.
• Eukaryotes have linear double-stranded DNA molecule. It has multiple ori-
gins of replication.
• Prokaryotes have single circular DNA molecule. It has single origin of
replication.
3. Unwinding of Double Helix
DNA molecule is a double-stranded helix. Its unwinding is a complex proce-
dure and is catalyzed by enzymes as follows:
• Helicase (unwindase) enzyme acts upon origin of replication. It catalyzes
splitting of hydrogen bonds between purines and pyrimidines of two DNA
strands. Both the strands are separated at Ori.
• The separated DNA strands are stabilized by single-stranded DNA-
binding proteins.
• Unwinding of DNA strands induces coiling tension on either side of origin of
replication. It results into the formation of supercoils in DNA molecule.
Topoisomerase
Template
Parental DNA
strand 3'
double SSB
strand Gyrase proteins RNA primer
5'
5' Leading strand

3'
5' Okazaki fragments
3' RNA primer
Replication 3'
fork
5'
Replication of DNA
Fig. 8.16 Replication of DNA
• Topoisomerase enzyme induces a nick in one strand of DNA to relieve coil-

ing tension. Later on, it seals the cut portions of DNA strand.
• Prokaryotes have gyrase enzyme. Its function is similar to action soft helicase
and topoisomerase.
• Separation of DNA strands proceeds toward both directions from origin of
replication. Separating strands appear like a Y-shaped structure and is
called as replication fork.
• Separated strands of DNA molecule serve as templates as in Fig. 8.16.
4 . Synthesis of RNA Primer
• RNA primer is a short chain of RNA. It is synthesized at 5′ end of DNA
template. Ribonucleotides are polymerized into RNA primer with the help of
primase enzyme.
• Number of RNA primers: there are two RNA primers, one on each DNA
strand. One RNA primer is formed at free end of DNA (3′ end of DNA strand)
template, and other RNA primer is synthesized at fork end of DNA (5′ end of
DNA strand) template.
• RNA primer helps to initiate synthesis of new strand. Afterward, RNA primer
is disassociated. The gap is filled by addition of deoxyribonucleotides through
action of DNA polymerase β and DNA polymerase I in eukaryotes and pro-
karyotes, respectively.
• DNA polymerase can continue the elongation of already initiated new
DNA strand.
5. Complementary Base Pairing
• Deoxyribonucleoside triphosphates attach to complementary nitrogen bases
on the DNA templates, for example, dATP pairs with thymine, dTTP pairs
with adenine, dGTP pairs with cytosine, and dCTP pairs with guanine nitrog-
enous bases. It is called complementary base pairing.
• Deoxyribonucleoside triphosphates are linked to nitrogen bases by formation
of hydrogen bonds.
6. Synthesis of New Strand of DNA
164 8 Nucleic Acids
• Deoxyribonucleoside triphosphates are held through hydrogen bonds with

complementary nitrogen bases on the DNA template.
• Deoxyribonucleoside triphosphates undergo hydrolysis into deoxyribo-
nucleoside monophosphates with release of pyrophosphates.
• Pyrophosphates undergo hydrolysis by pyrophosphatase enzyme to form
inorganic phosphate. In this reaction, energy is released.
• The energy is utilized in the formation of phosphodiester bonds between adja-
cent deoxyribonucleoside monophosphates on DNA template. This process
is called polymerization of deoxyribonucleoside monophosphates. It leads
to the formation of a new DNA strand on DNA template.
• In prokaryotes, DNA polymerase III and, in eukaryotes, DNA polymerase
δ catalyze synthesis of new DNA strands. Reaction requires Mg++ ions and
ATP molecules.
7 . Formation of Leading and Lagging DNA Strands
• DNA polymerase brings about synthesis of new strand in 5′ → 3′direction on
DNA template.
• Two DNA templates are antiparallel in nature. One template runs in
5′ → 3′direction, while the other template runs in 3′ → 5′direction. Therefore,
two new DNA strands are synthesized in opposite directions.
• Leading strand
–– Leading strand is formed on a DNA template which has polarity in
3′ → 5′direction.
• Leading strand is continuously elongated. Its 3′ end grows by addition of new
nucleotides. It is also called as continuous strand as in Fig. 8.16.
• Lagging strand
–– Lagging strand is formed on a DNA template which has polarity in
5′ → 3′direction.
• Lagging strand is discontinuously formed. It is due to exposure of a small
segment of DNA template over which new nucleotides are polymerized. In
this way, a short segment of new DNA strand is synthesized which is called
as Okazaki fragment as in Fig. 8.16.
–– Okazaki fragment (naming is based on scientist Reiji Okazaki who dis-
covered). Each fragment has nearly 200 base pairs in eukaryotes and 2000
base pairs in prokaryotes. A separate RNA primer is necessary for synthe-
sis of each Okazaki fragment. RNA primers are removed, and gaps are
sealed with deoxyribonucleotides through action of DNA polymerase β
and DNA polymerase I in eukaryotes and prokaryotes, respectively.
–– Individual Okazaki fragments are joined together by action of DNA ligase
enzyme.
• Lagging or discontinuous strand is composed of Okazaki fragments
joined together by DNA ligase.
8. Proofreading (Editing) and DNA Repair
Proofreading is a process of correcting errors in replication of DNA.
• Common error in DNA replication is the incorporation of incorrect nitroge-
nous base in growing DNA chain.
–– Nitrogenous bases in DNA molecule exist in common forms in which protons
occupy specific positions. These are keto forms. Due to a proton shift in a
8.10 Transcription 165
base, it is transformed from a common form into rare form which is called as
tautomerization. It is the most common cause for mismatch in base pairing.
–– The frequency of base-pairing mismatch is 0.001% (1 nucleotide in 10,000
nucleotides).
• In prokaryotes, DNA polymerase III can recognize mismatched nitrogenous
base in DNA chain. It is a 3′ → 5′ exonuclease enzyme. It excises incorrect
nucleotide from the end of chain in a direction opposite to DNA replication. It
inserts correct nucleotide. DNA ligase seals the repaired segment of DNA strand.
• In eukaryotes, DNA polymerase δ (3′ → 5′ exonuclease enzyme) performs
proofreading and repair.
• Proofreading corrects most of the base-pairing mismatch during the process
of DNA replication, while some are corrected after replication.
9 . DNA Helix Formation
• Newly formed DNA strands are coiled spirally to form a DNA duplex.
8.10 Transcription
Definition
Transcription is defined as enzyme-controlled multistep biological process of syn-
thesis of RNA from a template of DNA. Etymologically, transcript means “writing
consisted of same words as original.”
Overview
1 . Sense strand of DNA is transcribed and it is identical to a single strand of RNA.

2. DNA molecule contains genetic information. This information is preserved
through DNA replication. This information is expressed through transcrip-
tion and translation.
3. Transcription involves copying a template strand of DNA into a single-stranded
mRNA molecule. RNA molecule is complementary to template strand of
DNA and identical to non-template strand of DNA.
4. Transcription is a biological activity of rewriting an identical genetic
message.
• Transcription occurs during G1 and G2 phases in interphase of cell cycle.
8.10.2 Prerequisites of Transcription
RNA Polymerase
This enzyme is essential for polymerization of ribonucleotides. It catalyzes synthe-
sis of RNA from a template strand of DNA:
166 8 Nucleic Acids
• In prokaryotes, one type of RNA polymerase is present. It can catalyze transcrip-

tion of RNA.
• In eukaryotes, three types of RNA polymerases are present which are essential
for synthesis of different types of RNA molecules as follows:
–– RNA polymerase I
It catalyzes synthesis of rRNA except 5S ribosomal RNA.
–– RNA polymerase II
It catalyzes synthesis of mRNA and snRNA (small nuclear RNA).
–– RNA polymerase III
It catalyzes synthesis of tRNA, 5S RNA, and snRNA.
Ribonucleoside Monophosphates
• Ribonucleoside monophosphates constitute raw material for synthesis of RNA.
Transcription Unit
Transcription unit is a specific sequence in DNA which is involved in transcrip-
tion. It has following components such as:
• Promoter
Promoter is a particular sequence in DNA that initiates transcription. Promoter is
located upstream on DNA toward 5′ end of sense strand and 3′ end of antisense
strand. Promoter is located nearby transcriptional start site. The size of promoter
can vary between 100 and 200 base pairs. RNA polymerase and transcription
factors bind to promoter region.
• Enhancer
Enhancer is a specific sequence in DNA that activates transcription. It is located
at a distance of about 2000 to 1 million base pairs from gene to be transcribed.
Its position is either downstream or upstream of transcription start site. Enhancer
in eukaryotes binds with activators to increase chances of transcription.
Promoter and enhancer are together described as cis-acting elements.
Transcription factors bind with cis-acting elements.
• Terminator
Terminator is a specific sequence of DNA that terminates transcription and
brings about release of mRNA.
–– In prokaryotes, Rho factor is a protein which stops transcription. It brings
about dissociation of RNA polymerase from DNA and terminates transcription.
–– In eukaryotes, proteins associated with RNA polymerase II signal the termi-
nation of transcription as in Figs. 8.17 and 8.18.
• Structural gene
It is the gene which codes for the synthesis of RNA or proteins. Structural gene
has two strands that serve different functions such as:
–– Sense strand (+)
DNA strand that has 5′ → 3′ polarity is called as sense strand. It is also called
as coding strand or non-template strand.
Double stranded
helix (DNA Molecule)
5' 3'
3' 5'
Fig. 8.17 Double Stranded DNA Molecule and Cistron
5' 3'
3' 5'
[GENE] cistron
Fig. 8.18 Sites on Cistron
Promoter Transcription Termination

site site site
5' 3'
Coding strand
Core (sense strand)
enzyme RNA
(RNAP)
polymerase
Sigma factor 3' 5'

[GENE] Showing
cistron strands Template strand
Non-coding or
anti sense strand
Fig. 8.19 Attachment of RNA Polymerase (RNAP) to Promoter Region of Cistron
Sense strand of DNA has sequence (parallel) identical to sequence in RNA strand.
–– Antisense strand (─)
DNA strand that has 3′ → 5′ polarity is called as antisense strand. It is also
called as noncoding strand or template strand.
Antisense strand of DNA has sequence complementary (antiparallel) to
sequence in RNA. It acts as a template strand for synthesis of RNA strand as
in Fig. 8.19.
• Transcription factor
Transcription factor is a protein that binds with sequence of DNA. It controls
gene expression. Transcription factor binds with promoter. It can bring about
either upregulation or downregulation of transcription.
168 8 Nucleic Acids
8.10.3 Mechanism of Transcription
Steps in Transcription
1. Phosphorylation of Ribonucleoside Monophosphates

• Ribonucleoside monophosphates constitute raw material for synthesis of
mRNA. These are found in the nuclear sap. The four types are as follows:
–– AMP (adenosine monophosphate)
–– GMP (guanosine monophosphate)
–– UMP (uridine monophosphate)
–– CMP (cytidine monophosphate)
• Ribonucleoside monophosphates undergo phosphorylation to form ribonu-
cleoside triphosphates such as ATP, GTP, UTP, and CTP.
• Reaction is catalyzed by phosphorylase enzyme in the presence of ATP
molecules.
2. Initiation
• RNA polymerase binds to promoter on gene. Promoter is a sequence of gene
which starts transcription of a specific gene. They are located around tran-
scription start site of gene. They can be around 100 base pairs.
• Promoters in prokaryotes and eukaryotes are as follows:
• Promoters in prokaryotes
–– Pribnow box
Pribnow box in eukaryotes is called as TATA box. It is a specific sequence
of six nucleotides (TATAAT). It is located at ten nucleotides upstream from
transcription start site of DNA.
–– “35” sequence
It is a consensus sequence of TTGACA nucleotides. It is located at 35
nucleotides upstream of transcription start site of genome.
• Promoters in eukaryotes
–– TATA box
It is also called as Goldberg-Hogness box. It is located at 25 nucleotides
upstream of transcription start site of DNA.
–– CAAT box
It is a specific sequence of nine nucleotide bases (GGCCAATCT). This region
is located at 90 nucleotides upstream from transcription start site of DNA.
• In prokaryotes, single RNA polymerase can synthesize all types of RNAs. In
eukaryotes, specific RNA polymerases synthesize specific RNAs.
• In prokaryotes, RNA polymerase (holoenzyme) is made up of a core
enzyme (2α, 1β, and 1β′ polypeptide subunits) and a sigma factor.
A sigma factor helps in the recognition of promoter region of DNA.
• In eukaryotes, separate RNA polymerases (Pol I-rRNA, Pol II-mRNA, and
Pol III-tRNA) are necessary for transcription. RNA polymerase is associated
with general transcription factors (proteins).
General transcription factors help in recognition and binding with pro-
moter region of DNA.
RNA polymerase II is associated with TFIIA, TFIIB, TFIID, TFIIE, TFIIF,

and TFIIH.
A particular RNA polymerase binds to a specific promoter in DNA.
Activity of promoter can be increased or decreased by enhancers and silenc-
ers (sequence of nucleotides) which are placed either upstream or downstream
from promoter.
• RNA polymerase along with sigma factor or general transcription factor binds
to promoter region on gene and forms a RNA polymerase-promoter closed
complex (DNA is double stranded).
• Helicase enzyme brings about localized unwinding and separation of two
strands of DNA. Now RNA polymerase-promoter open complex is formed.
This complex has unwound DNA strands and is called as transcription
bubble as in Figs. 8.20, 8.21, and 8.22.
• RNA polymerase has binding site to bind with ribonucleoside triphosphate
(NTP). The first NTP is generally A or G nucleoside triphosphate. The first
NTP binds to RNA polymerase at transcription start site and undergoes
hydrogen bonding with complementary base of template strand. First NTP
binds through its 3′ end and its 5′ end is free.
• Selection of second NTP depends on the base pairing with sequence of tran-
scription start site. It forms H-bond with complementary base. RNA
polymerase brings about phosphodiester bond formation that results in an
initial RNA product.
3 . Elongation
• RNA polymerase moves downstream over template strand in 3′ to 5′ direc-
tion. It is coupled with unwinding of DNA duplex downstream toward 5′ end
of the template strand.
• Successive nucleoside triphosphates are added to 3′ end of newly synthesiz-
ing ribonucleotide chain by RNA polymerase. These NTPs form phosphodi-
ester bonds with already existing NTP.
• Sense strand helps to select incoming NTP. Around 35–40 NTPs are added in
a second.
Fig. 8.20 Release of Promoter Attachment of

Sigma factor from RNAP site RNAP Sense strand
5' 3'
RNA P
3' 5'
Sigma Template
strand
170 8 Nucleic Acids
Fig. 8.21 Showing Promoter Transcription Termination

release of sigma factor site site site
5' 3'
RNA P
3' 5'
Movement of RNAP
Sigma factor
released
Fig. 8.22 Showing Sense strand

transciption bubble
5' 3'
5' Nascent RNA 3'
U A A G C
A T T C G
3' 5'
RNAP
Uncoiled
Template DNA strands
strand [transcription bubble]
• The uncoiled DNA rewinds upstream of RNA polymerase toward 3′ end of

template strand as in Fig. 8.23.
4 . Termination
• Specific sequence present on template strand of DNA terminates elongation
of RNA molecule.
• As RNA polymerase approaches a specific sequence, Rho protein binds with
RNA and stops transcription.
• RNA molecule and RNA polymerase are dissociated from template strand.
• Single-stranded RNA transcribed from DNA template is called as primary
transcript as in Fig. 8.23.
8.11 Ribonucleic Acid (RNA) 171
Transcription Codind
bubble strand
Recoiling
DNA
5' 3'
3'
3' 5'
3
3''
3' Nascent RNA
5' 5' 3'
RNA RNAP
Uncoiling
5' DNA
Template
strand
Fig. 8.23 Synthesis of RNA
Important Terms
Gene Expression Gene expression is a biological process through which genetic
information within a gene is utilized in synthesis of gene product.
Gene Product
Gene products are biomolecules produced by gene expression. They are either a
polypeptide or a RNA molecule.
Transcription
Transcription is a biological process by which a template strand of DNA is copied
into single-stranded RNA molecule (primary transcript). It undergoes processing
(posttranscriptional modifications) to become functional RNA.
Single strand of RNA is complementary (antiparallel) to template strand of DNA.
Single strand of RNA is identical (parallel) to non-template strand of DNA.
• Transcription factor
A protein which binds with DNA template and controls gene expression
through promotion or suppression of transcription
• Transcriptional regulation
An act of controlling gene expression
• Transcription upregulation
Promoting the rate of gene expression
• Transcription downregulation
Repression of gene expression
8.11 Ribonucleic Acid (RNA)
8.11.1 Structure
RNA is a single-stranded polymer of ribonucleotides. RNA is an unbranched and

linear molecule.
172 8 Nucleic Acids
RNA is composed of three components such as:
• Phosphate group
• Ribose sugar (5C)
• Nitrogenous bases
RNA molecule contains four types of nitrogenous bases such as adenine, gua-
nine, uracil, and cytosine.
8.11.2 Types of RNA
Depending upon the function, RNA molecules are grouped into three types such as:
• mRNA
• tRNA
• rRNA
essenger RNA (mRNA)

M
The mRNA is a type of ribonucleic acid which contains genetic information
(message) in the form of codons from DNA to ribosomes for protein synthesis.
Occurrence
• The mRNA is transcribed in nucleus. It is translocated from nucleus to cyto-

plasm to control protein synthesis.
Types of mRNA
• It has numerous types depending upon the type of polypeptide chain.
Synthesis of mRNA
• The mRNA is synthesized by RNA polymerase II (in eukaryotes) and RNA poly-
merase in prokaryotes from template strand of DNA.
Structure
• The mRNA constitutes about 5% of the total ribonucleic acid of cell.
• It is a linear molecule. Its length is dependent on the length of polypeptide chain.
The mRNA is the longest among all RNA molecules.
• The mRNA carries genetic message (genetic code) from DNA in the form of a
unique sequence of three nucleotides, called as codon as in Fig. 8.23.
• Structurally, a mRNA is made up of about 500–1500 ribonucleotides. It has
five regions based on different functions such as:
–– Cap region (5′ end)
Its 5′ end is called as cap region. It contains a methylated guanylate nucleo-

tide. Cap region serves as an attachment site to bind with small subunit of
ribosome. It also functions to protect the mRNA from damage by ribonucle-
ase (RNase) as in Fig. 8.24.
Cap region is followed by a small noncoding region, and it is called as
leader segment. This region does not code for protein synthesis. It is rich
in A and U nucleotides.
–– Initiation codon region
This region contains an initiation or start codon. It is AUG and start codon is
universal in living organisms. In eukaryotes, AUG codes for methionine
(MET) as in Fig. 8.24. In prokaryotes, AUG codes for formylmethionine
(fMet).
This region marks the initiation of polypeptide chain synthesis.
–– Coding region
This region contains about 1500 ribonucleotides. This region contains codons
for amino acids. It is necessary for elongation of polypeptide chain.
–– Termination codon region
This region contains termination or stop codons to terminate the elongation of
polypeptide chain. Termination codons are UAG, UGA, and UAA.
A small noncoding region follows the termination codon region and is
called as called as trailer segment. This region is rich in A and U nucleo-
tides as in Fig. 8.24.
–– Tail region (3′ end)
Tail region is the 3′ end of mRNA. This end contains a sequence of
multiple adenosine monophosphate ribonucleotides which is called as
poly-(A) tail as in Fig. 8.24.
It protects mRNA from hydrolytic action of RNases. It also prompts
translocation of mRNA from nucleus to cytoplasm and protein
synthesis.
Function
• The mRNA carries genetic information from DNA regarding the sequence of
amino acids in polypeptide chain.
Leader Trailer
(non-coding region) (non-coding region)
5' Coding region 3'
Initiation Termination Poly A

codon codon cap
region region
Fig. 8.24 mRNA Molecule

174 8 Nucleic Acids
• In eukaryotes, a single gene (cistron) transcribes mRNA which controls synthe-

sis of one polypeptide chain (monocistronic).
• In prokaryotes, multiple genes transcribe a mRNA which controls synthesis of
more than one polypeptide chains (polycistronic).
ransfer RNA (tRNA)

T
It is a type of ribonucleic acid that serves to transfer activated amino acid from
cytoplasm to surface of ribosome for protein synthesis.
Occurrence
• The tRNA is located in nucleus. It is translocated to cytoplasm.
Types of tRNA
• There are about 60 tRNA molecules that exist.
Synthesis of tRNA
• The tRNA is synthesized by action of RNA polymerase III (eukaryotes) and

RNA polymerase (prokaryotes) from template strand of DNA.
Structure
• tRNA molecule constitutes about 15% of the total RNA of cell.
• tRNA is the smallest size among all three RNAs of cell.
• The shape of tRNA resembles the shape of a leaf of clover plant. Its clover leaf
shape was proposed by RW Holley in 1965. It is due to auto folding and base
pairing in tRNA molecule.
• Structurally, tRNA is made up of 70–95 ribonucleotide residues. It is folded
and undergoes base pairing to assume an L-shaped form with five arms as
described below:
• Each arm has stem (paired bases) and a loop (unpaired bases) except acceptor
arm that is without loop and variable arm that is without stem as in Fig. 8.25.
–– Anticodon arm or loop
In anticodon arm, stem contains five paired bases, and loop contains seven
unpaired bases. The three unpaired bases in loop are complementary to bases
in mRNA codons. Anticodon arm reads codons in mRNA and is essential
to recognize amino acids for activation and attachment to acceptor arm.
–– Acceptor arm
Acceptor arm is without a loop. In acceptor arm, 5′ end and 3′ end of RNA
molecule come closer to each owing to its folding. Stem contains seven to
nine nucleotide base pairs. Terminus of 5′ end has either guanine or cytosine
Fig. 8.25 T-RNA Carrier

molecule end
Amino acid OH
binding
site 3'
A
C
5' C
Amino acid
receptor arm
ΤΨU
Loop
Enzyme Stem
site
IV
I
III
D loop
Ribosomal
site
Anticodon Variable arm
arm
II Anticodon
loop
C C G
m-RNA
recognition
site
G G G
m-RNA
Codon
base. The 3′ end contains a sequence of three bases as CCA (cytosine-

cytosine-adenine). It is called as CCA tail. It has a free OH group at the tip as
in Fig. 8.24.
Activated amino acid attaches by its COOH group to OH group of CCA
tail.
–– D arm (loop)
This arm contains four base pairs in stem and seven unpaired bases in loop. D
loop contains a modified base called as dihydrouridine (5,6-dihydrouracil
base), a modified base with two hydrogen atoms in uracil.
This arm contains enzymes which are essential for attaching amino acid
with tRNA.
–– TΨC arm (loop)
This arm is located opposite to D arm. Its stem contains five paired bases. Its
loop contains a modified base called as pseudouridine.
176 8 Nucleic Acids
This arm is helpful in recognition of ribosomes.

–– Variable arm
• This arm is without a stem. Its loop contains five to six unpaired bases as in
Fig. 8.25.
Function
• The tRNA molecule carries anticodons to recognize codons of mRNA. It is
essential for recognizing amino acids.
• It also helps to transfer activated amino acids to surface of ribosomes for protein
synthesis.
ibosomal RNA (rRNA)

R
It is a type of ribonucleic acid that is structural component of ribosomes.
Occurrence
• It is present in small and large subunits of ribosomes in cytoplasm.
Synthesis of rRNA
• The rRNA is synthesized by RNA polymerase I (in eukaryotes) in nucleolus and
RNA polymerase (in prokaryotes) in cytoplasm.
Types of rRNA
• There are seven types of rRNA molecules such as:

–– Eukaryotes have 80S ribosomes.
–– Its 60S large subunit contains 28S rRNA, 5.8S rRNA, and 5S rRNA.
–– Its 40S small subunit contains 18S rRNA.
–– Prokaryotes contain 70S ribosomes.
–– Its 50S large subunit contains 23S rRNA and 5S rRNA.
–– Its 30S small subunit contains 16S rRNA.
Structure
• The rRNA molecule constitutes about 80% of total RNA of cell.
• It has highly variable length and its shape is highly folded.
• Structurally, rRNA is made up of polyribonucleotides containing nitrogenous
bases, ribose sugar, and phosphate residues.
• It has a single-stranded structure. In some regions, molecule is highly coiled to
form helix.
• In a helical region of molecule, bases are present in complementary pairs which
are held by hydrogen bonds.
• In non-helical region of molecule, bases are unpaired.
8.12 Translation (Protein Synthesis) 177
Function
• The rRNA forms subunits of ribosomes. They are helpful in protein synthesis.
8.12 Translation (Protein Synthesis)
Definition
Translation is a biological process in which genetic message contained in
mRNA directs the synthesis of polypeptide chain in ribosomes.
• Protein synthesis occurs on the surface of ribosomes either in cytoplasm or rough

endoplasmic reticulum.
8.12.2 Prerequisite of Translation
• Amino acids
Amino acids are raw material for protein synthesis. Human body makes use of
20 amino acids for biosynthesis of different types of proteins. Essential amino
acids are supplemented in diet, and nonessential amino acids are synthesized in
the body.
• Ribosomes
–– Ribosomes are called as protein factories. They are made up of rRNA and
proteins. Ribosomes exist in the cytoplasm in the form of two subunits such
as large and small subunits.
–– Before the initiation of protein synthesis, both subunits of ribosomes assem-
ble to form a ribosome in the presence of Mg++ ions. Further, multiple ribo-
somes group together on the strand of mRNA, and the structure is called as
polyribosomes or polysomes. Each ribosome is separated by a distance of
340 Å. The number of ribosomes in polysome is dependent on the length of
mRNA strand.
–– Large subunit of ribosome has three sites that perform different func-
tions such as:
A site: It is aminoacyl-tRNA site. This site attaches to tRNA carrying acti-
vated amino acid as in Fig. 8.25.
P site: It is peptidyl-tRNA site. It attaches to tRNA holding elongating poly-
peptide chain.
The tRNA that carries first amino acid methionine or formylmethionine
attaches to the P site of ribosome.
E site: It is the exit site. This site releases tRNA which enters cytoplasm and
binds with another activated amino acid as in Fig. 8.25.
178 8 Nucleic Acids
• RNA
Ribonucleic acids serve multiple important functions during protein synthesis.
–– mRNA carries genetic message.
–– tRNA carries anticodons to recognize and transfer activated amino acids.
–– rRNA is the structural basis of ribosomes.
• DNA
DNA is the master macromolecule that controls protein synthesis.
8.12.3 Mechanism of Translation
Protein synthesis is a complex biological process that is regulated by mRNA

molecule. It can be divided into three stages such as:
I nitiation of Polypeptide Chain

Depending upon the events involved in polypeptide biosynthesis, initiation stage
can further be subdivided into the following stages such as:
Phosphorylation of Amino Acids
• The mRNA codes for amino acids. A coded amino acid undergoes phosphor-
ylation to form amino acid-AMP complex with the release of pyrophos-
phate molecule. This complex is called as activated amino acid as in
Fig. 8.26.
• Reaction is catalyzed by aminoacyl-tRNA synthetase enzyme in the presence of
Mg++ ions. Each amino acid is activated by a separate enzyme.
(P site) (A site)
Peptidyl-tRNA amino acyl-tRNA
binding site binding site
(E site) Large submit of

Exit site for t RNA E P A ribosome
60S
RNA binding site
40S Small sub unit of

ribosome
Fig. 8.26 Sites on ribosome

Formation of tRNA-Amino Acid Complex
• The tRNA carries anticodons to recognize the coded amino acid.

• The COOH group of activated amino acid attaches to OH group of CCA sequence
at acceptor arm of tRNA. This process is called as charging of tRNA. The reac-
tion is catalyzed by aminoacyl-tRNA synthetase.
• Reaction produces tRNA-amino acid complex with release of a molecule of
AMP and aminoacyl-tRNA synthetase as in Fig. 8.26.
Aminoacyl-tRNA synthetase
Amino acid + ATP Amino acid–AMP Complex + Pyrophosphate
Mg++ ions
Aminoacyl-tRNA synthetase
Amino acid-AMP Complex + tRNA tRNA-amino acid Complex
+ AMP + Aminoacyl-tRNA synthetase
Formation of Translation Initiation Complex
• The subunits of ribosomes remain separated in cytoplasm.

• At the time of polypeptide synthesis, small subunit of ribosome binds with
leader segment on 5′ end of mRNA.
–– In prokaryotes, there is base pairing between ribonucleotides of rRNA and
mRNA.
–– In eukaryotes, 5′ cap end of mRNA helps in attachment of small subunit of
ribosome to leader segment.
• The mRNA carries initiation codon (AUG) which codes for methionine in
eukaryotes and formylmethionine in prokaryotes. The tRNA which carries acti-
vated methionine and formylmethionine has anticodons. There is hydrogen
bonding between anticodons of tRNA and codons of mRNA that helps to bind
initiator tRNA to mRNA as in Fig. 8.27.
• Large subunit of ribosome binds with small subunit in the presence of Mg++ ions.
• An assembly of ribosome, mRNA, and tRNA-amino acid complex is called
as translation initiation complex.
• Initiation factors and Mg++ ions are essential for the formation of translation
initiation complex.
• The tRNA-amino acid (methionine or formylmethionine) complex is located
at P site of ribosome. This marks the initiation of polypeptide chain synthe-
sis as in Fig. 8.27.
180 8 Nucleic Acids
Amino acy
Amino acid + ATP l tR
NA
Mg s
ppi + + yn
th
es
is
Amino acid - AMP-Enzyme
Activated amino acid complex
3' 3'
A A
C C
C C
5' 5'
AMP
Amino acyl tRNA

tRNA
Fig. 8.27 Charging of tRNA
longation of Polypeptide Chain

E
It is the successive addition of coded amino acids to the first amino acid on the sur-
face of ribosome to form a polypeptide chain.
Elongation of polypeptide chain can be described in the following steps such as:
• Peptide bond formation

–– The second tRNA-amino acid complex enters ribosome at A site. The tRNA
contains anticodons which help in its pairing with codons on mRNA through
hydrogen bonding.
–– The bond between COOH group of 1st amino acid and OH group in CCA of
1st tRNA is split. The COOH group of 1st amino acid at P site forms a peptide
bond with NH2 group of 2nd amino acid at A site. Reaction is catalyzed by
peptidyl transferase enzyme (ribozyme).
–– The 2nd tRNA located at A site carries a dipeptide (methionine amino acid/
formylmethionine amino acid).
–– The 1st tRNA is located at P site without amino acid as in Fig. 8.28.
• Translocation of tRNA
–– It is the movement of tRNA-amino acid complex from A site to P site on the
surface of ribosome.
mRNA binding site

Initiation
codon
AUG
5' 3' Small sub unit
ribosome
m-RNA
Initiation
factor
Initiation
codon
AUG
5' 3'
Small sub unit

ribosome
[mRNA - small subunit ribosome]
Methionine Amino acid

Fist tRNA
Anticodon on tRNA
Large subunit
P Initiation
UAC codon
E A
mRNA
AUG
5' 3'
Small
subunit
Fig. 8.28 Showing formation of translation initiation complex
–– The tRNA dipeptide moves from A site to P site. GTP is hydrolyzed to pro-
vide energy for the translocation of tRNA.
–– The 1st tRNA (uncharged) moves from P site to E site on ribosome. It is dis-
charged from E site to cytoplasm. It is again charged with another amino acid
as in Figs. 8.28 and 8.29.
182 8 Nucleic Acids
Methionine (MET)
Arginine (ARG)
P
A AG
E A
UAC
5' 3'
A UG
mRNA
Methionine
Arginine
P A
E
UAC A AG
5' 3'
A UG UU C
2nd tRNA
tRNA released
for reuse 3rd tRNA
Lysine
GG C
E P A
ME T A A G
5' 3'
A U G U U C GG C
Release
E P A factor
Polypeptide
released
5' 3' 5' 3'
UA A
Fig. 8.29 Elongation of polypeptide chain
One cycle of elongation of polypeptide chain includes:
• Attachment of successive tRNA-amino acid complex at A site on ribosome

• Formation of peptide bond between already existing amino acid residue at P site
and a successive amino acid residue at A site
• Translocation of uncharged tRNA from P site to E site
• Translocation of tRNA-growing peptide complex from A site to P site
This cycle takes about 0.1 s to complete. With every successive cycle, peptide
chain elongates by one amino acid residue.
)
ET
(M
ne G)
thi oni e (AR ine
Me inin th ion G)
Arg Me AR
in e(
Argin
AA
G
P
P
E A
UAC E
UAC AAG
5′ 3′
5′ 3′
m RNA Aug
Aug UUC
t RNA released for

reuse ] 3rd t RNA
NA
tR
2nd
ine
Lys
GG
C
E P A
MET-ARC
5′ 3′
AUG UUC GGC
Fig. 8.30 Diagram showing release of Nascent Polypeptide chain
As the ribosome moves in 5′ → 3′ direction on mRNA, it leads to expression of

all codons in the coding region of mRNA. Therefore, all coded amino acids are
bonded by peptide bonds to form a polypeptide chain.
Elongating polypeptide chain is kept attached to a particular ribosome.
ermination of Polypeptide Chain

T
• Ribosome reaches termination codon region at 3′ end of mRNA. Termination
codon stops addition of successive amino acid to elongating chain.
• A release factor attaches at A site of ribosome. It hydrolyzes a bond between
polypeptide chain and tRNA at P site of ribosome.
• Polypeptide chain is released from mRNA as in Fig. 8.30.
• Ribosome detaches from termination codon of mRNA. It dissociates into two
subunits as in Fig. 8.30.
184 8 Nucleic Acids
A newly synthesized polypeptide chain is called nascent polypeptide chain.

It has a primary protein structure as in Fig. 8.30.
8.12.4 Post-translational Modification of Polypeptide Chain
It is an elaborate biochemical process of altering nascent polypeptide chain after the

synthesis of polypeptide chain is complete.
Types of Post-translational Modifications
1. Proteolytic Splitting
• Nascent proteins have larger size (precursor) in comparison to active form of
protein. For example, parathormone and insulin are synthesized in precursor
forms. The proteins are proteolyzed in the Golgi body and release active pro-
teins. This process is also called as trimming.
2. Covalent Modification
• Covalent modification is the alteration in chemical structure of nascent pro-
tein through addition or removal of functional groups. It occurs by the fol-
lowing reactions such as:
–– Hydroxylation
It is the addition of hydroxyl groups in the nascent protein. In synthesis of
collagen, proline and lysine amino acids are hydroxylated into hydroxypro-
line and hydroxylysine. These reactions require ascorbic acid as coenzyme.
–– Glycosylation
It is the addition of glycosyl group to proteins containing serine, threonine,
and asparagine amino acid residues.
–– Iodination
It is the addition of iodine atom to tyrosine ring during synthesis of thyroxine.
–– Phosphorylation
It is the addition of phosphate group to proteins containing serine, threo-
nine, and tyrosine residues.
–– Carboxylation
It is the addition of CO2 molecule to glutamic acid residue in clotting factors.
Suggested Readings
London
Berg JM, Tymoczko JL, Stryer L, Clarke ND (2002) Biochemistry. W. H. Freeman, New York
Calladine CR, Drew HR, Luisi BF, Travers AA (2003) Understanding DNA: the molecule & how
it works. Elsevier Academic, Amsterdam
Davidson JN (1965) Biochemistry of nucleic acids, 5th edn. Wiley, New York
Korenberg A (1980) DNA replication. W. H. Freeman, New York
Enzymes
9
9.1 Definition
Enzymes are defined as highly specific biomolecules capable of catalyzing bio-

chemical reactions.
Commonly, enzymes are polypeptide in nature except a few RNA molecules
(Ribozymes) which possess enzymatic property.
Enzymes are synthesized by the cells of the body. They have catalytic potential
and increase the rate of a biochemical reaction.
9.2 Characteristics of Enzymes
• Enzymes are proteinaceous in nature.

• They are heat labile.
• They form colloidal solution.
• They have high specificity.
• They can accelerate a biochemical reaction.
• They can be denatured by heat, UV rays, X-rays, and alcohol.
• Enzymes can be simple proteins.
• Examples: lactase, sucrose, maltase, and malate isomerase
• Enzymes can be conjugated proteins. Such enzymes are called as
holoenzymes.
–– Holoenzyme has a protein part and nonprotein part as in Fig. 9.1.
–– Protein part of enzyme is called apoenzyme and nonprotein part is called as
prosthetic group.
Examples:
Aminotransferase + Pyridoxal phosphate
Glyceraldehyde-3-phospho dehydrogenase + NAD+
Glucokinase + Mg++

https://doi.org/10.1007/978-981-13-1035-5_9
186 9 Enzymes
Carbonic anhydrase + Zinc++
Alcohol dehydrogenase + Zinc++
• Enzymes can be monomeric and oligomeric in structure.
Examples:
Monomeric enzyme: lactase and glucokinase
Oligomeric enzymes: Alcohol dehydrogenase (dimer)
Lactate dehydrogenase (tetrameric)
• Multienzyme Complexes
These are stable clusters of more than one enzymes which are non-covalently
linked with each other. The complex catalyzes a biochemical reaction in a cas-
cade manner as in Fig. 9.2.
Pyruvate dehydrogenase complex catalyzes the conversion of pyruvic acid into
acetyl CoA-SH.
• Multienzyme: It is the enzyme that performs multiple catalytic functions. It is
due to presence of different domains on the enzyme. These domains have sepa-
rate catalytic function.
For example, bacterial DNA polymerase-1 has following properties:5′–3′

Deoxyribonucleotides polymerization activity3′–5′ Exonuclease activity
Fig. 9.1 Holoenzyme HOLOENZYME
APOENZYME + PROSTHETIC GROUP
(protein part) (non-protein part)
CO-ENZYME OR CO-FACTOR
Pyruvate dehydrogenase complex

=
Pyruvatedehydrogenase+Dihydrolipoyldehydrogenase+Dihydrolipoyl transacetylase
Thiamine pyrophosphate+Lipoic acid+CoA-SH+FAD+NAD+Mg++
Fig. 9.2 Pyruvate dehydrogenase complex

9.3 Nomenclature and Classification of Enzymes 187
9.3 Nomenclature and Classification of Enzymes
Enzymes act on the substances and transform them into products. The substances
which are transformed are called as substrates. The enzymes are named by addition
of suffix “ase” to the name of a substrate. For example, sucrose is converted into
glucose and fructose by enzyme sucrose. Enzymes like trypsin, chymotrypsin, and
pepsin are the exception to this rule of nomenclature of enzymes. Some enzymes
exist in inactive forms and are called as zymogen, for example, fibrinogen and pep-
sinogen. This method of nomenclature creates ambiguity.
The International Union of Biochemistry and Molecular Biology (IUBMB)
in 1964 adopted a system of enzyme classification. This system was based on the
type of chemical reaction which an enzyme can catalyze. It provided an Enzyme
Commission number (EC number) for every enzyme. According to IUBMB system
of enzyme classification,
• Each enzyme is described by EC number. It is followed by four digits sepa-

rated by points.
• First digit represents the enzyme class.
• Second digit represents subclass.
• Third digit represents sub-subclass.
• Fourth digit represents serial number of enzyme.
Example: Aminotripeptidase has nomenclature as EC3.4.11.4
• EC 3 represents hydrolase enzyme.

• EC 3.4 represents hydrolase enzyme that acts on peptide bond.
• EC 3.4.11 represents hydrolase enzyme that cleavages amino acid from
amino-terminal.
• EC 3.4.11.4 represents hydrolase enzyme that act on tripeptides.
According to suggestions of Nomenclature Committee by IUBMB, enzymes

can be categorized into six main classes, as described below:
• Class 1. Oxidoreductases
These enzymes catalyze oxidation and reduction reactions.
AH + B → A + BH (reduction)
A + O → AO (oxidation)
Examples: Lactate dehydrogenase, alcohol dehydrogenase, and glutathione
reductase
• Class 2. Transferases
These enzymes catalyze transfer of a group from one substrate to another.
AD + C → A + DC
Examples: Hexokinase, alanine transaminase, and aspartate transaminase
• Class 3. Hydrolases
These enzymes catalyze hydrolysis of peptides and glycosidic or ester bonds
by addition of water molecule.
188 9 Enzymes
AB + H2O → AOH + BH
Examples: Trypsin, pepsin, and esterase
• Class 4. Lyases
These enzymes catalyze cleavage of a large substrate without addition of
water.
Examples: Aldolase and fumarase
• Class 5. Isomerases
These enzymes catalyze isomerization of substrate.
Examples: Phosphohexoisomerase and retinal isomerase
• Class 6. Ligases
These enzymes catalyze the addition of two substrates.
Examples: DNA ligase and glutamine synthetase
• Six classes of enzymes can be remembered by OTH-LIL.
9.4 Enzyme Specificity
Definition
Enzyme specificity is the ability of enzyme to act upon a substrate and catalyze
a particular biochemical reaction.
It is the property of an enzyme. Enzyme specificity determines the ability of
enzyme to act upon one or more substrates. Higher the specificity, fewer the sub-
strates on which enzyme can act. It is dependent on three-dimensional structure of
active sites on the surface of enzyme.
Types of Enzyme Specificity
1. Absolute Specificity
Absolute specificity is a property of an enzyme when it acts upon only one par-
ticular substrate. It is a rare phenomenon. Enzyme with absolute specificity can
catalyze only a specific biochemical reaction.
Examples
• Urease catalyzes cleavage of urea.
• Lactase acts on lactose.
2. Group Specificity
Group specificity is a property of an enzyme when it acts on molecules contain-
ing specific functional groups.
Examples:
Trypsin acts on polypeptides which contain arginine and lysine.
Pepsin hydrolyzes peptide bonds among hydrophobic amino acids like, phe-
nylalanine, tryptophan, and tyrosine.
Hexokinase acts on 6-carbon containing monosaccharides.
Chymotrypsin hydrolyzes peptide bonds among aromatic amino acids.
3. Bond Specificity
Bond specificity is a property of an enzyme when it acts on specific type of
chemical bonds.
9.5 Mechanism of Enzyme Action 189
Examples:
Protease catalyzes hydrolysis of peptide bonds.
Lipase cleavages ester bonds.
Glucosidase acts on glycosidic bonds.
Group specificity and bond specificity are collectively called as relative
specificity.
Absolute specificity and relative specificity together constitute substrate
specificity.
4. Stereochemical Specificity
Stereochemical specificity is a property of an enzyme when it acts on specific stereo-
isomer. A substrate can be either laevorotatory or dextrorotatory, depending on its
optical property. Stereochemically active enzymes exhibit selectivity for optically
active substrate. Stereochemical specificity is also called as optical specificity.
Example:
d-amino acids and l-amino acids undergo oxidative deamination by d-
amino acid oxidase and l-amino acid oxidase enzymes.
5 . Reaction Specificity
Reaction specificity is a property of an enzyme when it catalyzes only a specific
biochemical reaction of a substrate. Any substrate can undergo various types of
biochemical reactions. Each reaction of a substrate is catalyzed by a particular
enzyme.
Examples:
Oxaloacetate undergoes condensation reaction with acetyl-CoA in the pres-
ence of citrate synthase enzyme.
Oxaloacetate is converted into aspartate by transaminase enzyme.
Oxaloacetate is decarboxylated and phosphorylated into phosphoenolpyru-
vate by PEP-carboxykinase enzyme.
9.5 Mechanism of Enzyme Action
Enzymes are organic molecules synthesized by living cells. They accelerate bio-
chemical reactions. Generally, the rate of enzyme-catalyzed reactions is around
103–1016 times faster than nonenzymatic reactions. Enzymes are highly specific and
can differentiate between optical isomers.
Several theories have been proposed to explain the mechanism of enzyme
action which are described below:
9.5.1 Theory of Activation Energy
Characteristics of Theory
• A biochemical reaction is accompanied by a continuous change in the energy

system.
190 9 Enzymes
• In ground state:
–– All molecules of a substrate must possess specific amount of kinetic energy.
–– Substrate molecules must undergo collision with each other.
–– Collisions must be in proper orientation. A molecule with its reactive side
must collide with the reactive side of another molecule. Otherwise, collisions
will be unproductive.
• Ground state is a stable state and has the lowest energy. Free energy of sub-
strate molecules is higher than product molecules in ground state. So the reaction
moves in a forward direction.
• Transition state:
–– It is a state of maximum energy along a reaction coordinate. It is a measure of
reaction progress in a reaction pathway.
–– Old bonds in substrate molecules are broken; new bonds are formed.
–– It is an unstable state.
• The difference in energy between ground state to transition state is called as
activation energy. It is the minimum amount of energy needed to transform all
molecules of a substrate from ground state to transition state as in Fig. 9.3.
• The rate of reaction is dependent on the magnitude of activation energy. The
higher the magnitude of activation energy, the lower the rate of a reaction.
• Enzyme binds with substrate and forms an enzyme-substrate complex.
Enzyme helps to orient the colliding molecules. So enzyme lowers the activa-
tion energy. Now more substrate molecules reach the transition state in a given
time period and are transformed into product. This accelerates the rate of
reaction.
LOWERING OF
ACTIVATION ACTIVATION
ENERGY ENERGY
WITH ENZYME
ACTIVATION
ENERGY
REACTION
WITHOUT ENZYME ENERGY
PRODUCTS
TIME
REACTANTS
REACTION
WITH ENZYME
Fig. 9.3 Diagram showing Activation Energy of Chemical Reaction

• Enzyme does not change the energy level of substrates and products. It does not
alter the equilibrium constant of a reaction as in Fig. 9.3.
• Examples:
–– Hydrolysis of sucrose by acid requires 26,000 cal/mol, whereas sucrase-
induced hydrolysis requires 10,000 cal/mol.
–– Nonenzymatic decomposition of hydrogen peroxide requires 18,000 cal/
mol, whereas catalase-induced decomposition requires 2000 cal/mol.
9.5.2 Enzyme-Substrate Complex Theory
In 1903, Victor Henri suggested that enzyme (E) binds with substrate (S) to form
enzyme-substrate (ES) complex.
Later on, in 1913, Leonor Michaelis and Maud L. Menten promulgated
Michaelis-Menten Enzyme Kinetics Theory of enzyme action.
Postulates of Michaelis-Menten Enzyme Kinetics Theory
• Enzyme (E) binds with substrate (S) to form an enzyme-substrate complex

(ES complex).
• This complex is formed through non-covalent forces like van der Waal’s forces,
ionic and hydrophobic interactions between enzyme and substrate.
• The ES complex is unstable and dissociates immediately to form product (P) and
enzyme as in Fig. 9.4.
9.5.3 Lock and Key Model of Enzyme Action
It was propounded by Emil Fischer in 1894. This model is also named as

“Fischer’s template theory.”
Products
Substrate
Active S
sites
E
Enzyme Enzyme Enzyme

substrate
complex
Fig. 9.4 Diagram showing Enzyme-Substrate Complex

192 9 Enzymes
Postulates
• In this model, lock is analogous to enzyme, while key is analogous to

substrate.
• This model describes that enzyme has predetermined shape and enzyme exhibits
rigidly in its shape.
• It provides a pre-shaped and rigid template to substrate molecule.
• As a key with proper shape fits into the lock, similarly, a substrate with compli-
mentary conformation can fit into the enzyme and forms enzyme-substrate com-
plex as in Fig. 9.5.
Limitations of Lock and Key Model
1. This model could not explain competitive and noncompetitive inhibition of

enzyme.
2. This model failed to explain the allosteric modulation of enzyme activity.
9.5.4 Induced-Fit Model
Introduction
Despite limitations, Fischer’s model was accepted for over 50 years by the scientist
community. The model explained scientific observations at that time.
In 1930, Haldane suggested that catalysis takes place in a small region of
enzyme. He called the region as “active site.”
In 1959, Daniel E. Koshland proposed induced-fit model for enzyme
action.
Active Compatible Active

site substrate site Substrate
Enzyme E S E S Enzyme E A No E- S Complex
Analog [Incompatible
substrate]
E - S Complex
Fig. 9.5 Diagram showing Lock and Key Model

Postulates of Induced-Fit Model
• This model is analogous to “hand in glove,” where hand represents substrate

and glove represents enzyme. As the hand approaches a folded glove, it opens up
and confirms proper hand-in-glove fit.
• This model assumes that enzymes are flexible and active sites are not
pre-shaped.
• The substrate molecule induces conformational changes in the active site. It
helps to orient catalytic amino acid residues in proper position. It optimizes
enzyme-substrate binding. It is necessary for substrate catalysis as in Fig. 9.6.
Active Site of Enzyme
• Enzyme is a macromolecule. Active site is a small region of enzyme where sub-

strate molecule binds.
• Active site is located in a cleft of enzyme. During folding of polypeptide chain,
internal cavities and surface clefts are formed.
• Active site has a three-dimensional structure possessing substrate-binding site
and catalytic site.
• A substrate binding site has large number of hydrophobic groups of amino acid
residues. These groups help in recognition of substrate. Substrate-binding site
attaches with substrate molecule through non-covalent forces.
• Catalytic site may have sulfhydryl group of cysteine and phenolic group of tyro-
sine. These residues bring about breakage of old bonds and formation of new
bonds in substrate molecule. These residues help to lower the activation energy.
• A substrate induces proper alignment of amino acid residues, while, a substrate
analog induces improper alignment of residues.
Enzyme E S E S
Active Substrate Enzyme - Substrate

site Complex
Fig. 9.6 Diagram showing Induced-Fit Model

194 9 Enzymes
9.6 Factors Regulating Enzyme Action
Enzyme activity is regulated by several factors. Therefore, rate of an enzymatic

reaction is affected by following factors.
9.6.1 Effect of Enzyme Concentration
• Rate of enzymatic reaction is directly proportional to enzyme concentration, pro-

vided the substrate concentration is infinite.
• Enzyme is the limiting factor in enzymatic reactions.
• Availability of active sites is increased by increasing the enzyme concentration.
More numbers of substrate molecules occupy active sites and are converted into
product.
• Graphic representation between rate of reaction and enzyme concentration
is “straight line” as in Fig. 9.7.
9.6.2 Effect of Substrate Concentration
• In the initial phase, rate of reaction is directly proportional to substrate concen-

tration. This point is called as ½ Vmax.
• At half maximal velocity (½ Vmax), about half of the enzymes are occupied by
substrate. Further increase in substrate concentration leads to full saturation of
enzymes. The rate of reaction attains maximal velocity (Vmax).
• Later on, reaction rate decreases and attains the steady state. Afterward, reaction
rate does not increase on increasing the substrate concentration.
• Graphic representation between rate of reaction and substrate concentra-
tion provides a “hyperbolic curve” as in Fig. 9.8.
Fig. 9.7 Diagram showing

effect of enzyme
concentration on rate of
reaction
Rate of reaction
Concentration of Enzyme
9.6 Factors Regulating Enzyme Action 195

effect of substrate
concentration on rate of
reaction
Vmax
Rate of reaction
V
Km
Concentration of Substrate
9.6.3 Effect of Product Concentration
• Substrates are converted into products in reversible reaction. As the concentra-

tion of products increase, the rate of reaction is slowed down. Further increase in
products concentration can lead to an equilibrium stage.
• Additional increase in product concentration can reverse the reaction rate.
9.6.4 Effect of Temperature
• Enzymes are thermolabile. The enzymatic activity is affected by temperature.

• A rise in temperature results into an increase in rate of reaction. Temperature
increases the kinetic energy of substrate molecules. They can easily reach transi-
tion state.
• A rise in 10 °C temperature doubles the rate of enzymatic reaction. This is called
as temperature coefficient (Q10).
• At a particular temperature, rate of enzymatic reaction is maximum and is called
as “optimum temperature.” In the human body, optimum temperature for enzy-
matic activity is 37 °C.
• Further increase in temperature results into decrease in rate of reaction. It is due
to denaturation of enzyme molecules at high temperature.
• Graphic representation between rate of reaction and temperature provides
a “bell-shaped graph” as in Fig. 9.9.
• Human body cannot withstand temperature changes over a wide range. In fever,
significant metabolic changes are observed.
9.6.5 Effect of pH
• The pH of medium affects the rate of enzymatic reaction.

196 9 Enzymes

effect of temperature on
rate of reaction
100% Rate of Reaction

Optimum Temp
0°C
0°C 37°C 75°C
Temperature (0C)
• A rise in pH of medium results into increase in rate of reaction, and it becomes

maximum at “optimum pH.” Enzymes in the human body exhibit maximum
activity at pH between (4 and 9). For example, optimum pH of pepsin, salivary
amylase, and trypsin are 1.6, 6.8, and 8, respectively.
• After optimum pH, rate of reaction decreases.
• The pH affects the orientation of catalytic groups of active site and substrate.
Very low pH inactivates hydrogen bonds in enzyme.
• Graphic representation between rate of reaction and pH of medium is a
“bell-shaped” graph as in Fig. 9.10.
9.6.6 Effect of Coenzymes and Cofactors
• Coenzymes are organic molecules and cofactors are metallic ions. Most of the
human body enzymes require cofactors and coenzymes for their activity.
• Coenzymes like FAD and NAD and cofactors like calcium, magnesium, and
manganese are necessary for enzyme-substrate formation.
• Rate of enzymatic activity increases in the presence of cofactors and
coenzymes.
9.6.7 Effect of Inhibitors
• The inhibitors decrease the rate of reaction. These substances attach on active
sites, and binding of substrate molecules is decreased.
• Heavy metals like mercury, gold, and cadmium decrease rate of reaction.
• Mercury inactivate the free –SH group of enzymes. It inhibits enzymatic
activity.
9.7 Enzyme Inhibition 197
Fig. 9.10 Diagram
showing effect of pH of 100%
medium on rate of reaction
100% Rate of Reaction

Optimum pH
0%
0 7.4 14
pH of Medium
9.7 Enzyme Inhibition
9.7.1 Definition
Enzymes undergo inactivation and denaturation by several chemical substances.

The substances which hinder enzymatic activity are called as “inhibitors,” and the
phenomenon is called as “enzyme inhibition.”
Depending on the nature of inhibitors and the mechanism involved, enzyme inhi-
bition can be three types.
9.7.2 Competitive Enzyme Inhibition Features
• The chemical structure of inhibitor resembles closely with structure of sub-

strate. Inhibitor is a structural analog of substrate molecule.
• Inhibitor has high affinity for active sites on which substrate binds. Inhibitor
competes with substrate for binding to active sites on enzyme.
• Inhibitor combines with enzyme and forms enzyme-inhibitor complex (EI com-
plex). This condition results into decrease in number of substrate molecules
which occupy active sites.
• The degree of competitive inhibition depends on the relative concentration of
inhibitor and substrate as in Fig. 9.11.
• This type of inhibition is also called as “reversible inhibition.” An increase in
substrate concentration helps to terminate competitive inhibition.
• In competitive inhibition, Michaelis constant (Km) is increased. The maximal
velocity (Vmax) remains unchanged.
198 9 Enzymes
I S Substrate S
Competitive Inhibited
inhibitor I
E-I Complex
E E
Enzyme Enzyme - inhibitor

complex
Fig. 9.11 Competitive Enzyme Inhibition
• Examples:
–– Succinic acid dehydrogenase oxidizes succinic acid into fumaric acid.
Substances like malonic acid and glutamic acid resemble structurally to
succinic acid and inhibit enzyme activity.
–– Bacteria utilize para-aminobenzoic acid (PABA) to synthesize folic acid.
Sulfonamide drug resembles structurally to PABA and binds to enzyme,
dihydrofolate synthetase. Thus drug inhibits the enzymatic activity.
–– Therapeutics makes use of competitive inhibition to treat methanol pois-
ing. Accidental ingestion of methanol has serious health effects. It is
metabolized by alcohol dehydrogenase enzyme. Ethanol is infused in
patient. It competes with methanol for the same enzyme and inhibits
metabolism of methanol in the human body.
9.7.3 Noncompetitive Enzyme Inhibition Features
• Noncompetitive inhibitor does not structurally resemble substrate.

• Noncompetitive inhibitor binds to sites other than active sites on the enzyme.
These sites are called as allosteric sites.
• Inhibitor binds with enzyme covalently and deforms the three-dimensional struc-
ture of enzyme. The enzyme is inactivated and cannot perform catalysis.
• Noncompetitive inhibition cannot be terminated by increasing substrate concen-
tration. So it is an “irreversible inhibition.”
• In noncompetitive inhibition, Michaelis constant (Km) remains the same. The
maximal velocity (Vmax) is decreased as in Fig. 9.12. Examples:
–– Fluoride ion inhibits activity of enolase enzyme. It interrupts glycolysis.
–– Cyanide is an inhibitor to enzyme cytochrome oxidase.
–– Mercury and iodoacetate inactivate: SH free groups in enzymes and
inhibit the activity of sulfhydryl group containing enzymes like cysteine.
9.7.4 Uncompetitive Enzyme Inhibition Features
• Inhibitor does not structurally resemble to substrate.

9.7 Enzyme Inhibition 199
Active
site Substrate E-S Complex
S S
Enzyme E E
Product
formed
Active Change in
site active site
S S
Enzyme E E
No product
I I formed
Domain
Non-competitor
inhibitor
Fig. 9.12 Noncompetitive Enzyme Inhibition
• Inhibitor does not bind with free enzyme. But it has high affinity for enzyme-
substrate complex and binds with ES complex.
• It is an irreversible inhibition. Inhibitor prevents product formation.
• In uncompetitive inhibition, Vmax and Km are decreased.
Example:
–– l-phenylalanine can inhibit human placental alkaline phosphatase
enzyme activity.
–– Lithium can prevent activity of enzyme inositol monophosphatase in
brain by uncompetitive inhibition.
9.7.5 Suicide Inhibition Features
• It is also called as “mechanism-based inhibition.” It is an irreversible

inhibition.
• Inhibitor is a substrate analog.
• It binds to an active site of enzyme. It is modified into a highly active inhibitor
by the enzyme. Thereafter, the modified inhibitor binds with active site cova-
lently and irreversibly, and there is formation of inhibitor-enzyme complex.
200 9 Enzymes
Example:
–– Drug allopurinol exhibits suicide inhibition of xanthine oxidase enzyme.
It is used in treatment of gout.
–– 5-Flurouracil shows suicide inhibition of thymidylate synthase enzyme.
–– Zidovudine shows suicide inhibition of HIV-1 reverse transcriptase
enzyme.
–– Acetylsalicylic acid is a suicide inhibitor of cyclooxygenase enzyme.
Allosteric Enzyme Modulation Features
• Allosteric enzyme is also called as regulatory or key enzyme. This enzyme

regulates a metabolic pathway in the body. Allosteric enzymes exhibit difference
in structure and activity from simple nonregulatory enzymes.
• Allosteric enzymes have an active site and an allosteric site. These are
located on different domains of the same enzyme.
• A substance which binds to allosteric site of an enzyme is called as modulator.
Depending on the structure of modulator, an allosteric enzyme can be homo-
tropic and heterotrophic as in Fig. 9.13.
• In homotropic enzyme, structure of substrate and modulator is identical, while
in heterotrophic enzyme, structure of modulator is different from structure of
substrate.
• Allosteric enzymes exhibit a sigmoidal graph between rate of reaction and sub-
strate concentration, while it is hyperbolic in simple nonregulatory enzymes.
Fig. 9.13 Allosteric Enzyme Modulation

Catalytic site
E
Allosteric site
Enzyme
E-S complex
E
A Allosteric activator
Absence of
E-S Complex
S
E
I Allosteric
inhibitor
9.8 Isoenzymes or Isozymes 201
• Modulator which enhances rate of reaction is called as stimulatory modulator as

in Fig. 9.13.
• Modulator which inhibits the rate of enzymatic reaction is called as inhibitory
modulator, and process is called as allosteric inhibition.
• In allosteric inhibition, modulator does not resemble structurally to substrate.
• Allosteric inhibitor binds to allosteric site of enzyme. It modifies the three-
dimensional shape of active site. It results into failure of substrate to bind to
active site. Allosteric inhibition is irreversible in nature as in Fig. 9.13.
• In allosteric inhibition, Vmax is decreased and Km is enhanced.
Example:
–– Phosphofructokinase is inhibited by ATP.
–– Glutamate dehydrogenase is inhibited by ATP.
–– Hexokinase is inhibited by ATP.
–– Citrate synthase is inhibited by ATP.
–– Pyruvate carboxylase is inhibited by ADP.
–– Carbamoyl phosphate synthetase II is inhibited by UTP.
9.8 Isoenzymes or Isozymes
9.8.1 Definition
As recommended by a committee appointed by the International Union of

Biochemistry in 1964,
Isoenzymes are the multiple forms of an enzyme existing in a single species which may
differ variously but catalyze a single biochemical reaction.
Example:
• Lactate dehydrogenase (LDH) has five isoenzymes.

• Creatine phosphokinase (CPK) has three isoenzymes.
9.8.2 Occurrence
• Isoenzymes are widely distributed among unicellular organisms, insects, plants,

amphibians, birds, and mammals.
• In humans, isoenzymes are present in serum and tissues.
9.8.3 Structure of LDH Isoenzymes
• LDH isoenzyme is made up of four polypeptide chains and it is tetramer. Each

polypeptide chain is described as either “H” or “M.”
• An isoenzyme of LDH contains definite number of H and M subunits. Depending
on different proportions, LDH shows five combinations.
202 9 Enzymes
Isoenzymes of LDH
Lactate dehydrogenase (LDH) enzyme exhibits 5 isoenzymes as LDH-1, LDH-2,
LDH-3, LDH-4, and LDH-5. These isoenzymes are described as follows:
LDH-1 has four “H” subunits and designated as (H4). It is predominantly found
in myocardial tissues.
LDH-2 has three “H” and one “M” subunits and designated as (H3 M1). It is
found in reticuloendothelial tissues.
LDH-3 has two “H” and two “M” subunits and designated as (H2 M2). It is found
in lung tissues.
LDH-4 has one “H” and three “M” subunits and designated as (H1 M3). It is
found in renal, pancreatic, and placental tissues.
LDH-5 has four “M” subunits and designated as (M4). It is predominantly found
in the liver and skeletal tissues.
Normal Value
• Normal serum concentration of LDH varies from 60 to 250 IU/L.

• Its concentration rises in acute myocardial infarction, damage to skeletal tissues,
acute hepatitis, pernicious anemia, and carcinoma.
9.8.4 Function of LDH
• All five isoenzymes have the same function.

• LDH brings about conversion of lactate to pyruvate. It is necessary for produc-
tion of energy through Cori’s cycle.
[E] + [S] [ES] [E] + [P]
(Enzyme) (Substrate) (Enzyme-substrate (Enzyme) (Product)

Complex)
• LDH is the key enzyme in anaerobic respiration. It also catalyzes conversion of

pyruvate into lactate in absence of oxygen.
All five Isoenzymes have same function
LDH brings about conversion of lactate to pyruvate. It is necessary for production of

energy through Cori’s cycle.
Lactic acid Pyruvic acid
NAD+ NADH + H+
LDH is the key enzyme in anaerobic respiration. It also catalyzes conversion of pyruvate
into lactate in absence of oxygen.
Pyruvic acid Lactic acid
NADH + H+ NAD+
9.9 Ribozymes 203
9.8.5 Clinical Importance
• Isoenzymes of lactate dehydrogenase have diagnostic value.

• In normal health, serum of an individual contains predominantly LDH-2
isoenzyme.
• In acute myocardial infarction (AMI), cardiac muscles are damaged. They
release LDH-1 isoenzyme in blood circulation and level of LDH-1 rises in serum.
It is helpful in diagnosis of AMI.
• In acute hepatitis, LDH-5 level predominates in serum.
• In muscle fatigue, concentration of lactate rises in skeletal tissues. It results in
acidosis. As a consequence, level of LDH-5 rises in serum.
• In malignancy, serum concentration of LDH increases.
9.9 Ribozymes
Definition
Ribozymes are ribonucleic acid molecules which can catalyze biochemical reactions.
Historical Aspect
• In 1967, Woese C., Crick F., and Orgel L. suggested the catalytic property of
RNA.
• In 1989, Cech T.R. and Altman S. discovered ribozyme and shared a Nobel Prize.
• In 1982, Kelly Kruger et al. coined the term Ribozyme.
Characteristics and Function of Ribozymes
• Ribozymes have highly diverse structure. Ribozymes are categorized into two
groups based on their size.
• Large ribozymes are composed of RNase P, group I, and group II introns. Large
ribozyme has a size varying from a few hundred nucleotides to 3000 nucleotides.
• Small ribozymes have size varying from 35 to 150 nucleotides.
• Ribozymes carry out RNA splicing. It is the editing of nascent mRNA transcript
into mature mRNA. This process cleavages introns and ligate exons in mRNA
transcript. Introns are the noncoding regions in mRNA transcript. They do not
code proteins. Exons are the coding regions of RNA.
• Helps in splicing of unwanted ribonucleotides from primary RNA transcript.
• Ribozymes require divalent magnesium ion (Mg++) for catalytic action.
Natural Ribozymes
RNase P, Group I self-splicing introns, Group II self-splicing introns (Spliceosome),
Hairpin ribozyme, and Hammerhead ribozyme
204 9 Enzymes
9.10 Lysozyme
Definition
Lysozyme is an antimicrobial, proteinaceous substance which provides non-
specific immunity against pathogens. It is also called as “muramidase” or “N-
acetylmuramide glycanhydrolase.”
Occurrence
• Lysozyme is distributed widely in plants and animals.

• In humans, lysozyme is abundantly found in secretions like tear, saliva, breast
milk, and mucus. Cytoplasmic granules of neutrophils and macrophages secrete
lysozyme.
• Egg white is rich in lysozymes.
Structure
• Lysozyme is composed of a single polypeptide chain of 129 amino acid

residues.
• Its molecular weight is 15,000.
• Under normal physiological condition, polypeptide chain of lysozyme is folded
to form a globular structure. It can unfold and refold rapidly. The polypeptide
chain possesses six sub-sites named as A, B, C, D, E, and F which bind to
substrate.
• Catalytic residues are located in between sub-sites D and E.
Function
• Lysozyme is an integral part of innate immune system.

• Lysozyme is a hydrolase. It cleavages 1,4 beta glycosidic bond between N-
acetylmuramic acid N-acetyl-d-glucosamine residues in peptidoglycans in bac-
terial cell wall. It kills bacteria.
• Lysozyme in human milk provides innate immunity to infants.
• Lysozymes in tears are antibacterial.
• Serum lysozyme level is a biomarker for diagnosis and prognosis of “sarcoid-
osis” disease.
Suggested Readings
Fersht A (1984) Enzyme structure and mechanism. W. H. Freeman, New York
Lipscomb WN (1983) Structure and catalysis of enzymes. Annu Rev Biochem 52:17–34
Plowman K (1971) Enzymes kinetics. McGraw-Hill, New York

Tanner NK (1999) Ribozymes: the characteristics and properties of catalytic RNAs. FEMS
Microbiol Rev 23(3):257–275
Walsh C (1979) Enzymatic reaction mechanism. W. H. Freeman, New York
White A, Handler P, Smith EL (1964) Principles of biochemistry, 3rd edn. The Blakiston Division,
McGraw-Hill, New York
Hormones
10
10.1 Hormones
The human body has a neurohumoral system. It plays a dominant role in communi-
cation and coordination among tissues. This system is comprised of a network of
neurons and hormones.
Hormones bind to hormone receptors located on target cells. They initiate a
chemical message which in turn is followed by a cascade of molecular events.
Hormones can influence physiological functions of organs, behavior of organism,
and metabolism of the human body.
10.1.1 Definition
Hormone is defined as an organic molecule that is synthesized in minute quan-

tity by specified tissues and transported by circulation to distant target tissues
to regulate their biochemical and metabolic functions.
Hormones act as the first chemical messenger and signaling molecules.
The word hormone is derived from the Greek word Hormacin which means
urge or excite.
Hormones are secretions of endocrine glands. These glands release secretion
directly into blood circulation. These glands are devoid of ducts.
10.2 Comparison and Contrast
Hormones
• They are synthesized by endocrine glands.

• They belong to a diverse chemical nature.

https://doi.org/10.1007/978-981-13-1035-5_10
208 10 Hormones
• They are produced in small quantity.

• They are translocated from site of synthesis to site of action.
• They are exhausted in metabolic reaction.
• They act as the first chemical messengers to regulate biological, metabolic, and
physiological functions of body.
• Hyper- or hyposecretion of hormones is manifested as hormonal disorder.
Enzymes
• They are synthesized by exocrine glands.

• They are protein in nature.
• They are also produced in small quantity.
• They are not translocated from site of synthesis.
• They remain unchanged in enzymatic reaction.
• They act as biocatalyst. Enzymes increase the rate of reaction.
• Deficiency of enzyme also manifests as a disorder.
Vitamins
• Vitamins are nutritionally important chemical substances (micronutrients). They

are supplemented with diet.
• Vitamins have diverse chemical nature.
• Vitamins are required in small quantity.
• They are distributed by circulation.
• Vitamins act as coenzyme in metabolic and enzymatic reactions.
• Deficiency of vitamins also manifests as disorder.
10.3 Classification of Hormones
10.3.1 Depending Upon Chemical Structure
Hormones can be classified into three groups as follows:
• Peptide/Protein Hormones
These hormones are peptide in nature. These hormones are hydrophilic. They are
transported in circulation in unbound state. They have short duration of action.
Peptide hormones are unable to pass across plasma membrane of target cells.
Structurally, they are either polypeptides or short peptides.
Examples: Insulin, glucagon, calcitonin, pituitary hormones, and
parathormone
10.3 Classification of Hormones 209
• Steroid Hormones
These hormones are lipid in nature. They are lipophilic. They are transported in
circulation in bound state with protein carriers. They have longer duration of
action. These hormones can pass through plasma membrane of target cells.
They are derived from cholesterol. Structurally, these hormones contain cyclo-
pentano-perhydro-phenanthrene nucleus, also called as sterane nucleus.
Examples: Adrenocorticosteroids, androgens, progesterone, and estrogens
• Amino Acid Derivatives
These hormones are derived from tyrosine amino acids. They bind with protein
carriers in blood circulation. They have longer duration of action.
Examples: T3 and T4 hormones (thyroid hormones), adrenaline, and nor-
adrenaline (catecholamines)
Peptide Hormones
Peptide hormones are made up of small chains of amino acids. They are pro-
tein in nature.
Peptide hormones along with secretory glands are enlisted as follows:
Peptide hormones along with secretory glands are enlisted as follows:
Antidiuretic Hormone
Oxytocin Hypothalamus - Pituitary Gland
Parathormone Parathyroid Gland
Calcitonin Thyroid Hormone
Insulin
Glucagon Pancreas (Islets of Langerhans)

Somatostatin
Human Chorionic Gonadotropin Placenta
Steroid Hormones
Steroid hormones are derived from cholesterol and have cyclopentano-per-
hydro-phenanthrene nucleus (sterane). These hormones are lipid in nature.
Steroidal hormones along with their secretory glands are enlisted as follows:
210 10 Hormones
Steroidal hormones along with their secretory glands are enlisted as follows:
Aldosterone
Cortisol Adrenal Cortex (Corticosteroids)
Corticosterone
11-Deoxycorticosterone
Testosterone
Dihydrotestosterone
Dehydroepiandrosterone Gonads (Sex Steroids)
Androstenedione
Estrogen
Progesterone
Amino Acid Derivative Hormones

The following hormones are derived from tyrosine amino acid.
Amine hormones along with their secretory glands are enlisted as follows:
Adrenaline
Nor-adrenaline Adrenal Medulla
Dopamine
The above hormones are called as catecholamine hormones.
Thyroxine (T4)
Tri-iodothyronine (T3) Thyroid Gland
10.3.2 Depending Upon Nature of Site of Action
• Trophic Hormones
These hormones act on endocrine glands. They control growth of target endo-
crine gland.
Examples: Anterior pituitary hormones (TSH controls proliferation of thy-
roid gland and gastrin controls proliferation of enterochromaffin cells in
gastric mucosa)
10.4 Mechanism of Action of Hormones 211
• Non-trophic Hormones
These hormones act directly on target cells and influence cell functions.
Examples: Insulin, glucagon, and thyroxine
10.4 Mechanism of Action of Hormones
Hormones are ligands (endogenous or exogenous molecules that bind to specific

biomacromolecules). Hormones act through receptors (biomacromolecules).
Depending upon structure and location, there are different receptors. Therefore,
hormonal action is mediated through receptor-specific mechanisms which are
described as follows:
1. Intracellular receptor-based mechanism of hormonal action:

• Type I nuclear receptor-based mechanism of hormonal action
• Type II nuclear receptor-based mechanism of hormonal action:
2. Cell-surface receptor-based mechanism of hormonal action
• cAMP as 2nd messenger-based mechanism of hormonal action
• cGMP as 2nd messenger-based mechanism of hormonal action
• Inositol 1,4,5-triphosphate as 2nd messenger-based mechanism of hor-
monal action
• Diacylglycerol as 2nd messenger-based mechanism of hormonal action
• Calcium as 2nd messenger-based mechanism of hormonal action
• Tyrosine kinase receptor-based mechanism of hormonal action
10.4.1 I ntracellular Receptor-Based Mechanism of Hormonal

Action
Definition
A receptor which is situated inside the cell is called as intracellular receptor.
Nuclear receptor is a type of intracellular receptor. It can bind with DNA and
control gene expression. Therefore, nuclear receptors belong to the family of
transcription factors. In human genome, 48 genes for nuclear receptors have been
identified.
Structure of Nuclear Receptor

Nuclear receptor is a transcription factor, and it has a molecular weight of nearly
70,000 daltons. It has the following domains discussed below.
N-terminal Domain
• This domain exhibits extreme variability in size and sequence of amino acid resi-
dues among different receptors. It modulates the process of gene transcription.
212 10 Hormones
DNA-Binding Domain
• DNA-binding domain has strictly conserved sequence. It contains two zinc fin-
gers (small supersecondary protein structures having coordination with zinc
ion). Zinc finger helps in binding with specific sequence of DNA, which is called
as hormone response element (HRE).
Hinge Region
• This domain is flexible and links DNA-binding domain with ligand-binding

domain (LBD).
Ligand-Binding Domain
• This domain has variable sequence. This domain attaches to ligand. It provides
surface for ligand-induced dimerization of receptor.
• It attaches with coactivator protein (protein which stimulates transcription of
RNA) and corepressor protein (protein which represses gene expression).
C-terminal Domain
• This domain has variable amino acid sequence in different receptors as Fig. 10.1.
Fig. 10.1 Structural
Organization of (Nuclear
receptor)
N – terminal
domain
DNA – Binding (DBD)

domain
Hinge region
Ligand binding (LBD)

domain
C – Terminal
domain
Based on the type of nuclear receptor, the two types of mechanisms of hormonal
action are as follows:
1. Type I nuclear receptor-based mechanism of hormonal action

2. Type II nuclear receptor-based mechanism of hormonal action
ype I Nuclear Receptor-Based Mechanism

T
It is described in the following steps.
Association of Receptor with Heat Shock Proteins (HSP)

In the absence of hormone (ligand), type I nuclear receptor is located in cytosol.
It is linked with heat shock proteins (proteins synthesized cell in stressful condi-
tions). Examples: Glucocorticoids and mineralocorticoids hormones
Dissociation of HSP
Lipid-soluble hormones (steroid hormones) can rapidly pass through plasma mem-
brane by simple diffusion. Within cytoplasm, hormone can either move in free
state or in bound state with carrier protein. It is transported intracellularly to recep-
tor to form hormone-receptor complex. Receptor undergoes conformational
changes, and it releases heat shock proteins.
Hormone-Receptor Dimerization
Hormone-receptor complex is a monomer. It binds with a similar monomer to form
a dimer (homodimerization).
Translocation of Receptor Dimer

Dimer is translocated to nucleus. Within the nucleus, the receptor binds directly to
hormone response element of DNA. Hormone response element is a short and spe-
cific sequence of DNA. It is located in promoter region of gene. It binds with hor-
mone-receptor complex.
Promoter region is a short sequence of about 100–1000 nucleotide base pairs
in length, located on sense strand of DNA towards 5′ side. It activates
transcription.
Receptor-DNA complex recruits coactivator proteins. They transcribe down-
stream DNA to mRNA which regulates synthesis of proteins as in Fig. 10.2.
Proteins control cellular functions (like proliferation, differentiation, maturation,
metabolism, survival, and apoptosis).
Examples of hormones: Androgens, estrogens, progesterone, and
glucocorticoids
ype II Nuclear Receptor-Based Mechanism

T
Type II nuclear receptor is located in the nucleus. In absence of ligand, it is associ-
ated with corepressor proteins.
214 10 Hormones
Hormone (steroid hormones)
(ligand)
Cell membrane
HSP
Heat
Cytosol
shock
nuclear receptor - hormone
protein
complex
(HSP)
Receptor
Type I dimerization
nuclear
receptor HRA
DBD RNA polymerase
DBD
Coactivator
Nuclear membrane
LBD
DNA
mRNA
mRNA
Protein
synthesis
Cell Function
Fig. 10.2 Mechanism of action of Type I nuclear receptor
Lipid-soluble hormone like thyroid hormone can easily pass through membrane
by facilitated diffusion. Within cytoplasm, it is translocated into nucleus. Hormone
binds with ligand-binding domain (LBD) of receptor to form hormone-receptor
complex (H-R complex). Receptor undergoes conformational changes and releases
corepressor proteins.
Hormone-receptor complex undergoes heterodimerization with retinoid X recep-
tor. The heterodimer attaches to HRE of DNA. Receptor recruits coactivator pro-
teins and RNA polymerase as in Fig. 10.3.
Heterodimer activates promoter of gene and transcribe mRNA. It controls pro-
tein synthesis which in turn regulates cell functions.
Examples: Thyroid hormone receptor, retinoid X receptor, and retinoic acid
receptor
Hormone
(Thyroid hormone)
Cell membrane
Cytoplasm
Nuclear pore
DBD
Hormone responsive
LBD
Coactivator
element [HRE]
Receptor
Type II N.R.
dimerization
DNA Nucleus
Corepressor Ribosome
Corepressor mRNA
detached mRNA
Protein
synthesis
mRNA
RNA polymerase
Cell Function
Fig. 10.3 Hormonal mechanism of action based on type II nuclear receptor
10.4.2 C
ell-Surface Receptor-Based Mechanism of Hormonal
Action
yclic AMP as 2nd Messenger-Based Mechanism

C
Cyclic AMP (cAMP)
• It is described as 3′,5′-cyclic adenosine monophosphate or 3′,5′-cyclic ade-

nylic acid. It is composed of adenine, ribose sugar, and phosphate group.
• Cyclic AMP differs from AMP in attachment of phosphate group. In cAMP,
phosphate group is attached at 3′and 5′carbon positions in cyclic manner in
ribose sugar, while in AMP, phosphate group is attached either attached at 5′C or
3′C in ribose sugar.
216 10 Hormones
• cAMP is a hydrophilic molecule and acts as 2nd messenger in cytosol.

• It is synthesized from ATP by action of adenylate cyclase enzyme and Mg++ ions.
It is hydrolyzed by 3′,5′ nucleotide phosphodiesterase enzyme.
Adenylate cyclase
ATP cyclic AMP + Pyrophosphate
Mg++
Phosphodiesterase
Cyclic AMP + H2O 5AMP
Mechanism
Activation of G-Protein-Coupled Receptor
• G-protein-coupled receptors:
–– They belong to the largest family of plasma membrane receptors (cell-surface
receptor or transmembrane receptor). They are integral proteins of plasma
membrane (proteins that span across the entire membrane).
–– The receptor has extracellular domain (N-terminal), transmembrane
domain (made up of seven transmembrane alpha-helices) and intracel-
lular domain (C-terminal).The seven transmembrane alpha-helices are
linked to three extracellular loops and three intracellular loops.
–– G-protein-coupled receptors are involved in the following three signal
transduction pathways as:
cAMP-dependent pathway
IP3-dependent pathway
DAG-dependent pathway
• Hormone binds with either N-terminal tail or extracellular loop or seven trans-
membrane alpha-helices (ligand-binding domain). Hormone induces conforma-
tional change in receptor molecule. Receptor enters in activated state.
Activation of G-Protein
• G-protein
–– G-protein is called as guanine nucleotide-binding protein. It acts as intracel-
lular molecular switch. It is “inactive” when bound to GDP and becomes
“active” when attached to GTP molecule.
–– It relays signal from external stimuli to interior of cell.
–– G-protein is a heterotrimeric protein. It is composed of α-, β-, and γ-subunits.
The β- and γ-subunits are tightly folded to form a stable dimer (Gβγ).
–– The Gα-subunit is associated with GDP in “inactive state.” The G-protein-
coupled receptor is bound to Gα-subunit of G-protein.
Hormone
(1st messenger)
Cell membrane G-Protein
Cytosol coupled
receptor
G-Protein (GS)
Adenyl cyclase
ATP GDP
GTP
2nd messenger cAMP Activated
G-Protein
Phosphorylates
protein
Cell function
Protein kinase Activated

(Inactive) Protein kinase
Fig. 10.4 CAM as second messenger based hormonal action
• Activated G-protein-coupled receptor binds with GTPase enzyme. This enzyme

in turn catalyzes dissociation of GDP from Gα-subunit of G-protein and attach-
ment of GTP molecule with Gα-subunit. This exchange of GDP with GTP on
Gα-subunit is brought about by activated G-protein-coupled receptor.
Therefore, it is called as guanine nucleotide exchange factor.
• G-protein enters in active state. It results into dissociation of Gα-subunit from
G-protein and G-protein-coupled receptor. Gα-GTP-subunit is released to affect
the functioning of target proteins as in Fig. 10.4.
Synthesis of cAMP
• Gα-GTP activates adenylate cyclase enzyme. It is located in the inner side of

the plasma membrane.
• Adenylate cyclase catalyzes conversion of ATP molecule into 3′,5′-adenosine
monophosphate (cAMP) molecule. Enzyme requires Mg++ for its activity.
• The cAMP acts as second messenger.
Role of cAMP
• Protein kinase A is an intracellular protein. It is a tetramer. In inactive state,

it is made up of two regulatory subunits represented as 2R associated with two
catalytic subunits represented as 2C.
• Cyclic AMP molecule attaches to protein kinase A and brings about separa-
tion of 2R subunits from 2C subunits. The 2C catalytic subunits are released and
influence phosphorylation of proteins in cytosol.
218 10 Hormones
• Catalytic subunit of protein kinase A induces phosphorylation of other proteins

in cytosol. It catalyzes transfer of phosphate group from ATP to serine and threo-
nine residues in protein molecules.
• Overall, protein kinase A-induced phosphorylation of proteins controls cell func-
tioning as shown in Fig. 10.4.
Example:
In the liver and skeletal tissues, glucagon induces release of cAMP and activa-
tion of PK-A. Protein kinase A converts inactive phosphorylase kinase into active
form. It in turn causes phosphorylation of phosphorylase B (inactive) into phos-
phorylase A (active). This phosphoprotein regulates breakdown of glycogen in
the liver and skeletal muscles.
yclic GMP as 2nd Messenger-Based Mechanism of Hormonal Action

C
The cGMP acts as second messenger in response to hormonal action. Synthesis of
cGMP follows analogous pathway as synthesis of cAMP after binding of hormone
to cell-surface receptor. It is briefly described as follows:
• Hormone (first messenger) binds to surface receptor located on external surface

of membrane.
• Hormone-receptor complex activates guanylyl cyclase enzyme located in the
inner surface of the membrane.
• Activated guanylyl cyclase enzyme acts on GTP as a substrate. GTP molecule is
bound to membrane.
• GTP is converted into cGMP which acts as second messenger. Protein kinase G
is an enzyme which is dependent on cGMP for its activity. Increase in cytosolic
concentration of cGMP activates protein kinase G.
• Activated protein kinase G in turn phosphorylates cellular proteins which control
cell functions.
Example: Phototransduction in rods is regulated by cGMP.
I nositol 1,4,5-Triphosphate (IP3) as 2nd Messenger-Based Mechanism

of Hormonal Action
• Inositol 1,4,5-triphosphate (IP3) is an organic compound. It is composed of
inositol ring and three phosphate residues which are attached to carbon atoms at
1,4,5 positions in the ring. It is a hydrophilic and polar molecule.
• Phosphatidylinositol (PI) is a class of phospholipids. It is located on the inner
portion of phospholipid bilayer in the plasma membrane. It has inositol as an
alcohol.
PI is phosphorylated by kinase enzyme in presence of ATP to form phosphati-
dylinositol-4-phosphate (PIP). It further undergoes phosphorylation by kinase
in the presence of ATP to form phosphatidylinositol-4,5-bisphosphate (PIP2).
PIP and PIP2 are the plasma membrane-bound phosphoinositides.
Mechanism
Activation of G-Protein-Coupled Receptor
• Hormone (first messenger) attaches to G-protein-coupled receptor. It is located

on the plasma membrane.
• Hormone induces conformational changes in receptor. It in turn activates plasma
membrane-bound Gq protein (a class of G-protein).
Activation of Phospholipase C
• Phospholipase C is a plasma membrane-bound enzyme. It exists in six isomeric

forms. It can cleavage phosphatidylinositol-4,5-bisphosphate.
• The α-subunit of Gq protein has the ability to activate phospholipase C enzyme
(PLC-β isozyme).
Cleavage of Phosphatidylinositol-4,5-Bisphosphate
• Phospholipase C (PLC-β) catalyzes hydrolysis of phosphodiester bond in

phosphatidylinositol-4,5-bisphosphate.
• There is formation of inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG)
molecules.
• DAG is a hydrophobic molecule. It remains associated with plasma membrane.
Release of IP3 (2nd Messenger)
• Inositol-1,4,5-triphosphate is a polar and hydrophilic molecule. It is released into

cytosol.
• It diffuses freely through the cytoplasm of cell. It moves toward smooth endo-
plasmic reticulum (SER) in a cell or sarcoplasmic reticulum in muscle fiber as in
Fig. 10.5.
Increase in Cytosolic Calcium Concentration
• Membrane of SER contains IP3receptors. These receptors are ligand-gated

calcium channels. Binding of IP3 molecule on its receptor results into the open-
ing of calcium channels. Thus Ca++ ions are released from SER into cytosol.
• Concentration of calcium in cytosol is increased from 0.1 μ moles to 1.0 μ
moles. Under normal condition, calcium concentration of cytosol is kept low
(0.1 μ moles) due to active export of calcium ions from cell by calcium pump.
• IP3 can additionally act on calcium channels in plasma membrane and induce
further increase in calcium concentration in the cell.
220 10 Hormones
Hormones
Phosphatidylinositol
-4-phosphate [PIP] PIP2
Hormone
Phosphatidyl
G-protein
-inositol [PI]
coupled receptor
Cell membrane
DAG G-protein
Ca++
DAG
Cytosol Hydrolysis
Activation I Protein kinase C
phospholipase C
Ca++Ca++
Calcium channel
IP3 Ca++Ca++Ca++ IP3 gated
Inositol
Ca++Ca++Ca++
1,4,5- Triphosphate
Ca++
IP3 Smooth
endoplasmic DAG
reticulum Activated
protein kinase C
Protein
phosphorylation
Regulation of
cell function
Fig. 10.5 DAG & IPT as second messengers based hormonal action
Activation of Protein Kinase
• Protein kinase is present in cytosol and involved in phosphorylation of proteins.

It transfers high-energy phosphate group from ATP to either tyrosine residue
(tyrosine kinase) or serine/threonine residues (protein kinase C).
• Protein kinase contains C1 and C2 domains at N-terminal. These domains can
bind with diacylglycerol and Ca++ ions, respectively. Protein kinase is kept inac-
tive due to binding of pseudosubstrate region (short sequence of amino acid resi-
dues) to catalytic domain.
• Cytosolic calcium concentration regulates activity of protein kinase in cyto-
plasm. Due to ↑ in Ca++ concentration, it binds at C2 domain of protein kinase and
dislodges pseudosubstrate region.
• Protein kinase is switched on as shown in Fig. 10.5.
Regulation of Cell Functions
• Activated protein kinase C catalyzes phosphorylation of cell proteins. The nature

of substrate protein is highly variable. It depends on the type of cell.
Phosphorylated proteins regulate cell functions.
Examples:
1. Protein kinase C activation in smooth muscle fibers in GIT causes con-
traction of muscle fibers.
2. Protein kinase C activation in neurons causes nerve cell excitation.
3. It induces secretion of saliva in salivary glands.
iacylglycerol as 2nd Messenger-Based Mechanism of Hormonal

D
Action
• Diacylglycerol (DAG) is an organic compound. It is composed of two fatty acid
residues attached to glycerol through ester bonding. DAG exists as 1,2-diacylg-
lycerol and 1,3-diacylglycerol. It is a lipophilic and nonpolar molecule.
• It is produced from hydrolytic cleavage of PIP2 by PLC-β. It is a hydrophobic
molecule and remains bound to plasma membrane.
• DAG attaches to C1 domain at N-terminal of protein kinase C. It activates enzyme
which in turn regulates cell functions.
• DAG acts synergistically with calcium ions on different domains on PK-C.
alcium as 2nd Messenger-Based Mechanism of Hormonal Action

C
Calcium Ions
• Calcium ions mediate through allosteric modulation of cell proteins. Calcium

ions act as a ubiquitous 2nd messenger. It controls a wide variety of bio-
chemical reactions and physiological functions.
• Low cytosolic calcium concentration.
–– Calcium ion is an important cation of extracellular fluid compartment. Its
concentration in ECF is 132 meq/L, while concentration of calcium ions in
cytosol is very low (0.1 μM). It is due to calcium pump in cytoplasm which
actively exports calcium ions from cytoplasm to ECF. Additionally, calcium
ions are transported into the smooth endoplasmic reticulum leading to deple-
tion of calcium in cytosol.
• Increase in cytosolic calcium concentration.
–– Phospholipase C catalyzes splitting of PIP2 into diacylglycerol and IP3 mole-
cules. DAG molecule remains in membrane, and IP3 molecule is transported
to cytosol. It binds with IP3-gated calcium channels in SER. Thus, calcium
ions are released into cytosol.
–– Indirectly, calcium channels in the membrane are opened, and calcium ions
are transported inside the cell.
–– These activities result into ↑ in calcium ions in cytosol to a level 0.5–
1.0 μM. Rise in calcium concentration triggers signals for various physi-
ological functions.
222 10 Hormones
Targets of Calcium Ions

Protein Kinase C is the target of intracellular calcium ions.
• Calcium ions bind at C2 domain of protein kinase C. It leads to activation of

PK-C.
• Activated PK-C in turn phosphorylates other proteins and regulates cell
functions.
Example: Calcium ions in matrix of mitochondria regulate activity of isoci-
trate dehydrogenase enzyme. It is a key regulatory enzyme in TCA cycle.
Calmodulin
• Calmodulin is calcium-modulated protein.
• It is a calcium ion-binding protein. It is made up of 148 amino acid residues.
Calmodulin has four calcium-binding regions. Each one is 12 amino acid resi-
dues long.
• Binding of calcium ions activate calmodulin. It undergoes conformational
changes which affect affinity of calmodulin to proteins.
• Activated calmodulin modulates protein kinases and phosphatases.
Example: Calmodulin regulates excitation-contraction coupling in smooth
muscle fibers. Calmodulin activates calcium-bound myosin light-chain
kinase enzyme. This enzyme in turn phosphorylates head of myosin light
chain. It causes excitation-contraction coupling and contraction of smooth
muscles.
Calcium ions as second messenger regulate secretion, contraction, transmis-
sion of nerve impulse at synapse, fertilization, regulation of nuclear pore, blood
clotting, and transcription.
First Messenger
• It is an extracellular molecule, for example, peptide hormone.

• It is unable to pass through the plasma membrane. It acts as a ligand and
binds with cell-surface receptor. It activates receptor (induces conformation
change in structure).
• Activated receptor brings about release of 2nd messenger by cell.
Second Messenger
• It is an intracellular biomolecule for signaling.

• It receives a signal (chemical message) from cell-surface receptor and relays
it to target molecule in cytosol or nucleus.
yrosine Kinase Receptor-Based Mechanism of Hormonal Action

T
Tyrosine kinase receptor is a cell-surface or transmembrane receptor. It is a
glycoprotein.
10.5 Hormones from Hypothalamus 223
Structure
• Most of the tyrosine kinase receptors are made up of single polypeptide chain
(monomer). However, insulin receptor is comprised of two alpha-chains which
are extracellular in position. These chains are interlinked by disulfide bridges.
Two alpha-chains are again attached to two transmembrane beta-chains by disul-
fide bridges.
• Tyrosine kinase receptor has three domains as:
–– Extracellular N-terminal region which contains many domains including
ligand-binding domain
–– Transmembrane helix
–– Cytoplasmic C-terminal region which has tyrosine kinase catalytic domain
Mechanism
• Hormone binds with ligand-binding domain of tyrosine kinase receptor on cell

membrane.
• Hormone-receptor complex induces receptor dimerization on membrane.
• Receptor dimer induces conformation change in the tyrosine kinase domain and
activates it.
• Activated tyrosine kinase catalyzes phosphorylation of tyrosine residues of the
other monomer in a dimer. It is called trans-autophosphorylation.
• Phosphorylated tyrosine kinase controls cell functions.
• For example, tyrosine kinase acts as a receptor for insulin, growth factors
like epidermal growth factor (EGF), platelet-derived growth factor (PDGF),
fibroblast growth factor (FGF), and nerve growth factor (NGF).
10.5 Hormones from Hypothalamus
Hypothalamus synthesizes factors that regulate release of hormones from pitu-

itary gland either through stimulation or inhibition of hormonal release.
These factors are peptides in nature and are called as hypothalamic
hormones.
Depending on action, they are designated as releasing hormones or inhibiting
hormones (statins).
Types of Hypothalamic Hormones

Six types of hypothalamic hormones are as follows:
• Thyrotropin-releasing hormone (TRH)

TRH is a tripeptide composed of pyroglutamate (derivative of glutamate), pro-
line, and histidine. It acts on the anterior pituitary and stimulates release of thy-
roid-stimulating hormone (TSH).
224 10 Hormones
• Corticotropin-releasing hormone (CRH)

CRH is a peptide of 41 amino acid residues. CRH acts on anterior pituitary and
stimulates release of adrenocorticotropic hormone (ACTH).
• Gonadotropin-releasing hormone (GRH)
GRH is a peptide of ten amino acid residues. It acts on the anterior pituitary and
stimulates release of gonadotropins (LH, FSH).
• Growth hormone-releasing hormone (GRH)
GRH is a peptide composed of 44 amino acid residues. It acts on anterior pitu-
itary and stimulates release of growth hormone. GRH is also named as
somatotropin.
• Growth hormone release-inhibiting hormone (GRIH)
GRIH is a peptide of 14 amino acid residues. It acts on anterior pituitary and
inhibits release of growth hormone. GRIH is also named as somatostatin.
• Prolactin release-inhibiting hormone (PRIH)
PRIH is dopamine. It acts on anterior pituitary and inhibits release of prolactin.
The hypothalamus controls the release of hormones and may be regarded as
master hormonal regulator.
10.6 Hormones from Pituitary Gland
Anterior Pituitary Hormones

The anterior pituitary is called as adenohypophysis. It produces tropins or tropic
hormones. These hormones act on endocrine glands and regulate their functions.
10.6.1 Prolactin
Prolactin is also called as mammotropin or lactogenic hormone or luteotropic

hormone.
Structure
• Prolactin is a polypeptide hormone.

• It is made up of 199 amino acid residues.
• Prolactin is homologous to growth hormone in sequence of amino acid
residues.
Secretion
• Prolactin is secreted by lactotrophs, acidophilic cells of the anterior pituitary

gland. Prolactin release-inhibiting hormone inhibits secretion of prolactin.
• Secretion of prolactin is stimulated by estrogen hormone. It is secreted in preg-
nancy and lactation.
10.6 Hormones from Pituitary Gland 225
Normal Serum Value
• In males, 2–10 ng/ml
• In pregnant females, 9–200 ng/ml
• In nonpregnant females, 2–20 ng/ml
Metabolic Functions
Mammotropic Effect
• Prolactin promotes growth of mammary gland and development of alveolar ducts

in breast. It promotes increase in size of breast in pregnancy.
Lactogenic Effect
• It enhances formation of milk proteins after child birth. It stimulates secretion of

milk.
Luteotropic Effect
• Prolactin maintains growth of corpus luteum.

• It stimulates secretion of progesterone from corpus luteum.
10.6.2 Thyroid-Stimulating Hormone
Structure
• Protein component is composed of α- and β-subunits.

• α-Subunit is made up of 92 amino acids. It is identical to gonadotropins (LH,
FSH, and HCG). β-Subunit is made up of 112 amino acids. It is biologically
active. Carbohydrate moiety is made up of oligosaccharides which are non-cova-
lently linked to α- and β-subunits.
Secretion
• TSH is a glycoprotein.
• It is secreted by basophilic cells of anterior pituitary gland.
Normal Serum Value
• Its normal value is 0.5–5 mIU/L.

226 10 Hormones
Metabolic Functions
TSH promotes synthesis of thyroid hormones through the following activities
as:
• TSH stimulates iodide pump and promotes transport of iodide from circulation
to follicular cells.
• TSH activates oxidation of iodide into active iodine.
• TSH stimulates generation of NADPH through HMP pathway.
• TSH promotes cleavage of iodinated thyroglobulin to release thyroid hormones.
Overall, TSH stimulates all stages of thyroid hormone synthesis.
10.6.3 Adrenocorticotropic Hormone
Secretion
• It is secreted by anterior pituitary gland.
Structure
• It is a polypeptide hormone. It is made up of 39 amino acid residues. At

N-terminal, initial 23 amino acid residues are biologically active.
• ACTH is secreted in the form of a precursor molecule called as proopiomelanocor-
tin peptide (POMC peptide). The precursor is made up of 260 amino acid residues.
• POMC peptide is proteolyzed into:
–– ACTH
–– β-Lipotropin
–– γ-Lipotropin
–– Endorphin
Normal Serum Value

In males, its value is 1–10 pmol/L.
In females, it is 1–6 pmol/L.
Metabolic Functions
• ACTH stimulates conversion of cholesterol into pregnenolone and synthesis of

corticosteroids.
• ACTH enhances release of insulin from pancreas.
• ACTH enhances release of melanin from melanocytes.
• It stimulates mobilization of fats in adipose tissues.
Gonadotropins
Gonadotropins regulate functioning of ovaries and testes. Gonadotropins are of two
types as:
• Follicle-stimulating hormone (FSH)

• Luteinizing hormone (LH)
10.6.4 Follicle-Stimulating Hormone (FSH)
Structure
• It is a glycoprotein. Protein component is made up of α- and β-subunits.

• α-Subunit is made up of 92 amino acid residues. Beta-chain is made up of 118
amino acid residues.
Normal Serum Value

In adult males, it is 2–15 mIU/L.
In adult females:
• Follicular stage, 3–9 mIU/L

• Ovulation stage, 3–18 mIU/L
• Luteal stage, 1–5 mIU/L
Metabolic Functions
In females
• FSH promotes growth of ovarian follicles.

• It stimulates maturation of graafian follicles.
• FSH promotes synthesis of estrogens.
In males
• FSH stimulates growth of testes.

• It increases growth of seminiferous tubules.
• It increases secretion of testosterone.
• FSH promotes proliferation of spermatocytes during spermatogenesis.
• FSH along with testosterone favors the maturation of spermatozoa (transfor-
mation of primary spermatocytes into secondary spermatocytes).
FSH and LH are essential for spermatogenesis in males.

228 10 Hormones
10.6.5 Luteinizing Hormone (LH)
Structure
• It is also called as interstitial cell-stimulating hormone (ICSH). It is a glyco-

protein. Protein component is made up of α- and β-subunits.
• α-Subunit is made up of 92 amino acid residues. Beta-chain is made up of 112
Normal Serum Value

In Males
• In adolescence, its value is up to 5 mIU/mL.

• In adult males, its value is 2–12 mIU/L.
In Females
• In puberty, its value is 2–30 mIU/mL.

• In menstrual phase
–– Follicular phase, up to 18 mIU/dL
–– Luteal phase up to 20 mIU/mL
Metabolic Functions
In females
• LH promotes final maturation of graafian follicles and release of ovum

(ovulation).
• LH induces release of estrogens from theca interna cells and granulose cells in
ovaries.
• LH promotes development of corpus luteum and release of progesterone.
In males
• LH stimulates interstitial cells in testes to release testosterone.
10.6.6 Growth Hormone
Growth hormone is also called as somatotropin or human growth hormone. It is a

peptide hormone. It regulates growth of cells and cell proliferation in human body.
Secretion
Growth hormone is synthesized by acidophilic cells in anterior pituitary gland.
These are specialized cells called as somatotropes.
Structure
• Growth hormone is made up of a single polypeptide chain. It is made up of 191

• Structurally, growth hormone is homologous to prolactin.
Normal Serum Value

Adult males, its value is <3 ng/mL.
Adult females, it is <8 ng/mL.
Children, it is <5 ng/mL.
Regulation of Growth Hormone

Secretion of GH is controlled by hypothalamic, anterior pituitary gland, liver, and
growth hormone as described below.
Hypothalamus
• It secretes growth hormone-releasing hormone (GHRH) and growth hormone-

inhibiting hormone (GHIH) to control secretion of GH.
Anterior Pituitary
It is under the control of the hypothalamus, liver, and serum growth hormone level.
Liver
• Growth hormone acts on hepatocytes and induces synthesis of somatomedin

C. This peptide hormone acts as:
–– Somatomedin C inhibits hypothalamus to release GHRH.
–– It stimulates release of GHIH from hypothalamus.
–– It inhibits pituitary gland to release GH.
Serum GH Level
Serum growth hormone level has feedback regulation on secretion of GH from ante-
rior pituitary. Increase in serum GH level activates release of GHIH from hypothala-
mus, and the mechanism is called as negative feedback mechanism.
In decreased serum GH level, it stimulates secretion of GHRH, and blood level
of GH is normalized.
Metabolic Functions
Effect on Protein Metabolism
• Growth hormone exerts protein anabolic effect through the following activities as:
–– It activates transcription of mRNA.
–– It stimulates uptake of amino acids by cells.
–– It activates polyribosome formation and promotes protein synthesis.
–– GH promotes positive nitrogen balance.
230 10 Hormones
GH and insulin have a similar anabolic effect on protein metabolism.

Effect on Carbohydrate Metabolism
• Growth hormone increases blood glucose level (hyperglycemia) through the

following activities as:
–– GH decreases glucose uptake by peripheral tissues.
–– GH reduces utilization of glucose by peripheral tissues.
–– GH promotes gluconeogenesis.
–– Increased secretion of GH leads to rise in blood glucose level. As a result,
secretion of insulin is increased from pancreas to control blood glucose level.
Growth hormone also stimulates beta-cells to release insulin. Continuous
secretion of GH exhausts beta-cells in the pancreas, and it causes deficiency
of insulin in blood. This stage is characterized by hyperglycemia, and this
effect of GH is called as diabetogenic effect.
GH and Insulin have an antagonistic effect on carbohydrate metabolism.

Effect on Lipid Metabolism
• Growth hormone induces lipolysis in adipose tissues through the following

activities as:
–– GH activates tissue lipase for breakdown of triglycerides in adipose tissues.
–– GH promotes mobilization of fatty acids in circulation and increases plasma-
free fatty acid concentration.
–– GH increases oxidation of fatty acids and promotes formation of ketone bodies.
Effect on Mineral Metabolism
• Growth hormone favors deposition of calcium and phosphate in the bone. It pro-
motes bone mineralization in growing children.
10.7 Hormone from Posterior Pituitary Gland
10.7.1 Antidiuretic Hormone (ADH)
It is a peptide hormone. It is also called as vasopressin or arginine vasopressin.
Secretory of ADH
• ADH hormone is synthesized by neurosecretory neurons in supraoptic nucleus of the

hypothalamus. Trace amount of ADH is synthesized by paraventricular nucleus of
the hypothalamus. These nuclei are located in anterior portion of the hypothalamus.
• The hypothalamus transports ADH in association with neurophysin II (carrier
protein) to posterior pituitary gland.
• ADH is stored along with neurophysin II in Herring bodies (terminal part of
axons from the hypothalamus) located in posterior pituitary.
• ADH is released into blood circulation through exocytosis.
10.7 Hormone from Posterior Pituitary Gland 231
Chemical Structure (Arginine Vasopressin)
• ADH is made up of nine amino acid residues.

• Its amino acid sequence is as follows:
–– Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2
• There is intrachain disulfide bridge (-S-S-) between the first and sixth cysteine
residues.
• At C-terminal of ADH:
–– Glycine amino acid is converted into primary amide, called as glycinamide.
–– It determines biological activity of ADH hormone as in Fig. 10.6.
Fig. 10.6 Antidiuretic hormone

CYS
TYR 1
2
ILE
3
GLN
4
ASN
5
CYS
6
PRO
7
ARG
8
GLY
9
232 10 Hormones
Normal Serum Value

It is 1–4 pg/mL.
Regulation of ADH Secretion

Angiotensin II
• It is a potent vasoconstrictor. It stimulates secretion of ADH.
Atrial Natriuretic Peptide
• It is a peptide hormone secreted by cardiac muscle fibers in the wall of auricles.

It inhibits secretion of ADH.
Cortisol
• It is a corticosteroid. It inhibits ADH secretion.
Extracellular Fluid Volume
• ↑ In ECV inhibits secretion of ADH.

• ↓ In ECV and blood volume (hypovolemia) stimulates secretion of ADH.
Replacement of arginine with lysine at eighth position will change nomenclature

of ADH as lysine vasopressin.
It is found in pigs, marsupials, and hippos.
Natriuresis
It is increase in excretion of sodium ions in urine. It leads to decrease in ECV.
Diuresis
It is increase in excretion of urine.
Natriuresis and diuresis result into excretion of large amount of urine except
that in natriuresis, urine has high amount of sodium ions.
Metabolic Functions
Antidiuretic Effect (Primary Function)
• ADH brings about reabsorption of water by distal convoluted tubules and col-
lecting ducts.
• It acts on V2 receptors located on cells of collecting ducts. It stimulates genera-
tion of cAMP (second messenger). Thereby, ADH increases water permeability
of cell membranes of cells of collecting ducts. Therefore, it brings about reab-
sorption of water from glomerular filtrate (antidiuresis).
10.7 Hormone from Posterior Pituitary Gland 233
• Urine becomes hypertonic with rise in concentration of sodium and chloride

ions. Volume of urine is decreased.
• ADH helps to regulate ECF volume.
Vasoconstrictor Effect (Subsidiary Function)
• ADH acts on V1 receptors located on smooth muscle fibers of blood vessels. It

acts through inositol triphosphate (IP3) pathway of signal transduction. It
increases calcium ions concentration in vascular smooth muscles.
• ADH causes vasoconstriction of blood vessels.
Urea-Retaining Effect
• ADH acts on collecting ducts in inner medulla of kidneys. It raises urea perme-
ability of cell membranes of collecting duct cells.
• ADH is responsible for retention of urea from filtrate. Urine becomes
hypertonic.
Applied Biochemistry
Diabetes Insipidus
• It is an endocrine disorder characterized by excretion of large quantity of urine

per day. It may go up to 20 L a day. Urine becomes hypotonic.
• Condition is attributed to diminished secretion of ADH (primary) or insensitivity
of renal tubules to ADH (secondary).
10.7.2 Oxytocin
It is a peptide hormone.
Secretion of Oxytocin
• Oxytocin is mainly synthesized by neurosecretory neurons in paraventricular

nucleus of the hypothalamus. Trace amount of oxytocin is synthesized in
supraoptic nucleus of the hypothalamus. These nuclei are located in anterior por-
tion of the hypothalamus.
• Oxytocin is released from the hypothalamus in association with neurophysin I
(carrier protein). Oxytocin and neurophysin I are transported via the hypothal-
amo-neurohypophyseal tract.
• Oxytocin and neurophysin I are stored in Herring bodies in posterior pituitary.
• Oxytocin is released into blood circulation.
234 10 Hormones
Chemical Structure (Oxytocin)
• Oxytocin is made up of nine amino acids residues.

• Its amino acid sequence is as follows:
–– Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2
• There is intrachain disulfide bridge (-S-S-) between the first and sixth cysteine
residues.
• At C-terminal of ADH:
–– Glycine amino acid is converted into primary amide, called as glycinamide.
–– It determines biological activity of ADH hormone as in Fig. 10.7.
Fig. 10.7 Oxytocin hormone

CYS
TYR 1
2
ILE
3
GLN
4
ASN
5
CYS
6
PRO
7
LEU
8
GLY
9
10.8 Parathormone (Parathyroid Hormone) 235
Normal Serum Value

It is between 3 and 5.5 pg/mL.
Metabolic Functions of Oxytocin

Milk Ejection Reflex
• It is a neuroendocrinal reflex that brings about release of milk from the mam-
mary glands into the nipples.
• Oxytocin hormone induces milk ejection reflex through the following
steps as:
–– Suckling of the nipple by the baby stimulates tactile receptors located in areo-
lar area of the breast.
–– Sensory impulses travel to the hypothalamus through the spinothalamic
tract.
–– Oxytocin is released from Herring bodies. It causes contraction of myoepi-
thelial cells located around mammary alveoli. The intra-alveolar pressure
rises. Milk is forced into milk ducts and is released in the nipples.
Effect on Uterus
In Pregnancy
• During pregnancy, activity of oxytocin is inhibited by estrogen and

progesterone.
• Toward the end of pregnancy, oxytocin is secreted in large amount. It acts on
the smooth muscle fibers. It causes contraction of the uterus.
• At the onset of labor, the cervix is dilated, and fetus moves down through the
cervix. The receptors in the cervix send impulses to the hypothalamus to release
oxytocin. It causes powerful and prolonged uterine contraction. Therefore, oxy-
tocin helps in child birth.
In Nonpregnancy Condition
• During coitus, vaginal tactile receptors send sensory impulses to hypothalamus

to secrete oxytocin.
• Oxytocin promotes contraction of the uterus. It brings about transport of sperms
to the fallopian tube.
10.8 Parathormone (Parathyroid Hormone)
Parathormone is a peptide hormone. It is essential for calcium homeostasis. It

is a hypercalcemic hormone.
236 10 Hormones
Secretion of Parathormone
• Parathormone is secreted by parathyroid glands. There are two pairs of parathy-

roid glands in the neck region. Each pair is located behind right and left lobes of
thyroid gland.
• Histologically, the parathyroid gland is made up of chief cells and oxyphil cells.
Parathormone is secreted by chief cells.
Chemical Structure
• Parathormone is a linear polypeptide which is composed of 84 amino acids.

• Alanine occupies N-terminal, and glutamine is present at C-terminal in poly-
peptide chain. From N-terminal, sequence of 1st to 34th amino acid is meta-
bolically active and is essential for action of parathormone on the kidneys and
bones.
• Biosynthesis of parathormone:
–– Pre-proparathormone (115 AA)
It is a large peptide molecule and called as precursor hormone. It is synthe-
sized within polyribosomes which are attached to rough endoplasmic reticu-
lum (RER) of chief cells.
Pre-proparathormone is made of 115 amino acid residues. It is translocated
into lumen of rough endoplasmic reticulum (RER). It undergoes successively
two proteolytic cleavages.
–– Proparathormone (90 AA)
Within lumen of RER, an enzyme named as signal peptidase splits a short
peptide of 25 amino acids from N-terminal of pre-proparathormone. Pre-
proparathormone is converted into proparathormone. It is made up of 90
amino acids. It is translocated into cisternae of Golgi bodies.
–– Parathormone (84 AA)
Within cisternae of Golgi bodies, an enzyme named as lipase B (trypsin
resembling enzyme) removes a peptide of six amino acids from N-terminal.
Proparathormone is converted into parathormone.
Parathormone (PTH) is stored in the secretory vesicle of Golgi bodies.
Regulation of Parathormone
Plasma calcium concentration is the key regulatory factor in secretion of
parathormone.
• ↓ Plasma calcium ion concentration stimulates release of PTH.

• ↑ Plasma calcium ion concentration inhibits release of PTH.
10.8 Parathormone (Parathyroid Hormone) 237
Signal Peptidase
It is an enzyme in endoplasmic reticulum that splits a signal peptide from the
N-terminal of a newly formed protein in ER.
Signal Peptide
It is also called as leader peptide or transit peptide.
It is a short peptide of 15–30 amino acid residues. It is located at N-terminal
of newly synthesized protein.
Signal peptide helps to translocate protein from one organelle (site of syn-
thesis) to another organelle for storage. Example: secretory proteins (hor-
mones, enzymes) and membrane proteins are translocated by co-translational
translocation.
Normal Serum Value

Its value is between 10 and 60 pg/mL.
Metabolic Functions
Parathormone ↑ plasma calcium ion concentration through its direct actions on
kidneys and bones and indirect action on intestinal mucosa.
Effect on Kidneys
PTH binds on receptors located on cell membranes of proximal convoluted tubules
and distal convoluted tubules. PTH is the first messenger and induces rise in concen-
tration of cAMP (2nd messenger). It regulates cellular actions. It has the following
effects on kidneys.
Calcium Ions
• PTH increases reabsorption of Ca++ ions from glomerular filtrate in DCT of renal
tubules. It decreases excretion of calcium ions.
• PTH results in hypercalcemia.
Phosphate Ions
• PTH decreases reabsorption of inorganic phosphates from glomerular filtrate in

PCT and DCT portions of renal tubules. It increases excretion of inorganic
phosphates.
• PTH results in hypophosphatemia.
• Hypophosphatemia in turn induces mobilization of inorganic phosphates from
the bones into plasma along with calcium ions.
Alpha-1-Hydroxylase Enzyme
• Enzyme is present in mitochondria of cells of PCT. Enzyme brings about hydrox-

ylation of 25-hydroxycholecalciferol into calcitriol. It stimulates absorption of
calcium ions in intestinal mucosa.
238 10 Hormones
Effect on Bones
PTH binds with specific receptors on membranes of osteoblasts, osteocytes, and
osteoclasts. It induces its action via ↑ cAMP. PTH has the following actions on
bones as:
• PTH increases calcium permeability of the membranes of osteoblasts and osteo-

clasts. It results into calcium mobilization from bones into blood circulation
(hypercalcemia).
• PTH activates osteoclasts in bones. These cells release proteolytic enzymes from
lysosomes which bring about dissolution of bone organic matrix. Calcium ions
diffuse into blood circulation (hypercalcemia).
Action on Intestinal Mucosa
• PTH has no direct action on intestinal mucosa owing to absence of receptors. It

stimulates hydroxylation of 25-hydroxycholecalciferol into calcitriol in kidneys.
Therefore, PTH acts through calcitriol on intestinal mucosa to enhance absorp-
tion of calcium ions (hypercalcemia).
10.9 Insulin
It is a peptide hormone and is a heterodimer. It is a key regulatory hormone for

blood glucose homeostasis and carbohydrate metabolism.
Secretion of Insulin
• Insulin is secreted by β-cells of islets of Langerhans in the pancreas.
Chemical Structure
• Insulin is made up of 51 amino acids. They are assembled into two polypeptide
chains.
• Chain A is made up of 21 amino acids, and chain B is composed of 30 amino
acids. Both chains are linked together by two disulfide bridges as:
–– First interchain disulfide bridge is present between seventh cysteine residue of
chain A and seventh cysteine residue of chain B.
–– Second interchain disulfide bridge is present between 20th cysteine residue of
chain A and 19th cysteine residue of chain B.
• One intrachain disulfide bridge is present in chain A. It links the 6th cysteine
residue to 11th cysteine residue in chain A as in Fig. 3.5.
10.9 Insulin 239
Insulin can never be administered orally. Low pH of stomach denatures

insulin by disruption of disulfide bridges. It is administered subcutaneously.
Biosynthesis of Insulin
Insulin is synthesized in the form of a large precursor molecule (single chain) in
beta-cells of islets of Langerhans. It undergoes proteolytic cleavages to form insulin.
It is described in the following steps.
Formation of Preproinsulin (109 AA)
• Synthesis of preproinsulin occurs in polyribosomes in beta-cells. Polyribosomes

are attached to the membrane of rough endoplasmic reticulum. It is made up of
109 amino acids.
• Preproinsulin contains a short peptide of 23 amino acid residues located at
N-terminal. It is called signal peptide.
• Signal peptide directs the translocation of preproinsulin from polyribosomes into
the lumen of rough endoplasmic reticulum.
Cleavage of Preproinsulin into Proinsulin (86 AA)
• Signal peptide in preproinsulin is cleavaged by signal peptidase enzyme (located

in membrane of RER).
• This cleavage results in conversion of preproinsulin into proinsulin. It is made up
of 86 amino acid residues.
• Proinsulin is translocated to cisternae of Golgi complex.
Conversion of Proinsulin into Insulin (51 AA)
• Proinsulin is composed of 86 amino acid residues which are arranged in three

domains in a single peptide chain as follows:
–– N-terminal domain of proinsulin (chain B).
–– C-terminal domain of proinsulin (chain A).
–– Connecting peptide: It is a short peptide (30–35 amino acid residues) that
connects two domains. It is called C-peptide. It contains dibasic amino acid
residues on both ends (31stArg-Arg32nd and 62ndLys-Arg63rd).
• Proinsulin is converted into insulin through hydrolytic action of two types of
protease enzymes as:
–– Action of endopeptidase on proinsulin It occurs inside golgi complex.
trypsin-like enzyme (endopeptidase) hydrolyses proinsulin at two points after
each dibasic amino acid pair. This results into release of connecting C-peptide
from proinsulin.
–– Action of exopeptidase on proinsulin
Carboxypeptidase B-like enzyme (exopeptidase) brings about cleavage of
amino acids from C-terminal in chain A and B as follows:
–– Two amino acids (62ndLys-Arg63rd) are cleavaged from C-terminal of chain A.
–– Two amino acids (31stArg-Arg32nd) are cleavaged from C-terminal of chain B.
240 10 Hormones
• Insulin and C-peptide are packed in equimolar concentration in vacuoles of

Golgi complex.
• Insulin undergoes dimerization by formation of hydrogen bond. It is formed
between hydrogen atom of amide group in phenylalanine of B25 and oxygen
atom of carbonyl group in tyrosine of A19.
• As concentration of insulin increases in vacuoles, three dimmers of insulin form
coordinative bond with two Zn++ ions. It results in crystallization of insulin and
is called as zinc insulin hexamer. Vacuoles are converted into secretory vesi-
cles containing zinc insulin crystals and C-peptide.
• Insulin is released by exocytosis.
Normal Serum Value

Its value is 30–180 pmol/L.
Metabolic Functions of Insulin
• Effect on Carbohydrate Metabolism

• Insulin decreases blood glucose level. It exerts hypoglycemic effect through
the following activities.
• ↑ Glucose Uptake by Peripheral Tissues
–– Glucose is actively transported across the peripheral tissues (adipose tissues,
mammary glands, kidneys, skeletal muscles). Insulin brings about prolifera-
tion of glucose transporters in cell membrane of peripheral tissues (extrahe-
patic tissues).
–– Glucose is permeable to hepatocytes. However, insulin can further enhance
glucose transport across hepatocytes.
↑ Glucose Utilization (Glycolysis)
–– Insulin stimulates formation of phosphofructokinase enzyme in peripheral tis-
sues. It is a rate-limiting enzyme in glycolysis. Therefore, insulin stimulates
utilization of glucose by peripheral tissues and enhances production of energy.
↑ Glycogenesis
–– Insulin activates glycogen synthase enzyme in hepatocytes and muscles. This
enzyme regulates glycogenesis. Therefore, insulin promotes glycogenesis in
the liver and skeletal muscles.
↓ Gluconeogenesis
Insulin decreases gluconeogenesis through the following activities as:
–– Insulin suppresses translation of PEP carboxykinase enzyme.
–– Insulin mediates through allosteric inhibition of fructose-1,6, bisphosphate
enzyme.
↓ Glycogenolysis
–– Insulin decreases glycogenolysis through the following activities as:
–– Insulin deactivates glycogen phosphorylase enzyme in the liver and muscles.
It is a rate-limiting enzyme in glycogenolysis.
• Effect on Lipid Metabolism
10.10 Glucagon 241
Insulin promotes synthesis of triglycerides in adipose tissues. It exerts lipo-

genesis effect through the following activities as:
↓ Lipolysis in Adipose Tissues
Insulin lowers lipolysis in adipose tissues through the following activities as:
–– Insulin deactivates tissue lipase.
–– Insulin stimulates phosphodiesterase enzyme. It decomposes cAMP in cyto-
sol and decrease activation of tissue lipase.
↑ Triglyceride Synthesis in Adipose Tissues
Insulin stimulates synthesis of triglycerides in adipose tissues through the
following activities as:
–– Insulin stimulates glucose uptake by adipose tissues. Surplus glucose is
diverted into synthesis of triglycerides in tissues.
–– Insulin induces synthesis of fatty acids in the liver which enter circulation as
free fatty acids. They are utilized by adipose tissues for triglyceride
formation.
↑ De Novo Synthesis of Fatty Acids in the Liver
Insulin promotes de novo synthesis of fatty acids in the liver. It mediates its
action through the following activities as:
–– Insulin activates pyruvate dehydrogenase complex and enhances oxidative
decarboxylation of pyruvate into acetyl CoA. It is necessary for de novo syn-
thesis of fatty acids in the liver.
–– Insulin promotes availability of NADPH through HMP shunt. It is necessary
for synthesis of fatty acids.
• Effect on Protein Metabolism
Insulin increases synthesis of proteins. Its protein anabolic effect is medi-
ated through the following activities as:
–– Insulin promotes transcription of mRNAs.
–– It increases polyribosomes formation by increasing availability of rRNA in
cytosol.
–– Insulin activates uptake of amino acids by body tissues. Conclusively, insulin
promotes translation of proteins on polyribosomes.
10.10 Glucagon
Glucagon is a peptide hormone. It is antagonistic to action of insulin. Glucagon

causes hyperglycemia.
Secretion of Glucagon
Glucagon is secreted by α-cells of islets of Langerhans in the pancreas.
Chemistry
Glucagon is a linear polypeptide hormone. It is composed of 29 amino acids. Its
molecular weight is 3485 daltons. Histidine is located at N-terminal and threonine
and is present at C-terminal in glucagon molecule as in Fig. 10.8. Glucagon does not
need zinc ion in crystallization.
242 10 Hormones
1 2 3 4 5 6 7 8 9 10
N– HIS SER GLN GLY THR PHE THR SER ASP TYR
Terminal
GLN ALA ARG ARG SER ASP LEU TYR LYS SER
20 19 18 17 16 15 14 13 12 11
ASP PHE VAL GLN TRP LEU MET ASN THR C–

21 22 23 24 25 26 27 28 29 Terminal
Fig. 10.8 Glucagon hormone
NH2-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-
Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-COOH
Biosynthesis of Glucagon
Proglucagon is synthesized in polyribosomes of alpha-cells. It is cleavaged by pro-
protein convertase-2 (endopeptidase) and carboxypeptidase (exopeptidase) and is
transformed into its active form, called as glucagon as in Fig. 10.8.
Normal Serum Value

Its value is 15–100 pg/mL in fasting condition.
Metabolic Functions
Glucagon is antagonistic to insulin in effects on protein, carbohydrate, and
lipid metabolism.

Glucagon increases blood glucose level (hyperglycemia). Its action is mediated
through the following activities.
–– ↑ Glycogenolysis
In the liver, glucagon activates phosphorylase b into phosphorylase a which
is responsible for glycogenolysis. It stimulates breakdown of glycogen.
Glucagon increases formation of glucose-6-phosphatase enzyme in liver.
Therefore, glucagon enhances liberation of free glucose molecules in liver.
In the skeletal muscles, glucagon has no effect on the glycogen in skeletal
muscles. It is due to the absence of glucagon receptors in muscle fibers.
–– ↑ Gluconeogenesis
In the liver, glucagon stimulates synthesis of PEP carboxykinase enzyme and
pyruvate carboxylase enzyme. Glucagon increases concentration of gluco-
genic amino acids in liver cells.
Overall, glucagon promotes gluconeogenesis from glucogenic amino acids
and lactic acid.
10.11 Thyroid Hormones 243
Glucagon exerts lipolytic effect on lipid metabolism through the following

activities.
–– ↑ Lipolysis of Tissue Triglycerides
Glucagon activates tissue lipase. It stimulates lipolysis of triglycerides in adi-
pose tissues. Its action is synergistic to thyroxine and antagonistic to insulin.
It increases production of fatty acids and glycerol.
–– ↑ Plasma FFA Concentration
Glucagon mobilizes fatty acids from tissues to blood circulation.
–– ↑ Ketogenesis
Glucagon promotes synthesis of ketone bodies in the liver from surplus fatty
acids.
Glucagon has a protein catabolic effect through the following activities.
–– ↑ Catabolism of Protein
Glucagon promotes breakdown of proteins in the liver. It increases concentra-
tion of amino acids in the liver. These are diverted into gluconeogenesis.
–– ↓ Protein Synthesis
Glucagon decreases synthesis of proteins.
• Effect on BMR
Glucagon enhances production of calories in association of thyroxine and corti-
costeroid hormones. It is called as calorigenic effect. It increases basal metabolic
rate by enhancing consumption of oxygen.
10.11 Thyroid Hormones
Thyroid hormones are catecholamines. They are derived from tyrosine amino acid.
Important catecholamine hormones derived from thyroid gland are as follows:
• Thyroxine (3′,5′,3,5-Tetra-iodothyronine), represented as T4

• 3′,3,5-Tri-iodothyronine, represented as T3
• 3′,5′,3-Tri-iodothyronine, represented as reverse T3
T4 T3
It constitutes 90% of total thyroid secretion It constitutes 9% of total thyroid secretion
It has high affinity to TBG and TBPA It has moderate affinity to TBG
Its biological activity is 1/4 to that of T3 Its biological activity is 4 times higher than T4
hormone
It has slow onset of action and longer It has rapid onset of action and short
duration of action duration of action
In blood circulation, 0.05% of total T4 is in unbound form. Remaining
amount of hormone is in bound form with carrier molecule.
In extrathyroidal tissues (liver, peripheral tissues, kidneys, and muscles), more
than 80% of circulation T4 is deionized by deiodinase enzyme into T3 hormone.
Thus T4 hormone acts as prohormone.
Under normal iodine intake,
Ratio of T4: T3 is 9:1.
244 10 Hormones
Secretion of Thyroid Hormones
• Thyroid hormones are secreted by follicular cells of thyroid follicles.

• Thyroid follicle is a spherical in shape. It is lined by cuboidal epithelial cells
called as follicular cells. They are endocrinal in nature.
• Thyroid follicles contain a central cavity (thyroid lumen). It is filled with a vis-
cous fluid called as colloid. It is enriched with thyroglobulin.
Chemistry
• T4 and T3 hormones are derived from iodinated tyrosine. Two tyrosine residues
are linked through oxygen atom (ether bonding) and form thyronine (C15H15NO4).
• Thyronine contains iodine atoms at C3 and C5 positions on inner aromatic ring.
• It contains iodine atoms at C′3 and C′5 positions on outer aromatic ring.
• The number of iodine atoms determines the nature of thyroid hormone as:
–– Iodination at all four positions (C′3, C′5, C3, C5) forms T4.
–– Iodination at three positions (3′,3,5) forms T3.One iodine atom is missing in
the outer aromatic ring.
–– Iodination at three positions (3′,5′,3) forms Reverse T3. One iodine atom is
missing in inner aromatic ring as in Fig. 10.9.
10.11.1 Biosynthesis
T4 and T3 hormones are synthesized by iodination of tyrosine residues in thyro-

globulin molecule. Biosynthesis of T4 and T3 hormones can be summarized in
the following two steps.
1. Synthesis of Thyroglobulin
2. Iodination of Tyrosine Residues
• Active uptake of Iodide
• Oxidation of Iodide
• Tyrosine Iodination
• Oxidative Coupling of Iodotyrosines
Synthesis of Thyroglobulin
• Thyroglobulin acts as prohormone.
• Chemically, thyroglobulin is a glycosylated protein (glycoprotein). It is a mac-
romolecule with molecular weight of 660,000 daltons.
• Thyroglobulin is composed of two units as:
–– A dimeric protein which is made up of two identical subunits. Each subunit
has a molecular weight of 330,000 daltons. Thyroglobulin molecule contains
about 140 tyrosine residues.
3′ I 3 I
O
HO O CH2 CH C OH
I H N
5I 5
3,5,3′,5′ – Tetraiodothyronine
[Thyroxine (T4)]
3′ I 3 I
H O
HO O C CH C OH
H N H
I
5
H
3,5,3′ – Triiodothyronine
[T3]
3′ I 3I
H O
HO O C CH C OH
I H N H
5′
H
3,3′,5′ – Triiodothyronine
[Reverse T3]
Fig. 10.9 Structure of thyroid hormones
–– A carbohydrate moiety which is made up of N-acetyl glucosamine, glucose,

galactose, and sialic acid. Carbohydrates constitute about 8% of the total
weight of thyroglobulin.
• Iodide weighs about 1% of total weight of thyroglobulin. About 70% of total
iodide in thyroglobulin is present in the form of iodinated tyrosyl residues as
monoiodotyrosine (MIT) and diiodotyrosine (DIT). Another 30% of total iodide
is present in the form of iodinated thyronyl residues as triiodothyronine and
tetraiodothyronine as in Figs. 10.10 and 10.11.
246 10 Hormones
Fig. 10.10 Oxidation of NADPH2 O2

iodide
NADP+ H2O2 IODIDE

I′
ACTIVE
H2O
IODINE
I+
FOOD
(Contains Iodine) Endothelium Thyroid Colloid

I2 Food intake follicular cell
Basolateral
membrane Nucleus
I2 Iodine
Capillary Follicular Apical
cell membrane
Oesophagus cytoplasm
Tyrobine
Stomach residues
I2 Na+ Na+
HCL Iodide pump Pendrin
I– Iodide I– Iodide trapping Cytosolic Thyro peroxidase

movement
Thyroglobulin
I– I– Active
Duodenum I– I– I–
Thyroid iodine
Iodination
Oxidation
I–
Vacuole hormone
Blood circulation
Blood
circulation
Iodide absorption
Endo-
Thyroglobulin
cytosis
Decomposition of
thyroglobulin
Iodinated
tyrosine
Fig. 10.11 Biosynthesis of thyroid hormone
Steps in Synthesis of Thyroglobulin
• It is synthesized on polyribosomes present in cytoplasm of thyroid follicular cells.

Polyribosomes are attached to the membrane of rough endoplasmic reticulum. After
synthesis of protein component, it is translocated into cisternae of Golgi complex.
• Within Golgi complex, glycosylation of protein component is completed.

Glycoprotein is packed in secretory vesicles, and they are budded off from cister-
nae of Golgi complex.
• Secretory vesicles translocate toward the cell membrane of follicular cells and
release glycoprotein into colloid by exocytosis.
Synthesis of T4 and T3 hormones is largely dependent on exogenous

supply of iodine which in turn is determined by hormone synthesis and
level of TSH.
I odination of Tyrosine Residues

Iodine is the most essential trace element in the human body. Normal iodine require-
ment for a healthy adult is 150 μg/day. Its requirement increases during early child-
hood period, puberty, and pregnancy. The human body contains about 20 mg of
iodine, and 80% of total iodine is concentrated in thyroid gland.
Dietary sources like vegetables and seaweeds are rich in iodine. Dietary iodine is
reduced into iodide in alimentary canal. Iodides are absorbed from intestinal mucosa
and are transported into blood circulation. In plasma, it exists as inorganic iodide
and is distributed in association with albumin.
Active Uptake of Iodide
• Thyroid follicles are supplied with blood capillaries. Normal plasma iodide con-
centration varies between 40 and 90 μg/L; however, thyroid iodide concentration
(T) is much higher than serum iodide concentration (S). It may vary between T/S
(10:1) and (100:10).
• Iodide is actively transported from plasma to the follicular cells. This influx of
iodide occurs against concentration gradient. It is called as iodide trapping.
• Active uptake of iodide is mediated by sodium-iodide symporter (NIS):
–– Sodium-iodide symporter is located in the basolateral membrane of follicu-
lar cell.
–– It is a transmembrane protein with molecular weight of 87,000 daltons. It is
involved in active cotransport of two Na+ ions with every iodide ion.
–– Sodium-iodide symporter is energy dependent. It relies on Na+-K+-ATPase
enzyme for its energy demand. ATP provides energy for NIS.
• Inhibitors of sodium-iodide symporter:
–– Thiocyanates and perchlorates are competitive inhibitors of NIS.
–– Cyanide inhibits NIS.
–– Cardiac glycosides (ouabain) inhibit NIS.
• Accelerator of sodium-iodide symporter (NIS):
–– Thyroid-stimulating hormone activates sodium-iodide pump.
• Iodide enters the follicular cells.
248 10 Hormones
Oxidation of Iodide
• Intracellular iodide moves toward the apical membrane of follicular cell.

• This transport of iodide across the cell is mediated by sodium-independent
chloride-iodide exchanger. It is called as pendrin. It brings about efflux of
iodide ions through the apical membrane of follicular cell as in Fig. 10.10.
• Activity of thyroperoxidase enzyme:
–– Thyroperoxidase is a metalloprotein. It located at the apical membrane of fol-
licular cell. It contains heme as a prosthetic group.
–– It is synthesized in RER of follicular cells.
–– Thyroperoxidase requires H2O2 for its activity. H2O2 is toxic in body tissues.
However, it is essential for oxidation of iodide and iodination of tyrosine
in follicular cells. Intracellular hydrogen peroxide is synthesized by a dual
oxidase enzyme (DUOX1 and DUOX2) which is located in the apical mem-
brane. Thyroperoxidase acts on hydrogen peroxide and reduces it into water
and oxygen.
–– At apical membrane-colloid interface, iodide and thyroglobulin are attached
at separate domains of thyroperoxidase enzyme. Released oxygen brings
about oxidation of iodide into active iodine as in Fig. 10.10.
Tyrosine Iodination
Tyrosine iodination occurs in colloid within thyroid lumen.
• At iodide-binding site of thyroperoxidase, active iodine is transported to tyro-

sine residue of thyroglobulin under the influence of thyroperoxidase enzyme.
• Iodination of tyrosine takes place at the third position of aromatic ring. It results
in the formation of monoiodotyrosine (MIT). Next iodination occurs at fifth
position of aromatic ring of tyrosine residue and forms diiodotyrosine (DIT).
• Under normal condition, MIT and DIT exist in equal concentration. However, in
deficiency of iodine intake, concentration of MIT exceeds concentration of DIT
in thyroid gland. The process of iodination of thyroglobulin is called as orga-
nization of thyroglobulin as in Fig. 10.10.
Oxidative Coupling of Iodotyrosines
• Two diiodotyrosine (DIT) molecules undergo oxidative coupling to form thy-

roxine. Reaction is catalyzed by thyroperoxidase enzyme. A molecule of alanine
amino acid is released in the coupling reaction. Alanine further undergoes deam-
ination to form ammonia and pyruvate.
• One molecule MIT undergoes coupling with one molecule of DIT to form
triiodothyronine with a release of alanine amino acid.
• One molecule of DIT undergoes coupling with one molecule of MIT to form
reverse triiodothyronine with release of alanine amino acid as in Fig. 10.10.
Internalization and Proteolysis of Thyroglobulin
• Pits are located on the apical membrane of follicular cells. Colloid containing
iodinated thyroglobulin enters into pits. These pits in turn invaginate to form
intracellular vesicles, and the process is called internalization or endocytosis.
• Endocytosis can be either receptor-mediated endocytosis or non-specific endo-
cytosis. Megalin is a surface protein located on apical membrane. It helps in
internalization of colloid as in Fig. 10.10.
• Vesicles are coated with megalin proteins. Vesicles contain iodinated thyroglobu-
lin (iodinated thyroglobulin; MIT, DIT, and T3 and T4 hormones).
• These vesicles shed their protein coat and are transformed into endosomes.
• Endosomes fuse with lysosomes. They contain hydrolytic enzymes which digest
thyroglobulin and liberate T4 and T3 hormones. These hormones pass through the
basal membrane of follicular cell and enter blood circulation.
• MIT and DIT are deiodinated in follicular cells. Iodine is reused in cells.
Transport
• Within blood circulation, T4 and T3 hormones bind with carrier proteins called
as thyroxine-binding proteins, which follow the two types as:
–– Thyroxine-binding prealbumin (TBPA)
–– Thyroxine-binding globulin (TBG)
• Hormones are transported in bound form to tissues.
Storage
• Thyroid hormones are stored in colloid in association with thyroglobulin.
Normal Serum Value

Free T4 is 10–25 pmol/L.
Total T4 is 60–160 mmol/L.
T3 is 1.5–3.5 mmol/L.
Metabolic Functions of Thyroid Hormones

Effect on Protein Metabolism
Thyroid hormones are protein anabolic hormones. They promote synthesis of
proteins in cells through the following activities as:
• These hormones stimulate mRNA synthesis.

• These hormones activate polyribosome formation and enhance synthesis of
proteins.
250 10 Hormones
In pathological condition, thyroid hormones are protein catabolic hormones:
• In hyperthyroidism, ↑catabolic activity results into muscle breakdown, wast-

ing, and muscle weakness. There is excessive formation of creatinine, and it
causes ↑ excretion of creatinine in urine (creatinuria).
• These manifestations are called as hyperthyroid myopathy.
Effect on Lipid Metabolism
• In adipose tissues:
–– Thyroxine activates tissue lipase enzyme. This hormone stimulates lipolysis
of tissue triglycerides. It increases plasma-free fatty acid concentration.
Thyroid lipolytic action is antagonistic to insulin.
• In the liver:
–– Thyroxine enhances biosynthesis cholesterol and phospholipids in the liver.
–– It promotes deposition of fats in the liver (fatty liver).
• In blood circulation:
Thyroxine decreases concentration of cholesterol in plasma. Its action is medi-
ated through the following activities as:
–– ↑ Synthesis of cholic acid and deoxycholic acid from cholesterol in the liver
–– ↑ Excretion of cholesterol through bile
Effect on Carbohydrate Metabolism

Thyroid hormones stimulate all activities associated with carbohydrate metabo-
lism as follows:
• In the small intestine, these hormones increase intestinal absorption of

glucose.
• They increase blood glucose level (hyperglycemia).
• In peripheral tissues, these hormones decrease insulin tolerance. Therefore,
they promote uptake and utilization of glucose by peripheral tissues. These hor-
mones stimulate glycolysis and TCA cycle and generate energy.
• In the liver:
–– Thyroid hormones increase glycogenolysis through activation of glucose-
6-phosphatase enzyme.
–– Thyroid hormones increase gluconeogenesis by activation of pyruvate car-
boxylase and PEP carboxykinase enzymes.
–– Thyroxine proliferates β-adrenergic receptors located on hepatocytes.
Therefore, it enhances glycogenolysis effect of adrenaline.
Effect on Basal Metabolic Rate
• Thyroid hormones have calorigenic effect. They stimulate consumption of oxy-

gen by tissues (except the brain, spleen, testes, and uterus) and increase the pro-
duction of energy. In thyroid hypersecretion, BMR can be increased to 50% of
the normal value.
10.12 Calcitonin 251
Effect on Vitamins
• Thyroxine is helpful in conversion of carotene into retinol.
10.12 Calcitonin
It is a peptide hormone. It is essential in calcium metabolism. It is a hypocalce-

mic hormone.
Secretion of Calcitonin
• Calcitonin is secreted by C cells found in thyroid gland. The C cells are also
called as parafollicular cells. These are specialized endocrinal cells.
Chemical Structure
• Calcitonin is a linear polypeptide. It is made up of 32 amino acids.

• At N-terminal, cysteine amino acid is present, while proline amino acid occu-
pies C-terminal of calcitonin. Proline is converted into primary amide and is
called as prolinamide.
• Interchain disulfide bridge links first cysteine residue with seventh cysteine resi-
due in the molecule as in Fig. 10.12.
Cysteine
I
S S
2 3 4 5 6 7 8 9 10
N– CYS GLY ASN LEU SER THR CYS MET LEU GLY
Terminal
20 HIS PHE LYS ASN PHE ASP GLN THR TYR THR 11
21 THR PHE PRO GLN THR ALA ILE GLY VAL GLY 30
H2N PRO ALA

32
C – Terminal
Prolinamide
Fig. 10.12 Structure of calcitonin

252 10 Hormones
Regulation of Calcitonin
Calcitonin is regulated by plasma calcium concentration.
• ↓ Plasma calcium ion concentration inhibits release of calcitonin.

• ↑ Plasma calcium ion concentration stimulates release of calcitonin.
Normal Serum Value

It is < 8 pg/L.
Metabolic Functions
Calcitonin ↓ plasma calcium ion concentration through its actions on the kidneys,
bones, and intestinal mucosa.
Calcitonin is antagonistic to parathormone.
Calcitonin binds to receptors on the cell membranes of bones cells and cells of renal
tubules. It induces rise in concentration of cAMP which in turn control cell activities.
Effect on the Kidneys
• Calcitonin acts on distal convoluted tubule and ascending limb of loop of Henle.
It decreases reabsorption of calcium ions and phosphate ions.
• Calcitonin results into hypocalcemia and hypophosphatemia.
Effect on Bones
• Calcitonin inhibits activity of osteoclasts and suppresses bone resorption. It

inhibits mobilization of calcium and phosphate ions from bone matrix into blood
circulation.
• Calcitonin promotes mineralization of bones.
Effect on Intestinal Mucosa
• Calcitonin inhibits hydroxylation of 25-hydroxycholecalciferol in kidneys and

synthesis of calcitriol. It indirectly decreases absorption of calcium from the
intestine.
10.13 Somatostatin
Somatostatin is a peptide hormone. It is a growth hormone-inhibitory hor-

mone or growth hormone-release inhibitory hormone.
Secretion of Somatostatin
Somatostatin is secreted by three organs in the body as:
• Hypothalamic Somatostatin
It is secreted by secretory neurons in ventromedial nucleus of hypothalamus. It
enters hypothalamo-hypophyseal tract and reaches anterior pituitary gland from
where it is released.
10.14 Adrenal Cortical Hormones (Corticosteroids) 253
• Pancreatic Somatostatin
It is secreted by delta (δ)-cells in the pancreas.
• Gastrointestinal Somatostatin
It is secreted by delta (δ)-cells in pyloric antrum in the stomach and duodenum.
Chemistry
It is a linear peptide. It is made up of 14 amino acids. Alanine amino acid is located
at N-terminal, and cysteine amino acid is present at C-terminal of somatostatin mol-
ecule. There exists an intrachain S-S bridge between the 3rd cysteine residue and
14th cysteine residue in the peptide chain.
Normal Serum Value

It is 8–20 pg/mL.
Metabolic Functions of Somatostatin
• Hypothalamic Somatostatin
It functions as regulator of release of growth hormone. It inhibits release of
growth hormone from anterior pituitary gland.
• Pancreatic Somatostatin
Somatostatin acts through paracrine signaling (communication between cells).
• It inhibits the following secretions as:
Insulin, glucagon, and pancreatic juice
• Gastrointestinal Somatostatin
Somatostatin inhibits secretory activity of parietal cells in the stomach through
paracrine signaling. It inhibits release of gastric secretion. Somatostatin from
duodenum enters hepatic portal vein and reaches systemic circulation. It acts on
target cells.
It inhibits the following secretions as:
Gastrin, secretin, cholecystokinin-pancreozymin, gastric inhibitory peptide, and
vasoactive intestinal peptide
Gastrointestinal somatostatin delays gastric emptying. It decreases gut motility.
10.14 Adrenal Cortical Hormones (Corticosteroids)
Corticosteroids are steroidal hormones which are secreted by adrenal cortex. They
are derived from cholesterol.
Corticosteroid hormones are characterized by the presence of cyclopentano-
perhydro-phenanthrene nucleus or also called as sterane nucleus.
Classification of Adrenocorticosteroids
They are classified into two categories based upon carbon skeleton and func-
tional activity.
254 10 Hormones
1. Depending Upon Carbon Skeleton

The two types of corticosteroids are as follows:
• C 21 corticosteroids
These hormones contain a total of 21 carbon atoms. The 17 carbon atoms in ring
“D” contain a side chain of 2 carbon atoms.
Examples:
Mineralocorticoids and glucocorticoids
• C19 corticosteroids
These hormones contain a total of 19 carbon atoms. These hormones contain
a keto group (C〓O) at position C17 and are called as 17-ketocorticosteroids
or 17-oxosteroids.
These hormones have androgenic functional activity.
Examples:
Androstenedione and dehydroepiandrosterone (DHEA)
2. Depending upon Functional Activity
The two types of corticosteroids are as follows:
• Glucocorticoids
These hormones are primarily involved in regulation of carbohydrates, addi-
tionally control metabolism of proteins and lipids, and have minor effect on
minerals metabolism and total body water (TBW).
Example:
Cortisol
It is C-21 corticosteroid. It contains three OH groups at C-11, C-17, and C-21
atoms. It is also called as 11-17-21-trihydroxycorticosterone as in Fig. 10.13.
Cortisol is the primary and most potent glucocorticoid hormone.
11-Deoxycortisol
It is a weak glucocorticoid. It is derived from cortisol.
Cortisone
It is a C-21 corticosteroid. It contains OH group at C-17 atom. There is dehy-
drogenation at C-11 atom. It is also called as
17-hydroxy-11-dehydrocorticosterone.
Corticosterone
It is a C-21 steroid. It contains OH groups at C-11 and C-21 atoms. It is also
called as 17-deoxycorticosterol. It has minor glucocorticoid activity.
• Mineralocorticoids
These hormones are mainly involved in regulation of mineral metabolism and
total body water (TBW).
Examples: Aldosterone, 11-Deoxycorticosterone (DOC),
11-Dehydrocorticosterone as in Fig. 10.13
Adrenocortical Sex Steroids
• These hormones are primarily involved in regulation of secondary sexual

characteristics.
Examples:
Androstenedione and dehydroepiandrosterone (DHEA)
H
H C H
C17 oxygenator H C20,C3 ketone
20 C = O
groups
C3 OH Group H C H H
O C4,C5 Double
H C H
17
H Bond
H3C C D H C H C D
A B A B
3
HO 5 C 5
4 = 4
O
C19 Steroid C21 Steroid
H H
H C H H C OH
C11 Dehydrogenator 2
20 C = O 20 C = O
O H3C HO H3C
OH OH
17 17
H3C 11 C D H3C 11 C D
A B A B
3 C
O O
Cortisone 17 – hydroxy – 11 – dehydro Cortisol 11,17,21 – Trihydroxy

corticosterone corticosterone
H H
H C H C11 Dehydrogenase H C OH
21 21
20 C = O 20 C = O
OH H3C O H3C
OH OH
17D 17
H3C 11 C H3C 11 C D
A B A B
3 3
O O
Corticosterone 11 – Dehydro corticosterone
Fig. 10.13 Hormones of adrenal cortex
Cortisol is the most essential and chief corticosteroid (glucocorticoid) hor-

mone which exists in free state in blood circulation. It is vitally important for
the functioning of the cardiovascular system, homeostasis, and immunological
response of body.
256 10 Hormones
10.14.1 Glucocorticoids
Biosynthesis
Corticosteroids are synthesized in adrenal cortex. It has three zones which are
specific for synthesis of individual group of corticosteroids as described below:
• Zona Fasciculata
It is the middle zone of adrenal cortex. Cells are arranged into bundles, also
called as zona fasciculata. This zone synthesizes chiefly cortisol (glucocorti-
coid) which controls glucose metabolism in adverse conditions like stress, fear,
fight, or flight.
This zone synthesizes small amount of androgens.
• Zona Glomerulosa
It is the uppermost zone of adrenal cortex which lies underneath renal capsule.
Ovoid cells are arranged into clusters.
This zone synthesizes aldosterone (mineralocorticoid) and is involved in reg-
ulation of mineral metabolism.
• Zona Reticularis
This is the innermost zone of adrenal cortex. It lies above the adrenal medulla.
Cells are arranged into a network-like pattern. This zone mainly synthesizes
cortical sex steroids. Small amount of cortisol is also produced.
Steps in Biosynthesis of Glucocorticoids

In Zona Fasciculata
• Intracellular Transport of Cholesterol

Cholesterol is the precursor for synthesis of corticosteroid hormones in adrenal
cortex. Cholesterol is present in cytosol of cells of adrenal cortex. Cholesterol
transport from outer mitochondrial membrane to inner mitochondrial membrane
is the rate-limiting step in synthesis of hormones.
Intracellular transport of cholesterol is mediated by a transport protein located in
adrenal cortex cells. It is called as steroidogenic acute regulatory protein
(StAR). This transport protein transfers cholesterol to inner mitochondrial
membrane.
• Conversion of Cholesterol into Pregnenolone
Inner mitochondrial membrane contains cytochrome P450 side-chain cleavage
enzyme (Cytochrome P450 SCC).
Cytochrome P450 SCC brings about two hydroxylations of cholesterol side chain at
C22 andC20 positions. First hydroxylation forms 22R-hydroxycholesterol. It
undergoes second hydroxylation to form 20-alpha-22R-dihydroxycholesterol.
Cytochrome P450 SCC requires molecular oxygen and reducing equivalents
(NADPH) for its activity. There is transfer of electrons from NADPH to cyto-
chrome P450 SCC through adrenodoxin and adrenodoxin reductase proteins.
• Cytochrome P450 SCC enzyme splits linkage between C20 and C22 in side chain
of cholesterol to form pregnenolone and isocaproic aldehyde.
• Conversion of Pregnenolone into 17-Hydroxypregnenolone
Pregnenolone is translocated to smooth endoplasmic reticulum. It undergoes
hydroxylation at C17 position to form 17-hydroxypregnenolone as in Fig. 10.14.
Reaction is catalyzed by 17alpha-hydroxylase. [It is also called as cytochrome
P450 17A1 or 17,20-desmolase. Enzyme has 17alpha-hydroxylase and 17,20-
lyase enzymatic activities. It is a key enzyme in the adrenal steroidogenic
pathway.]
• Conversion of 17-Hydroxypregnenolone into 17-Hydroxyprogesterone
Cholesterol
side chain clevge NADP+

Cytochrome
FAD
p450-
O2
NADPH2
17– Hydroxy 17– alpha–hydroxylase Isocaproic

pregnenolone Pregnenolone aldehyde
O2
dehydrogenase
dehydrogenase
NADPH2 NADP+
Isomerase
Isomerase
NAD+
NAD+
+
+
O2 NADPH
NADP+ 2
17– Hydroxy Progesterone

pregnenolone 17– alpha–hydroxylase
NADPH2
21–Hydroxylase
O2
NADPH+
11– Deoxycorticosterol
NADPH2
11– β – Hydroxylase
O2
NADPH+
Cortisol
Fig. 10.14 Biosynthesis of glucocorticoids

258 10 Hormones
17-Hydroxypregnenolone is converted into 17-hydroxyprogesterone by 3-beta-

hydroxysteroid dehydrogenase enzyme. It requires NAD+ as coenzyme to
accept hydrogen atoms.
• 3-Beta-hydroxysteroid dehydrogenase belongs to lass of oxidoreductase
enzymes. It is the single enzyme in adrenal cortical steroidogenic pathway
that does not belong to the family of cytochrome P450 enzymes.
• Conversion of 17-Hydroxyprogesterone into 11-Deoxycortisol
17-Hydroxyprogesterone undergoes hydroxylation to form 11-deoxycortisol.
Reaction is catalyzed by 21-hydroxylase enzyme located in the smooth endo-
plasmic reticulum. Enzyme requires molecular oxygen and NADPH for its
activity.
• Formation of Cortisol
11-Deoxycortisol is translocated to the inner mitochondrial membrane.
11-Deoxycortisol is converted into cortisol.
Reaction is catalyzed by 11-beta-hydroxylase enzyme. It requires molecular
oxygen and NADPH for its activity.
–– 17-Alpha-hydroxylase, 21-hydroxylase, and 11-beta-hydroxylase are
monooxygenase enzymes.
–– They belong to the family of cytochrome P450 enzymes. They require
molecular oxygen and NADPH and adrenodoxin protein.
Regulation by Adrenocorticotropic Hormone (ACTH)

ACTH regulates synthesis of glucocorticoid hormones through the following activi-
ties as:
• ACTH activates enzyme cholesterol esterase in cytoplasm. This enzyme pro-

vides cholesterol molecules for adrenal glucocorticoidogenesis.
• ACTH activates phosphogluconate dehydrogenase enzyme to provide ample
supply of NADPH.
Metabolic Functions of Glucocorticoids

• Glucocorticoids are antagonistic to insulin in carbohydrate metabolism.
They increase blood glucose concentration (hyperglycemia) through the follow-
ing activities as:
–– ↓ Uptake of glucose by peripheral tissues
Glucocorticoids inhibit activity of glucose transporters. It decreases transport
of glucose into muscles, adipose tissues. Concentration of glucose is increased
in plasma.
–– ↓ Utilization of glucose by peripheral tissues
Glucocorticoids decrease utilization of glucose in peripheral tissues. It dimin-
ishes glycolysis.
Therefore, glucocorticoids exhibit carbohydrate catabolic effect in

peripheral tissues.
–– ↑ Gluconeogenesis in the liver
It promotes formation of pyruvate carboxylase and fructose-1,6-bisphosphate
enzymes in the liver. It increases availability of glucogenic amino acids in the
liver.
Overall, glucocorticoids enhance gluconeogenesis in the liver.
Glucocorticoids stimulate formation of glycogen synthase enzyme. So it pro-
motes glycogen synthesis in the liver.
Therefore, glucocorticoids exhibit carbohydrate anabolic effect in liver.
Glucocorticoids Have Lipolytic Effect
Glucocorticoids activates tissue lipase enzyme. They stimulate lipolysis of tri-
glycerides in adipose tissues. Glucocorticoids promote mobilization of lipids
and increase free fatty acid concentration in plasma.
Glucocorticoids have both protein anabolic and catabolic effects.
–– Protein Catabolic Effect
In peripheral tissues, glucocorticoids increase breakdown of proteins and
diminish protein synthesis. They increase concentration of amino acids in
plasma.
–– Protein Anabolic Effect
In the liver, glucocorticoids promote synthesis of proteins by the following
activities as:
↑ Transcription of mRNA in hepatocytes
↑ Uptake of amino acids by hepatocytes
Promotes protein synthesis
Conclusively, glucocorticoids induce negative nitrogen balance on protein

metabolism.
Peripheral tissues Liver
Glucocorticoids induce catabolism Glucocorticoids induce anabolism
• ↓ Glucose uptake and utilization • ↑ Gluconeogenesis
• ↑ Glycogenesis
• ↑ Proteolysis • ↑ Protein synthesis
• ↓ Protein synthesis
• ↑ Free amino acids in plasma
• ↑ Lipolysis of triglycerides
• ↑ Free fatty acids in plasma
• Anti-inflammatory Effect
Endogenous cortisol in blood circulation does not have anti-inflammatory effect.
Therapeutic dose of cortisol induces potent anti-inflammatory effect through
the following activities as:
–– ↓ Release of pro-inflammatory cytokines
260 10 Hormones
Glucocorticoids suppress express of genes which are responsible for synthe-

sis of pro-inflammatory cytokines and key enzymes implicated in initiation
and continuation of inflammatory response in the body of host.
Glucocorticoids inhibit release of pro-inflammatory cytokines like TNF-α and
IL-1.
–– ↓ Capillary oermeability
Glucocorticoids reduce capillary permeability. Therefore, emigration of leu-
cocytes, especially neutrophils, is inhibited at the site of injury.
–– ↓ Activity of kallikrein
Kallikrein is a proteolytic enzyme for formation of bradykinin from kinino-
gen. Bradykinin mediates inflammation. Glucocorticoids inhibit synthesis of
bradykinin.
–– ↓ Proliferation of leukocytes at site ofiInjury
Glucocorticoids reduce proliferation of leukocytes like eosinophils, mono-
cytes, and neutrophils at site of injury.
–– ↓ Proliferation of fibroblasts
Glucocorticoids reduce proliferation of fibroblasts at site of injury. They
inhibit formation of collagen fibers.
–– ↓ Activity of phospholipase A2
Glucocorticoids inhibit lipolytic activity of phospholipase A2. They reduced
synthesis of prostacyclin, prostaglandin, thromboxane, and leukotrienes.
These endogenous substances are involved in mediation and maintenance of
inflammation.
Therapeutic cortisol is widely utilized in management of chronic inflammatory
disorders like multiple sclerosis, inflammatory bowel disease, rheumatoid
arthritis, and psoriasis.
• Anti-allergic Effect
Therapeutic glucocorticoids have anti-allergic effect through the following activ-
ities as:
–– Glucocorticoids inhibit proliferation of mast cells. They stabilize mast cells
and reduce degranulation of mast cells.
–– They prevent vasodilation by inhibiting release of histamine and bradykinin
from granules of mast cells.
–– They reduce capillary permeability.
Therapeutic cortisol and hydrocortisone that are used in treatment of anaphylaxis
(immediate hypersensitivity reaction, life-threatening) have profound anti-allergy
effect.
• Immunosuppressive Effect
Therapeutic glucocorticoids have immune-suppressive effect in viral, bacterial,
protozoan, and fungal diseases. They are widely used after organ transplantation
to minimize graft failure.
Glucocorticoids suppress proliferation of lymphocytes in the thymus, spleen,
and lymphoid tissues. They reduce the number of circulating lymphocytes.
• Effect on Gastrointestinal Secretions
Therapeutic glucocorticoids induce hypersecretion of gastric glands and pancre-

atic glands. The synthesis of HCl and pepsin is increased in the stomach. The
secretion of trypsin is enhanced in pancreatic juice.
Prolonged administration of glucocorticoids is associated with high predis-
position to peptic ulcer.
• Effect on the Bone
Therapeutic glucocorticoids suppress protein synthesis and organic bone matrix
formation. They prompt demineralization in bones. Prolonged use of glucocorti-
coids result into osteoporosis.
10.14.2 Mineralocorticoids
Mineralocorticoids are steroidal hormones which are chiefly concerned with

regulation of mineral metabolism and water balance.
Types of Mineralocorticoids
• Aldosterone
It is the principal mineralocorticoid hormone. It is a C21 steroid. It contains a
hydroxyl group at C11 position and an aldehyde group at C18 position as in
Fig. 10.13.
• 11-Deoxycorticosterone (DOC)
This is a weak mineralocorticoid. It produces effects similar to the effects of
aldosterone. It is synthesized in minute quantity.
• Corticosterone
• 11-Deoxycortisol
Biosynthesis of Mineralocorticoids
Mineralocorticoids are synthesized by cells of zona glomerulosa in adrenal cor-
tex. This zone has 18-hydroxylase enzyme and 18-hydroxysteroid dehydrogenase
enzyme which are essential for synthesis of mineralocorticoids. These enzymes are
absent in other two zones of adrenal cortex. Biosynthesis is described in follwoing
steps as:

Cholesterol is the precursor for synthesis of corticosteroid hormones in adrenal
cortex. Cholesterol is present in cytosol of cells of adrenal cortex. Cholesterol
transports from outer mitochondrial membrane to inner mitochondrial mem-
brane and is the rate-limiting step in synthesis of hormones.
Intracellular transport of cholesterol is mediated by a transport protein located in
adrenal cortex cells. It is called as steroidogenic acute regulatory protein
(StAR). This transport protein transfers cholesterol to inner mitochondrial mem-
brane as in Fig. 10.16.
262 10 Hormones

Inner mitochondrial membrane contains cytochrome-P 450 side-chain cleavage
enzyme (cytochrome P450 SCC).
Cytochrome P450 SCC brings about two hydroxylations of cholesterol side chain
at C22 and C20 positions. First hydroxylation forms 22R-hydroxycholesterol. It
Cytochrome P450 SCC requires molecular oxygen and reducing equivalents
Cytochrome P450 SCC enzyme splits linkage between C20 and C22 in side chain of
cholesterol to form pregnenolone and isocaproic aldehyde.
• Conversion of Pregnenolone into Progesterone
Pregnenolone is translocated to smooth endoplasmic reticulum. It is converted
into progesterone by 3-beta-hydroxysteroid dehydrogenase enzyme.
• Hydroxylation of Progesterone into 11-Deoxycorticosterone
Progesterone is hydroxylated to form 11-deoxycorticosterone. Reaction is cata-
lyzed by 21-hydroxylase.
• Conversion of 11-Deoxycorticosterone into Corticosterone
The 11-deoxycorticosterone is transported to inner mitochondrial membrane. It
is converted into corticosterone by 11-beta-hydroxylase.
• Hydroxylation of Corticosterone into 18-Hydroxycorticosterone
Corticosterone undergoes hydroxylation to form 18-hydroxycorticosterone.
Reaction is catalyzed by 18-hydroxylase enzyme.
• Formation of Aldosterone
The 18-hydroxycorticosterone undergoes oxidation to form aldosterone.
Reaction is catalyzed by 18-hydroxysteroid dehydrogenase.
• Forms of Aldosterone
It exists in blood circulation in two forms as:
–– Aldehyde form of aldosterone
Aldosterone contains an aldehyde group at C18 position in D ring as in Fig. 10.15.
–– Hemiacetal form of aldosterone
Aldosterone contains hemiacetal group at C11 position in C ring.
Metabolic Functions of Aldosterone
• Renal Reabsorption of Sodium Ions

Aldosterone acts on distal convoluted tubules and collecting tubules and induces
reabsorption of sodium ions from glomerular filtrate.
Aldosterone is highly important hormone for the regulation of sodium ions in
plasma and extracellular fluid (Fig. 10.16).
• Renal Reabsorption of Chloride Ions
Aldosterone also brings about reabsorption of chloride ions from filtrate. This
function of aldosterone is secondary to sodium reabsorption.
• Renal Excretion of Potassium Ions
Aldosterone induces excretion of potassium ions in urine. Every potassium ion is
exchanged with sodium ion.
Fig. 10.15 ■ H CH2– OH
O C C O
OH
11 18
H3C
C11 Hydroxyl Group (OH)

C18 Aldehyde Group (CHO)
O
Aldosterone
Cholesterol
Precursor to
aldosterone
Pregnenolone
O2
11 – Deoxycorticosterone
Dehydrogenase
NAD+ +
NADPH2 NADP+ NADPH2
Isomerase
11 – Beta –
O2 Hydroxylase
Progesterone NADP+
21 – Hydroxylase
18 – Hydroxy 18 – Hydroxylase
Corticosterone
corticosterone
Dehydrogenase
NADPH2 O2 NADP+
Aldosterone
Fig. 10.16 Biosynthesis of aldosterone

264 10 Hormones
• Regulation of Acid-Base Balance

Aldosterone promotes reabsorption of sodium ions from glomerular filtrate.
Reabsorption of each sodium ion is exchanged with tubular secretion of H+ ion
in filtrate. This function helps to maintain pH (hydrogen ion) of plasma and body
fluid.
Within the renal tubule, each reabsorbed sodium ion is exported to plasma in
association with one HCO3_ion. Therefore, NaHCO3 enters plasma, and it repre-
sents alkali reserve of the body.
These functions help to maintain acid-base balance of the body. The ↑ in secre-
tion of aldosterone is manifested as ↑ in plasma bicarbonate level. It is termed
as alkalosis, whereas ↓ in secretion of aldosterone, it is characterized as
↓plasma bicarbonate level and is termed as acidosis.
• Regulation of Blood Volume
Aldosterone promotes reabsorption of sodium ions, chloride ions, and bicarbon-
ate ions. It results in increased plasma osmolarity.
It is followed by increase in secretion of ADH from posterior pituitary gland. It
induces proportionate increase in reabsorption of water by renal tubules from
glomerular filtrate.
Overall, aldosterone maintains normal blood volume.
• Regulation of Extracellular Fluid Volume
Aldosterone also results into rise in osmolarity of extracellular fluid. It is due to
reabsorption of sodium and chloride ions.
The ↑ in electrolyte concentration of ECF is associated with increase in renal
reabsorption of water.
Hyperosmolarity of ECF stimulates the thirst center. Intake of water increases.
Overall, volume of extracellular fluid is increased.
• Effect on Exocrine Secretions
Aldosterone limits excretion of electrolytes in exocrine secretions. It helps to
conserve electrolytes in salivary, gastric, and intestinal glands.
Normal Serum Value

Cortisol
It is 15–70 μmol/L.
Aldosterone
It is 2–10 ng/dl.
10.14.3 Adrenal Medulla Hormones
Adrenal medullary hormones are called as catecholamines. They contain a cate-

chol ring (a benzene ring to which one hydroxyl group at C1 and other hydroxyl
group at C2 positions are linked) and amino group containing side chain.
Therefore, catecholamines are monoamines.
Types of Hormones
• Adrenaline (epinephrine)
• Noradrenaline (norepinephrine)
Biosynthesis
Catecholamines are synthesized in adrenal medulla and sympathetic neurons in cen-
tral nervous system.
Steps in Biosynthesis
• Conversion of Tyrosine into Dihydroxyphenylalanine (DOPA)

Tyrosine is precursor amino acid in synthesis of catecholamines. Tyrosine under-
goes hydroxylation to form 3,4-dihydroxyphenylalanine. Reaction is catalyzed
by tyrosine hydroxylase enzyme.
Reaction requires molecular oxygen. Biopterin (chemically related to folate) acts
as coenzyme in the reaction. Its biologically active form is called tetrahydrobi-
opterin that takes part in the reaction. It is oxidized into dihydrobiopterin during
hydroxylation reaction. Dihydrobiopterin reductase enzyme again reduces dihy-
drobiopterin into tetrahydrobiopterin utilizing NADPH as reducing equivalents.
• Formation of Dopamine
3,4-Dihydroxyphenylalanine undergoes decarboxylation to form dopamine.
Reaction is catalyzed by aromatic amino acid decarboxylase enzyme.
• Formation of Norepinephrine
Dopamine undergoes hydroxylation to form norepinephrine. Reaction is cata-
lyzed by dopamine beta-hydroxylase enzyme. Ascorbate acts as cofactor, and it
is oxidized into dehydroascorbate in hydroxylation reaction.
• Formation of Epinephrine
Norepinephrine is methylated to form epinephrine. Reaction is catalyzed by
phenylethanolamine N-methyltransferase enzyme. S-adenosyl methionine acts
as a methyl group donor, and it is converted into S-adenosyl homocysteine in
methylation reaction.
Storage
Epinephrine and norepinephrine are stored in chromaffin granules in the adrenal
medulla. These catecholamines are present in form of granules with size of 0.5 μ.
These hormones are released into blood circulation.
Normal Serum Value

Epinephrine
It is < 50 nmol/L.
Norepinephrine
It is < 2 nm/L.
266 10 Hormones
Metabolic Functions

Adrenaline and noradrenaline promote hydrolysis of glycogen and favor increase
in blood glucose concentration (hyperglycemia). These hormones produce
hyperglycemic effect through the following activities:
In the liver, adrenaline attaches with beta-adrenergic receptors located on cell
membrane of hepatic cells. It enhances concentration of cAMP in cytosol of
hepatocytes. Adrenaline activates phosphorylase b into phosphorylase a. This
enzyme is responsible for glycogenolysis in the liver. These hormones also stim-
ulate gluconeogenesis in the liver.
Overall, adrenaline and noradrenaline increase release of glucose from the liver
into blood circulation.
In skeletal muscles, adrenaline stimulates glycogenolysis through beta-receptor-
induced cAMP concentration.
The glycogenolytic effect of adrenaline is almost similar to that of glucagon in
the liver.
Adrenaline activates glycolysis in muscle fibers. It increases plasma lactate level.
Adrenaline and noradrenaline activate tissue lipase enzyme and promote lipoly-
sis of triglycerides in adipose tissues. These hormones increase transport of free
fatty acids into blood circulation. They increase plasma fatty acid
concentration.
• Effect on BMR
Adrenaline and noradrenaline increase consumption of oxygen and heat produc-
tion in the body. They increase BMR.
10.15 Hormones of Gonads
Gonadal hormones are steroidal in nature. They are derived from cholesterol.
They are characterized by the presence of sterane nucleus.
Gonadal hormones or sex hormones are secreted by the testes, ovary, corpus
luteum, placenta, and adrenal cortex.
Types of Sex Hormones

The three types of sex hormones are as follows:
• Androgens
Androgens are male sex hormones. They are C19 steroids. They contain methyl
groups at C10 and C13 positions.
Examples:
Testosterone, dihydrotestosterone, androstenediol, androstenedione, and
dehydroepiandrosterone (DHEA)
• Estrogens
Estrogens are female sex hormones. They are C18 steroids.
10.15 Hormones of Gonads 267
Examples:
Estrone, estriol, estetrol, and estradiol
• Progestogens
Progestogens are progestational hormones. They are C21 steroids.
Example:
Progesterone
10.15.1 Androgens
They are male sex hormones. They are highly important in the development of sec-
ondary sex characters in males. Androgens are secreted by testes.
A small fraction of androgens is found in females. They are present in circu-
lation. They are produced by conversion of androstenedione into testosterone
in peripheral tissues.
Biosynthesis
Androgens are synthesized by Leydig cells and zona reticularis in the adrenal cortex.
Steps in Biosynthesis of Androgens

Intracellular Transport of Cholesterol
–– Cholesterol is the precursor for synthesis of androgens. Cholesterol is present
in cytosol of Leydig cells in testes. Transport of cholesterol from outer mito-
chondrial membrane to inner mitochondrial membrane is the rate-limiting
step in synthesis of androgens.
–– Intracellular transport of cholesterol is mediated by a transport protein located
in Leydig cells. It is called as steroidogenic acute regulatory protein (StAR).
This transport protein transfers cholesterol to inner mitochondrial membrane.
Formation of Pregnenolone
–– Inner mitochondrial membrane contains cytochrome-P 450 side-chain cleav-
age enzyme (cytochrome P450 SCC).
–– Cytochrome P450 SCC brings about two hydroxylations of cholesterol side chain at
–– Cytochrome P450 SCC requires molecular oxygen and reducing equivalents
–– Cytochrome P450 SCC enzyme splits linkage between C20 and C22 in side
chain of cholesterol to form pregnenolone and isocaproic aldehyde.
–– Pregnenolone is translocated to smooth endoplasmic reticulum (SER).
• Conversion of Pregnenolone into Testosterone
It follows two pathways as shown below:
–– ∆4 Pathway or progesterone pathway
–– ∆5 Pathway or 17-alpha-hyroxypregnenolone pathway
268 10 Hormones
Steps in ∆4 Pathway or Progesterone Pathway

In SER, pregnenolone is changed into progesterone. Reaction is catalyzed by
3-beta-hydroxysteroid dehydrogenase enzyme.
• Hydroxylation of Progesterone
Progesterone undergoes hydroxylation to form 17-alpha-hydroxyprogesterone.
Reaction is catalyzed by 17-alpha-hydroxylase enzyme. The enzyme lyase splits
side chain of 17-alpha-hydroxyprogesterone to form androstenedione.
• Reduction of Androstenedione
Androstenedione undergoes reduction to form testosterone. Reaction is cata-
lyzed by 17-beta-hydroxysteroid dehydrogenase enzyme.
Steps in ∆5 Pathway or 17-alpha-Hyroxypregnenolone Pathway
• Conversion of Pregnenolone into 17-alpha-Hydroxyprogesterone

In SER, pregnenolone undergoes hydroxylation to form 17-alpha-hydroxypro-
gesterone. Reaction is catalyzed by 17-alpha-hydroxylase enzyme.
• Conversion of 17-alpha-Hydroxyprogesterone into Dehydroepiandrosterone
(DHEA)
Side chain of 17-alpha-hydroxyprogesterone is cleavaged by enzyme lyase to
form dehydroepiandrosterone.
• Reduction of Dehydroepiandrosterone
Dehydroepiandrosterone undergoes reduction to form androstenediol by enzyme
17-beta-hydroxysteroid dehydrogenase enzyme (Fig. 10.17).
Androstenediol undergoes isomerization to form testosterone as in Figs. 10.11
and 10.18.
• Dihydrotestosterone (DHT) is a biologically active form of testosterone.
• It is produced in peripheral tissues by reduction of testosterone catalyzed by
5-alpha-reductase enzyme.
–– Testosterone may be considered as prohormone; however, both hormones
exert metabolic effects on the human body.
Fig. 10.17 Testosterone H3C18 OH

17
13
H3C19
10 Methyl groups at C10,C13

C19 – Carbon skeleton
O
Cholesterol
Pregnenolone
Dehydrogenase
+
Isomerase
Progesterone 17 – ∝– OH – Progesterone
n – ∝ – Hydroxylase
Lyase
enzyme
Androstenedione
Dehydrogenase
Reductase
Dihydro testosterone Testosterone
Fig. 10.18 Biosynthesis of andrgens
Transport of Androgens
Testosterone and DHT bind to sex hormone-binding globulin in plasma. They are
distributed to target tissues.
Metabolic Functions
Testosterone and dihydrotestosterone (DHT) are metabolically active hormones.
They have the following effects on metabolism.

Testosterone and DHT are prominently protein anabolic hormones.
These hormones increase mRNA transcription and protein synthesis in accessory
glands (seminal vesicle, prostate gland) of males, bones, and skeletal muscles.
These hormones are involved in the growth of musculoskeletal system in males
during puberty.
These hormones reduce urinary excretion of nonprotein nitrogen. They exert
positive nitrogen balance. These hormones increase muscle mass and body
weight.
Androgens promote rise in low-density lipoproteins in plasma and decrease in
high-density lipoproteins in plasma. These hormones are implicated in high pre-
disposition to coronary artery disease in males than females. After menopause,
plasma level of estrogen is reduced, and women are equally inclined to heart
diseases.
270 10 Hormones
Androgens activate conversion of D-glucose into D-fructose in seminal vesicles.

These hormones activate synthesis of aldolase enzyme and promote glycolysis
and TCA cycle in seminal vesicle.
• Effect on Mineral Metabolism
Androgens reduce urinary excretion of nonprotein nitrogen (NPN). These hor-
mones limit urinary excretion of sodium ion, chloride ion, sulfate ion, phosphate,
and potassium. These hormones promote retention of NPN and other ions in
body tissues. This function prevents elevation of plasma NPN concentration.
• Effect on Bone Growth
Androgens promote mineralization of bones. These hormones increase forma-
tion of organic bone matrix and deposition of calcium and phosphate in bones
before closure of epiphyseal plates in long bones.
10.15.2 Female Sex Hormones
These hormones are steroidal in nature. They are synthesized from cholesterol.
Female sex hormones belong to two categories as:
Estrogens
• Estrogens are synthesized from ovarian follicles.
Progestogen
• Progesterone is synthesized by corpus luteum.
Estrogens
Estrogens are the predominant female sex hormones secreted by ovaries. These hor-
mones are involved in growth of female reproductive organs. They are responsible
for appearance of secondary sex characters in females.
Chemistry and Forms of Estrogens

Estrogens are found in the following three forms as in Fig. 10.19:
• Estriol
• β-estradiol
• Estrone
Metabolically active form of estrogen is β-estradiol. It is found in blood circula-

tion. β-estradiol and estrone are interconvertible.
β-estradiol is ten times more biologically active than estrone.
Estriol is an important metabolite of estrone. It is found in urine during
pregnancy.
OH
H3C18
OH
3
HO
Estriol
OH
H3C O
HO
Estradiol
3
HO
Estrone
Estrogens
CHO
C O
O
Progesterone
Fig. 10.19 Structure of female sex hormones
Structurally
• Estrogens are C18 steroids. They have sterane nucleus (nonlinear arrangement
A, B, C, and D rings).
• Ring A has aromatic character. These hormones contain –OH group at C3
position.
Biosynthesis
Estrogens are synthesized in graafian follicles and corpus luteum in the ovaries.
272 10 Hormones
Steps in Biosynthesis

Intracellular Transport of Cholesterol
–– Cholesterol is the precursor for synthesis of androgens. Cholesterol is present
in cytosol of theca interna and granulose cells in ovaries. Transport of choles-
terol from outer mitochondrial membrane to inner mitochondrial membrane
is the rate-limiting step in the synthesis of estrogens.
–– Intracellular transport of cholesterol is mediated by a transport protein located in
theca interna cells. It is called as steroidogenic acute regulatory protein (StAR).
This transport protein transfers cholesterol to inner mitochondrial membrane.
–– The inner mitochondrial membrane contains cytochrome-P 450 side-chain
cleavage enzyme (cytochrome P450 SCC).
–– Cytochrome P450 SCC brings about two hydroxylations of cholesterol side
chain at C22 andC20 positions. First hydroxylation forms
22R-hydroxycholesterol. It undergoes second hydroxylation to form
20-alpha-22R-dihydroxycholesterol.
–– Pregnenolone is translocated to smooth endoplasmic reticulum (SER of
theca interna cells).
–– In SER, pregnenolone is changed into progesterone. Reaction is catalyzed by
–– Hydroxylation of Progesterone
–– Progesterone undergoes hydroxylation to form 17-alpha-hydroxyprogesterone.
Reaction is catalyzed by 17-alpha-hydroxylase enzyme. The enzyme lyase
splits side chain of 17-alpha-hydroxyprogesterone to form
androstenedione.
–– Androstenedione is converted into testosterone by aromatic acid dehydroge-
nase enzyme.
–– Androstenedione and testosterone are precursor molecules for biosyn-
thesis of estrogens.
• Conversion of Androstenedione into Estrone
–– Androstenedione undergoes three successive hydroxylations at C19 posi-
tion, and hydroxylations are accompanied by elimination of C18 methyl group
to form estrone.
–– Reaction is catalyzed by aromatase enzyme. It belongs to the family of cyto-
chrome P450 enzymes (monooxygenase enzymes inducing hydroxylation dur-
ing steroidogenesis). Enzyme requires molecular oxygen and NADPH.
–– Estrone undergoes further hydroxylation to form 16-alpha-OH estrone cata-
lyzed by 16-alpha-hydroxylase enzyme.
–– 16-Alpha-OH estrone is reduced into estriol by reductase enzyme.

• Conversion of Testosterone into beta-Estradiol
Cholesterol
Pregnenolone
Progesterone
Androstenedione Testosterone
Aromatase
+
Dehydrogenase
NADPH2
NADPH2 Aromatase
O2
O2
NADP+
NADP+
Estrone Estradiol
Dehydrogenase
NADPH2
O2 16 – ∝ – Hydroxylase
NADP+
Reductase
16 – ∝ – Hydroxy estrons Estriol
Fig. 10.20 Biosynthesis of estrogens

274 10 Hormones
–– Testosterone undergoes three successive hydroxylations at C19 position, and

hydroxylations are accompanied by elimination of C18 methyl group to form
beta-estradiol as in Fig. 10.20.
–– Extra-graafian follicular tissues like the liver, skeletal muscles, and adi-
pose tissues can synthesize beta-estradiol and estrone utilizing circulat-
ing testosterone and androstenedione.
–– β-Estradiol is a predominant circualting estrogen.
Metabolic Functions

Estrogens exert protein anabolic effect.
These hormones activate mRNA transcription and protein synthesis in the bones,
uterus, and vaginal epithelium. Protein synthesis helps to prepare the uterus for
pregnancy.
Estrogens promote synthesis of triglycerides in adipose tissues (lipogenesis).
Estrogens are responsible for higher proportion of body fat.
Estrogens increase concentration of high-density lipoprotein in plasma. These
hormones decrease low-density lipoprotein fraction in plasma. Estrogens
decrease plasma total cholesterol level. Total cholesterol-lowering effect of
estrogens is antagonistic to testosterone. As a result, women have lower preva-
lence of coronary artery disease than men.
Estrogens stimulate synthesis of glycogen in cells of endometrium and vaginal
epithelium. These hormones promote glycolysis and generation of lactic acid in
the vaginal epithelium. Rise in lactate content of vagina helps to maintain low pH
(4.0) in vagina.
• Effect on Transhydrogenase Enzyme
Transhydrogenase enzyme catalyzes the following reaction as:
NADPH2 + NAD+ ⇔ NADP+ + NADH2
Estrogens activate transhydrogenase activity. It is necessary for oxidation of
NADPH2 and generation of NADH, which passes through electron transport sys-
tem to produce ATP.
After menopause, estrogen level is decreased. Transhydrogenase activity is
reduced. It results into utilization of NADPH in synthesis of triglycerides in
adipose tissues. It manifests as postmenopausal obesity in women.
• Effect on Bones
Estrogens promote protein synthesis in bones. These hormones activate organic
osteoid formation. Estrogens increase retention of calcium and phosphate ions in
the bone and favor mineralization of bones. Estrogens promote growth of bones
in females during puberty before closure of epiphyseal plates.
After menopause, plasma level of estrogens is reduced. It prompts demineralization
of bones. Menopause is associated with higher chances of osteoporosis in women.
• Estrogens Priming Effect
Estrogens activate gene expression for progesterone receptors on mammary

glands and the uterus. These hormones increase proliferation of progesterone
receptors. It is called priming effect of estrogen. Receptors are essential for effect
of progesterone on target tissues.
• Effect on Mineral Metabolism
Estradiol has a retentive effect on mineral metabolism. It retains sodium, chlo-
ride, calcium, and phosphate ions in body tissues. It exerts water retention effect.
Progesterone
Progesterone is a progestogen (steroidal hormone) which is synthesized by corpus
luteum in the ovary and placenta. It is also called as luteinizing hormone or gestagen.
Progesterone is additionally synthesized by adrenal cortex and testes.
Chemistry
Progesterone is a C21 steroid hormone. It contains methyl group at C10 and C13
positions.
Biosynthesis
Progesterone is synthesized as an intermediate metabolite in the steroidogene-
sis pathway of adrenal cortical hormones, androgens, and estrogens.
Cholesterol is a precursor molecule for biosynthesis of progesterone.

–– Cholesterol is the precursor for synthesis of progesterone. Cholesterol is pres-
ent in cytosol of cells. Transport of cholesterol from outer mitochondrial
membrane to inner mitochondrial membrane is the rate-limiting step in syn-
thesis of progesterone.
–– Intracellular transport of cholesterol is mediated by a transport protein located
in cells. It is called as steroidogenic acute regulatory protein (StAR). This
transport protein transfers cholesterol to inner mitochondrial membrane.
–– Inner mitochondrial membrane contains cytochrome-P 450 side-chain cleav-
age enzyme (cytochrome P450 SCC).
–– Cytochrome P450 SCC brings about two hydroxylations of cholesterol side chain at
–– Pregnenolone is translocated to smooth endoplasmic reticulum (SER).
In SER, pregnenolone is changed into progesterone. Reaction is catalyzed by
276 10 Hormones
Metabolic Functions
Preparation of Uterus for Implantation
• Progesterone increases synthesis of glycogen in endometrial cells. It increases lipid

store of endometrium. It boosts protein synthesis and activates proliferation of
endometrial cells. It increases proliferation of glandular tissues in endometrium.
• Overall, progesterone prepares uterus for implantation after fertilization.
• Progesterone is a pregnancy hormone.
• Progesterone inhibits secretion of luteinizing hormone (LH) from anterior
pituitary. It suppresses ovulation, and
• progesterone helps in the development of the breast and mammary glands.
It promotes development and growth of alveolar ducts in mammary glands. It
prepares mammary glands for lactation after child birth.
• Progesterone has thermogenic effect. It increases body temperature during
ovulation.
Placental Hormones
The placenta is a connecting organ between a growing fetus and the wall of uterus
in a mother’s body. It ensures gaseous exchange, blood supply, and waste elimina-
tion. Placenta also serves as endocrine organ.
Placenta secretes two hormones as follows:
Human chorionic gonadotropin hormone (HCG)
Chemistry
HCG is a glycoprotein.
Protein part of hormone is a heterodimer. It is made up of two subunits as:
• Alpha-chain
It is composed of 92 amino acid residues. This subunit is identical to LH, TSH,
and FSH hormones from the pituitary gland.
• Beta-chain
It is made up of 145 amino acid residues.
Carbohydrate part of hormone is composed of oligosaccharides which are

attached to asparagine and serine residues.
Biosynthesis
It is synthesized by syncytiotrophoblasts in the placenta. Syncytiotrophoblasts con-
stitute the outermost layer of cells which invade the wall of the uterus.
Metabolic Functions
• Maintenance of Corpus Luteum

HCG hormone acts on LHCG receptors in the ovary. It induces maintenance of
corpus luteum in the first trimester of pregnancy. It regulates secretion of proges-
terone through corpus luteum.
10.16 Applied Biochemistry 277
• Secretion of Testosterone
HCG stimulates growth of Leydig cells in fetal embryo. It induces secretion of
testosterone from fetal testes.
Normal Serum Value

Free testosterone
• Its value in males is 8–30 ng/100 mL and in females is up to 2 ng/100 mL.
Total Testosterone
• Its value in males is 280–1000 ng/100 mL and in females is 20–75 ng/100 mL.
Progesterone
• Its value in males is <1 ng/mL and in females during pregnancy is between

10 and 80 ng/mL.
Role of Estrogen and Progesterone in Inflammatory Gingivitis

During pregnancy, serum levels of estrogen and progesterone are elevated. These
hormones have inflammatory effect on the gingiva and periodontia.
• Elevated levels of progesterone and estrogen promote growth of microflora in

oral cavity. These hormones induce pro-inflammatory cytokines.
• ↑Progesterone probably alters proliferation of fibroblasts and collagen synthesis.
These changes aggravate inflammation in gingival and compromise repair in gin-
gival tissues in pregnancy.
• Additionally, iron deficiency coupled with folate deficiency in pregnancy further
retard healing of gingival tissues.
• Pregnancy-induced estrogen and progesterone level can cause gingivitis in 60%
of pregnant women. Poor oral hygiene is a chief predisposing factor to gingivitis
and periodontitis in association with increase in progesterone and estrogen levels
in pregnancy in women.
• Furthermore, use of oral contraceptives is also associated with prevalence of
chronic gingivitis and periodontitis in women.
• It is advisable to keep good oral hygiene by proper tooth brushing and use of
antiplaque mouth wash. It helps to minimize proliferation of causative organisms
and control aggravation of already-existing hormonal-induced inflammation in
gingivae and periodontia.
278 10 Hormones
Suggested Readings
London
Astwood EB (1968) Recent progress in hormones research. Academic, New York
Korenberg A (1980) DNA replication. W. H. Freeman, New York
Zachariasen RD (1993) The effect of elevated ovarian hormones on periodontal health: oral con-
traceptives and pregnancy. Women Health 20(2):21–30
Vitamins
11
11.1 Definition
Vitamins may be defined as organic compounds that are essential in minute

amounts for the attainment of normal growth and maintenance of general health.
11.2 Important Facts About Vitamins
• In 1912, a biochemist named Casimir Funk extracted a water soluble compound

from rice bran and named it “vitamine.” He thought that it was an “amine” which
was “vital” for life.
• Later research showed that all compounds were not amines. It was proposed by
Jack Cecil Drummond that the suffix “e” should be dropped from vitamine.
• Vitamins are essential nutrients.
• Vitamins differ from macronutrients. They do not produce energy in the body.
They are required in minute amounts unlike macronutrients such as proteins,
lipids, and carbohydrates which are necessary in large amounts.
• Vitamins are micronutrients because they are supplemented in minute amounts.
They are essential for normal physiological functions. Micronutrients include
vitamins and minerals.
• Vitamins differ from hormones. Most of vitamins are supplemented with diet.
Hormones are synthesized in the body by endocrine glands.
• Vitamins differ from enzymes. Enzymes are protein in nature. Enzyme catalyzes
biochemical reactions. Vitamins may act as cofactor in enzymatic reactions.
11.3 Classification of Vitamins
Based on the solubility, vitamins are classified into two groups as follows:

https://doi.org/10.1007/978-981-13-1035-5_11
280 11 Vitamins
1. Fat soluble vitamins

• They are soluble in nonpolar organic solvents.
• Bile salts help in the absorption of fat soluble vitamins from the intestine.
• They are stored in the liver.
Types: Vitamins A, D, E, and K
2. Water soluble vitamins
• They are soluble in water.
• They are absorbed easily.
• They are excreted in urine.
Types: Vitamin C and vitamin B complex
11.4 Fat Soluble Vitamins
11.4.1 Vitamin A
Vitamin A includes unsaturated organic compounds essential for living organisms.
History
• In 1816, physiologist F. Magendie observed that poorly nourished dogs devel-
oped corneal ulcer.
• In 1880, N. Lunin demonstrated the presence of a compound in milk that was
vital for nutrition.
• In 1911, W. Stepp proved fat soluble nature of vital substance in milk.
• In 1911, Hopkins proved the presence of vital factor in trace amount in milk that
was essential for nutrition and health. In 1918, vital factor in milk was declared
as vitamin A.
• In 1932, Paul Karrer demonstrated chemical structure of vitamin A.
• 1937, H. Holmes and R. Corbet extracted and crystallized vitamin A.
Chemical Structure
1. Forms of Vitamin A
It exists in nature in three distinct forms:
• Retinol
–– It is called as vitamin A (alcohol). Retinol exists in two forms such as
vitamin A1 and vitamin A2.
–– Vitamin A1 is mainly present in animal sources. It has only one double
bond in β-ionone ring. It has higher biological activity (60%) than vitamin A2.
–– Vitamin A2 (3-dehydro-retinol) is present in the liver of fresh water
fish. It is has two double bonds in β-ionone ring. Its biological activity is
less than half (40%) of the biological activity of vitamin A1.
–– Neo-vitamin A is the stereoisomer of vitamin A1. Neo-vitamin A has 70%
of the biological activity of vitamin A1.
–– Retinol form of vitamin A is predominant in animal body tissues. It is
esterified with long-chain fatty acids into “retinyl ester.”
• Retinal
–– It is called vitamin A aldehyde. Retinal and retinol can be interconverted
by retinal reductase enzyme.
11.4 Fat Soluble Vitamins 281
• Retinoic acid
–– It is called vitamin A acid. It is formed by oxidation of retinal. Retinoic
acid is nonconvertible into retinal or retinol as in Fig. 11.1
All three forms of vitamin A are called “retinoids.” They are found exclu-
sively in animal tissues.
2 . Structure
• Vitamin A has β-Ionone ring (cyclohexenyl) in its structure. It is
2,6,6-trimethyl cyclohexenyl ring. The ring contains three methyl groups and
one double bond.
• Isoprenoid chain is attached to β-Ionone ring. The isoprenoid chain has four
double bonds, two methyl groups, and “R” group which is linked to terminal
carbon.
• “R” group can be alcohol, aldehyde, or an organic acid as in Fig. 11.1.
3. Carotenoids (Provitamin A)
• Vitamin A is derived from carotenoids. They are provitamin A.
• Carotenoids are called “tetraterpenoids.” They possess four “terpene” units
(C10) or eight isoprene units and are C40 compounds as in Fig. 11.1.
Carotenoids are pigmented unsaturated organic compounds synthesized by
plants, algae, bacteria, and fungi.
• These compounds have red, yellow, and orange colors. Carotenoids are
exclusively found in plant tissues.
• Carotenoids are classified into two categories of organic compounds. The
compounds which do not contain oxygen are called “carotene.” The other
category of compounds containing oxygen is called “xanthophyll.”
• Carotene is derived from the Latin word carota that means carrot. They
are pigmented unsaturated group of hydrocarbons. Their general molecular
formula is C40Hx. Carotenes are tetraterpenes. Carotene exists in alpha,
beta, and gamma forms.
• Beta-carotene is a deeply reddish orange pigmented compound found in
plants and fruits.
–– It has two beta-ionone rings at both ends of molecule.
–– Beta-carotene is the most prominent carotenoid with “provitamin A”
activity.
–– Beta-carotene molecule is cleavage by beta-carotene 15,15′ oxygenase
(dioxygenase) enzyme into two molecules of vitamin A.
• Alpha-carotene and gamma-carotene.
–– They possess single beta-ionone ring. They have mild (30–40%) provita-
min A activity. They yield only single molecule of vitamin A by beta-car-
otene 15,15′ oxygenase enzyme.
• Beta-cryptoxanthin is another source for provitamin A.
–– It has around 30–40% of provitamin A activity. It yield single molecule of
vitamin A. It is related to xanthophyll category of carotenoids. It is
found in fruits like oranges and tangerines and in human blood.
Beta-ionone ring is absent in other carotenoids. They do not have vitamin
A activity. Animals including humans have capability to convert beta-
ionone ring (retinyl group) of carotene into retinol.
282 11 Vitamins
Site of Oxidation
CH3 CH3 CH3 CH3

CH3 CH3 CH3
CH = CH – C = CH – CH = CH – C = CH – CH =CH – CH = C – CH = CH – CH = C – CH = CH
CH3 CH3 B- Carotene CH3
B-Ionone
Oxidation Ring
B – Carotene dioxygenase
CH3 CH3 CH3
CH = CH – C = CH – CH = CH – C = CH – CHO
CH3
CH3
All trans – retinal
Retinal isomerase
CH3 CH3
11
CH = CH – C = CH – CH
12
CH3 CH
CH3
H3C – C
CH
CHO
11 – CIS – Retinal
CH3 CH3 CH3 CH3
CHO
CH3
RETINAL
NADPH2
NADP+
RETINAL REDUCTASE
H2O
NAD+
CH3 CH3 CH3
CH = CH – C = CH – CH = CH – C = CH – CH2OH
CH3
CH3 NADH2
Retinol
oxidation
RETINAL DEHYDROGENASE
RETINOIC ACID
Fig. 11.1 Interconversion of forms of Vitamin A

Table 11.1 RDA of vitamin A Age group RDA for vitamin A (IU retinol)
Children 1500–2000 IU
Adults 2500–3000 IU
Pregnant 3000 IU
women
Lactation 4000 IU
Source: (NIH 2017)
Retinol (1 IU) = 0.3 mcg retinol activity equivalents
Beta-carotene (1 IU) = 0.6 mcg retinol activity
equivalents
ietary Sources of Vitamin A

D
1. Animal Sources
Animal sources of food contain “retinyl palmitate” as a form of vitamin A
Example: milk, cheese, egg yolk, butter, and cod-liver oil.
2. Plant Sources
Plant sources of food contain carotenoids, chiefly as beta-carotene and beta-
cryptoxanthin as provitamin A Example: carrots; green leafy vegetables like
spinach, amaranth, broccoli, dandelions green, mustard greens, and lettuce; and
fruits like papaya, mango, oranges, tangerines, and sweet potato.
Spirulina is Cyanobacteria (blue-green algae) and is a seafood. It is a rich
source of vitamin A.
ecommended Dietary Allowance of Vitamin A

R
Recommended Dietary Allowance (RDA) is “the average quantity of a nutrient
which is sufficient to fulfill physiological needs of almost all (97.5%) healthy
individuals.”
RDA was developed by a committee comprising Lydia J. Roberts, Hazel
Stiebeling, and Helen S. Mitchell during the Second World War, and the committee
was set up by the US National Academy of Sciences (Table 11.1).
bsorption, Transport, and Storage of Vitamin A

A
Site and Process of Absorption
• Intestinal mucosa is the main site for absorption of vitamin A.

• Food contains carotenoids and retinyl ester. Carotenoids are incorporated into
mixed micelles. Carotenoids are absorbed by passive diffusion.
• Retinyl ester is hydrolyzed into retinal by cholesterol ester hydrolase enzyme. It
occurs in lumen of small intestine. Retinal is reduced into retinol by retinal
reductase enzyme. It is incorporated into mixed micelles. Retinol absorption
occurs by active transport (carrier-mediated transport).
• Inside enterocytes, retinol is re-esterified with long-chain fatty acids. Carotenoids
(beta-carotene, alpha-carotene, and beta-cryptoxanthin) are metabolized by beta-
carotene dioxygenase enzyme into retinol. The re-esterified retinol is released
into lacteals as chylomicrons. Retinyl ester is transported to the liver as in
Fig. 11.2.
284 11 Vitamins
VITAMIN A
Brush border surface

Food
Enterocyte
β – Carotene β – Carotene
O2
Retinal
Retinylesters Reduction
ROD Cell
Retinol
FFA
FFA Retino [Free Fatty Acid] Retinyl ester
All-Trans-Retinol
All-Trans-Retinal
Chylomicrons
Distribured
II-Cis-Retinal
Lacteals
circulation
Nucleus Blood
circulation
All-Tras-Retinol Liver
Retinol
+ Retinyl
Retinol binding palmitate
(RBP) protein
Storage form of Vit. A
Retinol-RBP
complex
RBP
Retinol Target cell
Retinic acid
Nuclear Retinol Nucleus

receptor
Transcription
Regulate cell function
Fig. 11.2 Vitamin A absorption, transport and regulatory function
Storage
• Vitamin A is stored in the liver as “retinyl palmitate.” Around 95% of vitamin A

is stored in the liver.
• It is released as retinol in blood circulation. Its normal plasma level is 18–60 μg
per 100 ml of blood.
Transport of Vitamin A in the Blood
• Retinol is transported in the blood in bound state. It binds with “retinol binding
protein” (RBP) in plasma and forms complex.
• Retinol-RBP complex circulates in the blood. It is carried to target cells. This
complex is attached to specific receptors on target cells. Retinol enters the
cell.
• Inside the cytoplasm, retinol binds to “retinoic acid binding protein,” and the
complex is carried to nuclear receptor on the surface of nuclear membrane. It
activates genes and controls cellular metabolism.
• Retinol acts similar to steroid hormone.
unctions of Vitamin A
F
Vitamin A has multiple functions. Its different forms have diverse functions. Retinal
is necessary for normal vision. Retinol acts as a steroidal hormone, and it regulates
protein synthesis. Retinol is necessary for cell differentiation and normal growth.
Retinoic acid is helpful in glycoprotein synthesis.
Diverse functions of vitamin A are described as follows:
Role of Vitamin A in Vision

Vitamin A contributes to structural integrity of photosensitive pigments of rods and
cones. It plays an important role in the “visual cycle” or “visual
phototransduction.”
Visual cycle or “visual phototransduction” is an elaborate neurochemical,
enzyme-controlled, cyclic process in which light energy is converted into elec-
trical energy (nerve impulse) by photoreceptors.
It was “George Wald” who described visual cycle in 1968. The visual cycle is
called “Wald’s visual cycle” or “rhodopsin cycle.”
Types of Photoreceptors in Human Eye

• The retina of the human eye contains two types of photoreceptors such as rods
and cones.
• Rods contain photosensitive pigment called “rhodopsin.” It is also called “visual
purple.” This word is derived from Greek words “rhodon” for rose due to pink
color and “opsis” for sight. Rods appear red due to rhodopsin.
• Rods have very high sensitivity to light, and they are helpful in vision in dim
light. This type of vision is called “night vision” or “scotopic vision.”
• Cones contain photosensitive pigment called “conopsin.”
–– Depending on type of conopsin, cones have three types.
–– Cones with “porphyropsin” are sensitive to red color at a wavelength of
665 nm.
–– Cones with “iodopsin” are sensitive to green light at a wavelength of 535 nm.
–– Cones with “cyanopsin” are sensitive to blue light at a wavelength of 445 nm.
–– Conopsin is sensitive to bright light, and it is helpful in vision in bright light.
This type of vision is called “color vision” or “photopic vision.”
286 11 Vitamins
Elements of WALD’s Visual Cycle

Structure of Rhodopsin
• Rhodopsin is a light-sensitive conjugated protein. It is located within membra-

nous discs in rods. Rhodopsin is composed of a protein called “opsin” and a
prosthetic group “retinal.”
• Opsin is a G-protein-coupled receptor. It is embedded in phospholipid layer of
membranous discs. It has seven transmembrane helices. Opsin is not
photosensitive.
• Retinal is a photosensitive moiety in rhodopsin molecule. It is also called “reti-
nene1.” The rhodopsin contains 11-cis-retinal as an isomer of retinal. It is
enclosed within seven transmembrane helices of opsin. The 11-cis-retinal is
linked covalently to amino group (–NH2) group of lysine residue in opsin with
protonated Schiff’s base (–NH+〓CH–).
• Dietary vitamin A and carotenoids are necessary for biosynthesis of
11-cis-retinal.
Photochemical Transformation in Rhodopsin
• A photon of light falls on rhodopsin. Light energy is absorbed by 11-cis-retinal,

and it undergoes photoisomerization into all-trans-retinal.
• The all-trans-retinal induces a series of conformational changes in opsin mole-
cule. A number of intermediates are produced. The unstable intermediates are as
follows:
–– Photorhodopsin is produced within femtoseconds (10−15 s).
–– Bathorhodopsin is produced within picoseconds (10−12 s).
–– Lumirhodopsin.
–– Metarhodopsin I.
–– Metarhodopsin II (photoactivated rhodopsin) contains deprotonated
Schiff’s base which is linked to all-trans-retinal. Its color changes from red
to yellow. Metarhodopsin II initiates phototransduction.
• The metarhodopsin II activates G-protein located inside the cytosol. It splits into
scotopsin and all-trans-retinal as in Fig. 11.3.
Phototransduction
• The G- proteins are guanine nucleotide-bound proteins. They are considered

as “molecular switches” present inside the cytosol. They help to transmit signals
from a stimulus outside the cell membrane to inside the cytosol.
• In rods and cones, “transducin” is the G-protein. It is composed of α-subunit,
β-subunit, and γ-subunit. These subunits are linked to internal surface of cell
membrane of rods.
• Activated transducin is bound to GTP, and it undergoes hydrolysis into GDP
with release of inorganic phosphate as in Fig. 11.3.
VITAMIN A
Light
Rhodopsin Batho Rhodopsin
Opsin + II CIS RETINAL

Lumi Rhodopsin
In Meta Rhodopsin I
Darkness
with in
Retina Meta Rhodopsin II
Within
OPSIN
Retina
Within retina
Retinal Within
II – CIS – Retinal All Trans Retinal
Isomerase Retina in
Acohol dehydrogenase
Darkness
Enters
Blood circulation
NADH2
Reaches
NAD+
Liver
Reaches Retina
WALD’S Within liver

VISUAL CYCLE
All – Trans Retinal
Liver Retinal isomerase
II – CIS Retinol
Liver NAD+ Acohol

dehydrogenase
NADH2
Enters blood circulation
II – CIS Retinol
Fig. 11.3 Wald’s visual cycle

288 11 Vitamins
• Transducin enters the cytosol and activates cyclic GMP phosphodiesterase

(cGMP) enzyme.
• Activated cGMP phosphodiesterase enzyme cleavages phosphodiester bonds in
cGMP. The enzyme hydrolyzes cGMP into 5’ GMP.
• The cGMP is the second messenger of cell. It is present in the cytosol of cells.
It helps to regulate physiological activities of cell.
• Cell membranes of rods have sodium ion channels. They are cGMP-gated.
• In darkness, high concentration of cGMP in rods always keeps the sodium
channels in open state. The sodium ions enter inside the rods through sodium
channels.
• “Dark current.” It is the constant influx of sodium ions inside rods. It is because
the sodium influx is maintained only when the rods are not stimulated as in dark-
ness. Sodium influx is responsible for a resting membrane potential of −40 mV
in rods. This is a unique characteristic of rods. Otherwise, resting membrane
potential of other sensory cells in the body is between −70 mV and
−90 mV. Therefore, sodium influx always maintains a state of mild depolariza-
tion in rods.
• Depolarized rods release glutamate neurotransmitter. The bipolar and ganglionic
cells in retina do not generate action potential.
• In light, activated cGMP phosphodiesterase enzyme cleavages phosphodiester
bonds in cGMP. The enzyme hydrolyzes cGMP into 5′ GMP, and concentration
of cGMP in cytosol of rods is decreased.
• A decline in cGMP concentration in cytosol closes the sodium channels. The
influx of sodium ions is stopped. As a result, resting membrane potential in rods
increases to −70 mV. This rise in electrical potential in cell membrane of rods is
called “hyperpolarization.”
Hyperpolarization in Rods
• Hyperpolarization results in closure of voltage-gated calcium channels in rods.

The release of glutamate at rods-bipolar cell synapse is inhibited.
• Inhibition of glutamate depolarizes bipolar cells in the retina. They release neu-
rotransmitter at bipolar-ganglionic cell synapse. This leads to generation of
action potential in ganglionic cells.
• The impulse (action potential) from ganglionic cells reaches optic nerve. It is
transmitted to visual center in cerebral cortex by optic nerve as in Fig. 11.3.
Regeneration of Rhodopsin
Rhodopsin is regenerated in the retina and liver through the action of enzymes.
• In the retina
–– This process occurs in darkness. The all-trans-retinol undergoes isomeriza-
tion into 11-cis-retinal by the activity of retinal isomerase enzyme.
–– 11-cis-retinal recombines with opsin to form rhodopsin.
• In the liver
–– The all-trans-retinal enters into blood circulation and is transported to the
liver.
–– It is reduced to all-trans-retinol by alcohol dehydrogenase enzyme in the
liver. The reduction requires NAD+ coenzyme and zinc as cofactor.
–– The all-trans-retinol is isomerized into 11-cis-retinol by retinol isomerase
enzyme.
–– 11-Cis-retinol is oxidized into 11-cis-retinal by dehydrogenase enzyme in the
presence of coenzyme, NADH2.
–– 11-Cis-retinal enters blood circulation and transported to the retina, and it
recombines with opsin to form rhodopsin.
Regeneration of rhodopsin completes the Wald’s visual cycle. Similar events

occur during phototransduction in cones.
Role in Color Vision

• Cones possess conopsin. It is composed of opsin and retinal moiety. Therefore,
vitamin A is necessary for daylight vision and color vision.
Role in Normal Epithelialization

• Retinol is necessary for normal epithelialization in the skin and mucous mem-
brane. It prevents excessive keratinization.
Role in Mucus Formation

• Retinyl phosphate is helpful in the formation of glycoproteins. These are struc-
tural components of mucus which is secreted by mucous membranes in body
cavities. Mucus keeps the surface of mucosa moist and healthy. Vitamin A pro-
tects mucosa from infection.
Role in Growth
• Retinoic acid behaves like steroid hormone. It binds to nuclear receptors. The
all-trans-retinoic acid binds to “retinoic acid receptors” (RARs), and 9-cis-
retinoic acid binds to “retinoid X receptor.” Retinoic acid regulates transcription
of genes. So retinoic acid regulates protein synthesis in the body.
• Therefore, retinoic acid is necessary for cell differentiation and growth of cells in
the body.
Role in Transferrin Synthesis

• The all-trans-retinoic acid has found to regulate protein synthesis. It enhances
synthesis of transferrin protein. It binds with iron in blood circulation.
Role in Immunity
• Vitamin A plays an important role in the development of immune system. It is
necessary for appropriate immune response against various infections.
• In experimental model on mice, it has been found that retinoic acid stimulates
proliferation and cytotoxicity of T cells.
290 11 Vitamins
• Retinoic acid can modulate activity of antigen-presenting cell.

• Retinoic acid can stimulate proliferation of B cells and antibody formation.
Role in Normal Reproduction

• The all-trans-retinoic acid is necessary for normal reproduction. The fact has
been proven by experiments on vitamin A-deficient (VAD) male and female rats.
The development of testes in VAD male rats was affected. The VAD female rats
were found to be sterile.
• Vitamin A is also necessary for normal embryo development.
Role in formation of Bones and Teeth

• Vitamin A has a role in the formation of bones and teeth. Retinoic acid activates
osteoblastic activity, and its deficiency decreases endochondral ossification.
Vitamin A as Antioxidant
• Antioxidant is a chemical compound that inhibits oxidation of biomolecules in
living system. They prevent lipid peroxidation and damage to proteins and
DNA. The beta-carotene scavenges free radicals in the body and acts as
antioxidants.
Vitamin A Has Anticancer Activity

• The all-trans-retinoic acid (ATRA) has found to have anticancer activity. It can
enhance anticancer effect of epigallocatechin-3-O-gallate (phytochemical in
green tea) on subcutaneous growth in mice model.
• The use of all-trans-retinoic acid has been approved by the US Food and Drug
Administration for the treatment of lymphoma and melanoma. The (ATRA) is
found to inhibit growth of tumor and metastasis.
itamin A Deficiency Disorders

V
1. Night blindness.
• Night blindness is the inability of an individual to see in dim light. It is the
initial manifestation of VAD.
• For normal visual cycle, a constant supply of vitamin A is necessary. It helps
to regenerate rhodopsin in the retina. Deficiency of vitamin A impairs rhodop-
sin regeneration and impairs the dark adaptation. It causes night blindness. It
is also called “nyctalopia.”
2. Xerophthalmia.
• It is characterized by dryness of the conjunctiva and cornea. Vitamin A defi-
ciency results in structural changes in lachrymal glands causing
xerophthalmia.
3. Bitot’s spot.
• Bitot’s spots are white, opaque, and triangular- or irregular-shaped lesions in
conjunctiva. They occur on either outer or inner side of the eye. It is due to
localized keratinization of conjunctiva. Lesion is named after French physi-
cian Pierre Bitot who described the lesion in 1863.
4. Keratomalacia.
• Corneal epithelium becomes keratinized and opaque. Keratomalacia is a
severe form of xerophthalmia which starts as xerosis of conjunctiva. This
condition progresses to xerosis of the cornea.
• Cornea undergoes ulceration and necrosis. The condition is irreversible.
• Keratomalacia is the leading cause of blindness among children in developing
world. VAD-associated blindness is preventable.
5. Deficiency of vitamin A causes keratinization in mucosa of respiratory tract.
The respiratory tract is susceptible to higher incidence of infections.
6. Deficiency of vitamin A leads to keratinization of urinary tract mucosa. It raises
incidence of calculi formation in the urinary tract.
7. Sterility in males.
• In mice model, VAD results in degeneration of germinal epithelium. It is
the cause of male sterility.
8. The skin becomes dry, scaly, and rough.
Hypervitaminosis A
It is the toxicity associated with consumption of excessive amount of vitamin A. It
is associated with following clinical manifestations:
• Hypervitaminosis A induces osteoclastic activity. Bone resorption and bone pain

are common symptoms. In infants and children, thinning of the skull bone (cra-
niotabes) occurs.
• Enlargement of the liver, nausea, and vomiting.
• Intracranial pressure is increased, headache.
• Incidence of fetal malformation in pregnant women.
• Yellow discoloration of the skin, peeling of the skin, hair loss, and pruritus.
• Loss of weight.
Applied Biochemistry
Role of Vitamin A in Oral Leukoplakia
Oral leukoplakia, according to WHO, can be defined as “a white patch which can-
not be diagnosed clinically or histopathologically as any other oral lesion.”
The cause of oral leukoplakia is uncertain. Smoking is associated with higher
incidence of the lesion. Chronic irritation in the mouth, alcohol consumption,
human papilloma virus, Candida albicans, and vitamin deficiency are the leading
predisposing factors for the disorder.
Oral leukoplakia has a premalignant potential without a particular histological
pattern.
Treatments of oral leukoplakia are dependent on the extension of lesion and its
histological pattern.
Surgical excision is indicated for localized lesions.
Retinoids are new drugs derived from vitamin A. They are administered orally
and applied topically. Retinoids act by inducing apoptosis of cancer cells.
292 11 Vitamins
The isotretinoin, 13-cis-retinoic acid, is vitamin A analog. It has the highest

potential in the prophylaxis of secondary tumors.
According to a study by Epstein and Gorsky (1999), 26 patients were adminis-
tered topically 0.05% tretinoin ointment four times a day, and it was continued for
3.5 years. Around seven to eight patients were cured of oral leukoplakia, and ten
patients experienced recurrence of lesion after the treatment was stopped.
According to a study by Piattelli et al. (1999), ointment containing 0.1% 13-cis-
retinoic acid was applied topically for 3 months. One patient was relived from the
lesion.
11.4.2 Vitamin D
History
• In 1919, Mellanby found that cod-liver oil has potential to prevent rickets in
experimental models on dogs.
• In 1922, McCollum named active ingredient of cod-liver oil as “vitamin D.”
• In 1931, Augus and coworkers isolated vitamin D and named “calciferol.”
• In 1950, its chemical structure was discovered by Otto Diels and Kurt Alders and
awarded Nobel Prize.
Chemical Structure
1. Forms of Vitamin D
• Vitamin D exists in five different forms such as vitamins D1, D2, D3, D4, and
D5. However, vitamins D2 and D3 are biologically important forms.
• Vitamin D1 is a combination of ergocalciferol and lumisterol in 1:1 ratio.
• Vitamin D2 is “ergocalciferol.”
–– It is synthesized from “ergosterol.”
–– Ergosterol is a sterol found in “ergot” fungi and yeast. The ergot fungi
belong to genus Claviceps. Ergosterol is a normal structural constituent of
cell membrane of fungi and yeast.
–– The plant “alfalfa” contains ergosterol. It is a perennial plant with flowers.
It is used as forage crop in North America and in countries like New
Zealand and South Africa.
–– Ergosterol is “provitamin D2.” It can absorb UVB (medium wave UV
rays with 290–315 nm wavelength) radiation. It undergoes photolysis.
–– Photolysis is characterized by activation of double bonds in B-ring due to
absorption of light photon. There is cleavage of bond between C9 and C10
and opening up of B-ring at C9–C10 position. It forms 9,10-secosteroid,
called “previtamin D2.”
–– Previtamin D2 exists in thermodynamically unstable cis and stable trans
forms. The cis form undergoes intramolecular rearrangement and con-
verted into “vitamin D2.”
• Vitamin D3 is “cholecalciferol.”
–– It is synthesized from “7-dehydrocholesterol.”
–– 7-dehydrocholesterol is the natural zoosterol. It is found in the epider-
mis (stratum basale and stratum spinosum) of the human skin. It is also
present in mammalian milk and insects
–– 7-dehydrocholesterol is provitamin D3. The UVB radiation can pene-
trate into the epidermis only. The 7-dehydrocholesterol can absorb UVB
radiation (290–315 nm wave length) and undergoes photolysis.
–– It is converted into thermodynamically unstable form, previtamin D3. It is
spontaneously photoisomerized into stable form, vitamin D3.
–– From the epidermis, vitamin D3 passes into capillaries in the dermis, and it
is transported to the liver.
• Active vitamin D3 is “calcitriol” (1,25-dihydroxycholecalciferol).
• Vitamin D4 is “22-dihydroergocalciferol.” It is found in mushrooms.
• Vitamin D5 is “sitocalciferol.” It is an important phytosterol.
“7-Dehydrocholesterol” and “ergocalciferol” are together referred to as
“provitamin D.”
2 . Structure
• Vitamin D belongs to fat soluble group of solid alcohols with fragmented
steroidal ring structure. These are called as “secosteroids.” The word is
derived from Latin words secare meaning to cut and stere for steroid mean-
ing solid and ol meaning alcohol as in Fig. 11.4.
• Secosteroids are subgroup of steroids. These are classified based on the cleav-
age of bond between carbon atoms. Cholecalciferol is 9,10-secosteroids. It
has broken bond between C9 and C10in B-ring.
• Provitamins D2 and D3 have some common structural features:
–– Both provitamins are C28 organic compound with molecular formula as
(C28H43OH).
–– They have “cyclopentano-perhydro-phenanthrene nucleus” and are made
up of four rings named as A, B, C, and D.
–– Hydroxyl group (OH) at C3.
–– C17 has a hydrocarbon chain.
–– Two double bonds between C5–C6 and C7–C8.
–– Ergocalciferol differs from cholecalciferol with the presence of addi-
tional methyl group at C24 and a double bond between C22 and C23 in
hydrocarbon chain as in Fig. 11.4.
ietary Sources of Vitamin D

D
1. Animal Sources
• Richest source: Fish liver oil is the best source of vitamin D3. The oil derived
from the liver of cod fish (cod is the name given to genus the Gadus of fishes)
is the richest source of vitamin D. One spoon full of cod-liver oil contains
around 1400 IU of vitamin D.
• Good source: Flesh of fishes like sardine, salmon, and tuna. Around 90 g of
salmon flesh contains about 450 IU of vitamin D.
294 11 Vitamins
21 C C 22
20 C23
C 26
18 C 25
12 17 C24
11 13 C 27
D S
C C 16
19
1 9
2 10 8
A B Uv
Ra
3 ys
HO 4 5 7
6 19
7 – Dehydrocholesterol CH2
[Present in Epidermis]
HO 5 7
Opening of
26 6
Steroid Ring at
18 25
C9-10 Positions
27
in Skin Secosterol
C5 19 [Cis form]
CH3
C 1
HO 3
C2
Cholecalciferol (D3)
En
[Trans form] ter
Re s Blo
ac
he od
sL
ive
r, u
nd
erg
oe
sH
OH yd
rox
25 yla
tio
no
f2
25 5C
En Hyd
zy
me rolas
s – es
Liv
C5 er
CH2
3 1 1 – ∝ Hydroxylase Enters circulations OH

HO in 25
OH C
2 Kidneys Reaches Kidneys
1;25 – Di hydroxy
cholecalciferol
[Calcitriol]
CH2
3
HO 25 – Hydroxy cholecalciferol
Fig. 11.4 Synthesis of Calcitriol (active Vitamin D3)
• Average source: Egg yolk (1 egg) and the liver of beef (80 g) contain around
40 IU of vitamin D. The unfortified whole milk (250 ml) contains around
40 IU of vitamin D.
• Poor source: Cheese (30 g) contains around 5 IU of vitamin D.
Table 11.2 RDA of Age group RDA for vitamin D (IU)

vitamin D Children 400–600 IU
Adult males 600 IU
Adult females 600 IU
Old aged (>50 years) 800 IU
Source: (NIH 2017)
1 IU = 0.025 mcg of cholecalciferol or ergocalciferol
2. Plant Sources
• Sun-exposed mushroom contains provitamin D2.
• Fortified foods like soya milk, whole cow milk, yogurt, cereals, and orange
juice are good sources of vitamin D for vegans.
ecommended Dietary Allowance of Vitamin D

R
Recommended Dietary Allowance (RDA) is “the average quantity of a nutrient which is
sufficient to fulfill the physiological needs of almost all (97.5%) healthy individuals.”
RDA was developed by a committee comprising Lydia J. Roberts, Hazel
Stiebeling, and Helen S. Mitchell during the Second World War, and the committee
was set up by the US National Academy of Sciences.
RDA of vitamin D has been established on the basis of minimum exposure to the
sun. The daily requirement of vitamin D for adults, pregnant women, and children
is given in Table 11.2.
bsorption, Transport, and Storage of Vitamin D

A
• Vitamin D is absorbed from the mucosa of the duodenum and jejunum.

• Bile salts are necessary for absorption of vitamin D.
Transport of Vitamin D
• It is transported from enterocytes in form of chylomicrons.

• It enters lacteals and blood circulation along with chylomicrons.
• In plasma, vitamin D binds to α2 globulin. It is transported to body tissues in
bound form.
Storage
• It is stored in the liver.
Biosynthesis of Calcitriol
Calcitriol is 1,25-dihydroxycholecalciferol. It is physiologically active form of vita-
min D. It is called “hormone.” Its biosynthesis involves the following steps:
1. Synthesis of Cholecalciferol in the Skin

• The 7-dehydrocholesterol is present in the epidermis (stratum basale and
stratum spinosum) of the human skin. It is considered as provitamin D3.
296 11 Vitamins
• The UVB radiation can easily penetrate the epidermis. The

7-dehydrocholesterol absorbs UVB radiation (290–315 nm wave length).
It undergoes photolysis.
• Photolysis of 7-dehydrocholesterol involves two steps:
–– Opening of B-ring: Absorption of light photon results in activation of
double bonds in B-ring of 7-dehydrocholesterol. There is a cleavage of
bond between C9 and C10. The B-ring at C9–C10 position opens up. It leads
to the formation of 9,10-secosteroid called “previtamin D3.”
–– Antarafacial sigmatropic hydrogen shifting: The previtamin D3 is a pre-
cursor to vitamin D3, and it exists in two isomeric forms as “cis previtamin
D3” and “trans previtamin D3.” The former isomeric form is thermody-
namically unstable. It undergoes spontaneous antarafacial sigmatropic
shifting of h ydrogen atom from C19 to C9 atom. It is an intramolecular
rearrangement of atoms. It results in the formation of thermodynamically
stable 9,10-secosteroid, called “vitamin D3.”
Previtamin D3 Vitamin D3 (Cholecalciferol)
• From the epidermis, vitamin D3 passes into capillaries in the dermal layer of
the skin. Ultimately, it is transported to the liver.
2 . Synthesis of 25-Hydroxycholecalciferol in the Liver
• Within the liver, cholecalciferol undergoes hydroxylation at C25 position. The
reaction is catalyzed by “25-hydroxylase enzyme” as in Fig. 11.4.
• 25-hydroxylase enzyme: It is a liver microsomal enzyme. It requires
NADPH, Mg++, and cytochrome P450.
• Cholecalciferol is converted into 25-hydroxycholecalciferol (25-HCC). It is
the chief storage form of vitamin D3 in the liver.
• A small amount of 25-hydroxycholecalciferol enters blood circulation. In
plasma, it binds with “vitamin D binding protein” and circulated to body tis-
sues. It reaches kidneys.
3. Synthesis of 1,25-Dihydroxycholecalciferol in Kidneys
• Within kidneys, the 25-hydroxycholecalciferol undergoes second hydroxyl-
ation at C1 position. The reaction is catalyzed by “1-alpha-hydroxylase
enzyme.” The enzyme is present in mitochondria of proximal convoluted
tubules as in Fig. 11.4.
• The enzyme requires ferredoxin reductase, cytochrome P450, and Mg++ ions.
• There is formation of 1,25-dihydroxycholecalciferol (1,25-DHCC).
• It is also called “calcitriol.” It is due to the presence of three hydroxyl groups
at C1, C3, and C25 positions in the molecule, and it regulates calcium
metabolism.
• Calcitriol acts analogous to steroid hormone.
Calcitriol is considered as “hormone,” while vitamin D3 (cholecalciferol) is
considered as “prohormone.”
Rationale: Following characteristics are put forward to justify above
statement:
• Cholecalciferol (vitamin D3) is produced in the skin on exposure to UVB light
of the sun.
• Calcitriol is synthesized in kidneys on enzymatic hydroxylation of

cholecalciferol.
• Calcitriol acts like steroid hormone. It acts on nuclear receptor.
• Like hormones, site of calcitriol synthesis is different from its site of action.
• Calcitriol acts on specific target tissues like hormones. It acts on the intestine,
bones, and kidneys.
• Synthesis of calcitriol is regulated by feedback inhibition.
• Calcitriol regulates calcium metabolism in association with parathyroid hor-
mone and calcitonin.
unctions of Vitamin D (Calcitriol)

F
Calcitriol acts on the intestine, bones, and kidneys in the following ways:
Effect of Calcitriol on the Intestine
• Calcitriol enhances intestinal absorption of calcium and phosphate. It acts

like a hormone as described below:
–– Calcitriol binds to nuclear receptor present in the cytosol of intestinal cells.
–– It forms calcitriol-receptor complex. This complex activates genes and regu-
lates transcription of m-RNA.
–– Calcitriol controls synthesis of “calcium binding protein” (CBP).
–– Calcium binding protein enhances uptake of calcium through intestinal cells.
• Calcitriol promotes degradation of “lithocholic acid” (LCA) in the intestine and
enhances its excretion. Lithocholic acid is a toxic bile acid that is produced in the
colon by bacterial action on chenodeoxycholic acid.
Effect of Calcitriol on Bones
• Calcitriol increases deposition of calcium and phosphate in bones.

• Calcitriol stimulates osteoblastic activity.
• It enhances hydroxyapatite content of bones.
• It is necessary for normal growth.
Effect of Calcitriol on Kidneys
• Calcitriol minimizes excretion of calcium and phosphate by renal tubules.

• It increases reabsorption of calcium and phosphate by renal tubules.
Effect of Calcitriol on Immunity
• Calcitriol acts on vitamin D receptor (VDR) which is located in immune cells.

• Calcitriol modulates innate immunity.
298 11 Vitamins
Effect of Calcitriol on Cancer Cells
• Calcitriol inhibits synthesis of hypoxia-inducible factor-1 and vascular endothe-

lial growth factor in cancer cells.
• Calcitriol inhibits angiogenesis in cancer cells. It is the formation of new blood
vessels in cancer cells. Calcitriol inhibits cancer cell proliferation.
eficiency Disorders of Vitamin D

D
Deficiency of vitamin D is not common in population as it is synthesized by the skin
on exposure to sunlight. However, the following predisposing factors are responsi-
ble for its deficiency in children and adults:
1 . Inhabitation in damp and congested places devoid of direct sunlight

2. Intake of diet poor in vitamin D
3. Chronic alcoholism
4. Renal and liver disorders
Vitamin D deficiency is manifested in following disorders:
Rickets
Rickets is a clinical condition characterized by skeletal deformities in bones of
growing children owing to impaired mineralization of bones.
The word “rickets” is derived from the Greek word “rachitis” which means
“in the spine.”
Clinical Features
Deficiency of calcitriol affects activity of osteoblasts. It is involved in the defec-
tive vascularization and incomplete mineralization of bones of growing children.
This condition affects bones before the closure of epiphyseal plates. Rickets has
following features:
• Bow Legs
–– Softening of ends of long bones
–– Softening of shaft of long bones due to defective mineralization
–– It causes long bone bending and bow legs
• Knock Knees
–– The condition is found in children between 2 and 5 years of age group.
–– In an upright standing position, the knees of both sides touch each other and
the feet are wide apart.
–– The knees and ankles are swollen.
• Hot Cross Bun Appearance of Fontanelles
–– Fontanelles are the membranous gaps between the cranial bones in infant
skull. They undergo ossification between 2 months and 24 months in normal
conditions.
–– In rickets, fontanelles do not calcify normally and remain open. They appear
like hot cross bun.
• Pigeon Breast
–– Pigeon chest is also called “pectus carinatum.” It is a deformity in the chest
bones.
–– It is characterized by outward positioning (protrusion) of sternum and ribs in
the chest.
–– It is due to excessive deposition of non-calcified organic bone matrix (oste-
oid) on the sternum and ribs.
–– Patients have difficulty in breathing.
• Rachitic Rosary
–– Rachitic rosary is appearance of beads over ribs.
–– It is characterized by the presence of nodules at costochondral joints in the rib
cage.
–– It is due to hypomineralization and excessive osteoid tissues over costochon-
dral joints.
Osteomalacia
Osteomalacia is the rickets in adults. It is a clinical condition characterized by soft-
ening of bones in adults owing to impaired mineralization of bones.
Diminished Innate Immunity
• Deficiency of calcitriol diminishes innate immunity.

• Calcitriol acts through vitamin D receptor on antigen-presenting cells of immune
system. Its deficiency inhibits differentiation and maturation of antigen-presenting
cells (APC). Immature APCs are unable to stimulate activation of T cells and B cells.
• Deficiency of calcitriol also suppresses secretion of interleukin-10 and interleu-
kin-12 which are necessary for activation of lymphocytes.
The word “osteomalacia” is derived from the Greek words “osteo” which
means “bone” and “malacia” which means “softness.”
Clinical Features
• Osteomalacia is associated with defective mineralization of bones due to decrease

in vitamin D, calcium, and phosphate levels.
• Initially, bone pain starts in lumbar region of the body. The involved bones
become sensitive to touch.
• Tenderness of bones in arms, legs, and spine.
• Proximal muscles of pelvic girdle become weak. It affects position of the body
during walking (gait). Patient assumes “waddling gait.”
• In progressive condition, bones become susceptible to pathological fracture.
Renal Osteodystrophy
Renal osteodystrophy is a condition characterized by degeneration of renal paren-
chyma and defective synthesis of calcitriol.
300 11 Vitamins
Clinical Features
• Chronic renal failure

• Bone pain and tenderness of bones
• Muscle weakness and tingling sensation over extremities
• Osteomalacia
• Hypocalcemia
Hypervitaminosis D
It is the toxicity associated with consumption of excessive amount of vitamin D. The
toxicity is induced by sustained hypercalcemia and hyperphosphatemia. Important
clinical manifestations are as follows:
• Nausea and vomiting and abdominal pain

• Constipation
• Loss of appetite, lethargy, and loss of weight
• Increased thirst and polyuria
• Increased probability of urinary lithiasis (kidney stone)
• Calcification of arteries, muscles, pulmonary bronchi, and gut mucosa
• Renal failure
Vitamin D3 (Calcitriol) and Health of Periodontal Tissues
Mineral Homeostasis
• Calcitriol influences calcium and phosphate homeostasis in the body. It regulates

mineralization of mandible and maxilla. So it influences bone density of jaw
bones.
• Calcitriol is necessary for normal bone remodeling.
• Calcitriol reduces alveolar bone resorption.
• In a clinical trial through randomization by (Garcia et al. 2011), it was observed
that supplementation of calcium and vitamin D improved severity of periodontal
infection.
Antibacterial Effect of Calcitriol
• Calcitriol acts through its vitamin D receptors located on immune cells. It inhib-
its release of pro-inflammatory cytokines through immune cells.
• Calcitriol also stimulates macrophages to release peptides. These peptides have
antibacterial effect on the microbes responsible for periodontal disease.
• Therefore, calcitriol minimizes incidence of inflammation and infection in peri-
odontal tissue.
11.4.3 Vitamin E
Vitamin E is a group of naturally occurring fat soluble organic compounds

with antioxidant activity.
Vitamin E includes:
1. Tocopherols (T)
2. Tocotrienols (T3)
They are collectively called as tocochromanol.

The word “tocopherol” is derived from the Greek words “tokos” which means
“birth,” “pherein” which means “to carry,”
and “ol” which means “alcohol.”
History
• Vitamin E is called “anti-sterility vitamin.”
• In 1922, the physician Dr. Evans and his assistant, Katherine S Bishop, recog-
nized the importance of fat soluble factor in diet for normal reproduction in rats
and named it as vitamin E.
• In 1936, alpha-tocopherol was isolated from wheat germ oil by Evans and his
coworkers.
• In 1938, the structure of vitamin E was described by Erhard Fernholz.
• In 1938, Paul Karrer synthesized vitamin E.
Chemical Structure
1. Forms of Vitamin E
• Vitamin E exists in nature in eight homologues.
• Four homologues belong to tocopherols and another four belong to
tocotrienols.
• Tocopherol homologues exist as alpha-tocopherol, beta-tocopherol, gamma-
tocopherol, and delta-tocopherol.
• Tocotrienol homologues exist as alpha-tocotrienol, beta-tocotrienol, gamma-
tocotrienol, and delta-tocotrienol.
2. Structure
• All compounds with vitamin E activity are synthesized by plants from homo-
gentisic acid.
• They have a common structural characteristic “6-chromanol ring.” It is a ben-
zodihydropyran as in Fig. 11.5.
• All vitamin E homologues are derived from 6-chromanol.
• Hydroxyl group is attached to C6 position at chromanol ring. It provides
hydrogen atom to free radicals. It is responsible for the antioxidant property
of vitamin E.
• A hydrophobic 16 carbon side chain is attached at C2 position of chromanol
ring.
302 11 Vitamins
5th methyl
group
CH3
C CH2
5 4
HO C CH2
6 3
2
C7 C (CH2)3 CH (CH2)3 CH (CH2)3 C CH3
H3C
8 O CH3
CH3 CH3 CH3
C 1
CH3
7th methyl
group
8–methyl
group
[5,7,8 – Trimethyltocol]
∝ – Tocopherol
Fig. 11.5 Alpha Tocopherol
• Tocopherols differ from tocotrienols in the structure of side chain. In tocoph-

erol, side chain (phytyl chain) is saturated, while in tocotrienols, side chain is
unsaturated with three double bonds at C3′, C7′, and C11′ positions.
• All vitamin E homologues differ by the number and position of methyl groups
which are attached to 6-chromanol ring.
• Examples:
–– Alpha homologues (tocopherol and tocotrienols) contain three (5, 7, 8)
methyl groups.
–– Beta homologues (tocopherol and tocotrienols) contain two (5, 8) methyl
groups.
–– Gamma homologues (tocopherol and tocotrienols) contain two (7, 8)
methyl groups.
–– Delta homologues (tocopherol and tocotrienols) contain one (8′) methyl group.
• All chemical forms of vitamin E are amphipathic molecules. The side chain is
lipophilic in nature, whereas the chromane ring is hydrophilic in nature.
• Each tocopherol has three chiral centers. First chiral center is positioned at
C2 in chromanol ring, and second and third chiral centers are positioned at C4′
and C8′ in side chain. Therefore, each tocopherol has (23) eight stereoisomers.
• Naturally occurring alpha-tocopherol exists in single stereoisomeric form
represented as RRR-α-tocopherol. Synthetic alpha-tocopherol is a race-
mic mixture (equal proportion) of eight stereoisomers and represented as
all-racemic or all-rac.
• Tocotrienols have single chiral center at C2 position.
• Biological activity and antioxidant property of all chemical forms of vitamin

E are not identical.
• Gamma-tocopherol: It is the most abundant form of vitamin E in human
diet. Its biological activity is 1/10th of alpha-tocopherol.
• Alpha-tocopherol: It has the highest antioxidant activity among humans. Its
reactivity toward reactive oxygen species is higher than other tocopherols and
polyunsaturated fatty acids.
ietary Sources of Vitamin E

D
1. Animal Sources
• Meat, milk, butter, and egg yolk
2. Plant Sources
• Tocopherols are abundant in cotton seed oil, sunflower oil, peanut oil, corn,
and soya oils.
• Wheat germ oil has the highest concentration of alpha-tocopherol. About 1
tablespoon full of oil has around 20 mg of vitamin E.
• Corn oil and soya bean oil are good sources of gamma-tocopherol.
• Tocotrienols are found in rice bran, barley, and palm oil.
ecommended Dietary Allowance of Vitamin E

R
Recommended Dietary Allowance (RDA) is “average quantity of a nutrient which
is sufficient to fulfill the physiological needs of almost all (97.5%) healthy
individuals.”
RDA was developed by a committee comprising Lydia J. Roberts, Hazel Stiebeling,
and Helen S. Mitchell during the Second World War, and the committee was set up by
the US National Academy of Sciences. Table 11.3 shows RDA of vitamin E.
bsorption, Transport, and Storage of Vitamin E

A
• Food contains free and esterified forms of tocopherols. Alpha-tocopherol is the

most active chemical form of vitamin E.
• Tocopherols are incorporated into mixed micelles. Esterified forms are hydro-
lyzed by pancreatic lipase. Bile salts are necessary for absorption of
tocopherols.
• Free tocopherols are absorbed in the mucosa of the duodenum and jejunum. The
absorption of tocopherols occurs by passive diffusion along with dietary lipids.
• Inside enterocytes, tocopherols are incorporated into chylomicrons.
• Tocopherols leave enterocytes and enter into lacteals and lymph vessels.
• Tocopherols along with chylomicrons pass into systemic circulation.
Table 11.3 RDA of vitamin E Age group RDA for vitamin E (IU)
Children 10–15 IU
Adults 20–28 IU
1 IU = 0.67 mg of alpha-d-tocopherol
304 11 Vitamins
Transport of Vitamin E in Plasma
• Within blood circulation, chylomicrons are catabolized by lipoprotein lipase

enzyme. Fatty acids and alpha-tocopherol are released. Alpha-tocopherol is dis-
tributed to the muscles, adipose tissues, cardiac muscles, and brain tissues.
• Dilapidated chylomicrons containing alpha-tocopherol are taken up by the liver.
• Within the liver, triglycerides and tocopherol are secreted into VLDL, and they
are exported from the liver into circulation.
• Within blood circulation, a few VLDL are catabolized by lipoprotein lipase and
are converted into LDL. The VLDL remnants with tocopherol are taken up by the
liver. Tocopherol is again secreted by the liver into IDL.
• Alpha-tocopherol has lipophilic nature. It is distributed along with lipoproteins
from the liver to muscle tissues and adipose tissues for storage.
Storage
• Alpha-tocopherol is stored in parenchymal cells of the liver. Trace amount of

alpha-tocopherol is also stored in stellate and Kupffer cells of the liver.
• It is also stored in skeletal muscles and adipose tissues.
unctions of Vitamin E
F
Antioxidant Function
• Vitamin E inhibits the synthesis of free radicals in the body.

• Predominantly, vitamin E scavenges free radicals from body tissues as in
Fig. 11.6. It acts by the following mechanisms:
–– The chromanol ring of tocopherol furnishes its hydrogen that neutralizes
the free radicals.
–– The “chromanol ring” of tocopherol is oxidized into “quinone ring.”
• Vitamin E prevents lipid peroxidation.
–– It protects integrity of cell membrane of body cells.
–– It protects integrity of mitochondrial membrane. Hence, it maintains oxida-
tive phosphorylation in the mitochondria.
–– It protects integrity of membranes of erythrocytes and prevents hemolysis.
• Vitamin E protects coenzyme A (CoA.SH) from free radicals by preventing
oxidation of sulfhydryl group.
• Vitamin E prevents oxidation of retinol.
Cardioprotective Activity
Vitamin E is helpful in prevention of coronary artery disease (CAD). It exerts its
cardioprotective function through following activities:
• Vitamin E inhibits synthesis of “vascular cell adhesion molecule” in endothelial

cells. So it inhibits “platelet adhesion” to vascular endothelium, and it also pre-
vents “platelet aggregation.”
VITAMIN E
Free radical
Peroxidation Phospholipid
of molecule
PUFA in membrane
Cell membrane
PUFA – PEROXL
FREE RADICAL
Breaks
CH3
O Peroxidation
CH3 Chain Reaction
H3 C
HO
CH3
PUFA in
Cell membrane
∝ – Tocopherol
Ascorbate [Reduced]
or
Glatathione [G ∼ SG]
O
CH3
O
Ascorbate [Oxidized]
or
Oxidized - tocopherol Glatathione [GS ∼ SG]
[Quinone]
Fig. 11.6 Mechanism of antioxidant function of Vitamin E

306 11 Vitamins
• Vitamin E increases synthesis of prostacyclin and nitric oxide from vascular

endothelial cells. These are potent vasodilators. They improve coronary artery
stenosis.
• Tocopherols and tocotrienols have been found to inhibit enzyme, 3-hydroxy-
3-methylglutaryl-CoA (3HMG-CoA) reductase. This enzyme is responsible
for biosynthesis of cholesterol by the liver. Therefore, vitamin E prevents
hypercholesterolemia.
• Overall, vitamin E prevents coronary artery stenosis and atherosclerosis which
are implicated in initiation and progression of CAD.
Anticarcinogenic Property
It has been demonstrated that gamma- and delta-tocopherols have anticariogenic
property.
• Gamma-tocopherol can inhibit proliferation of cancer cells in culture by

different mechanisms:
–– Gamma-tocopherol removes free radicals which can cause mutation of
genetic material of cell (DNA).
–– It inhibits synthesis of “cyclins” which is responsible for proliferation of
cancer cells.
–– It induces apoptosis in cancer cells.
• Gamma- and delta-tocopherols have better anticariogenic property than
alpha-tocopherol.
• Scientists have reported anticariogenic activity of delta- and gamma-tocopherols
in lung, colon, prostate, and breast malignancy.
However, various studies including case control and randomized trials con-
cerning the anticariogenic property of vitamin E have yielded mixed outcome.
Reproductive Activity of Vitamin E

Vitamin E has been found to be essential for normal health of seminiferous epithe-
lium in rats. Its deficiency has been associated with irreversible changes in the epi-
thelium. It could lead to sterility in male rats.
In female rats, vitamin E is necessary for normal growth of fetus in rat models.
Role in Immunity
Vitamin E is an immune modulator. It stimulates cell-mediated immunity and
humoral immunity in humans.
• It enhances phagocytosis by lymphocytes and macrophages in bacterial

infection.
• It stimulates differentiation of immature T cells.
It has been found in randomized human trial that vitamin E supplementa-

tion improved the cell-mediated immunity in healthy old-aged persons.
eficiency Disorders of Vitamin E

D
Vitamin E deficiency is responsible for many deficiency disorders in the body.
• Hemolysis of erythrocytes increases in deficiency of vitamin E. It is due to lytic

action of peroxides on the cell membrane of erythrocytes.
• Vitamin E deficiency is related to hemolytic anemia.
• Male rats suffer from sterility.
• Increased prevalence of fetus resorption in female rats.
• Hepatic necrosis.
–– Due to deficiency of amino acid cysteine in diet, the liver undergoes necrosis.
Vitamin E and selenium have protective role in hepatic necrosis.
• Muscular dystrophy.
–– Deficiency of vitamin E is related to muscular dystrophy (skeletal muscle
weakness and irreversible damage).
–– Its deficiency is responsible for increased lipid peroxidation in muscles and
increased synthesis of peroxides. It results in higher hydrolase activity which
damage muscles.
• Ataxia and vitamin E disorder (AVED).
–– It’s an inherited neurological degenerative disorder in which individuals have
deficiency of vitamin E in the body. It involves neurological manifestation.
Persons lack coordination in skeletal muscles, and their walking is affected
(ataxia).
Therapeutic Value of Vitamin E
• Vitamin E has preventive and curative role in nocturnal muscle cramps (painful
condition that occurs in the leg, thigh, or feet).
• Vitamin E improves intermittent claudication (cramp in calf muscle on walking
due to occluding blood vessels).
• Vitamin E is helpful in prevention of atherosclerosis. It is due to its potent anti-
oxidant and anti-inflammatory activities.
• Vitamin E also has a therapeutic role as anti-sterility vitamin.
• AVED improves with supplementation of vitamin E.
11.4.4 Vitamin K
Vitamin K belongs to a group of naturally occurring fat soluble organic com-

pounds possessing a common chemistry as “2-methyl-1,4-naphthoquinone.”
The generic name of vitamin K is derived from the German word “koagula-
tion.” It is due to its essentiality for synthesis of coagulation factors by the liver and
regulation of clotting.
308 11 Vitamins
History
• Henrik Dam and Schonleyder discovered an “antihemorrhagic factor” in chicks
around 1929 in Denmark. They observed that chicks suffered from prolonged
bleeding time after feeding with cholesterol deficient diet.
• H. Dam isolated vitamin K1 from alfalfa sprouts in 1939.
• E. A. Doisy isolated vitamin K2 in 1939.
• Dam and Doisy were awarded Nobel Prize in 1939.
Chemical Structure
1. Forms of Vitamin
Vitamin K occurs in nature in two forms which are derivative of naphthoqui-
none. The two forms are as follows:
• Vitamin K1
–– It is called “phylloquinone.” It is found in chloroplasts in leaves of plants.
–– Phylloquinone contains a methyl naphthoquinone ring. A phytyl chain
(partially saturated poly-isoprenoid alcohol) is attached to the ring.
–– Vitamin K1 is chemically 2-methyl, 3-phytyl, 1,4-naphthoquinone as in
Figs. 11.7 and 11.10.
–– It is derived from leaves of alfalfa.
–– It is also called “phytonadione.”
–– It is a yellow-colored thick oil.
• Vitamin K2
–– It is called “menaquinone.” It is synthesized by bacteria in the intestine.
–– Menaquinone contains a methyl naphthoquinone ring to which a phytyl
chain (unsaturated poly-isoprenoid chain with different carbon length) is
attached. The number of isoprene units may vary from 4 to 13 and vitamin
K2 is called as menaquinone-n in Fig. 11.8. For example, menaquinone-7
has seven isoprenoid units containing 35 carbons in side chain and desig-
nated as vitamin K2 (35).
–– Chemically, it is 2-methyl-3-difarnesyl-1,4-naphthoquinone.
–– It was isolated from decayed fish meal.
–– It is a yellow-colored thick oil.
• Vitamin K3
–– It occurs as synthetic analog of vitamin K, and it represents its third form.
It is called “menadione.”
CH3
CH3 CH3
CH2.CH C CH2 (CH2 CH2 CH CH2)3 H
Fig. 11.7 Phylloquinone (Vitamin K1)

Fig. 11.8 Menaquinone O
(Vitamin K2)
CH3
– C–CH ) – H
(CH2–CH– 2 6
CH3
O
Fig. 11.9 Menadione O
(Vitamin K3) CH3
–– Chemically, it is 2-methyl-1,4-naphthoquinone. It lacks the side chain

as in Fig. 11.9.
–– Vitamin K3 is water soluble.
–– It has higher activity than its natural counter parts.
–– It is used as parenterally in management of hypoprothrombinemia.
–– Vitamin K3 exists into another two forms called “menadiol” and
“menadioldiacetate.”
ietary Sources of Vitamin K

D
Vitamin K1 is present mainly in green leafy vegetables. Predominant sources are
spinach, alfalfa, cabbage, cauliflower, tomatoes, kale, turnip, and soya bean.
Vitamin K2 is mainly produced by intestinal bacteria in humans. The long-
chain menaquinones are primarily synthesized by gut bacteria in humans.
Chief menaquinones synthesized by intestinal bacteria are as follows:
• MK-10-13 are synthesized by Bacteroides.

• MK-8 by Enterobacter.
• MK-7 by Veillonella.
• MK-6 by Eubacterium lentum.
Vitamin K3 is man-made. It is a structural analog of vitamin K1 and K2

(Fig. 11.10).
310 11 Vitamins
Fig. 11.10 2–Methyl, 3–Hydroxy, O

1, 4–Naphthoquinone
2 CH3
C
4 C CH3
3
Table 11.4 RDA of Age group RDA for vitamin K (IU)

vitamin K Children 60 mcg
Adult males 100 mcg
Adult females 90 mcg
Source: (NIH 2017)
Daily requirement of vitamin K is between 50 and 100 mg
This amount of vitamin K (K1) is available in daily food.
The intestinal bacteria synthesize vitamin K2
Under normal conditions, deficiency of vitamin K has not
been reported among healthy persons
ecommended Dietary Allowance of Vitamin K

R
Table 11.4 shows RDA of vitamin K as follows:
bsorption, Transport, and Storage of Vitamin K

A
• Dietary vitamin K1 is incorporated into mixed micelles and is absorbed by

mucosa of small intestine. Within enterocytes, vitamin is incorporated into chy-
lomicrons. It is exported from enterocytes along with chylomicrons into lacteals
and transported into lymph vessels.
• Vitamin with chylomicrons enters systemic circulation through thoracic duct.
• The presence of bile salts is necessary for its absorption.
• Menaquinones are absorbed from gut mucosa and enter hepatic portal
circulation.
Transport of Vitamin K
• Phylloquinone and menaquinones are transported in the plasma along with

lipoproteins.
• Phylloquinone is the chief circulating form of vitamin K which is derived
from diet.
• Menaquinones, mainly MK-7, are present in the plasma in comparatively lower
concentration, and it is mainly derived from intestinal flora.
Storage
• Menaquinones, predominantly MK-7, are stored in the liver in large amount.

• Phylloquinone is stored in the liver in a negligible amount.
• Hepatic storage of phylloquinone is highly labile. It undergoes depletion within
2–3 days after the stoppage of dietary intake.
• Phylloquinone and menaquinones are also stored in small amounts in the bones
and adipose tissues.
unctions of Vitamin K
F
Role of Vitamin K in Carboxylation of Blood Clotting Factors
• The liver synthesizes blood clotting factors. The factors II, VII, IX, and X are
synthesized in inactive forms.
• These clotting factors undergo posttranslational modification with the help of
vitamin K as described below:
–– Vitamin K is changed into hydroquinone form in liver microsomes. The
reaction is catalyzed dehydrogenase enzyme in the presence of NADPH.
–– Hydroquinone acts as coenzyme for carboxylase enzyme.
–– Carboxylase brings about carboxylation of glutamic acid residue present
in clotting factors. Additional COOH group is incorporated at gamma carbon
position in glutamic acid.
–– It results in the formation of γ-carboxyglutamate.
–– In prothrombin, initial ten glutamate residues undergo carboxylation to form
γ-carboxyglutamate residues which combine with calcium ions to form
prothrombin-calcium complex. This complex attaches to phospholipids on
surfaces of platelets.
–– It ensures rapid conversion of prothrombin to thrombin and helps in blood
clotting.
–– Drug like dicumarol is an antagonist of vitamin K. The synthetic analog
like warfarin inhibits formation of γ-carboxyglutamate. They act as
anticoagulants.
Role in Oxidative Phosphorylation in Mitochondria
• Vitamin K acts as coenzyme in oxidative phosphorylation in the mitochondria.
eficiency Disorders of Vitamin K

D
Deficiency of vitamin K is unusual owing to its synthesis by intestinal bacteria as
well as intake of wide varieties of vegetables and oils. However, its deficiency
occurs in following conditions:
Prolonged Oral Administration of Broad-Spectrum Antibiotics
• Oral administration of broad-spectrum antibiotics like penicillin and cephalo-

sporin for prolonged period can inhibit growth of intestinal flora.
• It results in deficiency of menaquinones.
312 11 Vitamins
Malabsorption Syndrome
• Malabsorption is an inflammatory condition of intestinal mucosa.

• Absorption of dietary vitamin K is impaired.
• It causes deficiency of vitamin K.
emorrhagic Disorder in Infants

H
Deficiency of vitamin K is seen in malnourished infants.
It is attributed to following causes:
• Low count of colonic bacteria in infants.

• Distribution of vitamin K from the body of mother to fetus is limited.
• Breast milk contains low concentration of vitamin K.
Deficiency of vitamin K results in hypoprothrombinemia (fall in concentra-

tion of plasma prothrombin). Thus, bleeding time is prolonged.
Hemorrhagic disorder in infants can be of three types based on its onset:
1. Early onset
• Bleeding manifests within 24 h of birth of infants.
2. Classic
• Bleeding manifests within first week after birth of infants.
3. Late onset
• Bleeding manifests within 6 months after birth of infants.
Minor injury causes prolonged capillary bleeding. It is associated with discolor-

ation of the skin which is revealed during physical examination of infants.
11.5 Water Soluble Vitamins
11.5.1 Vitamin C
Vitamin C is a naturally occurring water soluble organic compound having

structural similarity to 6-C monosaccharide (l-glucose) with potent antioxi-
dant activity.
Vitamin C is called “ascorbic acid” owing to its “antiscorbutic property.”
Ascorbic is derived from the Latin word “scorbuticus” which stands for
“scurvy.”
History
• In 1490, Vasco da Gamma reported that his crew members fell sick with painful
gums and swelling of hands. They were treated with oranges and were cured.
11.5 Water Soluble Vitamins 313
• In 1600 ad, Sir Richard Hawkins, an English sea captain, reported that 10,000
sailors were severely affected by an amazing disease (scurvy) owing to dietary
deficiency. They died.
• In 1747, James Lind conducted a first trial on patients who suffered from scurvy.
He concluded that inadequate fruits and vegetables in diet was the main cause for
scurvy.
• In 1753, James Lind wrote a “Treatise on Scurvy.”
• In 1927, Zilva reported the presence of an “antiscorbutic factor” in lemon juice.
• In 1928, S. Gyorgi extracted an active ingredient from oranges and cabbage. It
was named “hexuronic acid.”
• In 1932, Waugh and King extracted vitamin C from lemon juice and crystallized it.
Chemical Structure
1. Forms of Vitamin C
• Vitamin C exists as d-ascorbic acid and l-ascorbic acid.
• d-Ascorbic acid lacks antiscorbutic activity and it is biologically
inactive.
• Vitamin C exists in nature as l-ascorbic acid as in Fig. 11.11.
2. Structure
• Its structure was established by E. L. Hirst, A. Szent-Gyorgyi, F. Michael,
and K. Kraft in 1933.
• Chemically, vitamin C (ascorbic acid) is a 2,3-enediol-l-gulonic
acid-g-lactone.
• Vitamin C has structure similar to l-glucose.
• It is an “enediol-lactone.”
• It has potent acidic property which is attributed to two hydroxyl groups
attached to C2 and C3 linked by a double bond.
Fig. 11.11 Forms of O

Vitamin C O
2H
C1
C1
HO C2 Oxidation
O O C2
Reduction O
HO C3
O C3
H C4
2H H C4
HO C5 H
HO C5 H
6CH2 OH
6CH2 OH
L-Ascorbic acid L-Dehydroascorbic acid

(Reduced state) (Oxidized state)
314 11 Vitamins
• Ascorbic acid can furnish two hydrogen atoms from hydroxyl groups and is
oxidized into “dehydroascorbic acid.” Ascorbic acid and dehydroascor-
bic acids are physiologically active. In the human body, ration of ascorbic
to dehydroascorbic acid is 15:1.
• Thus, it is a potent reducing agent and electron donor. It donates electrons
from double bond between C2 and C3. All biochemical activities of vita-
min C are due to its electron donation property.
• Oxidation of ascorbic acid renders it inactive. The oxidative process is
enhanced in the presence of copper and silver ions.
ietary Sources of Vitamin C

D
Plant Sources
• Indian gooseberry (Phyllanthus emblica) is the richest source of vitamin

C. It contains around 400–600 mg of ascorbic acid in 100 g of gooseberry.
• Citrus fruits like orange, lemon, and tangerine.
• Fruits like strawberries, kiwi, guava, and papaya.
• Vegetables like tomato, cabbage, and spinach.
ecommended Dietary Allowance of Vitamin C

R
Table 11.5 shows RDA of vitamin C as follows:
bsorption, Transport, and Storage of Vitamin C

A
• Humans lack the enzyme “l-gulono-lactone oxidase” necessary for conversion

of glucose to ascorbic acid. Therefore, vitamin C is supplied in diet.
• Dietary vitamin C is absorbed through intestinal mucosa.
Storage
• Vitamin C is not stored in the body.
Table 11.5 RDA of vitamin C Age group RDA for vitamin C

Infants (0–12 months) 40–50 mg (AI)
Preschool children 15–25 mg
(2–5 years)
Children (6–12 years) 25–45 mg
Adolescents (12–18 years) 65–75 mg
Adult male (>18 years) 90 mg
Adult female (>18 years) 75 mg
Pregnancy (>18 years) 85 mg
Lactation 120 mg
Old aged (>50 years)
Source: (NIH 2017)
AI adequate intake
Transport of Vitamin
• Vitamin C is distributed widely in body tissues by blood circulation.

• Organs like the adrenal gland, pituitary gland, liver, and brain have higher con-
centration of vitamin C owing to their higher metabolic activities.
• Vitamin C can cross placental barrier. It is supplied to the fetus in adequate
concentration.
Excretion
• Vitamin C is excreted in urine as such.

• It is metabolized into oxalic acid and diketogulonic acid. These metabolites are
excreted in urine.
unctions of Vitamin C
F
Antioxidant Activity
• Antioxidant is an element or compound that prevents oxidation of another bio-

molecules. Vitamin C is one of the potent water soluble antioxidant vitamins.
Others are Vitamin E and Vitamin A.
• It helps to remove free radicals from body tissues.
• It prevents body tissues from oxidative stress. Prominently, lipids, proteins, and
DNA are protected by vitamin C from oxidative damage.
• Free radicals are highly involved in the pathogenesis of coronary artery disease,
colon cancer, atherosclerosis, cataract, and other degenerative disorders of the
body. Vitamin C plays a preventive role in the.
Activity in Bone Metabolism
• Ascorbic acid stimulates osteoblasts and fibroblasts. It promotes synthesis of

osteoid formation (organic bone matrix).
• Osteoid is mineralization by deposition of hydroxyapatite crystals.
Activity in Collagen Synthesis
• Procollagen (unhydroxylated collagen) is made up of triple helix. It has three

polypeptide chains in which glycine is located at every third position.
• The chains are helically coiled in a left-handed direction. They in turn coil in a
right-handed direction to form a super helix (triple helix).
• Procollagen contains predominantly glycine and proline amino acids.
• Conversion of precollagen into collagen molecule requires hydroxylation of pro-
line and lysine residues.
• Hydroxylation reaction is catalyzed by prolyl hydroxylase and lysyl hydroxylase
enzymes.
316 11 Vitamins
• Ascorbic acid acts as coenzyme along with molecular O2 and Fe+++in hydroxyl-
ation process.
• Hydroxyproline and hydroxylysine provide stability to triple helix and convert
procollagen into stable collagen.
Activity in Cholesterol Metabolism
• Ascorbic acid is involved in the synthesis of bile acids from cholesterol. In defi-
ciency of ascorbic acid, in experimental guinea pigs, it has been found that syn-
thesis of bile acid is reduced.
• Ascorbic acid deficiency impairs activity of cholesterol-7 alpha-hydroxylase
enzyme in liver microsomes.
• Ascorbic acid is also helpful in regulation of normal serum cholesterol level (less
than 200 mg/dl). Dietary deficiency of ascorbic acid has been found to be associ-
ated with hypercholesterolemia.
Activity in Corticosteroid Synthesis
• Adrenal gland possesses high level of ascorbic acid in stressful condition.

• Adrenal cortex synthesizes corticosteroid hormones under the influence of ACTH.
• Ascorbic acid plays a role in the hydroxylation process during the synthesis of
corticosteroid hormone.
• Adrenal cortex contains high concentration of ascorbic acid. During stress, its
concentration decreases.
• In conditions like acute infection, fever, and renal and liver diseases, the level of
ascorbic acid in blood circulation is decreased. This indicates role of ascorbic
acid in combating infection and disease.
Activity in Catecholamines Synthesis
• Dopamine is converted into norepinephrine through hydroxylation in adrenal

medulla.
• Hydroxylation reaction is catalyzed by dopamine hydroxylase.
• Ascorbic acid acts as coenzyme in hydroxylation process.
Activity in Carnitine Synthesis
• Carnitine is helpful in the transportation of activated fatty acid from cytosol to

inside the mitochondria.
• Carnitine is synthesized in the liver through hydroxylation of gamma-
butyrobetaine by dioxygenase enzyme.
• Ascorbic acid acts as coenzyme in hydroxylation reaction along with
α-ketoglutarate and Fe+++.
Activity in Cellular Redox Reaction
• Ascorbic acid is helpful in oxidation-reduction biochemical reactions in the

body.
• It plays its role owing to reversibility of ascorbate-dehydroascorbate reaction.
• It acts as hydrogen donor in the redox reactions.
Activity in Electron Transport Chain
• Ascorbic acid is necessary for normal functioning of ETC in the mitochondria.

• It is essential for optimum activity of cytochrome oxidase enzyme, a constituent
of ETC.
Activity in Folic Acid Metabolism
• Ascorbic acid is necessary for conversion of folic acid into tetrahydrofolate by

enzyme dihydrofolate reductase.
• Ascorbic acid keeps dihydrofolate reductase in reduced form.
• Ascorbic acid and tetrahydrofolate are essential for maturation of erythrocytes.
Activity in Ferritin Synthesis
• Ascorbic acid is helpful in the synthesis of ferritin (storage form of iron in body
tissues).
• Ascorbic acid is also necessary for iron mobilization from iron store (ferritin).
Activity in Hemoglobin Metabolism
• Ascorbic acid is necessary for biodegradation of hemoglobin (released from

senile erythrocytes) into bile pigments.
• It is helpful in conversion of methemoglobin into hemoglobin.
Activity in Iron Absorption
• Plant-based dietary iron is present in ferric form.

• After intake of diet, ascorbic acid helps in the conversion of ferric form of iron
into ferrous form.
• Ascorbic acid combines with ferrous iron to form Fe-ascorbate complex. It is
water soluble.
• The complex is rapidly absorbed through intestinal mucosa.
318 11 Vitamins
Activity in Immunity Modulation
• Ascorbic acid promotes synthesis of immunoglobulins. They provide humoral

immunity.
• It stimulates phagocytic action of lymphocytes and macrophages.
Activity in Tryptophan Metabolism
• Ascorbic acid acts as cofactor in hydroxylation of tryptophan.

• Tryptophan is converted into serotonin (5-hydroxy-tryptamine). It is a mono-
amine and acts as neurotransmitter.
Activity in Tyrosine Metabolism
• Ascorbic acid acts as cofactor in hydroxylation by para-hydroxy-

phenylpyruvate.
• The hydroxylation reaction is catalyzed by p-hydroxy-phenylpyruvate
hydroxylase.
• Para-hydroxy-phenylpyruvate is converted into homogentisic acid.
eficiency Disorders of Vitamin C

D
Scurvy
• Scurvy is a chronic dietary deficiency disorder of vitamin C.

• It is prevalent among malnourished children, mentally challenged population,
and malabsorption syndrome.
It is characterized by the following conditions:
• Fragility of Capillaries
Capillaries become susceptible to rupture under normal pressure. It results in

bleeding in conjunctiva, retina, joint space, subperiosteal, and subcutaneous.
• Impaired Wound Healing
Wound healing is delayed. It is due to impaired collagen synthesis.
• Swollen Gums
Gums become swollen and painful. Gums bleed on slight pressure. Teeth become
loose.
• Impaired Osteoid formation
Activity of osteoblasts is decreased. Deposition of organic matrix (osteoid for-

mation) is decreased. Mineralization is decreased.
Formation of collagen and new capillaries is impaired.
The bone formed is weak and it is susceptible to fracture on mild pressure.
Anemia
Deficiency of vitamin C is associated with iron-deficiency anemia. It is called
microcytic hypochromic anemia. This type of anemia is generally caused by poor
absorption of dietary iron in the absence of vitamin C.
11.5.2 Vitamin B Complex
Vitamin B complex is a group of water soluble organic compound chemically

distinct from each other collectively endowed with coenzyme activity.
11.5.3 Vitamin B1 (Thiamine)
History
• In 2600 bc, the oldest Chinese medical treatise mentioned thiamine deficiency in
population.
• In 1884, K. Takaki conceptualized the disease beriberi to be caused by dietary
deficiency. He ordered to replace diet containing only rice with a diet enriched
with meat, wheat, and barley.
• In 1897, Dutch physicians named C. Eijkman and Grijns in Java induced a para-
lytic state resembling beriberi in chicken by feeding polished rice. Disease was
reversed by supplementing rice polishing in diet. He received Nobel Prize.
• In 1926, Dutch workers named as B. Jansen and W. Donald crystallized vitamin
B1 from rice bran.
• In 1935, R. R. Williams synthesized vitamin B1 and named it as “thiamine”
owing to the presence of “thiazole” and “amino groups.”
• In 1937, Lohman and Schuster extracted thiamine pyrophosphate from yeast.
Thiamine in the literature has been described as “aneurine” (which can cure
neuritis).
Thiamine is an “antineuritic vitamin” or “anti-beriberi factor.”
Thiamine is synthesized by bacteria, fungi, and plants. Animals derive thia-
mine from diet.
Thiamine is an essential nutrient for animals.
320 11 Vitamins
Chemical Structure
1. Forms of Thiamine
Thiamine (free form)
Thiamine pyrophosphate (bound form)
• It is abbreviated as TPP.
• It is a biological active form.
• It acts as coenzyme.
2. Structure of Thiamine
• Thiamine is a sulfur-enriched water soluble vitamin.
• Chemically, it is composed of two rings:
–– Pyrimidine ring: It is 2,5-dimethyl-4-aminopyrimidine.
–– Thiazole ring: It is 4-methyl-5-hydroxyethylthiazole.
–– Thiazole contains sulfur and nitrogen atoms, and it is a heterocyclic ring
(C3H3NS) as in Fig. 11.12.
• Rings are linked with methylene bridge.
• Thiamine is the exclusive compound with Thiazole ring in nature.
• Thiamine exists in bound form as “thymine pyrophosphate.” It acts as coen-
zyme. The hydroxyl group of thiamine undergoes esterification with two
phosphate residues to form thiamine pyrophosphate.
ietary Sources of Thiamine

D
1. Animal Sources
• Animal tissues contain phosphorylated thiamine (bound from).
• Good sources are meat, egg, and liver.
• Human milk and cow milk possess thiamine. It is present in trace amount
(0.04 mg/100 ml).
Methylene
bridge
3
H
3 4
N C C CH2 N C CH3
4
5
6
H3C C NH2 HC2 5 C CH2 CH2OH
C2 1
N1 S
Pyrimidine Thiazole
ring group
Fig. 11.12 Vitamin B1

2. Plant Sources
• Plant tissues contain thiamine in free form.
• Richest source is yeast.
• Good sources are cereals. Thiamine is present in variable amounts in different
fractions of grain.
–– Bran is good source of thiamine.
–– The “aleurone layer” in the bran is the richest in thiamine.
• Whole wheat and unpolished rice are good sources of thiamine.
• Additional sources are pulses, nuts and oil seeds, and green leafy vegetables.
Fractions of Wheat Grain
1 . Bran (15%). It is comprised of pericarp, testa, and aleurone.

2. Germ (2%).
3. Endosperm (83%).
Milling process removes bran (rich in fibers and vitamins) including germ frac-
tion from wheat grain leaving behind starchy endosperm.
ecommended Dietary Allowance of Thiamine

R
• RDA for Adults
–– Thiamine requirement is dependent on the quantity of carbohydrate con-
sumed per day.
–– It is 0.5 mg for adults for 1000 cal/day. Thiamine requirement is between 1.0
and 1.5 mg/day for adults.
• RDA for Children
–– It varies from 0.4 mg to 1.3 mg/day from infants to adolescents age groups.
• RDA for Pregnant/Lactating Women
–– Its requirement is 1.5 mg/day.
bsorption, Transport, and Storage of Thiamine

A
• Dietary thiamine from plant sources is present in free, whereas from animal
sources is present in bound form.
• Bound form of thiamine is hydrolyzed by pyrophosphatase enzyme in the intes-
tine, and thiamine is released.
• Thiamine is absorbed by enterocytes by the following two methods:
–– At low intraluminal concentration, thiamine is absorbed by carrier-
mediated process which is pH-dependent and Na+ independent.
–– At high intraluminal concentration, thiamine is absorbed by passive diffusion.
• Within Enterocytes (Intracellular)
–– Enterocytes contain “thiamine pyrophosphokinase enzyme.”
–– Enzyme brings about phosphorylation of thiamine into thiamine pyro-
phosphate (TPP).
322 11 Vitamins
• TPP is transported across cells (transcellular) from mucosa to serosa.

–– TPP undergoes dephosphorylation by phosphatase enzyme.
–– Free form of thiamine is released from small intestine. It is regulated by
Na+/K+ ATPase.
Storage
• Thiamine stored in the body is highly labile. Thiamine has high turnover rate.
• Thiamine storage is around 25–30 mg.
• Dietary deficiency of thiamine results into depletion body store within 2-3 weeks.
• Tissues with high thiamine storage are the heart, liver, and kidneys.
• Tissues with low thiamine storage are the brain and skeletal tissues.
Transport of Thiamine
• In blood circulation, thiamine binds to albumin. It is distributed to tissues in

complex state with albumin.
Excretion
• Thiamine is water soluble. Thiamine and its metabolites (thiazole and pyrimi-
dine) are excreted by the kidneys.
Functions of Thiamine
Thiamine pyrophosphate functions as coenzyme in biochemical reactions.
Oxidative Decarboxylation of Pyruvate
• Oxidative decarboxylation of pyruvate is catabolized by pyruvate dehydroge-

nase complex (PDH) enzyme into acetyl CoA. It occurs in aerobic glycolysis in
cells.
• TPP acts as coenzyme.
• TPP is essential in carbohydrate metabolism and energy production.
Oxidative Decarboxylation of α-Oxoglutarate (α-Ketoglutarate)
• Oxidative decarboxylation of α-oxoglutarate is catabolized by α-oxoglutarate

dehydrogenase into succinyl CoA.
• TPP is essential in citric acid cycle and energy production.
Non-oxidative Decarboxylation of Pyruvate
• Non-oxidative decarboxylation of pyruvate is catabolized by pyruvate carbox-

ylase into acetaldehyde. It occurs in yeasts.
Degradation of Branched-Chain Amino Acids (BCAAs)
• Valine, leucine, and isoleucine are branched-chain amino acids (BCAAs). They
undergo transamination in cytoplasm into α-keto acids.
• The α-keto acids are transported into the mitochondria and undergo oxidative
decarboxylation by branched-chain α-keto acid dehydrogenase complex (located
on inner mitochondrial membrane) into isobutyryl-CoA, 3-methylbutanoyl-CoA
and 2-methylbutanoyl-CoA.
Transketolation of Ribose-5-phosphate
• Transketolation of ribose-5-phosphate is catabolized by transketolase enzyme

into sedoheptulose and glyceraldehyde.
• TPP is essential in HMP pathway in glucose metabolism and energy production.
Conduction of Nerve Impulse
• Acetylcholine is synthesized from choline and acetyl CoA by choline acetyl-

transferase enzyme in nerves.
• TPP is essential in nerve impulse conduction.
Acts as Cholinomimetic
• Cholinergic neurons in brain have high concentration of thiamine.

• Thiamine enhances the cholinergic action of acetylcholine and also acts as cho-
linomimetic in the brain.
eficiency Disorders of Thiamine

D
Thiamine deficiency is caused by the following factors:
• Malnutrition
• Excessive intake of tea, coffee, and betel nuts (contain high amount of thiami-
nase enzyme)
• Chronic alcoholism
• Chronic disease like diabetes mellitus and liver cirrhosis
• AIDS
Metabolic Impairment
• Increased Plasma Pyruvate Level

324 11 Vitamins
–– Pyruvate accumulates in body tissues. Its plasma concentration is elevated

[normal value (0.7–1.5 mg/dl)].
–– In deficiency of thiamine, blood-brain barrier is compromised. Pyruvate
crosses blood-brain barrier. Brain functioning is impaired.
• Oxidative phosphorylation in the mitochondria and HMP pathway are neg-
atively affected.
• Synthesis of ATP and NADPH are reduced.
• Cellular activities are impaired.
• Thiamine deficiency impairs citric acid cycle. It has a leading role in metabolism
of carbohydrates, lipids, and amino acids.
• Its deficiency inhibits synthesis of ATP, NADPH, acetylcholine, and GABA
and hence impairs nerve conduction.
Beriberi
Beriberi is a thiamine deficiency disorder. It is prevalent in regions where people
consume polished rice as staple diet.
Beriberi is characterized by the following manifestations:
• Anorexia and loss of weight

• Nausea and vomiting
• Fatigue
• Pain in limbs
• Palpitation
It is distinguished into four categories depending on clinical manifestation:

Dry Beriberi
It is associated with peripheral nerve damage.
It is characterized by the following manifestations:
• Tingling sensation in upper and lower limbs (paresthesia)

• Pain in limbs
• Absence of deep tendon reflexes (test for normalcy of spinal cord and periph-
eral nervous system)
• Loss of motor neuron functioning in lower limbs resulting into paralysis
• Ataxia (neurological condition characterized by loss of coordination in skeletal
muscles movement). Walking difficulty and abnormal gait
• Anorexia, nausea, and vomiting
• Absence of edema
Wet Beriberi
It is associated with disorders of cardiovascular system.
• Increased heart rate (tachycardia)

• High systolic blood pressure
• Rapid and bounding pulse

• Congestive cardiac failure
• Difficulty in breathing on exertion (dyspnea)
• Edema of the feet, legs, face, and abdomen
• Anorexia, nausea, and vomiting
Infantile Beriberi
It is manifested in children between 2 and 3 years of age group.
• Hoarseness of voice in children.

• Anorexia, nausea, and vomiting.
• Loss of weight and child becomes marasmic.
• Tachycardia in children.
• Restlessness, lack of concentration, and poor temperament.
• Edema of legs.
• Convulsions in advanced stage of disease.
Gastrointestinal Beriberi
It is associated with manifestations of GIT:
• Accumulation of lactic acid in plasma

• Abdominal pain
• Nausea and vomiting
• Anorexia and weight loss
Wernicke’s Encephalopathy
It is a thiamine deficiency-associated neurological disorder. It is common in mal-
nourished population and alcoholics.
• Ophthalmoplegia [paralysis of ocular muscles (superior, inferior, medial, and

lateral recti, inferior and superior oblique muscles)] responsible for eye
movements
• Ataxia
• Confusion
Korsakoff Syndrome
It is associated with loss of neurons in the brain especially in alcoholics.
It is characterized by severe loss of memory without derangement in intellectual
capabilities:
326 11 Vitamins
• Anterograde amnesia (loss of ability to recall latest past events and generate new
memories, old memories remain intact)
• Retrograde amnesia (loss of ability to recall old memories, new memories can be
generated)
• Confusion
• Change in personality
Wernicke-Korsakoff Syndrome
It is a neurological condition in which clinical manifestations are mixed.
11.5.4 Vitamin B2
History
• In 1926, Goldberger and his coworkers observed a dietary factor to treat
pellagra.
• In 1932, Warburg and Christian discovered a yellow pigment from tissues that
was riboflavin.
• In 1933, Kuhn isolated riboflavin in pure form.
• In 1935, Paul Karrer described chemical structure of riboflavin.
Riboflavin is a deep yellow-colored compound and signifies “ribose” and

“flavin.”
It contains “ribitol” which is a sugar alcohol obtained by reduction of ribose.
It contains flavin nucleus, and its name is derived from the Latin word “fla-
vus” which means “yellow.”
Chemical Structure
1. Forms of Vitamin B2 (Riboflavin)
Riboflavin exists in animal- and plant-based sources in protein-bound form.
Active Forms of Riboflavin
• FMN (flavin mononucleotide also called as riboflavin-5′-phosphate)
• FAD (flavin adenine dinucleotide)
Around 80–90% of riboflavin in diet exists in FMN and FAD forms.
Certain enzymes act in the presence of these coenzymes. They are
called holoenzymes (flavoproteins), for example, succinate dehydroge-
nase, monoamine oxidase, etc.
Riboflavin acts as precursor in synthesis of FMN and FAD.
2. Structure
• Riboflavin is a deep yellow-colored water soluble organic compound.
• Chemically, riboflavin is 7,8-dimethyl-10-ribityl-isoalloxazine.
• It contains a flavin nucleus and d-ribitol.
• Flavin nucleus is 6,7-dimethyl-isoalloxazine. It is made up of three hetero-
cyclic rings linked together.
7α - Methyl CH2 – CHOH – CHOH – CHOH – CH2OH

Group
N9
N1
O
H3C
C
7
A B C
6
H3C C NH
N O
Benzene
6α - Methyl Ring
Group Pyrimidine Azine
Ring Ring
[6,7 – Dimethyl – 9 – D – Ribityl iso – Alloxane]

Riboflavin
[Vitamin B2]
• Flavin nucleus is linked to d-ribitol (ribose alcohol). The d-ribitol has an

open chain configuration. Its first carbon is attached to ninth position on the
flavin nucleus as in Fig. 11.13.
Riboflavin is a yellowish-orange water soluble vitamin which is

thermostable.
On exposure to UV rays, riboflavin is converted into lumiflavin which gives
greenish-yellow fluorescence.
ietary Sources of Riboflavin

D
1. Animal Sources
• Good sources are eggs, liver, kidneys, and lean meat (low-fat, high-protein
contents).
• Milk and cheese.
2. Plant Sources
Good sources are whole cereals, green leafy vegetables, pulses, and nuts.
Bacteria in colon synthesize free form of riboflavin. It is absorbed through
intestinal mucosa.
328 11 Vitamins
ecommended Dietary Allowance of Riboflavin

R
RDA for Infants and Children
• 0.5 mg to 1 mg/day
RDA for Adults
• 1.5 mg/day
bsorption, Transport, and Storage of Riboflavin

A
• Dietary riboflavin is covalently bound to flavoproteins.

• In the stomach
–– Hydrochloric acid causes hydrolysis of flavoproteins. It releases FMN and
FAD.
• In the intestine
–– Flavin adenine dinucleotide is converted into flavin mononucleotide by the
action of FAD pyrophosphatase enzyme.
–– Flavin mononucleotide is converted into free riboflavin by FMN phosphatase
enzyme.
• Free riboflavin is absorbed through mucosa of small intestine by the follow-
ing processes:
–– At low intraluminal concentration, riboflavin is absorbed by “carrier-
mediated” process which is pH-dependent and Na+ independent.
–– At high intraluminal concentration, thiamine is absorbed by passive
diffusion.
• Within enterocytes
–– Free riboflavin undergoes phosphorylation into flavin mononucleotide.
Reaction is catalyzed by flavokinase enzyme. It is ATP-dependent process.
–– FMN is transported across cells (transcellular) from mucosa to serosa.
• At serosa end of the small intestine, transcellular about 90% of FMN is dephos-
phorylated by alkaline phosphatase enzyme into riboflavin.
• Riboflavin enters hepatic portal vein and reaches the liver.
• Within the liver
–– Riboflavin is converted into FMN and FAD.
–– They are released into blood circulation.
Transport of Riboflavin
• In Plasma
–– Riboflavin is bound to plasma proteins like albumin and globulin. It is trans-
ported by blood circulation in bound form.
–– Plasma contains free riboflavin.
• Tissue Level
–– Free riboflavin can easily pass through cell membranes.
–– Its transportation occurs by passive diffusion at high concentration.
–– Its transportation occurs by carrier-mediated process.
–– Calcium and calmodulin (calcium-modulated protein) act as carriers of
riboflavin across cell membrane.
Absorption Inhibitors
Divalent metal ions as iron, zinc, copper, and manganese bind the riboflavin in
intestinal lumen. They inhibit mucosal absorption of riboflavin.
Alcohol inhibits mucosal absorption of riboflavin.
Storage
• Riboflavin is stored in the liver, heart, and kidneys.

• FAD is the predominant (80–90%) storage form of riboflavin.
• FMN is another storage form of riboflavin.
Excretion
• Primarily, riboflavin and its metabolites (hydroxymethylriboflavin, lumiflavin,

carboxymethylflavin) are excreted in urine.
• Small amount of riboflavin is excreted in stools (derived from unabsorbed ribo-
flavin produced by bacteria).
• Riboflavin is also excreted in milk.
Functions of Riboflavin
• Riboflavin is necessary for synthesis of coenzymes (FMN and FAD).
• FMN and FAD act as coenzymes in oxidation-reduction reactions involved in
various metabolic pathways. FMN and FAD accept two hydrogen atoms in the
reaction, and they are reduced to FMNH2 and FADH2.
• FMN acts as coenzyme in oxidation deamination of L-amino acids into ammonia
catalyzed by L-amino acid oxidase enzyme.
• FMN acts as coenzyme in electron transport chain with NADH dehydrogenase
enzyme in complex I.
• FAD acts as coenzyme in conversion of xanthine into hypoxanthine catalyzed by
xanthine oxidase enzyme in purine metabolism.
• FAD acts as coenzyme in conversion of succinic acid into fumaric acid catalyzed
by succinate dehydrogenase enzyme.
• FAD acts as coenzyme in conversion of glycine into glyoxylate catalyzed by
glycine oxidase enzyme.
• FAD acts as coenzyme in conversion of acyl CoA into alpha, beta-unsaturated
acyl CoA catalyzed by acyl CoA dehydrogenase enzyme.
• FAD acts as coenzyme in the conversion of α-ketoglutarate into succinyl CoA
catabolized by α-ketoglutarate dehydrogenase enzyme.
330 11 Vitamins
Deficiency Disorders of Riboflavin
Predisposing Factors to Riboflavin Deficiency

Riboflavin deficiency is rare in nature. The following factors predispose to its
deficiency:
Vegetarian Diet
• Deficiency of riboflavin is endemic. It is found in population who is strictly veg-

etarian and consumes little milk and milk products (vegans).
• Sportsmen who are dependent on vegetarian diet.
Poverty
• Population in slums and remote settings
Physiological Condition
• Pregnant women
• Lactating women
Chronic Alcoholics
• It is associated with poor intake of diet.
Diuretic
• Loop diuretics for prolonged period (furosemide).
Clinical Manifestations
Angular Cheilitis
• It is the inflammation of mucous membrane on the angle of the mouth.

• It is characterized by difficulty in mouth opening and cracking of corners of the
mouth.
Glossitis
• It is the inflammation of the mucosa of the tongue and papillary atrophy.

• It is manifested as redness of the tongue, burning sensation, and loss of taste.
Seborrheic Dermatitis
• It is the inflammation over the scalp.

• It is characterized by redness and dandruff over the scalp.
• Skin rash (redness and itch) appears on the skin of the face.
Redness and burning sensation in eyes

Normocytic and hypochromic or normochromic anemia
Anorexia (loss of appetite), nausea, and vomiting
Loss of body weight and fatigue
Peripheral Neuropathy
• It is the damage to peripheral nerves.

• It is characterized by pain, burning sensation, and tingling.
Deficiency manifestations of riboflavin are called ariboflavinosis.
Therapeutic Applications of Riboflavin
• Role in alleviating (tingling, burning sensations) in carpal tunnel syndrome

(compression on median nerve that passes through carpal tunnel in the
wrist)
• Role in alleviating symptoms in angular cheilitis and glossitis
• Role in management of seborrheic dermatitis and eczema
• Role in migraine treatment
11.5.5 Nicotinic Acid
History
• In 1867, nicotinic acid was prepared from nicotine through oxidation process.
• In 1915, Goldberger observed that pellagra could be produced in healthy persons
by feeding them on diet deficient of a particular factor, which was abundant in
milk and meat. He named the factor as pellagra-preventing factor (P-P factor).
It was nicotinic acid.
• In 1937, C. A. Elvehjem described the structure of nicotinic acid. He isolated P-P
factor from liver extract. He named it as vitamin B3 (due to its sequence in series
of vitamin B complex).
Chemical Structure
1. Forms and Structure of Nicotinic Acid
It exists in three forms:
• Niacin (nicotinic acid)
–– Chemically, it is pyridine-3-carboxylic acid.
332 11 Vitamins
Fig. 11.14 Nicotinic acid 4

(Niacin)
5 C–OH
3
6 2
• Nicotinamide (niacinamide)
–– Chemically, it is acid amide, pyridine-3-carboxylic amide.
• Inositol hexanicotinate (hexaniacinate)
–– Chemically, it contains six molecules of nicotinic acid attached to one
molecule of inositol in the center.
Nicotinamide exists in body tissues in metabolically active forms:
• Nicotinamide Adenine Dinucleotide (NAD+)
–– It is composed of nicotinamide + adenine + d-ribose sugar + two mole-
cules of phosphoric acid.
–– Nicotinamide adenine dinucleotide is also called diphosphopyridine
nucleotide (DPN).
• Nicotinamide Adenine Dinucleotide Phosphate (NADP+)
–– It is composed of nicotinamide + adenine + d-ribose sugar + two mole-
cules of phosphoric acid.
–– It has an additional phosphoric acid residue which is attached to C2 of
d-ribose sugar.
–– Nicotinamide adenine dinucleotide phosphate. It is also called triphos-
phopyridine nucleotide (TPN) as in Fig. 11.14.
Nitrogen atom in nicotinamide contains one unit positive charge. So they are
designated with + sign as suffix.
Nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide
phosphate act as coenzymes.
ietary Sources of Nicotinic Acid

D
1. Animal Sources
• Good sources are the liver, kidney, meat, and fish.
2. Plant Sources
• Rich sources are whole cereals (bran) and yeast.
• Good sources are nuts, peas, beans, lentils, and green vegetables.
• Poor sources are milk, eggs, and fruits.
ecommended Dietary Allowance of Nicotinic Acid

R
• It is around 5 mg/day.
RDA for Adults
• It is around 15–20 mg/day.
RDA in Pregnancy and Lactation
• It is around 20–25 mg/day.
bsorption, Transport, and Storage of Nicotinic Acid

A
Absorption
• Nicotinic acid and nicotinamide are absorbed from mucosa of small intestine.
Excretion
• Nicotinic acid and nicotinamide are excreted in urine.

• They are metabolized in the liver. Enzyme nicotinamide methyltransferase in the
liver transfers methyl group from active methionine to nicotinamide. Thus,
metabolite (N′-methyl nicotinamide) is excreted in urine.
unctions of Nicotinic Acid

F
Metabolically active forms, NAD+ and NADP+, act as coenzymes in metabolic
reactions in the body. They serve as coenzyme with holoenzymes in oxidation-
reduction reactions.
Hydrogen and Electron Transfer
• NAD+ and NADP+ are involved in hydrogen and electron transfer reactions.
• Both coenzymes accept hydrogen atom from metabolite. They are reduced and
metabolite is oxidized.
• At time of reduction of NAD+ and NADP+, each coenzyme accepts one hydrogen
atom and one electron from another hydrogen atom of metabolite.
• NAD+ and NADP+ are reduced into NADH and NADPH.
• H+ from the metabolite is released in tissues.
Lactate dehydrogenase
Lactic acid Pyruvic acid
NAD+ NADH + H+
Pyruvic acid Lactic acid
NADH + H+ NAD+
334 11 Vitamins
Enzymes Dependent on NAD+
• NAD+ acts as coenzyme in the conversion of ethanol into acetaldehyde catalyzed

by alcohol dehydrogenase enzyme.
• NAD+ acts as coenzyme in conversion of malate into oxaloacetic acid catalyzed
by malate dehydrogenase enzyme.
• NAD+ acts as coenzyme in the conversion of pyruvic acid into acetyl CoA cata-
lyzed by pyruvate dehydrogenase enzyme.
Enzymes Dependent on NADP+
• NADP+ acts as coenzyme in the conversion of glucose-6-phosphate into

6-
phosphogluconolactone catalyzed by glucose-6-phosphate dehydrogenase
enzyme.
eficiency Disorders of Nicotinic Acid

D
Niacin deficiency is caused by the following factors:
Deficiency of Tryptophan
• Tryptophan is necessary for biosynthesis of niacin.

• Population who has maize as staple diet suffers from niacin deficiency. The
maize protein (zein) lacks tryptophan amino acid. It leads to niacin
deficiency.
• Population who has sorghum as staple diet suffers from niacin deficiency.
Leucine amino acid in sorghum inhibits quinolinate phosphoribosyltransfer-
ase (QPRT) enzyme. It is responsible de novo synthesis of NAD+ coenzyme.
Administration of Antitubercular Drug (Isoniazid)
• Oral administration of isoniazid is responsible for diminished synthesis of pyri-

doxine. It causes tryptophan deficiency and it affects niacin level in the body.
Congenital Disorder
• Absorption of dietary tryptophan (nonpolar amino acids) is impaired in congeni-

tal disorder called Hartnup disease (autosomal recessive trait).
Its deficiency results in a disorder called pellagra (pelle means skin and agra
means rough).
Pellagra is characterized by three clinical manifestations:
Dermatitis
• It is a skin disorder.
• It is characterized by redness and scales on the face, feet, and ankles.

• Exposure to sunlight aggravates the condition.
Diarrhea
• It is characterized by water loose stools.

• Stools are mucus and or blood stained.
• Nausea, vomiting, and loss of weight are common manifestations with diarrhea.
Dementia
• It is characterized by loss of concentration, weak memory, and irritability.

• Behavior change.
• Delirium (disturbed state of mind with restlessness, incoherent speech, and
illusion (distorted perception) in advanced condition).
These cardinal manifestations of pellagra are called 3Ds.
Therapeutic Application
• It is helpful in the management of dyslipidemia (high serum level of LDL

and/or triglycerides).
• It is helpful in prophylaxis of atherosclerosis and coronary artery disease.
• It has a preventive role in nonalcoholic fatty liver disease in rats.
• Topical application of 1-methyl nicotinamide is helpful in the treatment of
rosacea (condition with progressive facial dermatitis).
11.5.6 Pantothenic Acid
History
• In 1938, R. J. Williams and R. W. Truesdail isolated pantothenic acid.
• In 1945, Lipmann discerned coenzyme A.
Pantothenic acid is derived from the Greek work “pantos” whose meaning is
“everywhere.” Pantothenic acid obtains its name owing to its abundance in cells
of plants and animals.
Pantothenic acid is also called as vitamin B5.
hemical Structure and Forms

C
1. Forms of Pantothenic Acid
Free Form
• Pantothenic acid is found in acid form.
• It is soluble in water.
• It is thermolabile.
336 11 Vitamins
Bioactive Form
• Bioactive form is called coenzyme A (co-acetylase).
• It is present in all body tissues.
• Coenzyme A is also found in bound state with proteins.
2 . Structure of Pantothenic acid
• It is composed of beta-alanine and pantoic acid (dihydroxy dimethyl butyric acid).
• Both units are linked together by peptide bond.
3. Structure of Coenzyme A
• Pantothenic acid has two linkages on either side.
• On one side, pantothenic acid is attached to adenosine-3-phosphate through a
pyrophosphate linkage.
• On other side, pantothenic acid is attached to beta-mercaptoethanolamine
(thiolethanolamine) through a peptide linkage.
• Coenzyme A is represented as CoA-SH.
• Terminal thiol group (-SH) of beta-mercaptoethanolamine is the reactive
site of coenzyme A as in Fig. 11.15.
Biosynthesis of Coenzyme A
Human, plant, and animal tissues can synthesize coenzyme A. Its synthesis follows
an elaborate biochemical process as:
• Phosphorylation
–– Pantothenic acid undergoes phosphorylation in the presence of ATP to form
4′-phsophopantothenic acid. Reaction is catalyzed by pantothenate kinase
enzyme.
• Synthesis
–– Cysteine is linked to 4′-phsophopantothenic acid in the presence of ATP and
enzyme phosphopantetheinyl synthetase to form 4′-phsophopantothenyl
cysteine.
• Decarboxylation
–– 4′-Phsophopantothenyl cysteine undergoes decarboxylation by phosphopan-
tetheinyl cysteine decarboxylase enzyme to form 4′-phsophopantetheine.
• Adenylation (AMPylation)
–– 4′-Phsophopantetheine undergoes adenylation (addition of AMP to protein
molecule) to form dephospho-CoA.
• Phosphorylation
–– Dephospho-CoA undergoes phosphorylation to form coenzyme A.
Fig. 11.15 Vitamin B3 CH3 OH

––
––
∝ β
CH2––C––CH––C–– NH––CH2––CH2––C––OH
γ
–
–
–
OH CH3 O O
Pantoic acid β-Alanine

Dietary Sources
Pantothenic acid is widely found in animals, plants, bacteria, and food.
Animal Sources
• Good sources are liver, kidneys, eggs, and yeasts.

• Average sources are chicken, milk, and fishes.
Plant Sources
• Good sources are cereals, legumes, and vegetables.
Royal jelly is the richest source of pantothenic acid.

Royal jelly is also a rich source of biotin and pyridoxine.
ecommended Dietary Allowance of Pantothenic Acid

R
Requirement of pantothenic acid among humans is uncertain.
• It is about 1–4 mg/day.
RDA for Adults
• It is about 5–10 mg/day.
Excretion
• Pantothenic acid is excreted in urine, sweat, and milk.
• Urinary excretion of pantothenic acid is between 2 and 4 mg/day.
unctions of Pantothenic Acid

F
Pantothenic acid in biologically active form serves multiple roles in human metabo-
lism as:
Synthesis of Acetyl CoA
• Coenzyme A links with acetate and forms acetyl CoA. It serves the following
functions:
–– There is a high-energy sulfur bond in acetyl CoA.
–– Acetyl CoA is involved in the synthesis of oxaloacetate formation in citric
acid cycle.
–– Acetyl CoA is involved in cholesterol formation.
–– Acetyl CoA is involved in acetylcholine synthesis.
–– Acetyl CoA is involved in synthesis of ketone bodies.
338 11 Vitamins
Synthesis of Succinyl CoA
• α-Ketoglutarate undergoes oxidative decarboxylation to form succinyl CoA

(active succinate). It serves the following functions:
–– Succinyl CoA is involved in the synthesis of heme.
–– Succinyl CoA is involved in utilization of ketone bodies by extrahepatic
tissues.
Beta Oxidation of Fatty Acids

Acyl synthase (thiokinase) is necessary for beta oxidation of fatty acids.
De Novo Synthesis of Fatty Acids

Pantothenic acid is a necessary component of acyl carrier protein which is involved
in de novo synthesis of fatty acids in extramitochondrial tissues.
Synthesis of Adrenocortical Hormones

Pantothenic acid is required for the formation of acetyl CoA and cholesterol. These
compounds are necessary for the synthesis of adrenocortical hormones in adrenal
cortex.
eficiency Disorders of Pantothenic Acid

D
Deficiency of pantothenic acid has not been observed among humans owing to its
abundance in plants and animals which constitute dietary sources of humans.
Deficiency Manifestations in Experimental Animals
• Dryness of the skin, thinning and loss of hair, and dermatitis

• Diarrhea, loss of appetite, and symptoms of gastritis
• Fatty liver
• Demyelination of peripheral nerves
Deficiency Manifestations in Human Volunteers

Deficiency of pantothenic acid was induced by the use of omega-methyl pantothe-
nate (antagonist) to human volunteers. The following manifestations were
observed:
• Burning feet syndrome

–– Tingling or burning sensation in the feet
–– Numbness in feet
–– Fatigue
• Loss of appetite, nausea, vomiting, and hyperacidity
• Loss of concentration, confusion, and irritability
11.5.7 Vitamin B6
History
• In 1930, Rudolf Peters induced skin lesions (rat acrodynia) in rats by feeding a
diet rich in thiamine and riboflavin but deficient of other nutrients.
• In 1934, Paul Gyorgy treated skin lesions in rats through a diet enriched with a
factor which he called vitamin B6.
• In 1938, Samuel Lepkovsky extracted and crystallized vitamin B6.
• In 1938, Leslie Harris and Karl Folkers showed pyridine derivative nature of
vitamin B6
Chemical Structure
Forms and Structure of Vitamin B6
Vitamin B6 exists in three forms:
• Chemically, vitamin B6 is 2-methyl-3-hydroxy-4,5-dihydroxymethyl

pyridine.
• Pyridoxine (pyridoxol) is a stable form.
• Another two forms are:
–– An aldehyde (pyridoxal)
–– An amine (pyridoxamine) as in Fig. 11.16.
• These forms are water soluble.
• They are damaged on exposure to UV light.
Vitamin B6 exists in two biologically functional forms:
• Pyridoxal phosphate
• Pyridoxamine phosphate
They act as coenzyme in metabolic pathways.
O
CH2OH Alcoholic group C–H Aldehyde group CH2.NH2 Amine group
4 4 4
HO
HO 3 5 CH2OH HO 5 CH2OH 3 5 CH2OH
H3C 2
6 H3C 6 H3C 2
6
1 N N1
Pyridoxine (pyridoxal) Pyridoxal Pyridoxamine
[Vitamin B6]
Fig. 11.16 Forms of Vitamin B6

340 11 Vitamins
Pyridoxal phosphate is synthesized in the brain, liver, and kidneys through phos-
phorylation as follows:
Pyridoxal kinase
Pyridoxine Pyridoxal phosphate
ATP ADP + Pi
ietary Sources of Vitamin B6

D
Vitamin B6 is produced by microbes and plants. It is also synthesized in human
colon by bacteria.
Dietary sources contain pyridoxine as the main form of vitamin.
1. Animal Sources
• Good sources are the liver, kidneys, eggs, meat, and fish.
• Poor source is milk.
2. Plant Sources
• Rich sources are yeast and whole cereals (bran).
• Good sources are nuts, pea, beans, lentils, and vegetables.
ecommended Dietary Allowance of Vitamin B6

R
• It is around 0.2–0.5 mg/day.
RDA for Adults
• It is around 1.5–2 mg/day.
• It is 2.5 mg/day.
RDA of pyridoxine is dependent on dietary intake of protein per day.
bsorption, Transport, and Storage of Vitamin B6

A
Absorption
• Dietary pyridoxine is absorbed through intestinal mucosa.

• After absorption, it enters enterohepatic circulation and reaches the liver.
• Pyridoxine undergoes phosphorylation by pyridoxal kinase and dephosphoryla-
tion by alkaline phosphatase enzymes.
Transport
• Pyridoxal-P in blood circulation binds to albumin, and it is distributed to body

tissues.
Storage
• Pyridoxine is stored mainly in muscles (70%).

• It is also stored in the liver (20%).
• Small amount of pyridoxine is stored in body tissues as pyridoxal-P.
Excretion
• Pyridoxine is excreted in urine.

• Metabolite of pyridoxine and pyridoxamine is 4-pyridoxic acid. It is excreted in
urine.
unctions of Vitamin B6
F
It acts as coenzyme in many metabolic reactions in the body as:
Arachidonic Acid Synthesis
• Arachidonic acid is synthesized from linoleic acid through action of desaturase

enzyme. Pyridoxal-P acts as coenzyme in the reaction.
CoA-SH Synthesis
• Pyridoxal-P acts as coenzyme in synthesis of CoA-SH from pantothenic acid.
Decarboxylation
• Amino acids undergo decarboxylation catalyzed by decarboxylase enzyme in

presence of pyridoxal-P as coenzyme.
• Histidine into histamine and CO2.
• Glutamic acid into gamma-aminobutyric acid (GABA) and CO2.
Glycogenolysis
• Glycogen is converted into glucose-1-phosphate catalyzed by phosphorylase

enzyme in the presence of pyridoxal-P as coenzyme.
Heme Synthesis
• Alpha-amino beta-keto adipic acid undergoes decarboxylation to produce

D-aminolevulinic acid (ALA). It is necessary for the formation of tetrapyrrole
rings in heme synthesis. It requires ALA synthase enzyme and pyridoxal phos-
phate as coenzyme.
342 11 Vitamins
Non-oxidative Deamination (Hydroxyl Group Containing Amino Acids)
• Hydroxyl group containing amino acids like serine and threonine undergoes non-
oxidative deamination by deaminase enzyme and pyridoxal-P as coenzyme.
Non-oxidative Deamination (Sulfur Containing Amino Acid)
• Sulfur containing amino acid like cysteine undergoes non-oxidative deamination

by desulfhydrase enzyme and pyridoxal-P as coenzyme.
Niacin Synthesis
• Kynureninase enzyme converts 3-hydroxy kynurenine into 3-hydroxy anthra-

nilic acid which is necessary for niacin synthesis.
• The activity of kynureninase enzyme is dependent of pyridoxal-P as coenzyme.
• In deficiency of pyridoxal-P, the 3-hydroxy kynurenine is converted into xanth-
urenic acid, and synthesis of niacin is impaired.
Sphingomyelin Synthesis
• Pyridoxal-P is necessary as coenzyme in synthesis of sphingomyelin.
Transamination
• Pyridoxal-P is necessary as coenzyme for catalytic activity of aminotransferases

(SGPT and SGOT).
Transsulfuration
• Pyridoxal-P is necessary as coenzyme in transsulfuration metabolic pathway. It

involves transfer of sulfhydryl group from homocysteine to cysteine (reverse
transsulfuration pathway in humans) and cysteine to homocysteine (direct trans-
sulfuration pathway in bacteria).
Deficiency Disorders of Vitamin B6
Factors Predisposing to Deficiency of Vitamin B6

Administration of Antitubercular Drug (INH)
• Isonicotinic acid hydrazide (INH) is an antitubercular drug.

• INH inhibits enzyme pyridoxal kinase which is necessary for phosphorylation of
pyridoxine.
• Another view is that INH complexes with pyridoxine to form hydrazone. It is
responsible incomplete phosphorylation of pyridoxine.
• Administration of INH in tubercular patients produces symptoms similar to defi-

ciency of pyridoxal-P.
• Severity of manifestations is dose dependent.
• Supplementation of pyridoxine helps to overcome deficiency symptoms.
The Use of Oral Contraceptives

Prolonged use of oral contraceptives results in deficiency of pyridoxal-P.
Chronic Alcoholics
• Ethyl alcohol is converted into acetaldehyde by alcohol dehydrogenase enzyme.

Acetaldehyde inhibits coenzyme activity of pyridoxal-P.
• It results in decrease in serum pyridoxal-P level in chronic alcoholics.
Malnutrition
• Dietary deficiency of vitamin B6 is common in poor population especially slum

dwellers.
• Population who consume polished rice as staple diet suffers from deficiency of
vitamin B6.
Anemia
• Pyridoxal-P deficiency results in diminished synthesis of heme. Serum iron

level is normal in the body. However, utilization of iron in erythropoiesis is
impaired.
• Pyridoxal-P deficiency causes microcytic hypochromic anemia. It is also
called sideroblastic anemia (characterized by abnormal erythroblasts
having a ring of iron granules loaded in mitochondria encircling the
nucleus).
Peripheral Neuritis
• Pyridoxal-P deficiency causes decreased synthesis of sphingomyelin. It results in

demyelination of nerves and inflammation of peripheral nerves.
• It is characterized by neuralgia, numbness, and burning sensation in extremities.
Dermatitis
• Pyridoxal-P deficiency causes decreased synthesis of niacin.

• It results in redness and scales on the skin.
344 11 Vitamins
Convulsions in Infants
• Pyridoxal-P deficiency results in decreased synthesis of neurotransmitters like

GABA, serotonin, and adrenaline.
• The activity of pyridoxal-P-dependent glutamic acid decarboxylase enzyme is
decreased. Thus, synthesis of GABA is diminished. It leads convulsions in infants.
11.5.8 Biotin
History
• In 1927, Boas found that rats that were fed upon large amount of raw egg white
suffered from symptoms of dermatitis and retarded growth and termed the disor-
der as egg-white injury. Later on, it was observed that fed based on cooked eggs
did not produce similar symptoms.
• In 1934, Lease and Parsons observed egg-white injury in chickens.
• In 1936, Kogl and Jonnis extracted a compound from yolk of dried egg and
called it biotin.
• In 1942, Du Vigneaud and colleagues described the structure of biotin.
• Biotin was termed as anti-egg white vitamin.
• Biotin is also called vitamin B7 or vitamin H (H for German words “haar and
haut” representing the hair and skin).
• Biotin is a sulfur-rich vitamin B complex.
• Factor responsible for egg-white injury was discovered as avidin
(glycoprotein).
• Avidin is a basic protein which binds with biotin to render it unavailable for
mucosal absorption.
Chemical Structure
1. Forms of Biotin
Biotin exists in two forms:
• Free Form
–– Biotin is a free form.
• Bound Form
–– Biocytin is a bound form of biotin with protein in tissues.
–– Biocytin is linked to lysine moiety by amide linkage.
Depending on Source
• Alpha-Biotin
–– It is found in egg yolk.
• Beta-Biotin
–– It is found in the liver.
2. Structure of Biotin
• Chemically, it is hexahydro-2-oxo-1-thieno-3-4-imidazole-4-valeric acid.
• Biotin is a heterocyclic monocarboxylic acid containing sulfur atom.
• Biotin is composed of imidazole ring and thiophene ring linked together.
• Valeric acid side chain is attached to thiophene ring as in Fig. 11.17.
CO2
Binding site O
C
HN 2 NH
1 3
Imidazole ring
HC CH
Thiophene 4 3
ring
5 2 CH CH2 CH2 CH2 C OH
H2C
O
1S
Site for binding

with lysine
Fig. 11.17 Biotin
ietary Sources of Biotin

D
1. Animal Sources
• Rich sources are egg yolk, liver, meat, kidneys, and milk.
2. Plant Sources
• Good sources are nuts, legumes, and almonds.
ecommended Dietary Allowance of Biotin

R
RDA of biotin for adults is around 100–300 μg/day.
bsorption, Transport, and Storage of Vitamin

A
Absorption
Transportation
Excretion
Functions of Biotin
Biotin acts as coenzyme with carboxylase enzyme. This enzyme catalyzes reactions
involved in carbon dioxide fixation. Biotin serves role in the following metabolic
reactions:
Role in Gluconeogenesis
• Pyruvic acid is converted into oxaloacetic acid by pyruvate carboxylase enzyme

in gluconeogenesis.. Pyruvate carboxylase activity is dependent on biotin as
coenzyme.
346 11 Vitamins
Role in Fatty Acid Synthesis
• Acetyl CoA is converted into malonyl CoA in fatty acid synthesis by acetyl CoA
carboxylase enzyme. Its activity is biotin dependent.
Carboxylation of Propionyl CoA
• Propionyl CoA is a metabolite in isoleucine, leucine, and valine amino acid

catabolism.
• It is the end product in oxidation of odd-chain fatty acids.
• Propionyl CoA undergoes carboxylation into methylmalonyl CoA by propionyl
CoA enzyme which is biotin dependent.
eficiency Disorders of Biotin

D
Biotin deficiency in humans is rare owing to its wide presence in plant and animals
dietary sources. Colonic bacteria also produce biotin.
However, biotin deficiency might arise due to following factors:
Prolonged Administration of Broad-Spectrum Antibiotics
• Administration of broad-spectrum antibiotics for prolonged period results in

damage of colonic bacteria.
Intake of Raw Eggs
• Intake of raw eggs (15–20 eggs per day) for prolonged period inhibits absorption
of dietary biotin. Avidin in egg white binds with biotin to form complex. Mucosal
absorption of biotin is inhibited.
Biotin Deficiency Manifestations
• Anorexia, loss of weight, nausea, and vomiting are common GIT

manifestations.
• Glossitis is the inflammation of tongue. It is associated with burning sensation
and loss of taste.
• Anemia is microcytic and hypochromic type.
• Fatigue, irritability, and depression are common CNS manifestations.
11.5.9 Folic Acid
History
• In 1931, Lucy Willis demonstrated that anemia in pregnancy could be treated
with an extract from brewer’s yeast.
• In 1941, H. K. Mitchell, E. E. Snell, and R. J. Williams isolated folic acid from
leaves of spinach.
• In 1943, B. Stokstad described chemical structure of folic acid.
Folate
• Folate is vitamin B9.

• It derives its name from the Latin word folium meaning leaf. It is found in
green leafy vegetables.
• Folate refers to water soluble, naturally occurring heterogeneous com-
pounds belonging to family of vitamin B complex.
• Folate is polyglutamates.
Folic Acid
• Folic acid is a synthetic form of folate. It is used for food fortification and
supplements.
• Folic acid has better stability in food and better digestibility in alimentary
canal than folate.
• Folic acid is monoglutamate.
orms and Chemical Structure

F
Forms of Folic Acid
Depending on number of glutamate residues attached to pteridine-PABA, it has the
following congeners:
• Monoglutamate (one glutamic acid residue)

• Polyglutamate (three to seven glutamic acid residues)
• Tetrahydrofolate (reduced folate)
–– It is a biological active variant of folate. It acts as coenzyme.
–– Four hydrogen atoms are attached to pteridine residue at positions 5, 6, 7, and 8.
–– Folic acid is reduced to 7,8-dihydrofolate by folate reductase enzyme. It is
again reduced to tetrahydrofolate by folate reductase. NADPH acts as reduc-
ing equivalent to donate hydrogen atoms to folate. Ascorbic acid acts as
cofactor in the reduction reaction as in Fig. 11.19.
• 5-Formyl Tetrahydrofolate (f5 FH4)
–– In 1948, it was found in liver extract and observed as growth factor for
Leuconostoc citrovorum and termed as citrovorum factor.
–– It is also called folinic acid or leucovorin.
–– It is a reduced folate with a formyl group at position 5 of pteroyl complex.
–– It is biological active form of folate and is used as an adjuvant with chemo-
therapeutic drugs like 5-fluorouracil and methotrexate in treatment of cancer.
It is also used in management of megaloblastic anemia.
–– It was extracted.
348 11 Vitamins
• 10-Formyl Tetrahydrofolate (f10 FH4)

–– It is called rhizopterin or Streptococcus lactis R factor (SLR).
–– It was extracted from the liver.
–– It contains formyl group at position 10 of pteroyl complex.
Structure of Folic Acid
• Folic acid derives its name from leaves in which (green leafy vegetables) it is
found in abundance.
• Folic acid is called pteroyl glutamic acid, vitamin B9, or folate.
• It is a water soluble heterogeneous compound.
• Chemically, folate is pteroyl glutamic acid (PGA). It is comprised of three
components:
–– Pteridine nucleus (made up of pyrimidine and pyrazine rings)
–– Para-amino benzoic acid (pteridine nucleus + para-amino benzoic
acid = pteroyl complex).
–– Glutamic acid (it exists in one to seven residues) as in Fig. 11.18.
ietary Sources of Folic Acid

D
Animal Sources
• Rich sources are the liver, kidneys, yeast, and meat.

• Good source is milk.
Plant Sources
• Sources are vegetables like spinach, cauliflower, peas, asparagus, broccoli, pea-
nuts, tomato juice, banana, papaya, and citrus fruit.
COOH
CH2
N 8N
CH2
H2N
7
2 H CH2
3 9 10
6
C CH2 NH C N CH
HN 5
O
COOH
OH
Pteridine nucleus Para-amino Glutamic
benzoic acid acid
Pteroic acid
Fig. 11.18 Structure of Folic acid

ecommended Dietary Allowance of Folic Acid

R
• It is 50 μg in infants and 100–300 μg for children.
RDA for Adults
• It is 400 μg for adults.
• Its RDA is 600 μg in pregnancy and 500 μg in lactation.
bsorption, Transport, and Storage of Folic Acid

A
Absorption
• Folate exists in polyglutamate form in natural foods. It is stable for limited

days. Preparation of food can destroy its biological activity. However, food forti-
fication is performed by addition of folic acid whose shelf life is higher than
folate and can be active for months.
• Polyglutamate is unable to pass through intestinal mucosa. It is cleavaged by
folate conjugase enzyme into monoglutamate. Folate conjugase is found in
mucosa of duodenum and jejunum.
• Proton-coupled folate transporter is present in brush border surface of duode-
num and jejunum. It supports proton and folate ions through intestinal epithe-
lium. Its activity is pH-dependent with optimum activity and is seen at pH of 5.5.
• Within Enterocytes
–– Small amount of monoglutamates is reduced into dihydrofolate and tetrahy-
drofolate by folate reductase enzyme.
–– Tetrahydrofolate undergoes methylation to form 5-methyl THF inside
enterocytes.
–– The 5-methyltetrahydrofolate escapes through the basolateral surface of
enterocytes and enters the liver through the hepatic portal vein.
Transportation
• The liver stores about 20% of 5-methyl THF, while the remaining 80% is
released into blood circulation.
• Within blood circulation
–– 5-methyl THF is present in circulation in three states:
Unbound form (30% of folates)
Bound form with albumin (66% of folates)
Bound with high affinity binders (3% of folates)
Excretion of folic acid
• About 2 μg of folate is excreted in urine.

• About 20% of dietary folate remains unabsorbed. It is excreted in stools.
• Trace amount of folate is excreted in saliva.
350 11 Vitamins
unctions of Folic Acid

F
1. Tetrahydrofolate serves as highly adaptable coenzyme in various metabolic reac-
tions in which it helps to transfer one-carbon unit. This is called folate-mediated
one-carbon metabolism.
Definition
One-carbon metabolism is the transfer of one-carbon unit from a donor
molecule to an acceptor molecule with the help of tetrahydrofolate as a
coenzyme.
One-carbon metabolism is responsible for the formation of different compounds

in the body.
Tetrahydrofolate is significantly involved in one-carbon metabolism.
Metabolically active tetrahydrofolate contains a reduced pteroyl complex with poly-
glutamates. The polyglutamates have three to seven glutamate residues linked
through γ-peptide bonds.
Occurrence
• One-carbon metabolism occurs in the cytoplasm and mitochondria.
Types of One-Carbon Units

One-carbon units which take part in metabolism are enlisted as follows:
Methyl unit (–CH3)
Hydroxymethyl unit (CH2OH)
Methylene unit (〓CH2)
Methenyl unit (–CH〓)
Formyl unit (–CH〓O)
Formimino unit (–CH〓NH)
One-carbon unit is linked covalently with tetrahydrofolate coenzyme either at N5
or N10, or both these positions in pteroyl complex are occupied by one-carbon units
as in Fig. 11.19.
Therefore, THF acts as an acceptor and a donor of one-carbon units in vari-
ous metabolic reactions.
Fig. 11.19 Showing inter- NH

convertibility of one-car- N H
bon unit carriers H2N 8
7 H
2
3 6 CH2 NH R
4
N
5 H
OH NH
[5, 6, 7, 8, –Tetrahydrofolate]
Sources of One-Carbon Units

The following compounds provide one-carbon units. They enter one-carbon pool of
the body.
• Choline and Betaine

–– Choline is a nitrogenous compound and a nutrient. It was extracted from the
bile of pig and ox. Chemically, choline is N,N,N-trimethylammonium com-
pound. It is a structural component of phospholipids like lecithin. It is present
as phosphatidylcholine. Choline can donate three methyl units to one-
carbon pool of the body.
–– Betaine is N-methylated amino acid. Chemically, betaine is N,N,N-
trimethylglycine (derivative of glycine).It was discovered in beet plant (roots
have rich concentration of sucrose). Betaine is a metabolite of choline and is
synthesized in the liver through choline oxidation. It can donate methyl unit.
–– Methyl units from choline and betaine are oxidized into hydroxymethyl
units. They are further oxidized in the presence of NADP into formyl
group. It is accepted by THF to form 5,10-formyl THF.
• Histidine provides formimino group, and THF acts as acceptor to form N5-
formimino tetrahydrofolate.
• Tryptophan provides formyl group, and THF acts as acceptor to form N10-
formyl THF.
• Conversion of serine to glycine provides hydroxymethyl group which is con-
verted into methylene group, and THF acts as acceptor to form N5,N10-
methylene THF.
• Oxidative deamination of glycine provides methylene group, and THF
accepts it to form methylene THF.
Utilization of One-Carbon Moiety

One-carbon units are used for biosynthesis of compounds in the body.
Tetrahydrofolate serves as coenzyme in these reactions.
Role of N5,N10-Methylenetetrahydrofolate
It is the most important compound, one-carbon metabolism.
Synthesis of N5-Methyltetrahydrofolate
N5,N10-methylenetetrahydrofolate undergoes reduction in the presence of NADH
to form N5-methyltetrahydrofolate. Reaction is catalyzed by methylenetetrahydro-
folate reductase.
Biosynthesis of Thymidine
N5,N10-methylenetetrahydrofolate acts as donor of one-carbon unit. It is
essential for methylation reaction in which 2-deoxy-uridine-5-monophosphate
(dUMP) is converted into 2-deoxythymidine-5-monophosphate (dTMP). Reaction
is catalyzed by thymidylate synthase enzyme. Therefore, N5,N10-
methylenetetrahydrofolate is essential for biosynthesis of thymidine (deoxy-
ribosyl-thymine) or thymine-deoxy-riboside.
Biosynthesis of Glycine from Serine
352 11 Vitamins
N5,N10-methylenetetrahydrofolate has a role in synthesis of glycine from serine. Beta

carbon of serine carries hydroxymethyl group which is cleavaged by THF-dependent
serine hydroxymethyl transferase enzyme into glycine and 5,10-methylene THF.
Oxidative Deamination of Glycine
Glycine is deaminated by multienzyme complex in the presence of pyridoxal phos-
phate. There are formation of NH3 and CO2 and release of methylene which is car-
ried by THF.
Role of 10-Formyl Tetrahydrofolate (f10 THF)

It can donate formyl group to metabolic reactions. It is synthesized from
5,10-methenyl THF through the action of methenyl THF cyclohydrolase.
Purine Biosynthesis De Novo
10-Formyl tetrahydrofolate donates formyl groups in purine biosynthesis. Formyl
groups provide C2 and C8 of purine ring.
Synthesis of Formyl-Methionyl-tRNA
10-Formyl tetrahydrofolate donates formyl group in the formylation process of methi-
onine. Formyl-methionyl-tRNA is essential for initiation of translation in prokaryotes
Conversion of Glycine into Serine
10-Formyl tetrahydrofolate donates formyl group to glycine and helps in biosynthe-
sis of serine.
Role of Methenyl THF

5,10-Methenyl THF acts as donor and acceptor of methenyl unit in metabolic reac-
tions. It is synthesized from 5,10-methylene THF via the action of methylene THF
dehydrogenase enzyme. It is also produced during breakdown of histidine (forma-
tion of 5-formimino THF which is converted into 5,10-methylene THF by
formiminotransferase-cyclodeaminase enzyme).
Methenyl THF and 5-formyl THF are found in cells. However, they do not
act as coenzymes.
Role of 5-Methyl THF

It is synthesized from N5,N10-methylenetetrahydrofolate by reduction. THF can
also accept methyl group from serine.
Conversion of Homocysteine into Methionine
5-Methyl THF provides its methyl unit to cobalamin and forms methylcobalamin. It
in turn transfers methyl group to homocysteine, and it is converted into
methionine.
Generally, deficiency of cobalamin is followed by insufficiency of folic acid. It
is due to failure of methyl group transfer to cobalamin and regeneration of THF as
in Fig. 11.20. This phenomenon is called as folate trap.
2. Folic acid is necessary for biosynthesis of DNA (de novo synthesis of thymine
and purine).
3. Folic acid is essential for proliferation of cells.
4. Folic acid is necessary for erythropoiesis.
N5, N10-Methylene THF
REDUCTASE ENZYME
N5-Methyl
THF
Vit B12
HOMOCYSTEINE
HOMOCYSTEINE METHYL TRANSFERAS

TETRAHYDROFOLATEMETHYL COBALAMIN
ONE-CARBON METABOLISM
METHIONINE
Fig. 11.20 Showing conversion of homocysteine into methionine
eficiency Disorders of Folic Acid

D
Deficiency of folic acid is generally found in the following conditions:
Poor Dietary Intake
• Dietary deficiency of folate is very common in developing countries. It is more

prevalent in vegetarians whose diet lacks vegetable and fruits.
354 11 Vitamins
Pregnancy
• Requirement of folic acid is increased in pregnancy and lactation. Therefore,

lack of folate supplementation results in folate deficiency.
Malabsorption Syndrome
• It is an inflammatory condition of intestinal mucosa owing to multiple factors. It

causes impairment in the absorption of dietary folates.
Drug Induced
• Drugs like phenobarbitone, phenytoin sodium inhibit absorption of dietary

folates.
Deficiency Manifestations
Megaloblastic Anemia
• Folic acid is necessary for DNA synthesis. Its deficiency results in impaired
DNA replication and megaloblastic anemia.
Impaired DNA Synthesis
• Folic acid is necessary for biosynthesis of purine and pyrimidine (dTMP). Its
deficiency results in decreased synthesis of DNA.
Neural Tube Defects
• They are birth defects in the spinal cord and brain. They are attributed to defi-
ciency of folate in mother. Neural tube defect is manifested as spina bifida (defect
in the spine) and anencephaly (little brain formation).
Homocysteinemia
• Folate deficiency results in increased plasma level of homocysteine (normal

4–10 mmol/L). Homocysteinemia is implicated in inflammation of blood vessels
and coronary artery disease.
Folate Therapy
• 1 mg of folate is administered orally for management of megaloblastic anemia.

Folate supplements are co-administered with vitamin B12 supplements.
11.5.10 Vitamin B12
History
• In 1934, William Murphy and George Minot discovered the effectiveness of liver
therapy in treatment of pernicious anemia.
• In 1964, D Hodgkin proposed structure of cobalamin.
• In 1965, Robert Woodward synthesized cobalamin.
orms and Chemical Structure of Vit. B12

F
Forms of Vitamin B12
• Hydroxocobalamin
Hydroxocobalamin is a type of vitamin B12 which is found in nature. It is synthe-
sized by bacteria.
• Cyanocobalamin
Cyanocobalamin is a synthetic analog of hydroxocobalamin. It contains a cya-
nide group. It is more stable than hydroxocobalamin. It is easily crystalized.
• Methylcobalamin
It is a form of cobalamin in which cyanide group is replaced with methyl group.
It is metabolically active analog of cyanocobalamin
• Cobamide
It is a highly active form of cobalamin in the body. It contains adenosyl group
linked to central cobalt atom via C → CO bond. It lacks cyanide ligand.
Cobamide exists in four forms:

1. Dimethyl-benzimidazole cobamide (DBC)
2. Benzimidazole cobamide (BC)
3. Adenyl cobamide (AC)
4. Methyl cobamide (MC)
Chemical Structure
• Vitamin B12 is water soluble essential micronutrient. It is called cobalamin.

• It is octahedral in shape. It has a stable cobalt-carbon bonded chemical structure.
• Cobalamin is made up of three components:
–– Corrin ring
–– Corrin ring has a tetrapyrrole structure. Four rings are numbered as I, II, III,
and IV. These rings are linked together by methylene bridges except ring I and
IV, which are linked directly without methylene bridge.
–– Cobalt atom
–– Cobalt is a heavy metal. It is positioned in the center of tetrapyrrole nucleus.
Cobalt atom is linked through coordinative bonding nitrogen atom in each
pyrrole ring. This is corrin ring.
–– DBI ring
356 11 Vitamins
H O H
N–C–C H H H
H H C–C–C–N
H 2C H H O H
A
C–H
H O H H
H3C H H H H
N H–C–H
H–N–C–C C–C–N
H H O H
D N CO N B
H O H H H H H
H–N–C–C–C C–C–C–N
H H N
H–C–H H H O H
H
H C
H–C–H C
O
A,B,C,D Pyrrole R
C H
H3C CH H2N – C – H2C – H2C rings I
H
O N
CH3
Nucleus
O O
N
Phosphate P CH3
Group
Dimethyl – Benzimidazole
O O CH3
N
OH
C C
H
H H
C C [Cynocabalamin]
O H Vitamin B12
HO – H2C
Ribose
–– The nitrogen atom of 5,6-dimethyl benzimidazole is linked to C5 of ribose

sugar, and it is termed as DBI ring.
–– DBI ring is attached to cobalt atom through its nitrogen atom and propionic
acid of IV pyrrole ring through phosphate bonding as in Fig. 11.21.
ietary Sources of Folic Acid

D
Animal Sources
• Richest source is the liver.

• Good sources are meat, fish, and eggs.
• Milk and milk products contain negligible amount of cobalamin.
Plant Sources
• Vegetables and fruits do not contain vitamin B12.
Microflora in human colon synthesizes cobalamin. It is unavailable for

absorption and is excreted in feces.
Microflora in human small intestine also produces cobalamin. Important
organisms are Klebsiella species and Pseudomonas.
ecommended Dietary Allowance of Cobalamin

R
• RDA of cobalamin for adults is 2 μg/day.
• RDA of cobalamin in pregnancy and lactation is 2.5 μg/day.
• RDA of cobalamin for children is 0.5–1.2 μg/day.
Absorption, Transport, and Storage of Folic Acid
Absorption
Release of Dietary Cobalamin in Stomach
• Dietary cobalamin is associated with protein in natural food sources. After intake
of food, hydrochloric acid in stomach helps to denature proteins and release free
cobalamin.
Formation of Haptocorrin-Vitamin B12 Complex in the Stomach
• Free cobalamin is liable to destruction by HCL. It complexes with a protein

called haptocorrin or R-protein. It is synthesized by salivary glands in the mouth.
However, low pH of the stomach favors formation of haptocorrin-vitamin B12
complex in stomach. Haptocorrin-B12 complex passes through the pylorus and
enters the proximal part of the duodenum.
Formation of Intrinsic Factor-Vitamin B12 Complex in the Duodenum
• Duodenum contains pancreatic protease. It is secreted by the pancreas. This

enzyme partly hydrolyzes the haptocorrin-vitamin B12 complex. Vitamin B12 is
released in the duodenum. Alkaline pH in duodenum favors association of intrinsic
factor of castle with vitamin B12 to form intrinsic factor-vitamin B12 complex. This
complex moves toward distal portion of the duodenum and reaches the ileum.
Receptor-Mediated Absorption
• Brush border epithelium of ileum contains cubilin and megalin receptors. They
facilitate absorption of intrinsic factor-vitamin B12 complex through intestinal
epithelium. Vitamin B12 is internalized and IF is released at cell surface.
358 11 Vitamins
Within Enterocytes
• Vitamin B12 attaches to transcobalamin-II within enterocytes. Complex is called

holo-transcobalamin-II. It enters the hepatic portal vein and reaches the liver.
Transport
• Cobalamin is transported in circulation in methylcobalamin form. It remains

associated with transcobalamin-II.
Storage of Vitamin B12
• Human body can store nearly 3000 μg cobalamin.

• The liver can store about 60% of total cobalamin, while the remaining 30% of
cobalamin is stored in muscles.
• Maximum liver storage of cobalamin is 4 mg.
Excretion
• Cyanocobalamin is excreted up to trace amount of 0.3 μg/day.
unctions of Vitamin B12

F
Conversion of Homocysteine into Methionine
• Methionine synthase enzyme catalyzes conversion of 5-methyltetrahydrofolate

into tetrahydrofolate. Enzyme requires cobalamin as cofactor for its activity.
• In the reaction, methyl unit is accepted by cobalamin to form methylcobalamin.
Methyl group is in active form and transferred to homocysteine to form methionine.
Conversion of Ribonucleotides into Deoxyribonucleotides
• Ribonucleotides are reduced into deoxyribonucleotides by ribonucleotides

reductase enzyme. It is an iron-dependent enzyme. Reaction occurs in the pres-
ence of vitamin B12.
• Ribonucleotide reductase acts on ribonucleoside 5′-diphosphates (ADP, UDP,
GDP, and CDP). Reduction occurs at 2’C of ribose sugar to form deoxyribonu-
cleoside 5′-diphosphates.
• In the reaction, NADPH provides hydrogen atoms for reductive synthesis of
deoxyribonucleotides.
Conversion of Deoxyuridine Monophosphate into Deoxythymidine

Monophosphate
• Deoxyuridine monophosphate (dUMP) is converted into deoxythymidine mono-

phosphate (dTMP) by thymidylate synthetase enzyme. The deoxythymidine
monophosphate is a monomeric structural component of DNA.
• Thymidylate synthetase is a cobalamin-dependent enzyme.
Thymidylate synthetase
5,10-methylenetetrahydrofolate + dUMP dihydrofolate + dTMP
• Cobalamin-dependent enzyme that catalyzed reductive methylation is an impor-

tant de novo pathway for the synthesis of deoxyribonucleotides.
Conversion of Methyl Malonyl CoA into Succinyl CoA
• Methyl malonyl CoA is a metabolite which is produced in beta oxidation of odd-

chain fatty acids like propionic acid. Methyl malonyl CoA is converted into suc-
cinyl CoA by methyl malonyl CoA mutase enzyme in the presence of vitamin B12.
Deficiency Disorders of Vitamin B12
Definition
Megaloblastic anemia is characterized by reduced count of erythrocytes with
appearance of megaloblasts in blood circulation.
Megaloblastic anemia is a type of macrocytic anemia.
Etiology
• Deficiency of folic acid

• Deficiency of vitamin B12
Pathogenesis of Megaloblastic Anemia
• Deficiency of either folic acid or vitamin B12 or both decreases the synthesis of
DNA in proliferating proerythroblasts (megaloblasts). It is due to the following
factors:
–– Diminished de novo synthesis of purine (folate deficiency).
–– Diminished conversion of dUMP into dTMP (folate deficiency). This leads to
reduction in dTTP formation in nuclear sap.
–– Reduced conversion of ribonucleotides into deoxyribonucleotides by reduc-
tase enzyme (cobalamin deficiency).
–– Reduced conversion of 5-methyl THF into THF, called folate trap (cobalamin
deficiency).
• Decreased DNA synthesis at S-phase manifests as:
–– Delayed unwinding of DNA strands and replication fork formation.
–– dUTP is incorporated into newly synthesizing DNA strands,
–– Ligation of Okazaki segments is delayed.
–– Failure to repair DNA strands and continuous fragmentation of DNA strands.
–– Prolongation of S-phase and delayed resting phase of megaloblasts.
–– Nucleus of megaloblast appears immature with open chromatin.
360 11 Vitamins
• However, synthesis of protein and RNA is normal in megaloblasts. Maturation of

cytoplasm is normal with adequate concentration of hemoglobin.
• Nuclear-cytoplasmic asynchrony:
–– It is the disproportionate nuclear to cytoplasmic ratio in proliferation
megaloblasts.
–– Maturation of cytoplasm continues normally, while nucleus remains imma-
ture. These megaloblasts have larger cytoplasmic volume in comparison to
normal megaloblasts. They have large size than normal.
–– Megaloblasts undergo macrocytosis.
• Pale color of the skin, nails, and conjunctiva.

• Inflamed and smooth appearance of the tongue owing to loss of papillae (glos-
sitis) and altered taste sensation.
• Loss of appetite.
• Loss of body weight.
• Muscular weakness.
• Patient suffers from diarrhea, steatorrhea (clay-colored stool due to the presence
of fat), abdominal distension, nausea, and vomiting.
• Difficulty in breathing and dizziness (dyspnea).
• Decrease memory, concentration, and restlessness.
• Increased predisposition to infections.
Hematological Manifestations
• Decrease in RBC count below 1 million per cubic millimeter. Anisocytosis (vari-
ation in size of RBC) and poikilocytosis (variation in shape of RBC) are well
marked in peripheral blood smear.
• Decrease in Hb concentration below 12 g%.
• ↑ in diameter of RBC (8.2 μm), while normal is 7.2 μm.
• ↑ in mean corpuscular volume to 100–160 μm3, while normal is between 77 and
94 μm3.
• ↑ in mean corpuscular hemoglobin to 50 pg, while normal is 28–32 pg.
• ↑ in reticulocyte count >4% while normal count is around 1%.
• ↓ in platelet count.
• The presence of hypersegmented neutrophils (neutrophil with five or more lobes
in nucleus, while normally three to four lobes are present in a nucleus) is a char-
acteristic feature of megaloblastic anemia.
• Increased hemolysis of RBC in the liver, spleen, and bone marrow.
Biochemical Manifestations
• Serum cobalamin concentration is <300 pg/mL.

• Fecal cobalamin concentration increases.
• Urinary cobalamin level is decreased owing to impaired absorption of cobalamin

from small intestine.
• Serum homocysteine and serum methylmalonic acid levels are increased.
• Increased serum indirect bilirubin level.
Bone Marrow
Aspiration from the bone marrow shows the following manifestations:
• Hypoxia in the bone marrow stimulates erythropoiesis. The bone marrow shows
hyperplasia.
• Presence of 70% proerythroblasts and early normoblasts (normal is 30%).
• Presence of 30% late normoblasts (normal is 70%).
It is termed megaloblastic hyperplasia of the bone marrow.
Macrocytic Anemia
Definition
Macrocytic anemia is a type of anemia characterized by presence of increased
size of erythrocytes in blood circulation.
Normal diameter of RBC is 7.2 μ, and normal mean corpuscular volume (MCV)
is between 80 and 90 μm3. In macrocytosis, the size of erythrocytes is increased to
8.4 μ, and their MCV is increased between 90 and 160 μm3.
Etiology
• Deficiency of folate or cobalamin or both

• Chronic alcoholism
• Liver disease
Types of Macrocytic Anemia

• Megaloblastic anemia is due to deficiency of folic acid or cobalamin. This type

of macrocytic anemia is associated with impaired synthesis of DNA.
Non-Megaloblastic Macrocytic Anemia
• This type of macrocytic anemia is caused by liver disease. Cell membranes of

erythrocytes have larger surface area than their volume. Under electron micro-
scope, erythrocytes appear as thin and bell shaped. They are termed as
leptocytes.
Alcohol-Induced Macrocytic Anemia
• Prolonged alcohol consumption might have deleterious effect on the bone mar-
row. The erythrocytes have round shape and larger size than normal.
362 11 Vitamins
Pernicious Anemia (Addison Anemia)

It is characterized by reduced count of erythrocytes along with the presence of meg-
aloblasts in blood circulation and hyperplasia of the bone marrow.
The word “pernicious” means fatal/injurious/harmful.
Historical Aspect
• In 1855, a physician named Thomas Addison described pernicious anemia. He

was uncertain about the cause of anemia and called it idiopathic anemia.
• In 1872, a physician in Germany, Anton Biermer, coined the term pernicious
anemia, owing to severity of disease and lack of proper treatment.
• Studies of G. H. Whipple, G. R. Minot, and P. Murphy proved that a diet rich in
liver to patients could cure pernicious anemia. In 1934, they were awarded with
Nobel Prize.
Etiology
• Primarily, secretion of intrinsic factor of castle is decreased in the stomach.

• Secondarily, absorption of dietary vitamin B12 is impaired. It leads to its defi-
ciency in plasma.
Pathogenesis
• Autoimmunity is directed against gastric mucosa and parietal cells. There is

depletion of intrinsic factor which leads to impaired absorption and deficiency of
vitamin B12.
• Synthesis of DNA is impaired. It leads to pernicious anemia (a type of megalo-
blastic anemia).
Clinical Manifestation of Pernicious Anemia

These are the same as seen in megaloblastic anemia.
• Intrinsic factor (IF) of castle is a glycoprotein. It is secreted by parietal (oxyn-

tic) cells of gastric mucosa. It helps in the absorption of dietary vitamin B12.
• Due to atrophic gastritis (Ch. inflammation of gastric mucosa with loss of
glandular activity), parietal cells are damaged and antibodies are produced
against IF.
• It leads to either decrease or absence of IF in the stomach. It results in defi-
ciency of vitamin B12.
Folate Trap
• Deficiency of cobalamin is responsible for folate trap. Therefore, deficiency of

cobalamin is associated with folate insufficiency.
Increased Plasma Homocysteine Level
• Deficient cobalamin causes rise in plasma homocysteine level. It is due to

impaired methylation of homocysteine. It is excreted in urine and the condition
is called homocystinuria.
Impaired Myelin Synthesis
• Impaired methylation of homocysteine is responsible for deficiency of methio-

nine and S-adenosyl methionine (SAM). Therefore, methylation of phosphati-
dylethanolamine to form phosphatidylcholine does not occur. Choline deficiency
is manifested as impaired synthesis of myelin. Demyelination is the cause of
neurological lesions of CNS.
Achlorhydria
• Gastric atrophy results in damage of parietal cells. The secretion of HCl is either
reduced or absent. It affects digestion of food.
Congenital Pernicious Anemia

It is prevalent in neonates. Its occurrence is limited. It is an inherited trait and is
characterized deficiency of intrinsic factor in new born babies. Intrinsic factor is
helpful in absorption of dietary cobalamin.
High-Energy Facts
• Isoprene/isoprenoid is five-carbon skeleton-based unsaturated branched-

chain hydrocarbon with molecular formula (C5H8) as CH2〓C(CH3)–
CH〓CH2. It is a colorless volatile liquid produced by conifers and citrus
plants. The liquid has strong aroma. Its polymeric compounds are constitu-
ent of natural rubber. Biologically important compounds like carotene, reti-
nol, tocopherols, lanosterol, and squalene are derived from isoprene units.
• Terpene and terpenoid are volatile unsaturated hydrocarbons found in
essential oils of conifers and citrus plants. These are derived from isoprene
units. Terpenes have multiples of isoprene units as (C5H8)n. Based on
number of isoprene units, terpenes are classified as:
• Hemiterpenes, monoterpenes, diterpenes, and tetraterpenes contain single,
two, four, and eight isoprene units.
• Physical Manifestation of Bleeding
–– Petechiae
–– Purpura
–– Ecchymosis (Bruise)
–– Hematoma
364 11 Vitamins
Suggested Readings
Epstein JB, Gorsky M (1999) Topical application of vitamin a to oral leukoplakia—a clinical case
series. Cancer 86(6):921–927
Garcia NM, Hildebolt CF, Miley D, Dixon DA, Couture RA, Anderson Spearie CL, Langenwalter
EM, Shanon WD, Deych E, Mueller C, Civitelli R (2011) One-year effects of vitamin D and
calcium supplementation on chronic periodontitis. J Periodontol 82(10):25–32
Gorsky M, Epstein JB (2002) The effect of retinoids on premalignant oral lesions—focus on topi-
cal therapy. Cancer 95(6):1258–1264
Gupta A (2017) Role of vitamin B12 and folic acid in nutritional anemia. In: Nutritional anemia in
preschool children. Springer-Nature, Singapore
Institute of Medicine, Food and Nutrition Board (2001) Dietary reference intakes for vitamin a,
vitamin k, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel,
silicon, vanadium, and zinc. National Academy Press, Washington, DC
Lodi G, Sardella A, Bez C, Demarosi F, Carrassi A (2002) Systematic review of randomized trials
for the treatment of oral leukoplakia. J Dent Educ 66(8):896–902
National Institutes of Health. Vitamin A: A fact sheet. 2017. https://ods.od.nih.gov/factsheets/
VitaminA-HealthProfessional/
National Institutes of Health-Office of Dietary Supplements. Nutrient recommendations: dietary
reference intake. U. S. Department of Health and Human Services, Bethesda. 2017. https://ods.
od.nih.gov/Health_Information/Dietary_Reference_Intakes.aspx
National Institutes of Health-Office of Dietary Supplements. Vitamin A. U. S. Department
of Health and Human Services, Bethesda. 2017. https://ods.od.nih.gov/factsheets/
VitaminA-HealthProfessional/
National Institutes of Health-Office of Dietary Supplements. Vitamin D. U. S. Department
of Health and Human Services, Bethesda. 2017. https://ods.od.nih.gov/factsheets/
VitaminD-HealthProfessional/
Piattelli A, Fioroni M, Santinelli A, Rubini C (1999) Bcl-2 expression and apoptotic bodies in
13-cis-retinoic acid (isotretinoin)-topically treated oral leukoplakia: a pilot study. Oral Oncol
35(3):314–320
Stone WL, Krishnan K, Campbell SE, Qui M, Whaley SG, Yang H (2004) Tocopherols and the
treatment of colon cancer. Ann N Y Acad Sci 1031:223–233
World Health Organization (1999) Thiamine deficiency and its preventive and control in major
emergencies. WHO, Geneva
Part III
Metabolism
Digestion and Absorption of Proteins
12
Dietary proteins are building elements of the living body. Proteins are primarily
necessary for generation and regeneration of body tissues. Proteins have higher
impact on growth than lipids and carbohydrates. 1 g of protein provides 4 calories
of energy.
A healthy adult person should consume around 0.8 g/kg body weight of protein
per day. It is around 560 g in adult male who has 70 kg of body weight. Its daily
requirement increases during pregnancy and lactation. Children in age group
between 1 and 5 years require 30–40 g of protein daily. Old-aged persons, convales-
cents, and persons suffering from chronic diseases like cirrhosis and renal failure
require higher quantity of protein. The carbohydrates are important component of
human diet.
12.1 Dietary Sources
1. Plant Sources
• Cereals, pulses, peas, beans.
• Nuts like walnuts, almonds, cashew, and Brazil nuts are enriched with high-
quality of proteins.
• Spirulina is the richest protein source. Its dry weight contains around
60–70% of proteins. It contains almost all essential amino acids.
• Seeds from plants like flax, pumpkin, hemp, and sesame are rich in protein,
PUFA, and minerals.
2. Animal Sources
• Milk and milk products are good source of calcium and protein. Milk contains
whey and casein proteins.
• Egg contains 6–8 g of protein.
• Fish and liver are sources of protein.
• Pork, beef, and chicken provide high-quality amino acids.

https://doi.org/10.1007/978-981-13-1035-5_12
368 12 Digestion and Absorption of Proteins
12.2 Digestion of Proteins
12.2.1 Digestion in Oral Cavity
• Proteolytic enzymes in oral cavity are absent. Protein digestion does not start in
oral cavity. Cooking of food brings about denaturation of proteins. They are mas-
ticated in the mouth and swallowed as bolus.
12.2.2 Digestion in Stomach
• Gastric juice contains important proteolytic enzymes. They are present in inac-
tive form called as “zymogen.”
• Proteolytic enzymes in stomach are as follows:
–– Pepsinogen
–– Rennin
–– Gelatinase
• Pepsinogen is an inactive proteolytic enzyme secreted by chief cells of gastric
mucosa.
• It is converted into pepsin by HCl in the stomach. The activated pepsin can cata-
lyze conversion of pepsinogen into pepsin, called as “autocatalysis.”
HCl
Pepsinogen Pepsin
(Zymogen) (Active form)
Pepsin (Autocatalysis)
Pepsinogen Pepsin
(Optimum pH 2.0)
• Optimum pH for catalytic activity of pepsin is 2.0. It is maintained by secretion

of hydrochloric acid.
Pepsin Action on Proteins

• Pepsin is an endopeptidase. It can cleavage peptide bonds formed between car-
boxylic groups of aromatic amino acids like phenylalanine, tyrosine, and trypto-
phan with amino groups of another amino acids.
• It liberates proteoses and peptones.
Pepsin
Proteins Proteoses + Peptones
• Pepsin cannot digest keratin protein, fibroins from silk, and mucoprotein.
• Pepsin is denatured at pH >5.0.
12.2 Digestion of Proteins 369
Pepsin Action on Casein of Milk

• Casein is a phosphorus-containing protein of milk. It is present in soluble form
(caseinogen) in milk.
• Pepsin is the chief milk protein-digesting enzyme in humans. Pepsin acts on
soluble casein and converts it into soluble paracasein and whey protein.
• In the presence of calcium ions, soluble paracasein is precipitated into calcium
paracaseinate (insoluble). It is called as “milk curdling.”
Pepsin in adults or Rennin in infants
Casein Paracasein + Whey Protein (Proteose)
(Soluble) (Soluble)
Paracasein + Calcium ions Calcium Paracaseinate
(Soluble) (Insoluble coagulum or curd)
Action of Rennin on Milk

• Rennin is also named as “chymosin.” It is a proteolytic enzyme. It is present in
the stomach of infants. It is absent in adult humans. It is also found in “proren-
nin” form in the stomach of calves.
• Optimum pH for rennin activity is 2.0.
• Rennin acts on casein protein in milk and converts it into calcium paracaseinate.
Its activity is similar to pepsin.
Action of Gelatinase enzyme

• It cleavages gelatin protein in diet into polypeptides. Gelatin is used in bakery to
prepare jellies, cakes, desserts, and other products. It has gelling action. It is
digested in the stomach by gelatinase enzyme.
12.2.3 Digestion in the Small Intestine
• The duodenum receives pancreatic juice and intestinal juice. They contain many
proteolytic enzymes.
Proteolytic Enzymes in Pancreatic Juice
–– Trypsin
–– Chymotrypsin
–– Carboxypeptidase
–– Elastase
–– Collagenase
Proteolytic Enzymes in Intestinal Juice

–– Enterokinase
–– Aminopeptidase
–– Tripeptidase and dipeptidase
Action of Proteolytic Enzymes

1. Trypsin
• It is the main proteolytic enzyme in pancreatic juice. It is secreted by acinar
cells of the pancreas in “trypsinogen” form.
• Trypsinogen is activated by an enzyme called as “enterokinase.” This enzyme
is secreted by intestinal glands.
• Trypsin also catalyzes conversion of trypsinogen into trypsin, and the process
is called as autocatalysis.
Enterokinase/Ca++
Trypsinogen Trypsin
Trypsin (autocatalysis)
• The optimum pH for activity of trypsin is 7.8.

• Trypsin activates chymotrypsinogen into chymotrypsin.
• Trypsin is an endopeptidase. It can hydrolyze into proteoses and peptones
into polypeptides, tripeptides, and dipeptides.
• Trypsin also has mild proteolytic activity on casein protein at pH 7.8.
• Trypsin can activate fibrinogen and proelastase into fibrin and elastase.
2 . Chymotrypsin
• It is secreted by the pancreas in chymotrypsinogen form. It is activated by
trypsin into chymotrypsin. It is a powerful protein-splitting enzyme.
• The optimum pH for activity of chymotrypsin is 7.8.
• Chymotrypsin is an endopeptidase. It can hydrolyze proteins into polypep-
tides, tripeptides, and dipeptides.
• It has mild hydrolytic action on casein protein.
Trypsin and Chymotrypsin

Native Proteins Polypeptides + Tri and Dipeptides
3. Carboxypeptidase
• Carboxypeptidase is secreted by the pancreas in “pro-carboxypeptidase”
form. It is activated by trypsin.
• Carboxypeptidase is an exopeptidase. It hydrolyzes terminal peptide bond at
the carboxy-terminal end of polypeptides and tripeptides. It liberates amino
acids.
12.2 Digestion of Proteins 371
• Pancreatic juice contains two types of carboxypeptidases as A and

B. Carboxypeptidase A is a zinc-containing metalloprotein.
• The optimum pH for carboxypeptidases A and B is 7.8.
• Carboxypeptidase A splits peptide bond at C-terminal involving aromatic
amino acids like phenylalanine, tyrosine, and tryptophan.
• Carboxypeptidase B splits peptide bond at C-terminal involving basic amino
acids like arginine and lysine.
• Both carboxypeptidases cannot split dipeptides.
Carboxypeptidase
Polypeptides Amino acids
4. Elastase
• Elastase is secreted by the pancreas in “proelastase” form. It is activated by
trypsin.
• The optimum pH for its activity is 7.8.
• It splits elastin protein. It hydrolyzes peptide bonds linked to carboxylic group
of neutral aliphatic amino acids like glycine, valine, and alanine.
• It liberates peptides.
5. Collagenase
• Collagenase is secreted by the pancreas.
• It splits collagen protein. It liberates peptides.
6. Enterokinase
• Enterokinase is also called as “enteropeptidase.” It is secreted by intestinal
glands. It is localized in brush border surface of intestinal mucosa.
• It is an endopeptidase.
• It activates trypsinogen into trypsin in the presence of calcium ions.
7. Aminopeptidase
• Aminopeptidase is secreted by intestinal glands in the duodenum and jeju-
num. It is an exopeptidase.
• It cleavages terminal peptide bonds at amino-terminal end of polypeptides
and tripeptides.
• It cannot split dipeptides.
• It liberates amino acids.
8. Tripeptidase and Dipeptidase
• These enzymes split tripeptides and dipeptides. They liberate free amino
acids in the lumen of the intestine.
Mixture of aminopeptidase, tripeptidase, and dipeptidase in intestinal juice is

called as “Erepsin.” It digests polypeptides, tripeptides, and dipeptides into
free amino acids.
12.3 Absorption of Amino Acids
Dietary proteins are digested into constituent amino acids in the lumen of the intes-
tine. Amino acids are absorbed from the mucosa of the small intestine. They enter
the hepatic portal vein and reach the liver. A small fraction of tri- and dipeptides
remain undigested in the lumen of the intestine. They can enter through brush bor-
der surface of mucosal cells of the small intestine. Within the enterocytes, they are
hydrolyzed by tri- and dipeptidases into amino acids.
12.4 Mechanism of Absorption
Based on the isomeric form of amino acids, there are two mechanisms involved in
the absorption of amino acids. They are as follows:
12.4.1 Passive Diffusion of Amino Acids
• This mechanism involves diffusion of amino acids through the mucosa of the
small intestine.
• This mechanism favors concentration gradient.
• d-amino acids are absorbed by passive diffusion. It is a slow absorption
process.
12.4.2 Active Transport of Amino Acids
• This mechanism involves active transportation of amino acids through the

mucosa of small intestine.
• l-amino acids are absorbed by active transport.
• Active transport is dependent on energy. The ATP is utilized in the transport of
amino acids.
• The process requires a protein carrier and sodium ions.
• Carrier molecule is located on the outer surface of microvilli of the small intes-
tine. It links with l-amino acid and sodium ion and passes through the intestinal
membrane. It is a co-transport method.
• Carrier moves to inner surface of microvilli and dissociates from l-amino acid
and sodium ion.
• Sodium ion is pumped out by sodium pump.
• Amino acids pass through enterocytes and enter portal circulation.
12.5 Clinical Significance 373
12.4.3 Influence of Meister Cycle (Gamma-Glutamyl Cycle)

in Amino Acid Absorption
Glutathione is a tripeptide. It is a γ-glutamyl-cysteinyl-glycine molecule. According

to Meister, glutathione is actively involved in the translocation of active group of
l-amino acids except l-proline amino acid. It transports amino acids into tissues of
the kidneys, seminal vesicle, brain, and small intestine.
The process repeats in a cyclic pathway in which glutathione is regenerated. A
brief detail about the Meister cycle has been given below:
• Glutathione interacts with l-amino acid in the presence of sodium ion, and
gamma-glutamyl-amino acid complex is formed. The reaction is catalyzed by
gamma-glutamyl transferase enzyme.
Gamma-Glutamyl transferase
Glutathione + L-amino acid γ-glutamyl-amino acid complex + Cysteinyl-Glycine
• γ-Glutamyl-amino acid complex is broken into L-amino acid and 5-Oxo-Proline

compound by gamma-glutamyl cyclo-transferase enzyme. L-amino acid is
absorbed through the membrane.
Gamma-Glutamyl Cyclo-transferase
γ-Glutamyl-Amino acid complex L-amino acid + 5-Oxo-Proline
• 5-Oxo-Proline is converted into L-Glutamic acid by 5-Oxo-Prolinase enzyme. It

combines with cysteinyl-glycine to form glutathione.
5-Oxo-Prolinase
5-Oxo-Proline L-Glutamic acid
L-Glutamic acid + Cysteinyl-Glycine Glutathione
12.5 Clinical Significance
1. Allergy to Dietary Proteins

• Dietary proteins may induce immunological response in selected individuals.
Generally, dietary proteins are digested into amino acids which are not
antigenic.
• For a protein to become antigenic, it must be an oligopeptide of minimum 6–7
amino acid residues. These peptides cannot be absorbed through intestinal
mucosa.
• These antigenic peptides pass in between the mucosal cells (paracellular) and
enter blood circulation, for example, gluten-induced antibodies and antibod-
ies to the colostrum.
2 . Protein-Losing Enteropathy
• In the condition, there is excessive loss of plasma proteins through the intes-
tinal mucosa. It is due to inflammation of gut mucosa. The plasma protein
level is decreased.
3. Oxoprolinuria
• It is a clinical condition characterized by excessive excretion of pyrogluta-
mate in urine. It is called as pyroglutamate aciduria.
• It is due to deficiency of 5-oxoprolinase enzyme.
• Whey protein is the mixture of globular proteins found in whey. These

proteins are alpha-lactalbumin, beta-lactoglobulin, and immunoglobulins.
–– Whey is the liquid that is left after the milk has been coagulated. It
contains lactose, minerals, and whey proteins. Cow’s milk contains
80% casein and 20% whey proteins, while human breast milk has 60%
whey proteins and 40% casein.
• Rennin is also called as “chymosin.” It is a proteolytic enzyme secreted by
mucosa of abomasum in ruminants. It acts on casein. Rennin is also
secreted by chief cells of stomach in infants. Its secretion is high in early
infancy, and it is replaced by pepsin at the end of infancy. In adult humans,
rennin is absent. Rennin coagulates or curdles milk.
–– Renin is a proteolytic enzyme secreted by cells of the juxtaglomeru-
lar apparatus of kidneys. Renin converts angiotensinogen into angio-
tensin I.
• Endopeptidase is an enzyme that cleavages peptide bond within protein
molecule. It cannot split terminal peptide bonds, for example, trypsin, chy-
motrypsin, elastase, and pepsin.
–– Exopeptidase is an enzyme that cleavages terminal peptide bonds
either from amino- or carboxy-terminal.
• Carboxypeptidase is an enzyme that cleavages peptide bond at carboxy-
terminal of a polypeptide chain.
• Aminopeptidase is an enzyme that cleavages peptide bond at amino-
terminal of a polypeptide chain.
Suggested Readings
Harper HA (1979) Review of physiological chemistry, 17th edn. Lange Medical, New York
Kleiner IS, Orten JM (1966) Biochemistry, 7th edn. Mosby, St. Louis
Latner AL, Cantarow A, Trumper M (1975) Clinical biochemistry, 7th edn. W. B. Saunders,
Philadelphia
Mazur A, Harrow B (1971) Textbook of biochemistry, 10th edn. W. B. Saunders, Philadelphia
Murray RK et al (1999) Harper’s biochemistry. Lange Medical, New York
Murray RK et al (2003) Harper’s illustrated biochemistry, 26th edn. Lange Medical, New York
Oser BL (ed) (1965) Hawk’s: physiological chemistry, 14th edn. Mc-Graw Hill, New York
Robins RJ, Davies DD (1981) The role of glutathione in amino-acid absorption. Lack of cor-
relation between glutathione turnover and amino-acid absorption by the yeast Candida uti-
lis. Biochem J 194(1):63–70 Available at: h ttp://https://www.ncbi.nlm.nih.gov/pmc/articles/
PMC1162717/?page=1
Metabolism of Proteins and Amino Acids
13
Proteins are integral component of human diet. They represent about 12 kg dry
weight of the body in a healthy person of 70 kg weight. Proteins are composed of
20 amino acids.
Dietary proteins are hydrolyzed by digestive enzymes in the alimentary canal.
Constituent amino acids are absorbed through intestinal mucosa. Free amino acids
are transported to the liver through the hepatic portal vein. They enter blood circula-
tion and are distributed to tissues. Tissue proteins undergo proteolysis to release free
amino acids which enter blood circulation.
Fats and carbohydrates have definitive storage in body tissues. Unfortunately, the
body lacks storage proteins to supply amino acids as is performed by tissue triglyc-
erides and glycogen in the liver and skeletal muscles.
Amino acids are either supplemented by food or provided by breakdown of tis-
sue proteins or synthesized in body tissues.
Surplus amino acids in the body are catabolized in different phases. Initially,
amino group is either transaminated or deaminated that results into formation of
alpha-keto acids and ammonia. In second phase, ammonia is transformed into urea
and excreted. Carbon skeleton of alpha-keto acid is metabolized into intermediate
metabolites. They generate calories.
13.1 Amino Acid Pool
Definition
Amino acid Pool is defined as the Labile store of free amino acids from different
sources together constitutes amino acid pool.
Cells, blood circulation, and extracellular fluid contain free amino acids which
together constitute amino acid pool.

https://doi.org/10.1007/978-981-13-1035-5_13
378 13 Metabolism of Proteins and Amino Acids
Amino acid pool is comprised of nearly 90–100 g of amino acids. The amino
acid pool represents a small fraction of total body proteins which constitute
nearly 12 kg in a hypothetical person of 70 kg weight.
Amino acids differ from carbohydrates and lipids in terms of storage in tissues.
Carbohydrates are stored in skeletal muscles and the liver in the form of glycogen.
Lipids are stored in adipose tissues in the form of triglycerides. Unfortunately,
amino acids are not stored in body tissues.
Amino acid pool is maintained by following two factors:
1. Sources providing free amino acids to amino acid pool

2. Sources utilizing free amino acids from amino acid pool
13.1.1 Sources Providing Free Amino Acids into Amino Acid Pool
• Dietary proteins
–– Proteins in diet are important source of amino acids to amino acid pool.
–– It has been observed that 35–50 g of tissue proteins are wasted daily from the
body.
–– It is recommended that about 35–50 g of dietary proteins should be consumed
per day. It helps to augment the protein loss.
–– Dietary proteins are hydrolyzed in gut to release free amino acids.
–– Free amino acids are absorbed by mucosa of the small intestine.
–– Dietary proteins supply free amino acids to the pool. They also provide essen-
tial amino acids to the body.
–– It represents positive nitrogen balance.
–– However, daily recommended intake of proteins should be around 50 g per
day. Protein intake in developed countries is much higher (50–100 g per day).
• Proteolysis of tissue proteins
–– Tissue proteins are continuously degraded and resynthesized through a well-
regulated mechanism. It is called as turnover of tissue proteins. The rates at
which different proteins are catabolized and resynthesized are highly variable
and depend on type of cell.
–– Glucocorticoids regulate proteolysis of tissue proteins. The proteolytic
enzymes are contained within lysosomes in cells. The mobilized amino acids
from tissue proteins enter amino acid pool and thereafter are transported to
the liver for resynthesis of proteins.
–– The following are pathological conditions:
Acute infections like diarrhea, influenza, typhoid fever
Chronic conditions like tuberculosis, malabsorption syndrome, celiac disease
Burns
Proteolysis of tissue proteins is tremendously increased in diseases.
Released amino acids are transported into the liver where they are resynthe-
sized into acute-phase C-reactive protein and alpha-antitrypsin.
13.1 Amino Acid Pool 379
However, synthesis of plasma proteins like albumin and globulin by the liver
in diseased state declined.
Glucocorticoids regulate proteolysis of skeletal muscles in burns, diarrhea,
and typhoid conditions. On an average, protein loss is about 0.5–1.0 g per kg
body weight per day.
• Biosynthesis of nonessential amino acids
–– Nonessential amino acids are synthesized from the intermediate metabolites
in various metabolic pathways. For example, aspartate and alanine are synthe-
sized by transamination reaction.
–– Amino acid like cysteine is synthesized from essential amino acid, methio-
nine (methionine → homocysteine + serine → cystathione → cysteine).
–– In normal health, input to amino acid pool is in equilibrium with output.
–– Amino acid pool is in dynamic equilibrium.
–– Body has positive nitrogen balance.
13.1.2 Sources Utilizing Free Amino Acids from Amino Acid Pool
Amino acid pool is depleted by the following three sources:
• Biosynthesis of tissue proteins

• Structural proteins like actin, myosin, albumin, and hemoglobin are synthesized
from amino acids of AA pool.
• Biosynthesis of functional proteins like enzymes and hormones is performed
from amino acid pool.
• Biosynthesis of nonprotein nitrogenous substances like urea, creatine, creati-
nine, and xanthine takes place from amino acid pool.
–– Catabolism of amino acids from amino acid pool takes place by deamina-
tion and decarboxylation as in Fig. 13.1.
• Conversion of amino acids into glucose and fatty acids.
A cell can utilize fixed number of amino acids from amino acid pool for its func-
tions. Conversely, it furnishes the same number of amino acids to the amino acid
pool. Such a cell is considered to be in dynamic equilibrium and human body
has nitrogen balance.
Catabolism of Amino Acids

Catabolism of amino acids is essentially focused on the removal of nitrogen
from amino acids. The alpha amino group prevents oxidation of amino acids.
After liberation of nitrogen, carbon skeleton (alpha-keto acid) of amino acid is
metabolized through various pathways.
Three important biochemical mechanisms are described as follows:
1. Transamination
2. Deamination
3. Transdeamination
METABOLISM OF AMINO ACIDS
Tissue protein Total Body

Formation of Daily
break down Tissue-proteins
non-essential dietary proteins
300–400 gms/day 10–12 kg in adults
amino acids (50–80g) day
Absorption
Digestion
Loss of Tissue protein
Amino Acid Pool
amino acids (30g/day) synthesis
100g 300–400 gms/day
in urine
Urea uric acid

creatinine
ATP Synthesis Synthesis Formation of

(10% of calories of Non-Protein
requirement 1 day) Carbohydrates Nitrogenous
substances
(Purine pyrimidine, Creatline
porphyrins)
Fig. 13.1 Sources and utilization of Amino acids from pool
13.2 Transamination
Definition
Transamination is a biochemical process in which an amino group (-NH2) from
the amino acid is transferred to the α-keto acid to form new amino acid and
α-keto acid.
Characteristics of Transamination
• Transamination involves transfer of an amino group from donor amino acid to

recipient alpha-keto acid. It is an intermolecular transfer of amino group as in
Fig. 13.2.
• In transamination, ammonia is not formed.
• Donor amino acid is converted into new alpha-keto acid. Recipient alpha-keto
acid is converted into new amino acid.
• Transamination is a reversible process.
• In transamination, α-amino group of amino acid is transferred to α-keto acid.
Exception: δ-amino group of ornithine is transaminated.
• Alpha-keto acids, namely, glutarate, oxaloacetate, and pyruvate, act as recipient
of amino group.
• Few amino acids, namely, proline, hydroxyproline, lysine, and threonine, do not
undergo transamination.
13.2 Transamination 381
H COOH H COOH
Transaminase
H2N C COOH + C=0 H 2N C COOH + C=0
Pyridoxal
R1 R2 phosphate R1 R2
a - amino acid a - Keto acid

New New
(Donor) (Recipient)
a - amino acid a - keto acid
AMINO ACID PYRIDOXAL
(DONOR) PHOSPHATE AMINO ACID
(NEW)
TRANSAMINASE
TRANSAMINASE
KETO ACID KETO ACID
PYRIDOXAMINE
(NEW) (RECIPIENT)
PHOSPHATE
ALANINE
TRANSAMINASE
ALANINE PYRUVATE
+ +
α – KETO GLUTARATE serum glutamate GLUTAMATE
pyruvate transamianse
ASPARTATE
TRANSAMINASE
ASPARTATE OXALOACETATE
+ +
α – KETO GLUTARATE GLUTAMATE
serum glutamate
oxaloacetate transaminase
Fig. 13.2 Transamination (a) mechanism of transamination (b) alanine transaminase (c) aspartate
transaminase
Site of Occurrence
• Primarily, transamination occurs in the liver, heart, kidneys, and brain tissues.
• However, transamination can take place in all body tissues.
Enzymes for Transamination
• Transamination is catalyzed by transaminase enzyme. It is also called as ami-

notransferase. Transaminases are widely distributed in cytoplasm and mito-
chondria of cells. Transaminases can be two types:
• Group transaminases
These enzymes catalyze transamination of a group of structurally related amino
acids.
• Substrate-specific transaminases
These enzymes catalyze specific amino acids.
Example:
1. Alanine aminotransferase (ALT) or also called as serum glutamate pyruvate
transaminase (SGPT)
2. Aspartate aminotransferase (AST) or also called as serum glutamate oxalo-
acetate transaminase (SGOT)
Coenzyme for Transamination

Pyridoxal phosphate acts as coenzyme in transamination. It is attached by covalent
bonding to ε-amino group of lysine residue in transaminase enzyme.
Mechanism
Transamination occurs in two steps as follows:
• Step I
Formation of pyridoxamine phosphate
–– Donor amino acid interacts with pyridoxal phosphate bound to transaminase
enzyme. Amino group is transferred to pyridoxal phosphate and results in
formation of pyridoxamine phosphate.
–– Donor amino acid is converted into new alpha-keto acid as in Fig. 13.2a.
• Step II
Transfer of amino group from pyridoxamine phosphate to recipient alpha-
keto acid
–– Pyridoxamine phosphate transfers its amino group to recipient alpha-keto
acid. There is formation of new amino acid. Pyridoxal phosphate is regener-
ated as in Fig. 13.2a.
Mechanism of Substrate-Specific Transaminases
• Alanine Aminotransferase (ALT)

ALT is abundantly found in the cytoplasm of hepatocytes. It is also present in the
heart and kidneys. Alanine transaminase catalyzes transfers of alpha-amino
group of alanine amino acid to alpha-ketoglutarate. Transamination results in the
formation of glutamate as new amino acid and pyruvate as new alpha-keto acid
as in Fig. 13.2b.
• Aspartate Aminotransferase (AST)
AST is abundantly found in cardiac muscle fibers and liver cells. It is found in
the cytoplasm and mitochondria. It is also present in the kidneys and intestine.
Aspartate transaminase catalyzes transfer of alpha-amino group from aspartate
to alpha-ketoglutarate to form glutamate as new amino acid and oxaloacetate as
new alpha-keto acid as in Fig. 13.2c.
13.3 Deamination 383
Biological Significance
• Transamination provides carbon skeleton of amino acids after removal of

amino group. It is called as alpha-keto acids. Carbon skeleton of amino acids
is variously catabolized as follows:
–– Transamination (ketogenic amino acid) provides acetyl CoA. It serves as
a precursor for cholesterol synthesis. Example: leucine
–– Transamination (glucogenic amino acid) provides pyruvate. It is utilized in
TCA cycle and gluconeogenesis. Example: alanine, glycine, cysteine, and serine
–– Transamination (glucogenic amino acid) provides oxaloacetate. It is uti-
lized in TCA cycle. Example: aspartic acid and asparagine
–– Transamination (glucogenic amino acid) provides alpha-ketoglutarate. It
is utilized in TCA cycle. Example: glutamate, glutamine, arginine, histidine,
proline, and hydroxyproline
–– Transamination (glucogenic amino acid) provides succinyl CoA. It is uti-
lized in TCA cycle. Example: valine, methionine, and threonine
• Transamination helps to synthesize nonessential amino acids. For example,
pyruvate is converted into glutamate.
• Transamination helps to convert surplus amino acids into another amino
acid which is low in amino acid pool. It helps to normalize the amount of nones-
sential amino acids.
• For clinical significance, transamination helps in the diagnosis and progno-
sis of liver and heart diseases. Transaminases are intracellular enzymes. Their
plasma concentration is maintained in a reference range. In liver diseases, hepa-
tocytes are damaged. Transaminases are released in blood circulation, and their
plasma concentration is elevated. This act is utilized in diagnosis and determin-
ing the outcome of disease.
13.3 Deamination
Definition
Deamination is a biochemical process of conversion of amino acid into alpha-
keto acid with the liberation of ammonia.
Site of Occurrence
Deamination occurs in the liver, kidneys, and brain tissues.
13.3.1 Types of Deamination
Deamination is of two types which are as follows:
Oxidative Deamination
It is the release of ammonia through breakdown of amino acid in the presence
of molecular oxygen.
Enzymes for Oxidative Deamination

Two enzymes are present in cells which deaminate D-amino acids and L-amino
acids. They are as follows:
• d-Amino acid oxidase (DAAO)

–– This enzyme catalyzes oxidative deamination of d-amino acids.
–– Enzyme is a flavoprotein (Fp)-dependent enzyme. It requires FAD as coen-
zyme for its activity.
–– In a latest research, DAAO has been shown to be associated with control of
glutamate secretion in CNS. Concentration of DAAO in plasma was found to
be much higher in schizophrenia patients as revealed in postmortem
examination.
• l-Amino acid oxidase (LAAO)
–– This enzyme catalyzes oxidative deamination of l-amino acids.
–– Enzyme is a flavoprotein (Fp). It requires FMN as coenzyme for its activity.
–– LAAO is limited to tissues of the liver and kidneys.
Mechanism of Oxidative Deamination

It occurs in the following two steps:
• Dehydrogenation of amino acid

Amino acid undergoes dehydrogenation to form α-imino acid. Hydrogen atoms
are accepted by flavoprotein (Fp), and it is reduced into FpH2 (FADH2 and
FMNH2) as in Fig. 13.3.
• Spontaneous hydrolysis of α-imino acid
The α-imino acid is an intermediate metabolite. It is an unstable compound. It
undergoes spontaneous hydrolysis (breakdown of a bond by addition of water
molecule) to form α-keto acid with loss of a molecule of ammonia (NH3) as in
Fig. 13.3.
• Decomposition of H2O2
Reduced flavoprotein (FpH2) is further oxidized into (Fp) by supply of molecular
oxygen. This activity results into formation of H2O2. It is toxic to tissues, and it
is decomposed into water and oxygen by catalase enzyme as in Fig. 13.3.
Non-oxidative Deamination
It is the deamination of amino acids in the absence of molecular oxygen in cells.
Non-oxidative deamination takes place by substrate-specific enzymes.
Depending on types of amino acids, non-oxidative deamination has the follow-
ing three types:
Deamination of Hydroxy Amino Acids

Hydroxy amino acids like serine, homoserine, and threonine undergo non-oxidative
deamination. Reaction is catalyzed by serine dehydratase enzyme (l-hydroxy
amino acid dehydratase). It is located in cytoplasm of the liver.
Enzyme requires pyridoxal phosphate as coenzyme.
13.3 Deamination 385
H2O
se
Catala
H2O2
O2
FMN FMNOH2
H
α
R C COOH R C COOH
H2N L - Amino acid oxidase HN
L - Amino acid α - Imino acid

H2O
R C
COOH
NH3
α - Keto acid
Fig. 13.3 Oxidative deamination
Fig. 13.4 Non- H2O

oxidative deamination
(Amino acid
of hydroxy amino acids H2C–OH dehydratase)
–
H–C–N–H Pyridoxal
H–C–H
H Phosphate
C–N–H
COOH
H
Serine
H2O COOH
NH3
CH3
Imino Acid
– –
C=O
COOH
Pyruvic Acid
• Serine is converted into imino acid with release of water molecule. Imino
acid undergoes hydrolysis to form pyruvate with release of a molecule of
ammonia as in Fig. 13.4.
Deamination of Sulfur-Containing Amino Acids

Sulfur-containing amino acids like cysteine and homocysteine undergo non-
oxidative deamination as in Fig. 13.5. Reaction is catalyzed by cysteine desulfhy-
drase enzyme.
Enzyme requires pyridoxal phosphate as coenzyme.
• Cysteine is converted into imino acid with release of H2S. Imino acid
undergoes hydrolysis to form pyruvate with release of a molecule of
ammonia.
Deamination of Histidine
• Histidine is converted into urocanic acid with release of a molecule of ammo-

nia. Reaction is catalyzed by histidase enzyme as in Fig. 13.6.
Fig. 13.5 Non-oxidative H2C∼SH

deamination of sulfur- Desulfhydra
HC–N–H se
containing amino acids
(Cysteine & Homo H
Cysteine ) COOH H2S
H3C
C = NH
COOH
Imino Acid
H 2O
NH3
Pyruvic acid
Fig. 13.6 Non-oxidative Histidase

deamination of Histidine Histidine Urocanic acid
NH3
13.4 Transdeamination 387
13.4 Transdeamination
Definition
The α-amino groups from most of the l-amino acids are transferred to α-ketoglutarate.
Reactions are catalyzed by group transaminases and substrate-specific transaminases.
There is formation of glutamate. It is the highly abundant amino acid in amino acid pool.
Glutamate rapidly undergoes oxidative deamination to form α-ketoglutarate with
release of a molecule of ammonia.
Glutamate is the center stage of amino acid catabolism. It is either oxida-
tively deaminated or acts as donor of amino group in synthesis of glutamine
amino acid.
Transdeamination is the sequential transamination of l-amino acids (transfer of
amino group to α-ketoglutarate) to generate glutamate which in turn successively
proceeds to oxidative deamination to regenerate α-ketoglutarate with liberation
amino group in the form of a molecule of ammonia in body tissues.
Site of Occurrence
It occurs in most of body tissues.
Enzymes
Transamination occurs by transaminases.
Oxidative deamination of glutamate occurs by l-glutamate dehydrogenase
enzyme.
• l-Glutamate dehydrogenase
It is a zinc-dependent metalloenzyme.
It is found in mitochondria of most of body tissues.
Enzyme is substrate specific (acts on l-glutamate).
Enzyme requires NAD+ or NADP+ as coenzyme.
It is an allosteric enzyme. Its activity is inhibited by ATP and NADH. Its activity
is stimulated by ADP.
Mechanism
• Step I
Synthesis of l-Glutamate
–– Transamination of l-amino acids brings about synthesis of l-glutamate in
tissues.
• Step II
Dehydrogenation
–– l-Glutamate undergoes dehydrogenation with loss of two hydrogen atoms.
Reaction is catalyzed by l-glutamate dehydrogenase in the presence of
NAD+ as coenzyme. Hydrogen atoms are accepted by coenzyme, and it is
reduced into NADH2. l-Glutamate is converted into α-imino-glutarate as in
Fig. 13.7.
Transamination
Alpha Amino Acids
α-ketoglutarate
COOH
– – – –
H–C–H
H–C–H
H–C–NH2
COOH
COOH L-Glutamate
– – – –
α-ketoglutaric acid
H–C–H L-glutamate
NAD+
H–C–H Dehydrogenase
(Deamination)
α C= O NADH2
COOH
COOH
NH3
H–C–H
– – –
H–C–H
H2O
α C – NH
COOH
α-imino-glutaric acid
Fig. 13.7 Transdeamination
Hydrolysis
–– The α-imino-glutarate is an unstable compound. It undergoes rapid hydrolysis
to form α-ketoglutaric acid with loss of a molecule of ammonia.
–– Transdeamination is an important pathway to catabolism of N of amino acids
with disposal of ammonia as in Fig. 13.7.
13.5 Urea Cycle
Definition
Urea cycle is the cyclic biochemical pathway in which ammonia is converted
into urea.
Ammonia is a nitrogenous compound. It is produced through different catabolic
pathways. Ammonia is transformed into urea which represents the main excretory
form of nonprotein nitrogenous compound in the body.
13.5 Urea Cycle 389
Characteristics of Urea Cycle
• Urea cycle is also called as Krebs-Henseleit cycle.

• Urea cycle is called as ornithine cycle.
• Urea cycle involves five enzyme-catalyzed reactions.
• In urea, first nitrogen atoms are derived from ammonia, and second nitrogen
atom is derived from alpha-amino group of aspartic acid as in Fig. 13.9.
Site of Occurrence
• Urea cycle takes place in the liver.

• In the kidneys, incomplete urea cycle takes place. Kidneys lack arginase enzyme
for the formation of urea from arginine.
13.5.1 Steps in Urea Cycle
Urea cycle can be described in the following five steps:

Mitochondrial reactions
1. Formation of carbamoyl phosphate

2. Synthesis of citrulline
Cytosolic Reactions
3. Synthesis of argininosuccinate
4. Synthesis of arginine
5. Synthesis of urea
ormation of Carbamoyl Phosphate

F
• This reaction occurs in mitochondrial matrix of hepatocytes.
• Ammonia molecule undergoes condensation with a molecule of CO2 to form
carbamoyl phosphate. Two ATP molecules are utilized in reaction.
• Reaction is catalyzed by carbamoyl phosphate synthetase-I (CPS-I) enzyme.
–– Carbamoyl phosphate synthetase-I is located in mitochondria of liver cells. It
catalyzes urea synthesis.
–– Carbamoyl phosphate synthetase-II is located in cytosol of liver cells. It cata-
lyzes synthesis of pyrimidine.
• Carbamoyl phosphate synthetase-I is an allosteric enzyme. It is the key regula-
tory enzyme in urea synthesis as in Fig. 13.8.
Synthesis of Citrulline
• This reaction occurs in mitochondrial matrix of liver cells.
• Carbamoyl group is transferred to amino group of ornithine molecule. There is
formation of citrulline.
p
2A+
Pi
NH+4 2ADp +
Mg++
Carbamoyl
CO2 phosphate
synthetase I
H–N–H
C=O
O
O–P–O
H2N
O
C O
CARBAMOYL PHOSPHATE NH
+
H N H2 TR CH2
AN ORN
–
CH2 SC IT CH2
AR HIN
NH2 (UREA)
BA
CH2 M H C H
E
OY
CH2
UREA CH2
LA
CYCLE
SE
+
HC NH3 CH-NH3
– –
COO COO
ORNITHINE CITRULLINE
O
C
–
COO
H2O CH2 N+ H 2
NH2
H C NH C ATP
SE TE
ASE
AMP+Pi
A
– NH
SYN UCCIN
COO
GIN
CH2 –
COO
THA
H2
AR
S
N
CH2
NH
INO
CH2
+
C
2
ARG
CH2 +
CH NH3
NH
+
CH 2
CH – N H3 COO
–
CH
2
COO–
CH
ASPARTATE
CH
ARGINO
-N
CO
H3
+
SUCCINATE
O
–
ARGININE COO
– MA OAA
LA
CH TE
CH TCA
COO
– CYCLE CO2
FUMARATE
Fig. 13.8 Urea cycle

13.5 Urea Cycle 391
H2N-C=O-NH2
1st N-atom from Ammonia
C-atom from CO2
2nd N-atom from Aspartic acid
Fig. 13.9 Sources of atoms in Urea
• Reaction is catalyzed by ornithine carbamoyl transferase enzyme. It is also

called as ornithine transcarbamylase.
• Citrulline is transported across mitochondrial membranes to cytosol. It occurs by
a carrier protein as in Fig. 13.8.
• Citrulline is excreted in milk.
Synthesis of Argininosuccinate
• This reaction occurs in cytosol of liver cells.
• Citrulline undergoes condensation with a molecule of aspartic acid to form
argininosuccinate as in Fig. 13.8.
• Reaction is catalyzed by argininosuccinate synthetase enzyme.
• One molecule of ATP is hydrolyzed to provide two high-energy phosphate bonds.
ATP is converted into AMP.
Synthesis of Arginine
• Argininosuccinate is cleavaged into a molecule of arginine and fumarate as in
Fig. 13.8.
• Reaction is catalyzed by argininosuccinate lyase enzyme.
• Fumarate has inhibitory effect on argininosuccinate lyase. However, it is pre-
vented by channelizing fumarate into mitochondria to take part in TCA cycle. It
is converted into malate and oxaloacetate. Transamination of oxaloacetate fur-
ther regenerates aspartic acid.
Synthesis of Urea
• Arginine undergoes cleavage to form a molecule of urea and ornithine as in
Fig. 13.8.
• Reaction is catalyzed by arginase enzyme.
• Ornithine reenters mitochondrial matrix for reuse.
13.5.2 Biological Significance
Ammonia Detoxification
• Urea cycle is highly involved in conversion of toxic ammonia into nontoxic urea.
It is excreted in urine. Therefore, urea cycle helps to regulate ammonia plasma
concentration.
Synthesis of Arginine
• Arginine is a conditionally essential amino acid. It is synthesized by the liver,

kidneys, and intestinal mucosa. In kidneys, arginine cannot be decomposed into
urea due to the lack of arginase enzyme. Therefore, arginine from the kidneys
and intestine can be utilized in synthesis of proteins.
13.5.3 Regulation of Urea Cycle
Dietary Intake of Proteins
• Protein-rich diet results into increase in concentration of glutamate. It is a sub-

strate for ammonia synthesis. Hence, synthesis of urea is increased after con-
sumption of protein-rich diet.
N-Acetyl Glutamate (NAG)
• N-Acetyl glutamate activates allosterically the CPS-I enzyme. It is the rate-

limiting step in urea synthesis.
Arginine and Glutamate Concentration
• N-Acetyl glutamate is synthesized from glutamate and acetyl CoA by N-acetyl

glutamate synthetase enzyme. Both arginine and glutamate activate allosterically
synthesis of N-acetyl glutamate synthetase enzyme.
Fumarate
• Fumarate is an inhibitor of argininosuccinate lyase enzyme.
13.5.4 Clinical Significance
isorders of Urea Cycle

D
In healthy person, plasma concentration of urea ranges between15 and 40 mg/100 ml.
The deficiency of enzymes involved in urea cycle disturbs normal synthesis of urea.
The following conditions are caused by genetic deficiency of any one of the enzymes
as follows:
Hyperammonemia Type I
• Disorder is caused by inherited deficiency of CPS-I. There is increased plasma

ammonia level (hyperammonemia).
Hyperammonemia Type II
• Disorder is caused by deficiency of ornithine transcarbamylase. There is

increased plasma ammonia level (hyperammonemia).
Citrullinemia
• Disorder is caused by deficiency of argininosuccinate synthetase. There is

Argininosuccinic Aciduria
• Disorder is caused by argininosuccinate lyase. It is a rare condition. There is

Hyperargininemia
• Disorder is caused by deficiency of arginase. There is increased plasma ammonia

level (hyperammonemia).
Suggested Readings
London
Latner AL, Cantarow A, Trumper M (1975) Clinical biochemistry, 7th edn. W. B. Saunders,
Philadelphia
Lehninger AL (1978) Biochemistry, 2nd edn. Kalyani, Ludhiana
Oser BL (ed) (1965) Hawk’s: physiological chemistry, 14th edn. McGraw-Hill, New York
Digestion and Absorption
of Carbohydrates 14
14.1 Definition of Digestion

Digestion is the physicochemical process of conversion of nondiffusible form of food
into diffusible form in the alimentary canal.
Carbohydrates are important component of human diet. A healthy adult person

requires around 2800 cal/day. Calorific value of carbohydrates is 4 cal/g. It is rec-
ommended that around 60% of calories should be obtained from carbohydrates. A
person should consume around 450 g of carbohydrates per day. However, carbohy-
drates are the major dietary component in economically weaker section of society.
Carbohydrates provide 90% of daily calories.
Plant Sources of Carbohydrates
• Cereals like rice, wheat, maize, oats, and millets are source of starch.
• Fruits like banana, mango, apple, grapes, orange, and dates are rich in starch,
fructose, and glucose.
• Vegetables like potatoes, sweet potatoes, carrots, beets, and cassava are rich
sources of starch.
• Honey is rich in fructose.
• Jaggery is a highly concentrated product from sugarcane juice. It contains around
50–60% sucrose. Jaggery is a traditional dessert in India and Asia.
• Cold drinks and beverages contain fructose and sucrose.
• Nondigestible carbohydrates like cellulose and pectin in vegetables and fruits.

https://doi.org/10.1007/978-981-13-1035-5_14
396 14 Digestion and Absorption of Carbohydrates
Animal Sources of Carbohydrates
• Milk contains lactose.

• Meat, sardines, salmons, sea food, and eggs are almost carbohydrate free.
• Shellfish contains 5% carbohydrates.
• Organ liver contains 5% carbohydrates.
14.3 Digestion of Carbohydrates
Digestion is an enzymatic hydrolytic process. Carbohydrate digestion starts in oral

cavity, and it is completed in the small intestine. It occurs in different portions of
alimentary canal.
14.3.1 Digestion in Oral Cavity
• Oral cavity contains saliva. It is rich in “salivary amylase.” This enzyme is also
called as “ptyalin.”
• Salivary amylase acts on dietary starch and glycogen. It requires chloride ions
and pH 6.7 for catalysis of polysaccharides.
• Starch and glycogen are converted into dextrin, maltose, and maltotriose.
• Dietary substances are masticated by teeth, and this physical process helps in
proper mixing of salivary enzyme with food particles.
• Partially digested food is called as “bolus,” and it is swallowed and enters the
stomach.
• In the stomach, activity of salivary amylase stops due to decline in pH of the
medium (1.5–3).
Salivary amylase
Dietary Starch and Glycogen Dextrin + Maltose + Maltotriose
Cl- ions / pH (6.7)
14.3.2 Digestion in the Stomach
• Gastric juice does not contain enzyme for carbohydrate digestion.

• Hydrochloric acid in the stomach may hydrolyze sucrose into glucose and
fructose.
14.3.3 Digestion in the Duodenum
• Duodenum part of the small intestine is important for digestion. It receives pan-
creatic juice from the pancreas. It is rich in “pancreatic amylase.”
14.4 Absorption of Carbohydrates 397
• The enzyme splits starch, glycogen, and dextrin into maltose.

• Its optimum activity requires Cl ions as cofactor and pH 7.4.
Pancreatic amylase
Dietary Starch, Glycogen and Dextrin Maltose
Chloride ions/pH 7.4
14.3.4 Digestion in the Small Intestine
• Intestinal juice contains disaccharide-splitting enzymes. The optimum pH is 7.4

for intestinal enzymes.
• Enzyme intestinal amylase cleavages alpha-1,4 glycosidic bonds in disaccha-
rides and oligosaccharides. It liberates glucose.
• Enzyme maltase acts on maltose molecules and liberates glucose molecules.
• Enzyme lactase splits lactose into glucose and galactose.
• Enzyme isomaltase cleavages alpha-1,6 glycosidic bonds in limit dextrin mole-
cules. It liberates glucose and maltose molecules.
• Enzyme sucrase splits sucrose molecules and liberates glucose and fructose.
14.4 Absorption of Carbohydrates
14.4.1 Definition
Absorption is a physiological process of passage of digested food from the lumen of the
alimentary canal into blood circulation.
14.4.2 Rate of Absorption of Monosaccharides
• Only monosaccharide form of carbohydrates is absorbed from lumen of the

small intestine.
• Dietary carbohydrates are digested into monosaccharides like glucose, galactose,
fructose, and mannose.
• Monosaccharides have different rate of absorption from intestinal lumen.
• C.F. Cori in 1933 studied rate of absorption of glucose and other monosaccha-
rides from lumen of small intestine in experimental albino rats. According to
Cori’s study:
–– Galactose absorption is the fastest among monosaccharides.
–– Galactose and glucose are the most rapidly absorbed monosaccharides.
–– Fructose and mannose have intermediate rate of absorption.
–– Xylose and arabinose have the slowest rate of absorption among all monosac-
charides in intestinal lumen. Comparative rate of absorption has been depicted
in Fig. 14.1.
Arabinose
Monosaccharides Xylose
Mannose
Fructose
Glucose
Galactose
0 20 40 60 80 100 120
Rate of absorption
Fig. 14.1 Showing rate of absorption of monosaccharides
14.4.3 Mechanism of Monosaccharide Absorption
bsorption by Simple Diffusion

A
Features
• It is the movement of sugar molecules from the region of higher concentration
(lumen of the intestine) to blood circulation through mucosal cells.
• Simple diffusion is dependent on concentration gradient.
• Small amount of all monosaccharides are absorbed by simple diffusion.
• Predominantly, arabinose and xylose are absorbed by simple diffusion.
bsorption by Active Transport

A
Features
• It is a process of movement of sugar molecules against concentration gradi-
ent across the plasma membrane of the intestine in the presence of a protein
carrier and energy.
• Glucose and galactose are rapidly absorbed by active transport mechanism.
• Comparative higher rate of absorption of glucose and galactose is due to active
transport.
• Active transport is the chief process which is involved in absorption of monosac-
charides from the lumen of small intestine. Active transport of monosaccharides
can be explained by Crane hypothesis as described below:
Crane Hypothesis of Active Transport

Features
• In 1960, R. K. Crane proposed “sodium-glucose cotransport hypothesis” for
absorption of glucose through intestinal mucosa.
–– Brush border epithelium of the small intestine contains specialized protein
molecule which helps in transport of glucose. It is called as “carrier
protein.”
–– Carrier protein is a transmembrane protein molecule.
–– Active transport of carbohydrates (monosaccharides) requires the presence of
sodium ions in the lumen of small intestine.
14.4 Absorption of Carbohydrates 399
Carrier Intestinal
Enterocyte
(symporter) lumen
Na+
Na+
G
Glucose
Na+ Glucose (G)

ADP
ATP Na+
Na+
k+
k+
Na+ – k+ –ATPase
Brush border
surface of enterocyte
Microvillus
Nucleus
Basolateral Cytoplasm
surface
Fig. 14.2 Digestion and absorption of carbohydrates
–– This is an ATP-dependent mechanism.

–– On the surface of brush border epithelium of small intestine, sodium ion
attaches to carrier protein molecule. This binding results in conformational
change in carrier protein molecule. Thereafter, glucose binds to carrier pro-
tein at another site.
–– Carrier protein molecule passes across plasma membrane of the small intesti-
nal epithelium and enters inside the epithelial cell (enterocyte) as in Fig. 14.2.
This method of active transport is called as Co-transport.
–– Crane sodium-glucose co-transport hypothesis explains the role of con-
comitant oral administration of sodium and glucose in oral rehydration
therapy in diarrhea and cholera.
Role of Glucose Transporters (GLUT) in glucose absorption
• Glucose transporters are the membrane-bound protein molecules. They help in

transport of glucose across the plasma membrane of cells.
• Glucose transporters are found in different body tissues.
• They are numbered from GLUT-1 to GLUT-14, depending upon the tissues in
which they are located.
Important Glucose Transporters
• GLUT-1
–– It is found in the RBC, retina, fetus, and blood-brain barrier.
–– It is helpful in the uptake of glucose.
–– GLUT-1 activity is independent of insulin.
• GLUT-2
–– It is found in epithelial cells of the small intestine, liver, and pancreas.
–– GLUT-2 helps in glucose transport through intestinal epithelial cells. GLUT-2
activity is independent of insulin.
• GLUT-4
–– It is found in adipose tissues and muscles.
–– GLUT-4 activity is under control of insulin hormone.
bsorption by Facilitated Transport

A
• It is a process of passive movement of sugar molecules in favor of concentra-
tion gradient across the plasma membrane of the intestine in the presence of
a transmembrane integral protein.
Features
• Fructose and mannose are absorbed by facilitated transport.
• It occurs in the presence of a transmembrane integral protein molecule.
• It occurs independent of ATP.
• It occurs in favor of concentration gradient.
Facilitated transport of monosaccharides from the lumen of small intestine can
be explained by ping-Pong model as described below:
Ping-Pong Model of Facilitated Transport

• Transmembrane integral proteins span across the plasma membrane of the
intestine.
• Transmembrane protein has two conformations.
–– Ping conformation
–– Pong conformation
• In pong conformation, transmembrane protein molecule is exposed to sugar
molecules in the lumen of the intestine. Sugar molecules bind to its extrinsic
surface. They are transmitted across the lipid bilayer and reach intrinsic surface
of transmembrane protein. Pong conformation of protein changes to ping
conformation.
• In ping conformation, sugar molecules are delivered to interior of cells. Again,

transmembrane protein mutates to pong conformation.
Lactose Intolerance
• This disorder is due to deficiency of lactase enzyme in intestinal epithelium. The

deficiency may be congenital and acquired.
• Congenital lactase deficiency is rare in occurrence. Infants cannot digest lac-
tose in milk. Its symptoms are:
–– Diarrhea
–– Distension of abdomen
–– Abdominal cramps and flatulence
–– Wasting
• Acquired lactase deficiency occurs due to environmental factors and struc-
tural changes in GIT.
–– Lactase activity is normal in early life.
–– It is a common condition among population in Southeast Asia.
–– Lactase activity is decreased in older age.
–– It results into diarrhea, flatulence, and abdominal cramps.
Suggested Readings
Crane RK (1960) Intestinal absorption of sugars. Physiol Rev 40:789–825
Ramasubbu N, Paloth V, Luo Y, Brayer GD, Levine MJ (1996) Structure of human salivary
α-amylase at 1.6 Å resolution: implications for its role in the oral cavity. Acta Crystallogr Sect
D Biol Crystallogr 52(3):435–446
Thorens B, Mueckler M (2010) Glucose transporters in the twenty-first century. Am J Physiol
Endocrinol Metab 298(2):E141–E145
Metabolism of Carbohydrates
15
15.1 Introduction
Carbohydrates are synthesized by green plants utilizing solar energy. Primary con-
sumers are mainly dependent on plant-based foods. Carbohydrates are the major
energy source in plant-based food substances. Living organisms derive energy eas-
ily from carbohydrates. Glucose is the prime carbohydrate in the human body as a
source of energy. Glucose is the major carbohydrate that is involved in carbohydrate
metabolism in humans.
15.2 Glycolysis
Definition
Glycolysis is defined as series of biochemical reactions involved in conversion
of glucose or glycogen into pyruvate or lactate with the release of energy.
Glycolysis is derived from Greek words:
Glycose means sugar.
Lysis means decomposition.
Site of Occurrence
• Glycolysis occurs in cytosol in almost all cells of the body.

• Brain tissues and erythrocytes are exclusively dependent on glycolysis for fulfill-
ing energy requirement.

https://doi.org/10.1007/978-981-13-1035-5_15
404 15 Metabolism of Carbohydrates
Characteristics of Glycolysis
• Glycolysis is also called as Embden-Meyerhof-Parnas pathway.

• It was described by Embden and Meyerhof in 1940.
• Glycolysis occurs in aerobic as well as anaerobic conditions.
• Glycolysis is chief glucose metabolism in erythrocytes for energy generation
(RBCs lack mitochondria; fatty acids cannot be oxidized; TCA cycle cannot
operate; glucose is anaerobically decomposed into lactate for energy).
• Glycolysis is the obligatory pathway to provide energy to brain tissues (glu-
cose is completely oxidized into CO2 and water). In conditions like fasting and
starvation, brain tissues depend on ketone bodies and lactic acid for energy
synthesis).
Steps in Glycolysis
Glycolysis occurs in a cascade of biochemical reactions. Each one is enzyme con-
trolled. These reactions are described in the following steps:
1. Phosphorylation of glucose
• Glucose is freely permeable into liver cells. Glucose uptake in cardiac mus-
cles, adipose tissues, and skeletal muscles is regulated by insulin.
• Glucose undergoes phosphorylation to form glucose-6-phosphate.
• Reaction requires one molecule of ATP. It donates phosphate group and is
converted into ADP.
• Mg++ ions act as cofactor in the reaction.
• Reactions are catalyzed by kinase enzyme whose nomenclature is based on
its presence in tissues as:
–– Glucokinase. This enzyme is found in the liver. It catalyzes phosphoryla-
tion of only glucose. Due to its high affinity to glucose, it is not inhibited
by glucose-6-phosphate.
–– Hexokinase. This enzyme is found in all body tissues. It phosphorylates
hexoses like glucose, mannose, or fructose. It is inhibited by
glucose-6-phosphate.
• Glucose-6-phosphate is the KEY compound across glucogenesis, gluconeo-
genesis, glycogenolysis, HMP shunt, and uronic acid pathway.
• Glucose phosphorylation is an irreversible step as in Figs. 15.1 and 15.2a, b, c.
2. Isomerization of glucose-6-phosphate
• Glucose-6-phosphate undergoes isomerization into fructose-6-phosphate
as in Figs. 15.1 and 15.2a, b, c.
• Reaction is catalyzed by phosphohexose isomerase and Mg++ ions.
3. Phosphorylation of fructose-6-phosphate
• Fructose-6-phosphate undergoes phosphorylation into fructose-1,6
bisphosphate as in Figs. 15.1 and 15.2a, b, c.
• One ATP molecule is consumed in reaction and is converted into ADP. Mg++
ions act as cofactor.
15.2 Glycolysis 405
Metabolism of carbohydrates
Hexokinase Phosphohexo
or Isomerase
Glucose Glucose – 6 – Phosphate Fructose – 6 – Phosphate
Glucokinase
e
Mg++ as
kin
ucto
ATP ADP ofr AD
P
s ph
Pho
P
AT
Fructose – 1, 6 – Bis phosphate
Glycerol
ATP
Glycerol kinase
Aldose
ADP
Glycerol – 3 – P
Phosphotriose
Dihydroxy isomerase Glyceraldehyde – 3 – Phosphate
Acetone
Dehydrogenase Phosphate NAD+ Glyceraldehyde – 3 – P
Dehydrogenase
NADH+ H+
NAD+ NADH+ H+
1,3 – Diphospho glycerate
ADP
Phosphoglycerate kinase
ATP
3 – Phospho glycerate
Phosphoglycerate
Mutase
Enolase
H2O Mg++
Phospho Enol Pyruvate

ADP Pyruvate kinase
Mg++
ATP
Pyruvate (Enol) Pyruvate
(Keto) Spontaneously
Fig. 15.1 Showing glycolysis
• This is an irreversible step.

• Reaction is catalyzed by phosphofructokinase.
–– Phosphofructokinase. It is a key regulatory enzyme in glycolysis.
–– It is an allosteric enzyme. Its enzymatic activity is inhibited by ATP mol-
ecule and citrate molecule and activated by AMP molecule.
O O
=
C–H C–H
ADT ATD
H – C – OH Mg++ H – C – OH
HO – C – H Hexokinase HO – C – H
or
H – C – OH H – C – OH
Glucokinase
H – C – OH H – C – OH O
6CH 2 – OH 6CH2 – O – P – OH
Glucose OH
Glucose – 6 – P
Phosphohexo
isomerase CH2OH
Glucose – 6 – Phosphate
=
C=O
HO – C – OH
H–C–H
H – C – OH
H – C – OH O
6CH2 – O – P – OH
OH
Fructose – 6 – P
CH2 – O – P – OH
=
ATP ADP C–O OH

mg++
Fructose – 6 – Phosphate HO – C – OH
Phosphofructo kinase
H – C – OH
H – C – OH O
6CH2 – O – P – OH
OH
Fructose – 1,6, Bis Phosphate
Fig. 15.2 Glycolysis
15.2 Glycolysis 407
Fructose – 1, 6 – Bis phosphate
Aldolase
=
1 CH – OH 1C
2 –H
2 C=O
Phosphotriose 2
O H – C – OH
isomerase O
=
3 CH2 –O – P – OH
=
3 CH2 – O – P – OH
OH OH
Dihydroxy acetone Glyceraldehyde – 3 – P
Phosphate
=
Glyceraldehyde – 3 – P
1 COO – P – OH2
Dehydrogenase
Glyceraldehyde – P– P H – C – OH OH
2
O
=
3 CH2 – O – P – OH
NAD+ NADH+H+
O OH
=
HO – P – OH 1,3 – Bisphospho
glycerate
OH
Inorganic
phosphate
Phospho glycerate COOH

kinase
1,3 – Bis–phospho glycerate H – C – OH
O
=
CH2 – O – P – OH
ADP ATP
OH
Phospho glycerate 1 COOH

O
mutase 2
=
3 – Phospho glycerate HC – O – P – OH
3 CH OH OH
2
Fig. 15.2 (continued)
Enolase
2 – phosphoglycerate COOH
O
Mg++
C – O – P – OH
H2O
H – C – H OH
Phospho enol
pyruvate
Pyruvate Kinase
Phospho Enol pyruvate COOH
ADP
ATP C=O
H–C–H
Enol pyruvate
Spontaneous
Enol pyruvate COOH
C=O
H–C–H
H
Pyruvate
4. Splitting of fructose-1,6 bisphosphate

• Fructose-1,6 bisphosphate (6C) compound is cleavaged into two com-
pounds, namely, dihydroxyacetone phosphate (3C) and glyceraldehyde-
3-phosphate (3C), as in Figs. 15.1 and 15.2a, b, c.
• Reaction is catalyzed by aldolase (lyase enzyme) as in Figs. 15.1 and 15.2a,
b, c.
• Dihydroxyacetone phosphate is a ketotriose, and glyceraldehyde-3-phosphate
is an aldotriose. These triose phosphates are interconvertible by phosphotri-
ose isomerase.
Dihydroxyacetone phosphate is converted into glyceraldehyde-3-phosphate.
It results into formation of two molecules of glyceraldehyde-3-phosphate
from one molecule of glucose.
• Bromohydroxyacetone phosphate resembles structurally to dihydroxyacetone
phosphate.
It competitively inhibits phosphotriose isomerase. This leads to inhibition of
glycolysis.
5. Oxidative phosphorylation of glyceraldehyde-3-phosphate
• Glyceraldehyde-3-phosphate is oxidized and phosphorylated into
1,3-bisphosphoglycerate as in Figs. 15.1 and 15.2a, b, c.
15.2 Glycolysis 409
• Reaction is catalyzed by glyceraldehyde-3-phosphate dehydrogenase. It

requires NAD+ as coenzyme. It is reduced to NADH with a release of one H+.
• Inorganic phosphate (Pi) provides phosphate group for phosphorylation.
• Each NADH passes through ETS and generates three ATP molecules
(3 × 2 = 6 ATP).
6. Substrate-level phosphorylation
• 1,3-Bisphosphoglycerate is a high-energy phosphate molecule, and it is
converted into 3-phosphoglycerate as in Figs. 15.1 and 15.2a, b, c.
• Its high-energy phosphate group at first position is transferred to ADP to form
ATP. This process is called substrate-level phosphorylation (synthesis of
ATP without passing through electron transport chain, while synthesis of
ATP through oxidation of NADH and FADH in ETS is called as oxidative
phosphorylation).
• Reaction is catalyzed by phosphoglycerate kinase in the presence of Mg++
ions as cofactor.
7. Conversion of 3-phosphoglycerate
• 3-Phosphoglycerate is converted into 2-phosphoglycerate as in Figs. 15.1
and 15.2a, b, c.
• Reaction is catalyzed by phosphoglycerate mutase.
• Enzyme transfer phosphate group from third position to second position. It is
internal rearrangement of molecule.
8. Conversion of 2-phosphoglycerate
• 2-Phosphoglycerate is converted into phosphoenol pyruvate (PEP).
• Reaction is catalyzed by enolase in the presence of Mg++ ions as cofactor.
• There is loss of a water molecule.
• PEP has high-energy phosphate enolic group.
• Fluoride inhibits enolase (sodium fluoride is added in collected blood sam-
ple for blood-glucose estimation test; it prevents glycolysis in the sample)
9. Substrate-level phosphorylation
• Phosphoenol pyruvate is converted into pyruvate.
• High-energy phosphate group in PEP is transferred to ADP to form ATP.
• Reaction is catalyzed by pyruvate kinase.
Energetic in Glycolysis (One Molecule of Glucose)

Aerobic glycolysis
Synthesis of Consumption of
Glycolysis step ATP ATP
Phosphorylation of glucose Nil 1 ATP
Phosphorylation of fructose-6-phosphate Nil 1 ATP
Oxidative phosphorylation of 2 NADH Nil
glyceraldehyde-3-phosphate 2 × 3 = 6 ATP
Conversion of 1,3-bisphosphoglycerate into 2 ATP Nil
3-bisphosphoglycerate
Conversion of phosphoenol pyruvate into pyruvate 2 ATP Nil
Total synthesis Total consumption
+10 ATP −2 ATP
In aerobic glycolysis
• Net gain = 8 ATP
In anaerobic condition
• Two NADH molecules are produced in oxidative phosphorylation of

glyceraldehyde-3-phosphate.
• NADH molecule must be oxidized into NAD+ through electron transport chain in
mitochondria. It is the NAD+ coenzyme that can sustain glycolysis. However, in
anaerobic condition, NADH cannot be oxidized into NAD+. Additionally,
cytosolic pool of NAD+ is limited. These events lead to discontinuity in sup-
ply of NAD+ for glycolysis.
• In order to continue glycolysis in anaerobic condition, pyruvate is reduced
into lactate in cells.
• Reaction is catalyzed by lactate dehydrogenase in the presence of NADH
coenzyme.
• In the reaction, two NADH molecules are oxidized into two NAD+.
Pyruvate Lactate
NADH + H+ NAD+
• NAD+ generated in pyruvate reduction is utilized by cells in oxidative phos-

phorylation of glyceraldehyde-3-phosphate. This activity maintains glycoly-
sis in anaerobic condition.
Synthesis of Consumption of
Glycolysis step ATP ATP
Phosphorylation of glucose Nil 1 ATP
Phosphorylation of fructose-6-phosphate Nil 1 ATP
Conversion of 1,3-bisphosphoglycerate into 2 ATP Nil
3-bisphosphoglycerate
Conversion of phosphoenol pyruvate into pyruvate 2 ATP Nil
Total synthesis Total consumption
4 ATP 2 ATP
Net gain = 2 ATP (4 − 2 = 2)
In anaerobic glycolysis
• Net gain = 2 ATP

15.2 Glycolysis 411
Site of Occurrence of Anaerobic Glycolysis

Erythrocytes
• Erythrocytes lack mitochondria. Glucose undergoes anaerobic glycolysis in

erythrocytes. They need continuous supply of glucose for energy need.
• Lactate accumulates in erythrocytes.
• It is transported to the liver for oxidation into pyruvate.
Skeletal muscles
• Skeletal muscles have high-energy demand owing to rapid contraction.

• In strenuous physical exercise, energy demand of tissues is increased. Oxygen
supply to contracting muscles is limited.
• In this relative anaerobic condition, anaerobic glycolysis occurs in skeletal mus-
cles. Accumulated lactate is cycled to the liver through blood circulation.
Regulation of Glycolysis
Glucokinase enzyme
• Glucokinase phosphorylates glucose into glucose-6-phosphate in the liver.

Glucokinase has low affinity to glucose molecules. It is controlled by feedback
mechanism. Decrease in blood glucose level inhibits glucokinase enzyme which
sequentially inhibits glycolysis. Adequate concentration of glucose promotes
glucokinase.
Hexokinase enzyme
• Hexokinase enzyme phosphorylates hexoses in tissues. Hexokinase has high

affinity for glucose. It is also controlled by feedback mechanism. Decrease in
glucose concentration activates hexokinase and sequentially promotes glycoly-
sis. This mechanism is responsible for supply of energy to the brain, cardiac
fibers, and skeletal fibers.
Phosphofructokinase enzyme
• Phosphofructokinase is the key regulatory enzyme in glycolysis. It is alloste-

rically inhibited by ATP and citrate molecules. It is activated allosterically by
AMP molecule.
Pyruvate kinase enzyme
• Pyruvate kinase is a key regulatory enzyme in glycolysis. It catalyzes dephos-

phorylation of phosphoenol pyruvate into pyruvate. It is an irreversible step. It is
activated by insulin and inhibited by glucagon hormone.
Insulin hormone
• Insulin hormone activates phosphofructokinase enzyme, pyruvate kinase

enzyme, and hexokinase enzyme (glucokinase).
Glucagon hormone
• Glucagon hormone inhibits phosphofructokinase enzyme, pyruvate kinase

enzyme, and hexokinase enzyme (glucokinase).
15.3 Pyruvate Metabolism
Pyruvic acid (pyruvate) is α-keto acid. It is produced in the body in the following
metabolic pathways:
• Pyruvate is produced in glycolysis.

• Pyruvate is produced by oxidation of lactic acid.
• Pyruvate is produced by transamination of amino acids.
• Pyruvate is produced as in Fig. 15.3.
Pyruvate is actively transported through inner membrane of mitochondria.

It enters the mitochondrial matrix. It undergoes oxidative decarboxylation.
Glycolysis
Glucose
Glucose Gluconeogenesis
P Lactate
L. Dehydrogenase Y
Lactate R Transamination
U Alanine
Transamination V
Alanine Gluconeogenesis
A Glucogenic
T amino acid
Pyruvate carboxylase Oxaloacetate
E
Oxalo decarboxylase
Oxalo acetate
acetate
CO2
Pyruvate
dehydrogenase
complex
Acetyl coa
Fig. 15.3 Pyruvate metabolism

15.3 Pyruvate Metabolism 413
Oxidative Decarboxylation of Pyruvic Acid (Pyruvate)

In aerobic condition
• Pyruvate (3C) is oxidized and decarboxylated to form (2C) compound as acetyl

CoA-SH.
• Reaction occurs in the mitochondrial matrix.
• Reaction is catalyzed by pyruvate dehydrogenase.
Pyruvate dehydrogenase
• It is a multienzyme complex called as pyruvate dehydrogenase complex.

• It is found in the mitochondrial matrix.
• Its high concentration is found in cardiac muscles and kidneys.
• Pyruvate dehydrogenase complex is a cluster of three types of enzymes:
–– Pyruvate decarboxylase
–– Dihydrolipoyl dehydrogenase
–– Dihydrolipoyl transacetylase
• Pyruvate dehydrogenase requires six prosthetic groups for catalysis:
–– Lipoic acid
–– Thiamine pyrophosphate (TPP)
–– CoA-SH
–– NAD+
–– FAD
–– Mg++
Steps in Oxidative Decarboxylation of Pyruvate
1 . Enzyme-bound thiamine pyrophosphate activates pyruvate in the presence of Mg++.

2. Activated pyruvate undergoes decarboxylation by pyruvate decarboxylase bound
to TPP. Active pyruvate is converted into alpha-hydroxyethyl thiamine pyro-
phosphate (active acetaldehyde).
3. Active acetaldehyde is converted into acetylated lipoate by lipoate transacetylase
bound to lipoic acid coenzyme.
4. Acetylated lipoate is converted into acetyl CoA and reduced lipoate in presence
of CoA-SH.
5. FAD+ oxidizes reduced lipoate into oxidized lipoate with formation of FADH2.
6. FADH2 transfers hydrogen atoms to NAD+ to form NADH + H+ which enters
ETS chain to generate two ATP molecules.
Energetic of Oxidative Decarboxylation of Pyruvate
• Two pyruvate molecules are produced by glycolysis.

• Oxidative decarboxylation of pyruvate generates two acetyl CoA molecules
and two NADH.
• Two NADH molecules generate six ATP molecules.
• Energetics in oxidative decarboxylation of pyruvate
–– +6 ATP
15.4 Citric Acid Cycle
Definition
Citric acid cycle is defined as a series of biochemical reactions occuring inside
mitochondria of aerobic organisms that are essential for synthesis of energy in the
form of ATP. It is termed as Citric acid Cycle because citric acid is the first metabo-
lite that is produced as a sequence of chemical reactions.
Site of Occurrence
• Citric acid cycle occurs in mitochondrial matrix in eukaryotes.
Characteristics of Citric Acid Cycle
• Citric acid cycle was described by Sir Hans Krebs in 1937.

• Citric acid is the first metabolite in the cyclic process, and it is named as citric acid
cycle. It is also called as Krebs cycle based on the name of the discoverer. It is addi-
tionally termed as tricarboxylic acid cycle as citric acid has three carboxylic groups.
• The cyclic pathway starts with synthesis of citric acid utilizing acetyl CoA and
terminates with regeneration of citric acid accompanied by production of CO2,
water, and ATP molecules.
• TCA cycle is the common metabolic pathway involving oxidation of lipids,
carbohydrates, and proteins. Amino acids are metabolized into intermediate
metabolites of TCA cycle. Fatty acids and glucose are oxidized to form acetyl
CoA. Overall, diffusible form of macronutrients is converted into acetyl CoA
which in turn is oxidized into CO2 with release of energy through TCA cycle.
• TCA cycle is absolutely amphibolic pathway. It includes catabolic as well as
anabolic reactions as follows:
–– Anabolic reactions
Aspartate is synthesized from oxaloacetate.
Alpha-ketoglutarate can be used for synthesis of glutamate.
–– Catabolic reactions
Steps in Citric Acid Cycle
1. Condensation
• Oxaloacetate (4C) compound undergoes condensation with acetyl CoA to
form citric acid as in Fig. 15.4.
• Reaction is catalyzed by citrate synthetase.
• CoA-SH is released in the reaction.
2. Isomerization of citric acid
• Citric acid undergoes isomerization into isocitric acid as in Fig. 15.4.
• Reaction is catalyzed by aconitase.
15.4 Citric Acid Cycle 415
• Process occurs in two steps:

–– Dehydration of citrate
This reaction is catalyzed by aconitase enzyme. Citric acid is dehydrated
to form cis-aconitate.
–– Hydration of cis-aconitate
Hydration of cis-aconitate forms isocitrate.
• Dehydration and hydration reactions are catalyzed by aconitase.
3. Oxidative decarboxylation of isocitrate
• Isocitrate (6C) undergoes oxidation and decarboxylation to form
α-ketoglutarate (5C) as in Fig. 15.4.
• Reaction is catalyzed by isocitrate dehydrogenase in the presence of NAD+.
Process occurs in two steps:
–– Oxidation of isocitrate
Isocitrate is oxidized to form oxalosuccinate. Reaction delivers two hydro-
gen atoms which are accepted by NAD+. It is reduced in NADH + H+.
–– Decarboxylation of oxalosuccinate
Oxalosuccinate is decarboxylated to form α-ketoglutarate. A molecule of
CO2 is released.
Both reactions are catalyzed by isocitrate dehydrogenase.
4. Oxidative decarboxylation of α-ketoglutarate
• α-Ketoglutarate (5C) is oxidized and decarboxylated to form succinyl CoA
(4C) as in Fig. 15.4.
• Reaction is catalyzed by α-ketoglutarate dehydrogenase complex in presence
of NAD+ and CoA-SH.
• α-Ketoglutarate dehydrogenase complex is a multienzyme complex. Reaction
is similar to oxidative decarboxylation of pyruvate into acetyl CoA-SH.
5. Conversion of succinyl CoA into succinic acid
• Succinyl CoA-SH undergoes hydrolysis to form succinic acid as in Fig. 15.4.
• Reaction is catalyzed by succinate thiokinase.
• Thioester bond in succinyl CoA-SH is cleavaged to release high-energy phos-
phate group.
• Guanidine diphosphate (GDP) is phosphorylated into GTP with release of
CoA-SH.
• GTP molecule phosphorylates one molecule of ADP into ATP by nucleoside
diphosphokinase.
6. Oxidation of succinic acid
• Succinic acid is oxidized to form fumarate as in Fig. 15.4.
• Reaction is catalyzed by succinate dehydrogenase in the presence of FAD+ as
coenzyme.
• Reaction releases a pair of hydrogen atoms which are accepted by FAD+.
• FADH2 generates two ATP molecules through oxidative phosphorylation in
ETS chain.
• Succinate dehydrogenase is exclusive enzyme that is attached to the mito-
chondrial membrane.
Oxidative
PYRUVATE CH3 CO ∼ S. COA O
Decarboxylation
ACETYL COA H2 – C – C – OH
O
COA – SH HO – C – C – OH
COOH O
H2 – C – C – OH
C=O
Citrate synthase CITRATE
CH2
Aconitase
COOH H2O
CH – COOH
Oxaloacetate
C – COOH
CH2 – COOH
Malate Dehydrogenase
+ +
NADH H
CIS – ACONITATE
H2O ACONITASE
+
NAD
HO C COOH
H C COOH
O
CH2 COOH
HO – CH – C – OH
O ISOCITRATE
H – CH – C – OH +
NAD
Isocitrate
Malate Dehydrogenase
+ +
NADH H
O
C – COOH
HO2 H – C – COOH
Fumarase
CH2 – COOH
Oxalo succinate
++
Mh Isocitrate
O Dehydrogenase
H – C – C – OH CO2
O
O
HO – C – C – OH
C – COOH
H
H – C – CH
Fumarate
CH2 – COOH
∝ – Ketoglutarate
Succinate Dehydrogenase
COA – SH ∝ – Keto
FADH2
glutarate
CO2 Dehydrogenase
H +
O NAD
FAD
H – C – C – OH
+ +
P NADH H
H – C – C – OH AT
+
P O
H COA ∼ SH GD
C ∼ COOH
ADP
Succinate
H– C – H
GTP
GDP+Pi CH2 – COOH
Succinyl coa
Succinate thiokinase
Fig. 15.4 Citric acid cycle

15.4 Citric Acid Cycle 417
7. Hydration of fumarate
• Fumarate (4C) undergoes hydration into malate (4C) as in Fig. 15.4.
• Reaction is catalyzed by fumarase.
8. Dehydrogenation of malate
• Malate (4C) undergoes dehydrogenation to form oxaloacetate (4C) as in
Fig. 15.4.
• Reaction is catalyzed by malate dehydrogenase in the presence of NAD+.
• It is reduced into NADH + H+.
Energetic of TCA Cycle
Steps in TCA cycle Synthesis of ATP

Oxidative decarboxylation of 6 ATP (2 NADH × 3)
isocitrate
Oxidative decarboxylation of 6 ATP (2 NADH × 3)
α-ketoglutarate
Conversion of succinyl CoA into 2 ATP
succinic acid
Conversion of succinate into fumarate 4 ATP (2 FADH × 2)
Conversion of malate into 6 ATP (2 NADH × 3)
oxaloacetate
Total gain of ATP in TCA cycle
• 24 ATP
Energetic of Glucose Catabolism

• Net gain in glycolysis
–– 8 ATP
• Net gain in oxidative decarboxylation of pyruvate
–– 6 ATP
• Net gain in TCA cycle
–– 24 ATP
Net gain = 38 ATP

Total energy production in glycolysis
1 ATP = 7300 cal
Oxidation of one molecule of glucose
• 7300 × 38 = 277400
Regulation of TCA Cycle

TCA cycle is regulated by the following factors which are described as follows:
Citrate synthase
• Citrate synthase catalyzes synthesis of citrate in TCA cycle. ATP molecule is an

allosteric inhibitor of citrate synthase enzyme.
ATP molecule
• Increased demand of ATP molecules in cell promotes TCA cycle. It is due to

close association between synthesis of NADH in TCA cycle and oxidation of
NADH in electron transport chain.
Hypoxia
• Decreased partial pressure of oxygen in circulation and tissue hypoxia result in

accumulation of NADH2 and FADH2 in cells. These coenzymes retard citric acid
cycle.
Isocitrate dehydrogenase
• ADP molecule is an allosteric stimulator of isocitrate dehydrogenase enzyme. It

promotes TCA cycle.
Toxins (arsenite, fluoroacetate, malonate)
• Toxins inhibit mitochondrial enzyme and retards TCA cycle. It is as follows:

–– Arsenic
Arsenic is a heavy metal. It is a noncompetitive inhibitor of alpha-ketogluta-
rate dehydrogenase enzyme.
–– Fluoroacetate
It is a noncompetitive inhibitor of aconitase enzyme.
–– Malonate
Malonate is a competitive inhibitor of succinate dehydrogenase enzyme.
15.5 Shuttle System
Definition
Shuttle system is comprised of a pair of substrates which are interconvertible
through dehydrogenase enzymes.
Significance of Shuttle System
• NADH2 is produced in glycolysis. They are impermeable to mitochondrial mem-

brane. But oxidation of NADH2 occurs in mitochondrial matrix. Shuttle system
serves to transfer reducing equivalents from NADH2 molecule to mitochondrial
matrix across mitochondrial membranes.
15.5 Shuttle System 419
Types of Shuttle Systems

There are two types of shuttle systems operating in human tissues as follows:
• Alpha-glycerophosphate shuttle system

• Malate shuttle system
Steps in Working of Alpha-Glycerophosphate Shuttle System
• This shuttle system is less prominent in human body tissues. It is important in

liver tissues.
• In cytosol
–– Dihydroxyacetone phosphate is present. It is reduced into
alpha-glycerophosphate.
–– Reaction is catalyzed by alpha-glycerophosphate dehydrogenase enzyme.
This enzyme is NADH2-dependent. Coenzyme is oxidized into NAD+.
–– Alpha-glycerophosphate moves across mitochondrial membranes into matrix.
• In mitochondrial matrix
–– Alpha-glycerophosphate is oxidized into dihydroxyacetone phosphate.
Reaction is catalyzed by alpha-glycerophosphate dehydrogenase enzyme.
This enzyme in mitochondria is flavoprotein dependent.
–– Reducing equivalents are accepted by FAD and are converted into FADH2.
–– FADH2 enters electron transport chain and produces two ATP molecules.
Significance
• Alpha-glycerophosphate shuttle system is less significant for human tissues.

NADH2 produces two ATP molecules through this system.
• One molecule of glucose after entering into glycolysis and TCA cycle pro-
duces 36 ATP molecules in alpha-glycerophosphate shuttle system.
Steps in Working of Malate Shuttle System
• This shuttle system is highly important for human tissues for production of ATP
molecules.
• In cytosol
–– Oxaloacetate is reduced into malate. Reaction is catalyzed by malate dehy-
drogenase enzyme. It is NADH-dependent enzyme in cytosol. NADH2 mole-
cule provides reducing equivalents for reduction, and it is oxidized into NAD+.
–– Malate crosses the mitochondrial membranes and enters the matrix.
• In mitochondrial matrix
–– Malate is oxidized into oxaloacetate. Reaction is catalyzed by malate dehy-
drogenase enzyme. This enzyme in mitochondria is NADH-dependent.
–– NAD+ coenzyme accepts reducing equivalents and reduced into NADH2
which in turn enters ETC and releases protons.
Significance
• One molecule of glucose after entering into glycolysis and TCA cycle pro-
duces 38 ATP molecules in malate shuttle system.
• This is a universal shuttle system for the oxidation of NADH2 and synthesis of
ATP molecules.
15.6 Hexose Monophosphate Shunt (HMP)
Definition
Hexose monophosphate shunt is an alternate carbohydrate metabolic pathway
for the oxidation of glucose.
• HMP shunt is also called as pentose-phosphate pathway (PP pathway) or

Warburg-Dickens-Lipman pathway.
Site of Occurrence
• HMP shunt chiefly occurs in tissues which are actively involved in synthesis of
lipid and nucleotides.
• It occurs in the liver, erythrocytes, adipose tissues, adrenal cortex, gonads, lactat-
ing mammary glands, cornea, and lens.
Characteristics
• HMP shunt is mainly an anabolic pathway unlike glycolysis which is a catabolic

pathway.
• HMP shunt involves oxidation of glucose like glycolysis; however NADH and
ATP are not generated in HMP. It serves to generate NADPH called as reducing
equivalents.
• In HMP shunt, hydrogen atoms are accepted by NADP coenzyme and not NAD+
coenzyme as in glycolysis.
• Oxidation of glucose results in formation of CO2 in HMP shunt, while it is not
formed in EMP pathway.
• HMP pathway and EMP pathway occur in cytosol of cells.
Steps in HMP Shunt

This pathway occurs in two separate phases:
• Phase A – conversion of hexose into pentose

• Phase B – conversion of pentose into hexose
15.6 Hexose Monophosphate Shunt (HMP) 421
Biochemical Reactions in Phase A
1. Phosphorylation of glucose
Glucose is phosphorylated into glucose-6-phosphate. Reaction is catalyzed by
hexokinase enzyme. One molecule of ATP is hydrolyzed into ADP to provide
high-energy phosphate to glucose as in Fig. 15.5.
2. Oxidation of glucose-6-phosphate
Glucose-6-phosphate is dehydrogenated to form a cyclic ester called as
6-phosphogluconolactone as in Fig. 15.5.
Reaction is catalyzed by glucose-6-phosphate dehydrogenase. Two hydrogen
atoms are liberated and accepted by coenzyme NADP. It is reduced into NADH2.
3. Hydrolysis of 6-phosphogluconolactone
The cyclic ester, 6-phosphogluconolactone, is unstable. It undergoes nonenzy-
matic hydrolysis to form 6-phosphogluconic acid as in Fig. 15.5.
4. Oxidation of 6-phosphogluconic acid
Oxidation of 6-phosphogluconic acid forms ribulose-5-phosphate as in Fig. 15.5.
Reaction is catalyzed by 6-phosphogluconate dehydrogenase. Coenzyme NADP
accepts two hydrogen atoms and reduced into NADPH2.
5. Conversion of ribulose-5-phosphate
Ribulose-5-phosphate is catalyzed by two distinct enzymes. Ribulose-5-
phosphate epimerase converts ribulose-5-phosphate into xylulose-5-phosphate.
Another enzyme ribulose-5-phosphate isomerase converts ribulose-5-phosphate
into ribose-5-phosphate as in Fig. 15.5.
Biochemical Reactions in Phase B
1. Transketolation
Ribose-5-phosphate reacts with xylulose-5-phosphate to form sedoheptulose-7-
phosphate and glyceraldehyde-3-phosphate as in Fig. 15.5.
Reaction is catalyzed by transketolase.
2. Transaldolation
Sedoheptulose-7-phosphate and glyceraldehyde-3-phosphate react together to
form erythrose-4-phosphate and fructose-6-phosphate as in Fig. 15.5.
Reaction is catalyzed by transaldolase.
3. Transketolation
Erythrose-4-phosphate reacts with xylulose-5-phosphate to form fructose-6-
phosphate and glyceraldehyde-3-phosphate as in Fig. 15.5.
Reaction is catalyzed by transketolase.
Fructose-6-phosphate and glyceraldehyde-3-phosphate are converted into
glucose-6-phosphate.
Kinase
Glucose Glucose – 6 – P
Mg++
6 molecules + Glucose – 6 – P
ADP ADP NADP
Dehydrogenase
NADPH2 Mg++
6 – phosphoglucono
lactone
H2O
NADPH2 NADP+ Hydrolase
Phosphogluconolactone
Dehydrogenase
Ribulose – 5 – Phosphate 6 – phosphogluconate
6 molecules CO2 6 molecules

e Ep
as im
er era
om se
Is
Ribose – 5 – Phosphate xylulose – 5 – Phosphate

4 molecules
2 molecules
2 mol
Ribose – 5 – Phosphate + xylulose – 5 – Phosphate

(2 mol) T (2 mol)
ke ra
to ns
2 mol
la TPP
se
Sedoheptulose – 7 – Phosphate + Glyceraldehyde

(2 mol) 3 – phosphate
Tra
Transketolase
ns (2 mol)
ald
ola
se
Fructose – 6 – Phosphate + Erythrose – 4 – phosphate

(2 mol) (2 mol)
Fructose – 6 – Phosphate + Glyceraldehyde – 3 – phosphate

(2 mol) (2 mol)
Fig. 15.5 Hexose monophosphate shunt
Significance of Hexose Monophosphate Shunt
• Synthesis of NADPH
NADPH is the reducing equivalent which is produced in HMP pathway. It
donates hydrogen atoms in various anabolic reactions in tissues as:
–– Cholesterol synthesis
–– “De novo” synthesis of fatty acids
15.7 Gluconeogenesis 423
–– Synthesis of steroid hormones

–– Uronic acid pathway in synthesis of ascorbic acid
• Synthesis of pentoses
D-ribose is synthesized in HMP pathway. It is a structural component of nucleic
acid.
• Synthesis of CO2
Carbon dioxide is produced in HMP pathway. It is used in synthesis of purine
and fatty acids.
HMP shunt liberates fructose-6-phopshate and glyceraldehyde-3-phosphate
which are utilized in EMP pathway for synthesis of ATP.
• Maintenance of RBC integrity
In RBCs, reduced glutathione (G-SH in glutathione peroxidase) is helpful in decom-
position of hydrogen peroxide into water and oxygen. Glutathione is oxidized (G-S-
S-G) in reaction. NADPH is essential to convert oxidized glutathione into reduced
glutathione. NADPH prevents oxidative damage to erythrocytes by H2O2.
• Helpful in phagocytosis
NADPH is necessary for synthesis of superoxide radicals which destroy foreign
substance in macrophages.
15.7 Gluconeogenesis
Definition
Gluconeogenesis is an anabolic process in which glucose is synthesized from
non-carbohydrate substances.
Site of Occurrence
• The liver is the chief organ for gluconeogenesis.

• It also occurs in the kidneys.
Gluconeogenesis from Pyruvate

Pyruvate is produced in glycolysis. It is a metabolic product in transamination of
amino acids. Pyruvate is converted into glucose through the following steps:
• Pyruvate undergoes carboxylation to form oxaloacetate. Reaction is catalyzed by

pyruvate carboxylase. One molecule of ATP is hydrolyzed to ADP with release
of inorganic phosphate.
• Oxaloacetate undergoes simultaneously decarboxylation and phosphorylation to
form phosphoenol pyruvate. Reaction is catalyzed by phosphoenol pyruvate car-
boxykinase. One molecule of GTP is hydrolyzed into GDP with release of CO2.
• Phosphoenol pyruvate is reversibly converted into fructose-1,6-bisphosphate.
• Fructose-1,6-bisphosphate is converted into fructose-6-phosphate by fructose-
1,6-bisphosphatase enzyme. This is a hydrolysis reaction with release of inor-
ganic phosphate.
• Fructose-6-phosphate is reversibly isomerized into glucose-6-phosphate by

phosphoglucoisomerase.
• Glucose-6-phosphate is converted into glucose by glucose-6-phosphatase enzyme.
Gluconeogenesis from Glucogenic Amino Acids
• Glucogenic amino acids undergo transamination and deamination. They are

metabolized into intermediates of TCA cycle.
• Intermediates are finally converted into oxaloacetate and pyruvate which enters
gluconeogenesis pathway.
Gluconeogenesis from Glycerol
• Mobilization of fat in adipose tissues releases fatty acids and glycerol. Glycerol
is transported to the liver by blood circulation.
• In the liver, glycerol is phosphorylated into glycerol-3-phosphate by glyceroki-
nase enzyme. Adipose tissues lack above enzyme.
• Glycerol-3-phosphate undergoes oxidation to form dihydroxyacetone phosphate
by glycerophosphate dehydrogenase enzyme. Coenzyme NAD+ is reduced into
NADH2.
• Dihydroxyacetone phosphate isomerizes into glyceraldehyde-3-phosphate by
phosphotrioisomerase.
• Glyceraldehyde-3-phosphate undergoes gluconeogenic reactions to form glu-
cose as in pyruvate to glucose conversion.
15.8 Gluconeogenesis from Lactic Acid (Cori’s Cycle)
Definition
It is metabolic pathway in which lactate synthesized in skeletal muscles is con-
verted into glucose in the liver.
The pathway was discovered by Carl Cori and Gerty Cori.
Site of Occurrence
• Cori’s cycle occurs in the liver and skeletal muscles.
Steps in Cori’s cycle

Steps in Cori’s cycle can be divided into two groups:
Steps in skeletal muscles
• Skeletal muscles store glycogen to serve a source of energy. Skeletal muscles are
actively contractile tissues as in Fig. 15.6.
• In skeletal muscles, glycogen is decomposed into glucose-1-phosphate which is
converted into glucose-6-phosphate by phosphoglucomutase enzyme. The
glucose-6-phosphate undergoes glycolysis to generate ATP for muscular activity.
15.8 Gluconeogenesis from Lactic Acid (Cori’s Cycle) 425
Glucose Glucose Glycogenesis Glucose
Glucose 6 – P
Gluconeogenesis
Liver
Skeletal muscle fibre

Pyruvate
COOH
Blood
C=O
Pyruvate
CH3
NADH2
Lactate
NAD+
dehydrogenase Lactate
NAD+ dehydrogenase
NADH2 COOH
Lactate
CHOH
Lactate
CH3
Lactate
Fig. 15.6 Cori’s cycle
• Under normal muscular activity, supply of oxygen is adequate in muscles. The

glucose-6-phosphate is aerobically converted into pyruvate which enters TCA
cycle to produce ATP molecules.
• Under strenuous muscular contractility, oxygen supply to muscles is
decreased. Pyruvate molecule is reduced into lactate by lactate dehydroge-
nase enzyme in the presence of NADH2.
• Pyruvate reduction yields NAD+ molecules which are essential to maintain gly-
colysis in anaerobic condition.
• Accumulation of lactate in skeletal muscle can cause muscle cramps. To avoid it,
lactate diffuses rapidly into blood circulation. Lactate is transported to the liver.
Skeletal muscles are unable to convert lactate into glucose as in Fig. 15.6.
Steps in the liver
• In the liver, lactate is oxidized into pyruvate by lactate dehydrogenase enzyme in

the presence of NAD+ coenzyme.
• Pyruvate is converted into glucose.
• Liver glucose is either stored as glycogen or released into systemic circulation as
in Fig. 15.6.
Significance of Cori’s Cycle
• Cori’s cycle is an important pathway for gluconeogenesis. It is helpful in uti-

lizing and converting lactate (waste metabolite) into glucose.
• Cori’s cycle helps in prevention of lactic acidosis (accumulation of lactate in
tissues, type of metabolic acidosis)
15.9 Glycogenolysis
Definition
It is a biochemical process of breakdown of glycogen into glucose in body tissues.
Site of Occurrence
• Glycogenolysis occurs in the liver and skeletal muscles.
Steps in Glycogenolysis
Cleavage of alpha-1 → 4-glycosidic linkage
• Glycogenolysis is started by phosphorylase enzyme. It is the key regulatory

enzyme for breakdown of glycogen into glucose-1-phosphate. This enzyme
splits alpha-1 → 4-glycosidic linkages in glycogen chains. After every cleavage,
glycogen molecule becomes shorter by one glucose residue. The cleavage of
alpha-1 → 4-glycosidic linkages continues till four glucose moieties remain on
either side of glycogen chain. Phosphorylase enzyme cannot split
alpha-1 → 6-glycosidic linkages as in Fig. 15.7.
Phosphorylase enzyme
It is found in liver cells as well as in skeletal muscle fibers.
–– Liver phosphorylase enzyme
–– Liver contains two forms of enzyme:
1. Phospho-phosphorylase enzyme:
It is an active form of enzyme and exists in phosphorylated form.
2. Dephospho-phosphorylase enzyme:
It is an inactive form and exists in dephosphorylated form.
–– Muscle phosphorylase enzyme
Skeletal muscles contain two forms of enzyme:
1. Phosphorylase a
It is an active form of enzyme.
2. Phosphorylase b
It is an inactive form of enzyme.
Glucagon hormone has no effect on muscle phosphorylase.
Pyridoxal phosphate as coenzyme (four molecules) is essential for muscle
phosphorylase.
15.9 Glycogenolysis 427
∝ – 1 – 4 –Glycosidic bond
Glycogen
1 15
19
10
Pi
ase ∝ – 1 → 6 –Bond
phoryl
hos
en p
cog
Gly
4 – Glucose Glycogen + GLUOCSE – 1 – PHOSPHATE

Residues
{ One glucose
residue shorter }
1 5
9
4
ing
ra nch Four glucose
eb e
D zym residues on one side of ∝– 1 6 – bond
En
Glucose – 1 – Phosphate
Phosphogluco Mg++
mutase
Glucose – 6 – P
Glucose – 6 – Phosphatase
Glucose
Fig. 15.7 Glycogenolysis
Cleavage of alpha-1 → 6-glycosidic linkage
• Debranching enzyme called as amylo-1-6-glucosidase splits 1 → 6-glycosidic

linkage between a branch chain and main chain of glycogen. It liberates a residue
of glucose as in Fig. 15.7.
• Phosphorylase and debranching enzymes cleavage glycogen into glucose-1-
phosphate residues.
Conversion of glucose-1-P into glucose-6-phosphate
• Glucose-1-p is converted into glucose-6-P by activity of phosphoglucomutase

enzyme as in Fig. 15.7.
Fate of glucose-6-phosphate
• Glycogen is hydrolyzed into glucose-6-P residues in the liver and skeletal tissues.
• In the liver, glucose-6-phosphatase enzyme dephosphorylates glucose-6-P into
free glucose molecules. This enzyme is also found in renal tubules. Glucose from
liver cells enters systemic circulation and results into hyperglycemia.
• In skeletal tissues, glucose-6-phosphatase enzyme is not found. Therefore,
G-6-P is not converted into glucose molecules. Glucose-6-phosphate enters gly-
colysis cycle and is converted into pyruvate and lactate.
Regulation of Glycogenolysis
Role of phosphorylase enzyme
• It is a key enzyme in glycogenolysis.

• Rise in cyclic AMP in cell activates protein kinase enzyme. It activates phos-
phorylase kinase b (inactive form) into phosphorylase kinase a (active form).
• Phosphorylase kinase a in turn activates dephospho-phosphorylase into phospho-
phosphorylase. It promotes glycogenolysis.
Role of hormones
• Thyroxine, epinephrine, and glucagon stimulate synthesis of cAMP in cytosol

and promote glycogenolysis.
• Glucagon promotes glycogenolysis in the liver only.
Role of calcium ions
• Rate of glycogenolysis in muscles is higher than liver cells during contraction.

• Phosphorylase kinase enzyme in muscles has four subunits. Its beta subunit has
the ability to bind with Ca++ ions and calmodulin (calcium-modulated protein),
and its activity is enhanced. Calcium ions promote glycogenolysis in muscles.
15.10 Glycogenesis
Definition
Glycogenesis is the biochemical process of formation of glycogen from glucose
in body tissues.
15.10 Glycogenesis 429
Site of Storage of Glycogen

Glycogen is a storage form of carbohydrate in human body. It is stored in liver cells
and skeletal tissues.
• In the liver, about 70–100 g of glycogen is stored. This quantity of glycogen

constitutes 4–6% weight of liver (normal weight = 1.8 kg).
• In skeletal tissues, about 240 g of glycogen is stored, and it represents around
0.7% of the weight of skeletal muscles in the body (weight of skeletal
muscles = 35 kg).
• Total quantity of glycogen storage is between 300 and 350 g in an adult person
with 70 kg of weight of the body.
Site of Occurrence
• It takes place mainly in the liver and skeletal tissues. However, glycogenesis can
also occur in all body tissues.
Steps in Glycogenesis
Phosphorylation of glucose
• Glucose is phosphorylated into glucose-6-phosphate. Reaction is catalyzed by

glucokinase enzyme (liver) and hexokinase enzyme (skeletal tissues).
• Reaction occurs in the presence of Mg ions, and one molecule of ATP is con-
sumed in reaction as in Fig. 15.8a, b.
Conversion of G-6-P into G-1-P
• Glucose-6-P is converted into glucose-1-phosphate through action of phospho-

glucomutase enzyme in the presence of Mg++ as in Fig. 15.8a, b.
Formation of uridine-diphosphate-glucose
• Glucose-1-phosphate is transferred to uridine triphosphate (UTP) molecule to

form uridine diphosphate glucose (UDP-G).
• Reaction is catalyzed by UDP-G pyrophosphorylase enzyme.
• A molecule of inorganic pyrophosphate is liberated in the reaction. It is cleav-
aged into inorganic phosphate by pyrophosphatase enzyme as in Fig. 15.8a, b.
Transfer of glucose residue to glycogen primer
• Glucose residue from UDP-G is transferred to glycogen primer. Its presence is

essential for initiation of glycogen synthesis.
• Glycogen primer
–– It is present in cytosol of the cell and it is also called as glycogenin.
–– It is a glycoprotein. Its protein (dimeric) component is made up of two similar
monomers. Each monomer is attached to oligosaccharide chain of seven glu-
cose residues.
–– Glycogen primer of glycogenin serves as a primer and an autocatalytic gly-
cosyltransferase enzyme (brings about transfer of carbohydrate moiety
from activated nucleotide sugar molecule to glycosyl acceptor molecule).
ATP ADP
Mg++
Glucose Glucose – 6 – Phosphate
Glucokinase
or
Hexokinase
Mg++
Glucose – 6 – P Glucose – 1 – Phosphate
Phosphogluco mutase
UDP – Glucose pyrophosphorylase

Glucose – 1 – P UDP –
(UDP – Glucose)
UTP PPi
Glycogen synthase
UDP –
(UDP – Glucose) Glycogenin
OH
UDP
Glycogenin
OH
Glucose
1 2 3 4
Glycogen primer
14 UDP –
Glycogen
Synthase
14 UDP ∝ – 1,4 Bonding
18
1 5 10 15
[Glycogen Chain]
Fig. 15.8 Glycogenesis
15.10 Glycogenesis 431
∝ – 1, 4 – Bonding
9
13
1 5 10
Main branch
Branching ∝ – 1, 6 – Bonding
by
Glucosyl Side Chain
∝ – 4 – 6 – Transferase 18
Enzyme
Elongation [Glycogen synthase]
Branching
Continues
Glycogen
–– First glucose residue from UDP-G is transferred to nonreducing end of glyco-

gen primer and forms alpha-1,4-glycosidic bond. UDP is released for its
reuse.
Elongation of glycogen chain
• Glycogen chain elongates by successive addition of glucose residues at nonre-

ducing end of glycogen primer through formation of alpha-1,4-glycosidic
bonds.
• Glycogen synthase is the key regulatory enzyme that controls elongation of gly-
cogen chain as in Fig. 15.8a, b.
• Glycogen synthase
–– It is a glycosyltransferase enzyme.
–– It catalyzes transfer of glucose moiety from UDP-G to glycogen chain by

formation of alpha-1,4-glycosidic bond.
–– Glycogen synthase is unable to initiate formation of glycogen chain. It
requires a glycogen primer to accept glucose moieties from UDP-G and start
de novo synthesis of glycogen chain.
–– Glucose-6-P acts as an allosteric stimulator of glycogen synthase enzyme.
Branching of glycogen chain
• After the elongation of glycogen chain up to at least 11 glucose residues, branch-

ing of glycogen chain takes place by action of branching enzyme. It is called as
amylo-1,4 → 1,6-transglucosidase enzyme.
• Branching enzyme transfers six glucose residues from alpha-1,4 chain to another
chain by formation of alpha-1,6-glycosidic bond. This short chain of six glucose
residues serves as a branch on another chain. The growth of branch chain contin-
ues by addition of glucose residues by alpha-1,4 bonding.
• Glycogen synthase and branching enzymes act together in the synthesis of gly-
cogen molecule as in Fig. 15.8a, b.
Regulation of Glycogenesis
Glycogen synthase
• Glycogen synthase is the key regulatory enzyme in the synthesis of glycogen. It

exists in active glycogen synthase and inactive glycogen synthase in tissues. The
active GS promotes glycogenesis and inactive GS inhibits glycogenesis.
• These two forms are interconvertible through action of cyclic AMP.
• Active GS is changed into inactive GS by phosphorylation, while inactive GS is
converted into active GS by dephosphorylation.
Protein kinase
• This enzyme is found in cytosol of cells. Protein kinase activity is dependent on

cyclic AMP. The enzyme has two regulatory subunits (R2) and another two cata-
lytic subunits (C2) and is designated as (R2 C2).
• An increase in concentration of cyclic AMP in cytosol leads to detachment of
two regulatory subunits from catalytic subunits, and protein kinase is activated.
• Activated protein kinase catalyzes phosphorylation of glycogen synthase and con-
verts active form into inactive form of enzyme. This process inhibits glycogenesis.
Insulin
• Insulin promotes hydrolysis of cyclic AMP by phosphodiesterase enzyme and

converts it into AMP. Insulin decreases concentration of cAMP in cytosol.
15.11 Inherited Disorders of Glycogen Metabolism 433
• Insulin promotes conversion of inactive GS into active form and stimulates gly-
cogenesis in skeletal tissues and liver cells.
Glucose and glycogen concentration
• Increased concentration of glucose in cells promotes glycogenesis through posi-

tive feedback mechanism.
• Increased concentration of glycogen in cells inhibits glycogenesis by negative
feedback mechanism.
Glucocorticoid hormones
• Glucocorticoid hormones increase synthesis of glycogen synthase in the liver.

These hormones stimulate glycogenesis in liver cells.
15.11 Inherited Disorders of Glycogen Metabolism
Definition
A group of disorders related to glycogen metabolism which are inherited from
parents to offsprings are called as glycogen storage diseases.
Types of Glycogen Storage Diseases (GSD)

They are classified into six types as follows:
• Type I glycogen storage disease

–– This type of disorder is called as Von Gierke’s disease.
–– It is attributed to deficiency of glucose-6-phosphatase enzyme in hepato-
cytes and intestinal mucosa cells.
–– The disorder is an autosomal recessive trait.
Clinical manifestations
Hepatocytes, renal tubules, and intestinal mucosal cells contain large amount
of glycogen. It is not converted into glucose due to deficiency of glucose-6-
phosphatase. It led to hypoglycemia.
Excessive oxidation of fats leads to ketosis and fatty liver.
Children suffer from stunted growth.
Prognosis is poor. Children die in younger age.
• Type II glycogen storage disease
–– It is called as Pompe’s disease.
–– Disorder is due to deficiency of maltase enzyme.
–– It is an autosomal recessive trait.
Excessive amount of glycogen is stored in the liver, cardiac muscle fibers,
skeletal muscles, and smooth muscle fibers.
Enlargement of heart (cardiomyopathy).

Prognosis is poor. Affected children die in age of 9 months to 2 years due to
cardiac failure.
• Type III glycogen storage disease
–– This disorder is called as Forbes’ disease.
–– It is due to deficiency of debranching enzyme.
Glycogen cannot be completely degraded due to deficiency of debranching
enzyme.
Excessive accumulation of limit dextran (altered structure of storage gly-
cogen) is found in the liver, heart, and skeletal muscles. Disorder is also called
as limit dextrinosis.
Enlargement of liver (hepatomegaly).
Weakness and wasting of muscles (myopathy).
Prognosis is average. Affected individuals can survive to adulthood.
• Type IV glycogen storage disease
–– Disorder is called as Andersen’s disease.
–– It is due to deficiency of branching enzyme.
Excessive accumulation of unbranched glycogen chains (abnormal glycogen)
is found in the liver, heart, kidneys, and skeletal muscles. This abnormal gly-
cogen resembles amylopectin, and disorder is also called as
amylopectinosis.
Enlargement of the liver, spleen, and heart is commonly observed in affected
individuals.
Cirrhosis of the liver and heart failure are the commonest consequences in
sufferers.
Prognosis is poor. Affected children survive 3–4 years of life.
• Type V glycogen storage disease
–– Disorder is called as McArdle’s disease.
–– It is due to deficiency of phosphorylase enzyme in skeletal muscles.
Excessive accumulation of glycogen is observed in skeletal muscles.
Weakness of muscles, muscle cramps.
Prognosis is average.
• Type VI glycogen storage disease
–– Disorder is called as Hers’ disease.
–– It is due to deficiency of phosphorylase enzyme in the liver.
Excessive glycogen is stored in the liver.
Enlargement of liver.
Prognosis is average.
15.12 Disorders of Carbohydrate Metabolism 435
15.12 Disorders of Carbohydrate Metabolism
15.12.1 Diabetes Mellitus
Definition
Diabetes mellitus is an endocrine disorder associated with relative or absolute
deficiency of insulin.
It is a clinical condition characterized by increased blood glucose level. Today,
diabetes mellitus has emerged as the major cause of morbidity among young and
old-aged population alike. This condition is the main predisposing factor to diabetic
retinopathy (injury to retina), atherosclerosis (thickening of blood vessels), coro-
nary artery disease, cerebral stroke, and chronic renal failure.
15.12.2 Types of Diabetes Mellitus
I nsulin-Dependent Diabetes Mellitus (IDDM)

It is also called as type I diabetes mellitus or juvenile onset diabetes mellitus.
Occurrence
IDDM occurs early in life. It appears in adolescent age group. Both children and
adults can be affected by the disease. Patients need administration of insulin. These
individuals have high tendency to develop ketoacidosis.
Prevalence
IDDM affects 5–10% of diabetic young population.
Etiopathogenesis
IDDM is characterized by decreased synthesis of insulin by beta cells of islets of
Langerhans in the pancreas. Insulin plasma level is decreased.
• T-cell-mediated autoimmunity
–– Majority of the cases of IDDM are caused by autoimmunity against beta
cells. The T cells destroy beta cells of the pancreas. The affected person has
to rely upon insulin throughout their life owing to deficiency of insulin
production.
–– Heredity is also implicated in the pathogenesis of IDDM. Genetically prone
individuals have higher tendency to develop diabetes mellitus than normal
persons. Viral infection, obesity, and fat-rich diet become the predisposing
factors in the onset of diabetes mellitus in genetically susceptible persons.
• Idiopathic
–– Idiopathic type I diabetes mellitus has not ascribed any specific cause for its
pathogenesis. It occurs spontaneously in predisposed individuals.
–– Its prevalence is high among male African-American population. It has also
been observed in other ethnic population.
–– Patients with idiopathic type I diabetes develop ketoacidosis rapidly.

Individual characteristics appear similar to patients of type II diabetes.
–– Idiopathic type I diabetes patients do not show autoimmune markers in blood
circulation.
on-insulin-Dependent Diabetes Mellitus (NIDDM)

N
It is also called as type II diabetes mellitus or maturity onset diabetes mellitus.
Occurrence
It appears late in life. It affects persons in middle age group and generally above
40 year of age. These individuals have less tendency to develop ketoacidosis.
Prevalence
NIDDM is prevalent in nearly 90% of diabetic population.
Etiopathogenesis
• NIDDM is characterized by nonresponsiveness of body tissues to insulin. It

is called as insulin resistance. As a compensatory mechanism, pancreas
secretes more amount of insulin to regulate blood glucose level. This con-
dition is called as hyperinsulinemia. It is associated with type II diabetes
mellitus.
• Insulin resistance is responsible for reduced glucose uptake and its utilization by
peripheral body tissues.
• There is manifestation of increase in blood glucose level (hyperglycemia).
• Predisposing factors include central obesity (↑ waist to hip ratio), alcohol con-
sumption, sedentary lifestyle, stress, and intake of high fat and carbohydrate
diet.
estational Diabetes Mellitus

G
Gestational diabetes mellitus appears during pregnancy. It affects nearly 5–10% of
pregnant women. It is associated with decreased insulin secretion in pregnancy.
The condition disappears after termination of pregnancy.
Clinical Manifestation of Diabetes Mellitus

Hyperglycemia
• Blood glucose level is increased. It is a characteristic manifestation of diabetes

mellitus.
Glycosuria
• It is the excretion of glucose in urine. Glucose is excreted by tubules when blood

glucose level is higher than renal threshold for glucose (160–180 mg/100 ml).
Polyuria
• It is the passage of large amount of urine by individual (>2 L/day). It is another

cardinal sign of diabetes mellitus.
• Frequency of micturition is also increased.
Polydipsia
• It is the excessive thirst in individuals who suffer from diabetes mellitus and
diabetes insipidus. It is followed by intake of large amount of water.
Polyphagia
• It is the increased appetite in individuals who suffer from diabetes mellitus. It is

associated with intake of large amount of foods repeatedly. Despite polyphagia,
there is weight loss in affected individuals. It is owed to impairment in glucose
utilization by body tissues.
Asthenia
• Physical weakness, lethargy, and inability to perform routine activities are com-
mon symptoms of diabetic patients.
Recurrent infection
• Owing to poor nutritional status of diabetic patients, immunity is compromised.

It leads to recurrent viral and bacterial infections in the body. Skin infections like
boils, diabetic foot, and infection of upper respiratory tract are common infec-
tions. These patients are predisposed to tuberculosis.
• Polyuria, polydipsia, and polyphagia are cardinal signs of diabetes mellitus
15.12.3 Laboratory Investigation of Diabetes Mellitus
stimation of Blood Glucose Level

E
• Blood glucose estimation is an important test to confirm diagnosis of diabetes
mellitus.
• According to the WHO diabetes diagnostic criteria:
• Normal fasting blood glucose level should be <100 mg/dl.
• Normal post-prandial (2 h after food intake) blood glucose level should be
<140 mg/dl.
• Impaired fasting blood glucose level should be 110–125 mg/dl.
• Impaired post-prandial blood glucose level should be ≥140 mg/dl.
lucose Tolerance Test

G
It determines the ability of body tissues to utilize carbohydrate after an intake of a
given amount of glucose.
Types of Glucose Tolerance Test
Oral Glucose Tolerance Test

Preparation of Patient
• Patient is advised to abstain from eating or drinking at least 8–12 h before test.
• Patient should be alert physically and mentally.
Procedure of Test
• A baseline fasting blood sample is collected.

• Patient is asked to drink a solution of 75 g of glucose (recommended by WHO
for adults) dissolved in 250 ml of water within 5 min.
• After every 30 min, five blood samples are collected.
• All six samples are estimated to determine blood glucose level. A graph is plot-
ted for six values of blood glucose concentration against time and it is called
glucose tolerance curve.
Interpretation
Normal Glucose Tolerance Curve
Characteristics
• Fasting blood glucose level should be < 110 mg/dl.

• At 1 h period, blood glucose level should be < 180 mg/dl. It is the highest peak
of blood glucose concentration. It should not exceed the renal threshold for
glucose.
• At 2.5 h period, fasting blood glucose level (<110 mg/dl) should be obtained.
Diabetic Glucose Tolerance Curve
• Fasting blood glucose level is elevated (>110 mg/dl). Fasting glucose level
between 110 and 125 mg/dl indicates borderline impaired glucose tolerance.
• At 1 h period, blood glucose level rises >180 mg/dl. The highest peak of blood
glucose concentration is obtained after 1 h.
• At 2.5 h period, fasting blood glucose level is not obtained. It confirms
hyperglycemia.
Intravenous Glucose Tolerance Test

It is indicated in condition of malabsorption of glucose from alimentary canal.
Indications
• Coeliac disease
• Environmental enteropathy
• Hypothyroidism
Procedure
• A dose of 3 g/kg of weight of glucose is administered intravenously in 50% solu-

tion in 5 min.
• Baseline blood sample and half hourly five blood samples are taken.
Interpretation of glucose tolerance curve is the same as in OGTT.
stimation of Glycated Hemoglobin (HbA1C)

E
Glycated hemoglobin (HbA1C) offers the best test for monitoring long-term
blood glucose level in diabetic patients. It does not help to diagnose diabetes
mellitus. It provides a blood glucose level of previous 3 months.
Interpretation of Glycated Hemoglobin Test

WHO diabetes diagnostic criteria
• HbA1C value < 6% is indicative of very good blood glucose control.

• HbA1C value < 7% is indicative of adequate blood glucose control, and
WHO has recommended a cutoff (6.5%) for diagnosis of diabetes mellitus.
• HbA1C value ≥ 8% is indicative of poor blood glucose control.
Glycation
• It is nonenzymatic attachment of glucose with protein.

• It occurs between free glucose moiety and polypeptide chain of hemoglobin in
blood circulation. Reducing end of glucose attaches covalently to N-terminal of
amino acid in chain.
• It is not physiological process. Glycated Hb predisposes to generation of higher
amount of free radicals within RBCs.
• It serves as a biomarker to determine level of blood glucose over an extended
period of 2–3 months in diabetic patients.
Glycosylation
• It is an enzymatic addition of carbohydrate moiety to a specific region of

polypeptide chain.
• It occurs as posttranslation modification.
• Glycosylation is necessary for rendering structural integrity and functional
patency to protein molecules.
Suggested Readings
Bender DA (2004) Introduction to nutrition and metabolism, 3rd edn. Taylor & Francis Group,
Philadelphia
Nelson DL, Cox MM (2004) Principles of biochemistry, 4th edn. W.H. Freeman and Company,
New York
Rosenthal MD, Glew RH (2009) Medical biochemistry – human metabolism in health and disease.
Wiley, Hoboken, NJ
Digestion and Absorption of Lipids
16
Lipids are important source of energy for living organisms. A healthy adult person
requires around 2800 calories per day. It is recommended that around 20–35% of
daily calories should be furnished by dietary lipids. Calorific value of fats is (9
calories/g). An adult person should consume around 60–90 g of fats per day. Intake
of calories from saturated fatty acids and trans fatty acids should be <10% and 2%
of total calories, respectively, per day.
Plant Sources of Lipids
• Vegetable oils like cottonseed oil, sunflower oils, mustard oil, rapeseed oil,
canola oil, and soya oil.
• Vegetable sources of lipids are rich in MUFA and PUFA.
• They are superior in quality to animal sources of lipids.
Animal Sources of Lipids
• Milk, butter, eggs, meat, cod liver oil, and pork.
16.1.1 Digestion of Triglycerides
Human diet contains a variety of lipids. Triglycerides constitute around 90–95% of

the total dietary lipids. They are the predominant lipids that are stored in body tis-
sues. Triglycerides serve as stored form of lipids.

https://doi.org/10.1007/978-981-13-1035-5_16
442 16 Digestion and Absorption of Lipids
igestion in Oral Cavity

D
• Saliva contains lingual lipase. It is secreted by “Von Ebner’s glands” located
on dorsal surface of the tongue.
• The optimum pH for lingual lipase is between 4.0 and 4.5.
• Milk fat (butter) contains butyric acid (C4) fatty acids. Lingual lipase digests it
into glycerol and fatty acid.
• Lingual lipase hydrolyzes milk fats in the absence of bile salts.
• Enzymatic activity of lingual lipase continues in the stomach.
• It digests about 20–30% of dietary triglyceride (short chain fatty acids) into
monoglyceride, diglyceride, and fatty acid. Milk fat (butter) contains butyric acid
(C4). Lingual lipase favorably digests milk fat.
Lingual Lipase
Milk fat (Butter) Diglyceride + Monoglyceride + Butyric acid
Optimum pH 4.0
Digestion in the Stomach
• Gastric juice contains “gastric lipase.” It is secreted by chief cells of gastric

mucosa in fundus of the stomach.
• The optimum pH for gastric lipase is between 3.0 and 6.5. This is a weak lipo-
lytic enzyme. It is due to high pH (1.5–2.5) of the stomach.
• Gastric lipase hydrolyzes lipids containing short-chain fatty acids like milk fat
and egg yolk.
Lingual lipase and gastric lipase are acidic lipases which exhibit catalytic activ-
ity at acidic pH, in the absence of bile salts, and colipase.
• Fats retard the movement of food from the stomach to duodenum. This condition
is called as “gastroparesis” or “delayed gastric emptying.”
igestion in the Small Intestine

D
• Intestinal juice contains bile salts, pancreatic lipase, and intestinal lipase.
• Bile salts are secreted by the liver.
• Role of Bile Salts
–– Bile salts are sodium taurocholate and sodium glycocholate secreted in
bile.
–– Lipids are insoluble in aqueous medium in the intestinal lumen. But alkaline lipase
is a water-soluble enzyme. This difficulty is solved by emulsification of fats.
–– Bile salts lower the interfacial tension. It helps in emulsification of fats in
duodenum.
–– “Emulsification of fats” – Immiscible large fat drops are fragmented into
minute fat droplets in the duodenum. Fat droplets are suspended in aqueous
medium in duodenum. This process is called as emulsification.
–– Therefore, bile salts act as a “hydrotrope.”
16.1 Dietary Sources 443
Digestion of lipids
–
1CH –O–C– R1 1CH
2 –OH
o 2
o
–
–
R1
–
–
2CH–O–C– R2 2CH–O–C– R2
Lipase o +
–
–
o
–
–O–C– R3
–
3CH COOH
–
3CH –O–C– R3 H 2O 2
2
Triacylglycerol 2,3–Diacylglycerol
Free fatty
acid
Lipase
H2O
o
CH2–OH
–
CH2OH CH2–O–C– R2 o
Lipase Lipase
–
– –
CHOH
–
–
CHOH H2O CH2–O–C– R2 + R3
CH2OH
–
–
–
CH2OH CH2OH COOH
Glycerol 1–Monoacyl 2–Monoacyl Free fatty
+ glycerol glycerol acid (FFA)
R2 – COOH
FFA
Fig. 16.1 Hydrolysis of trialcylglycerol
–– Surface area of a fat drop is between 1 and 3 μm. After emulsification, it increases
around a thousand times. Lipase gets a larger surface area for its activity.
–– Bile salts help to attach a molecule of pancreatic lipase with two mole-
cules of colipase.
• Role of Pancreatic Lipase
–– Pancreatic lipase (steapsin) is secreted by the pancreas. It is the important
alkaline lipase for hydrolysis of triglycerides as in Fig. 16.1.
–– The optimum pH for pancreatic lipase is between 7.4 and 8.5.
–– This enzyme hydrolyzes ester bonds in triglycerides.
–– Cleavages ester bond at position-α – Initially, it cleavages ester bond at
position-α and liberates α-fatty acid. Triglyceride is converted into
α’,β-diglyceride.
–– Cleavages ester bond at position-α – Later on, it cleavages ester bond at
position-α. This action converts α’, β-diglyceride into β-monoglyceride.
–– Isomerization of β-monoglyceride into α-monoglyceride – The compound
β-monoglyceride offers resistance to hydrolysis by pancreatic lipase. It is
isomerized into α-monoglyceride by isomerase enzyme. It is digested into
glycerol and fatty acid.
• Role of Colipase
–– It is a coenzyme for lipase. It is secreted from the pancreas in a procolipase
form. It is activated by trypsin.
–– Colipase is required for optimum enzymatic activity of pancreatic lipase.
• Role of Calcium Ions

–– Calcium ions precipitate free fatty acids into calcium soap. They increase
lipase activity.
16.1.2 Digestion of Cholesteryl Ester
• Cholesterol is present in diet in free state and bound state as cholesteryl ester
(10–15%). The liver also secretes free cholesterol in bile.
• Pancreatic and intestinal juices contain “cholesterol esterase.” It is a carboxyl
ester hydrolytic enzyme.
• Cholesterol esterase is a non-specific ester-splitting enzyme. It can cleavage ester
linkage at all three, namely, α, β, and α’ positions. It can digest triglycerides,
cholesteryl ester, and phospholipids.
• Its activity is enhanced by the presence of bile salts. The enzyme cleavages ester
bond in cholesteryl ester. It liberates cholesterol and free fatty acids.
16.1.3 Digestion of Phospholipids
• Pancreatic juice contains phospholipase A2.

• This enzyme exists in inactive form (zymogen) in pancreatic juice. It is acti-
vated by trypsin.
• Activated phospholipase A2 cleavages ester bond in phospholipids (lecithin).
• This enzyme converts phospholipid into free fatty acids and lysophospholipid
(lysolecithin).
Digestion End Products of Dietary Lipids
• About 70–75% diglycerides + monoglycerides

• About 20–25% glycerol + free fatty acids
• Free cholesterol
• Lysophospholipids
• Phospholipids
16.2 Absorption of Lipids
Absorption of digestion end products of lipids occurs in three stages. They are as
follows:
1. Luminal Stage
In this stage, digestion products of lipids exist in emulsified form in the lumen of
the intestine. They migrate toward brush border surface of enterocytes.
2. Cellular Stage
In this stage, digestion products of lipids pass through the membrane of enterocytes.
16.2 Absorption of Lipids 445
3. Transportation Stage
In this stage, lipids enter the lymph vessels and blood circulation.
Many theories have been proposed to explain the absorption of digestion end
products of lipids. Broadly, theories fall into two categories as complete hydrolysis
theory and partial hydrolysis theory of lipid absorption.
16.2.1 Lipolytic Theory
This theory was proposed by Pfluger, Verzer, and Mcdougall. Dietary lipids are
completely hydrolyzed into glycerol and free fatty acids. The free fatty acids are
precipitated into soaps. Water-soluble soaps can easily enter enterocytes.
16.2.2 Frazer’s Partition Theory
According to Frazer’s partition theory, dietary lipids are partially hydrolyzed into
monoglycerides, diglycerides, and free fatty acids. They form emulsion in the pres-
ence of bile salts. They can enter enterocytes.
16.2.3 Bergstrom Theory
It was proposed by Bergstrom in 1982. This latest theory explains the absorption of
digestion products of lipids. The theory has the following postulates:
1. Unstirred Water Layer

• The brush border surface of intestinal epithelial cells (enterocytes) is covered
by a thick layer of water in the lumen of the intestine. This water layer is
called as “unstirred water layer” as in Fig. 16.4. This water layer is not
mixed with the fluid content of the rest of the intestinal lumen.
• Digestion products of lipids should cross this layer to reach brush border
surface of enterocytes.
2. Formation of Mixed Micelle
• “Hydrophilic Colloidal Aggregates” are called as micelle.
• Micelles are two types as “pure micelles” which are made up of bile salts only
and “mixed micelles” which contain bile salts and digestion end products as
in Figs. 16.2 and 16.3.
• Micelles have spherical shape with size between 3 and 6 nm. They are much
smaller than the size of fat drops which have the size between 1000 and
3000 nm.
• Formation of micelle starts when the concentration of bile salts in intestinal
lumen exceeds “critical micellar concentration” (CMC). It is the concentra-
tion of bile salts in lumen of the intestine above which formation of micelles
starts.
Bile salts
Aquous medium
Hydrophilic head Phospholipid

Hydrophobic tail molecule
Fig. 16.2 Micelle
Mixed micelle
Phospholipid
Monoglyceride
Long chain
Aquous
Fatty acid
medium
Bile salt
Cholesterol
Fig. 16.3 Mixed micelle
• Mixed micelle is composed of monoglycerides, diglycerides, cholesterol,

and long-chain fatty acids along with bile salts, calcium salts of fatty
acids, and bicarbonates as in Fig. 16.3.
• Mixed micelle has an external surface and internal core. External surface of mixed
micelle is made up of polar and hydrophilic heads of bile salts, and internal core
is made up of nonpolar and hydrophobic tails of bile salts. A mixed micelle con-
tains around 20–45 bile salt molecules. The external surface of micelle is soluble
in the fluid present in the lumen of the intestine. Its internal core is soluble in
monoglycerides, diglycerides, cholesterol, and long-chain fatty acids.
3 . Passive Diffusion of Digestion Products of Lipid
• Hydrophilic surfaces of micelles can easily pass through unstirred water
layer. They carry digested lipids to brush border surface of enterocytes. This
is the site of lipid absorption.
• Micelles align along the brush border surface of enterocytes. Digestion prod-
ucts of lipid are absorbed by passive diffusion through brush border surface of
enterocytes.
• Bile salts are dissociated from micelles. They are reabsorbed in the ileum
and enter hepatic portal vein to reach the liver. Again, bile salts are
secreted in bile and reach the duodenum. This is called enterohepatic
circulation as in Fig. 16.4.
Fat Drop
Bile sacts
Liver
Unstirred Micelle
wate layer
Lipase
He
Hepatic
epatic
Enterocyte
portal
SCFA
vein
LCFA
Smooth endoplasmic reticulum
Resynthesis
Mono
T.G.
glyceride
Glycerol
Chylo
Blood Micelle
microns
vessel
Bile Apical
Lacteal Basolateral salts membrane
Nucleus
enterohepatic membrane
Reabsorbed
circulation
in ileum Brush border
surface
SCFA - Short chain fatty acid - L.C.F.Acid

LCFA - Long chain fatty acid - Cholesterol
- Bile salt - Glycerol
- Phospholipid
Fig. 16.4 Absorption of lipids in entreocyte

4. Resynthesis of Triglyceride, Cholesterol Ester, and Phospholipid Inside

Enterocytes
• Digestion products of lipid are transported to smooth endoplasmic reticulum
inside the enterocytes as in Fig. 16.4.
• Within smooth endoplasmic reticulum, long-chain fatty acids are activated
into fatty acyl CoA by acyl CoA synthetase enzyme. Two molecules of fatty
acyl CoA combine with monoglyceride and form triglyceride. This is called
as “monoacylglycerol pathway.”
• Lysophospholipid combines with long-chain fatty acid and is reconverted into
phospholipid by acyltransferase enzyme. Free cholesterol is again re-esterified
into cholesterol ester by cholesterol acyltransferase enzyme.
5. Formation of Chylomicrons
• Chylomicrons are triglyceride-rich endogenous hydrophilic lipoprotein
particles. The word is derived from the Greek words “chylos” (juice or milky
fluid) and “micron” (very small particle).
• Triglycerides, cholesterol ester, and phospholipids aggregate together in the
presence of apolipoprotein in smooth endoplasmic reticulum to form
chylomicrons.
• Chylomicron has a central core and an external surface. The central core
contains nonpolar triglycerides and cholesterol ester molecules. They are cov-
ered by a single layer of polar phospholipid and cholesterol molecules. This
monolayer of phospholipids is associated with integral proteins called apoli-
poprotein B48.
• The size of the chylomicrons is between 30 and 60 nm. Chylomicron contains
around 88% triglycerides, 7% phospholipids, 3% cholesterol, and 2%
apo-B48.
• Chylomicrons are released into cisternae of Golgi bodies. They undergo mod-
ification by addition of apolipoprotein-A1 and glycosylation of apolipoporo-
tein-B48. Chylomicrons are budded from Golgi bodies into cytosol of
enterocytes.
6. Transport of Chylomicrons
• From inside the enterocytes, chylomicrons are exported from the basolateral
surface of enterocytes by exocytosis. They move through interstitial spaces
enterocytes. Chylomicrons enter into lacteals present in intestinal villi.
• From lacteals, chylomicrons pass into lymph vessels.
• From lymph vessels, chylomicrons enter into the thoracic duct (also called as
left lymphatic duct, which arises at 12th thoracic vertebrae and drains lymph
and chyle (digested fats)).
• From the thoracic duct, chylomicrons pass into systemic circulation at the
level of the left subclavian vein. These chylomicrons are called as “nascent
chylomicrons.”
7. Fate of Chylomicrons
• Nascent Chylomicrons. They enter the blood circulation and called as circu-
lating chylomicrons.
• Circulating Chylomicrons. They undergo delipidation in the blood circula-

tion. Lipoprotein lipase (LPL) brings about hydrolysis of triglycerides in chy-
lomicrons. The LPL is an enzyme located on the surface of endothelial cells
in capillaries. The released fatty acids are distributed to adipose tissues and
skeletal muscles where they are stored.
• Chylomicron Remnants. The remaining particles receive apolipoprotein
C-II and apolipoprotein E. The resultant particles are called as chylomicron
remnants.
• After a fat-rich diet, the concentration of chylomicrons in blood rises. The
plasma appears milky white due to chylomicrons.
• Chylomicron remnants are taken up by the liver from circulation. The triglyc-
erides of chylomicrons are hydrolyzed in the liver, and they are called as
“very low density lipoprotein (VLDL).” They are exported from the liver and
undergo delipidation by vascular lipoprotein lipase in circulation.
• VLDL are converted into LDL as in Fig. 16.4.
Absorption of SCF and MCF
• Length of fatty acid chain determines the absorption of fatty acids in the small
intestine.
• Short-chain fatty acids (<6C) and medium-chain fatty acids (6–12C) enter intes-
tinal epithelial cells (enterocytes) from lumen of the intestine.
• From the inside of the enterocytes, they directly enter the hepatic portal vein and
reach the liver.
• SCF and MCF are metabolized in liver cells. They are not stored in adipose
tissues.
• Absorption of SCF and MCF is independent of bile salts and micelles.
Absorption of Glycerol
• Glycerol is absorbed from the lumen of the small intestine. It passes into the
hepatic portal vein and enters the liver as in Fig. 16.4.
• Glycerol is converted into glycerol-3-phosphate in the liver. It can either enter
into glycolysis or is utilized in gluconeogenesis.
Absorption of Cholesterol
• Cholesterol in free form is absorbed from lumen of the intestine. It enters entero-
cytes and undergoes esterification to form cholesterol ester.
• It is incorporated into chylomicrons and enters lacteals.
Absorption of Phospholipids
• Phospholipids are absorbed from lumen of the intestine as lysophospholipids.

They enter enterocytes and are restructured as phospholipids. They are incorpo-
rated into chylomicrons and are exported from enterocytes.
• Small amount of phospholipids are also absorbed from lumen of the intestine due
to their amphipathic nature.
1. Steatorrhoea
• It is a clinical condition characterized by excessive amount of fats in feces.
• Steatorrhoea is due to obstruction in the flow of bile, malignancy of pan-
creas, celiac disease, or tropical sprue (malabsorption syndrome in tropical
regions).
• Inadequate bile salts or pancreatic lipase results in impaired digestion of
dietary fats. They remain unabsorbed and are expelled from the intestine
along with stool.
2. Chyluria
• It is a clinical condition characterized by excessive fats in the urine. It has a
milky appearance.
• The condition is due to an abnormal passage between the urinary tract and
lymphatic system of the intestine. This is called as “chylous fistula.”
Suggested Readings
Bender DA (2004) Introduction to nutrition and metabolism, 3rd edn. Taylor & Francis, Philadelphia
Ching K (2008) Fatty acids in foods and their health implication, 3rd edn. CRC Press, Boca Raton
Hamosh M (1990) Lingual and gastric lipases. Nutrition 6(6):421–428 [Abstract]
Harper HA (1979) Review of physiological chemistry, 17th edn. Lange medical publisher,
New York
Iqbal J, Hussain MM (2009) Intestinal lipid absorption. Am J Physiol Endocrinol Metab
296:E1183–E1194
Kleiner IS, Orten JM (1966) Biochemistry, 7th edn. Mosby, St Louis
Murray RK, Granner DK, Mayes PA, Rodwell VW (1999) Harper’s biochemistry. Lange Medical
Publisher, New York
Murray RK, Granner DK, Mayes PA, Rodwell VW (2003) Harper’s illustrated biochemistry, 26th
edn. Lange Medical Books, New York
Nelson DL, Cox MM (2004) Principles of biochemistry, 4th edn. W.H. Freeman and Company,
New York
Rosenthal MD, Glew RH (2009) Medical biochemistry – human metabolism in health and disease.
Wiley, Hoboken, NJ
Metabolism of Lipids
17
17.1 Introduction
Lipids are stored in adipose tissues. Lipids are main reservoir of fuel for physiologi-
cal activities of human body. Stored lipids are always in a dynamic state. Lipids are
constantly decomposed and resynthesized depending on the calorie requirement,
age, metabolism, dietary intake, gender, and diseases. Tissue lipase hydrolyzes the
tissue triglycerides under the influence of hormones. Free fatty acids are released
into blood circulation. Under normal condition, serum free fatty acid concentration
varies between 6 and 15 mg/100 ml.
Sources of Free Fatty Acids in Plasma

Many sources liberate free fatty acids into blood circulation. These sources are as
follows:
Mobilization of Tissue Fat
• Tissue fat from adipose tissue constitutes the richest source of free fatty acids.
Tissue lipase degrades tissue fat and releases fatty acids.
Chylomicrons and Very Low Density Lipoprotein
• Chylomicrons and VLDL in blood circulation are decomposed by lipoprotein

lipase and release fatty acids.
Dietary Fats
• Triglycerides from diet are hydrolyzed by lipase in the lumen of the intestine.
Fatty acids are absorbed and enter blood circulation. Fatty acids in plasma are

https://doi.org/10.1007/978-981-13-1035-5_17
452 17 Metabolism of Lipids
distributed in bound form with albumin (albumin-FFA complex). Plasma has

nearly 30 mg/100 ml of free fatty acids in a postabsorptive state.
17.2 Oxidation of Fatty Acids
Serum plasma concentration of fatty acids varies between 10 and 25 mg/100 ml in

postabsorptive state. Free fatty acids combine with albumin as albumin-FFA com-
plex. Fatty acids are distributed to body tissues in complexed state by blood
circulation.
Fatty acids have a high turnover rate. They have very short half-life of around
1–3 min. Their uptake by body tissues is rapid, and fatty acids are actively oxidized
within tissues. Hormones regulate plasma fatty acid concentration.
17.2.1 Types of Fatty Acids Oxidation
Alpha Oxidation
Definition
• Alpha oxidation of long-chain fatty acids is the oxidation at alpha carbon atom
of fatty acid that results in the removal of one carbon atom (carbon atom next to
carboxylic group called as alpha carbon) from fatty acid.
Site of Occurrence
• Alpha oxidation of long-chain fatty acids takes place in brain cells.
Mechanism
• It is the main mechanism for oxidation of phytanic acid. It is found in milk

and milk products and animal fat. Phytanic acid is also formed from plant-based
food sources. Chlorophyll in plants is hydrolyzed into phytol in human body.
Phytol is oxidized into phytanic acid.
• Long-chain fatty acid undergoes hydroxylation by alpha-hydroxylase enzyme to
form 2-hydroxy fatty acid. It undergoes oxidative decarboxylation to form long-
chain fatty acid with one carbon atom shorter than parental fatty acid.
• Reactions occur in the presence of molecular oxygen and NADPH2.
Significance
• Alpha oxidation in brain tissues results in formation of hydroxy fatty acids like
cerebronic acid. It is structural component of brain cerebrosides.
• It serves to synthesize odd-chain fatty acids in brain tissues. They are essential
for synthesis of sphingomyelin.
17.2 Oxidation of Fatty Acids 453
Omega Oxidation
Definition
• It is the oxidation of medium-chain fatty acid at omega carbon atom (last carbon
atom of methyl group in hydrophobic side chain distant from COOH group).
Site of Occurrence
• Omega oxidation occurs in smooth endoplasmic reticulum in the liver and

kidneys.
• It also occurs in bacteria. It is an alternate metabolic pathway to beta oxidation.
Mechanism
• Omega oxidation of fatty acid was proposed by Verkade and colleagues.

• Medium-chain fatty acid undergoes hydroxylation at omega carbon atom to form
omega hydroxyl fatty acid. Reaction is catalyzed by omega hydroxylase enzyme.
• Oxidation of hydroxyl group at omega carbon atom takes place by alcohol dehy-
drogenase enzyme in the presence of NAD+ coenzyme which forms aldehyde
which again undergoes oxidation to form carboxylic group. Medium-chain fatty
acid is converted into dicarboxylic acid.
• Dicarboxylic acid can enter beta oxidation at both ends.
Beta Oxidation
Definition
Beta oxidation of fatty acid is the biochemical process involving oxidation at beta
carbon atom (second carbon atom from carboxylic group) of fatty acid chain that
results in cleavage of two carbon residue (acetyl CoA) from fatty acid chain.
Historical Aspect
• Beta oxidation of fatty acids was proposed by Franz Knoop in 1904.

• Knoop attached phenyl residue to omega carbon of fatty acids. Dogs were fed
diet rich in such fatty acids. He observed phenyl derivative of fatty acids in the
urine of dogs.
• Knoop fed diet rich in phenyl propionate (odd carbon atoms) to dogs and detected
excretion of hippuric acid (benzoic acid conjugation with glycine) in urine.
While, he fed diet rich in phenylbutyrate (even carbon atoms) to dogs and
detected excretion of phenylacetate in urine.
• On the basis of above observations, Knoop proposed beta oxidation of fatty
acids.
Site of Occurrence
• Fatty acids undergo beta oxidation in the liver, kidneys, cardiac muscles, adipose
tissues, lungs, and gonads.
• Brain tissues, RBCs, and adrenal medulla cannot oxidize fatty acids.
Enzymes
• Initial step in beta oxidation occurs in cytosol as the enzymes are located in
cytosol.
• Beta oxidation proper occurs in mitochondrial matrix as enzymes are located in
mitochondrial matrix.
Steps in β-Oxidation of Fatty Acids

Beta oxidation of fatty acids is carried in three distinct steps:
1. Activation of Fatty Acids (Cytosol)

2. Transport of Activated Fatty Acids into Mitochondria
3. Beta Oxidation Proper (Mitochondria)
Activation of Fatty Acids

Activation of fatty acids involves two biochemical reactions in stages. Process
requires a molecule of ATP, coenzyme A, and Mg++ ions as in Fig. 17.1.
• In the first stage, fatty acid interacts with ATP to form acyladenylate.
• In the second stage, acyladenylate combines with coenzyme A and forms Acyl CoA.
• Reaction is catalyzed by Acyl CoA synthetase (thiokinase).
Activation process consumes two high-energy phosphate bonds. ATP is con-

verted into pyrophosphate which is hydrolyzed by inorganic pyrophosphatase to
form phosphate.
R–CH2–CH2–CH2–CH2– COOH Long chain fatty acid
ATP CoA∼SH
Pyrophosphatase PPI + AMP Mg++ Acyl CoA synthetase
2Pi
H2O
R–CH2–CH2–CH2–CH2– CO∼S. CoA

Acyl CoA
Fig. 17.1 Activation of fatty acid

• Activation of fatty acids occurs in cytosol.

• Activated fatty acid is called Acyl CoA.
Types of Acyl CoA Synthetases

1. Acetyl CoA Synthetase
• It is a short-chain fatty acid activation-catalyzing enzyme.
• It catalyzes (C2) acetic acid and butyric acid (C4).
2. Medium-Chain Synthetase
• It is a medium-chain fatty acid activation-catalyzing enzyme.
• It catalyzes C4–C12 fatty acids.
3. Long-Chain Synthetase
• It is a long-chain fatty acid activation-catalyzing enzyme.
• It catalyzes C12–C22 fatty acids.
Transport of Activated Fatty Acids into Mitochondria

Activated fatty acids, Acyl CoA, require transportation across mitochondrial mem-
branes for beta oxidation proper as in Fig. 17.2.
Inner membrane of mitochondria is impermeable to Acyl CoA. Its transport is
carried by a carrier molecule. It is called as carnitine.
Carnitine
• Chemically, it is β-hydroxy-γ-trimethyl aminobutyrate.

• Carnitine is found in the milk, meat, liver, skeletal muscles, and yeast.
• Carnitine is made up of two amino acids, namely, lysine and methionine.
• It is synthesized in the liver and kidneys.
Mechanism of Transportation
• Acyl CoA reacts with carnitine on the outer surface of the inner mitochondrial
membrane and forms acyl-carnitine. Reaction is catalyzed by carnitine acyltrans-
ferase I (CAT I). CAT-I is located on the outer surface of the inner mitochondrial
membrane.
• Acyl-carnitine is translocated across the inner mitochondrial membrane. It enters
the mitochondrial matrix.
• Inner surface of inner mitochondrial membrane contains carnitine acyltransfer-
ase II (CAT-II). This enzyme catalyzes acyl-carnitine into Acyl CoA and carni-
tine. This step requires coenzyme A.
• Carnitine is released which enters cytosol for reuse as a carrier.
• Two pools of coenzyme A in cells
Cytoplasm
Long chain fatty acid

CoA∼SH
ACYL-CoA
Syntetase ATP
AMP+PPI
Mg++
ACYL-CoA R–CH2–CH2–CH2–CH2–Co∼S.CoA
nd rial
ocho e
r mit n
Oute embra
m
CH2+ γ β α
H3C–N–CH2–CH–CH2–COOH
–
CH3 OH
Carnitine + ACYL-CoA ACYL carnitine
Carnitine acy transferase I

I
CAT
Cytosolic size of
inner membrane
Translocase
CAT II
Carnitine
Matrix Acyl
size of transferase Carnitine Acyl Carnitine
membrane II CAT II
Acyl CoA CoA∼SH
Mitochondria
Matrix
β−oxidation of
acyl CoA
Fig. 17.2 Transport of Acyl CoA with carnitine

Cytosol coenzyme A
–– This coenzyme is helpful in activation of fatty acids in cytosol. It is a prepara-
tory phase before transport into mitochondria.
Mitochondrial coenzyme A
–– This coenzyme is helpful in activation of fatty acids in mitochondrial matrix.
It is a preparatory phase before beta oxidation proper.
β-Oxidation Proper
It occurs in sequential cycles. Each cycle of oxidation liberates two carbon residues
from fatty acids (acetyl CoA). This phase is described in the following four steps:
1. Oxidation
Acyl CoA is oxidized into α,β-unsaturated acyl CoA. It has double bond between
α and β carbon atoms. Reaction is catalyzed by acyl CoA dehydrogenase enzyme.
Reaction requires coenzyme FAD which accepts hydrogen atoms and is reduced
into FADH2. It is transported across ETS chain to produce 2 ATP molecules.
2. Hydration
α,β-unsaturated acyl CoA undergoes hydration to form β-hydroxyacyl
CoA. Reaction is catalyzed by enoyl CoA hydratase.
3. Oxidation
Enoyl CoA hydratase undergoes oxidation to form β-keto acyl CoA. Reaction is
catalyzed by β-hydroxyacyl CoA dehydrogenase enzyme.
4. Cleavage
β-keto acyl CoA undergoes splitting to form acetyl CoA with release of new acyl
CoA which is two carbon atoms shorter than parental acyl CoA. Reaction is
catalyzed by β-keto acyl CoA thiolase.
The new acyl CoA reenters beta oxidation cycle to produce another acetyl CoA,
and the process continues till complete oxidation of participating fatty acid as in
Fig. 17.3.
Energetic in Beta Oxidation

Oxidation of Even Carbon Chain Fatty Acids
Even chain fatty acid like palmitic acid (C15H31COOH) undergoes beta oxidation
and yields the following important facts:
• Seven cycles of beta oxidation for palmitic acid

–– In each cycle, five ATP molecules are formed from oxidation of NADH2 and
FPH2 in ETC.
–– Total production is 35 ATP molecules in 7 cycles (5 × 7 cycles).
• Production of eight Acetyl CoA residues in beta oxidation of palmitic acid
–– One Acetyl CoA residue enters TCA cycle and produces 12 ATP.
–– A total of 96 ATP molecules are produced (12 × 8 Acetyl CoA residues).
γ β α O
R–CH2–CH2–CH2–CH2 C~S–CoA
Acyl – CoA
Acyl CoA
FP
Dehydrogenase
FP.H2
β α O
R–CH2–CH2–CH–CH–C~S–CoA
α, β – Unsaturated
Acyl CoA
Enoyl H 2O
CoA
hydratase
OH O
R–CH2–CH2–CH–CH2–C~S–CoA
β α
β – Hydroxy acyl CoA
β – OH – Acy NAD+
CoA
Dehydrogenase NADH+ H+
O O
R–CH2–CH2–C– CH2–C~S–CoA
β α
β – Keto acyl CoA
Thiolase CoA~SH
O O
TCA CH3–C~SO–CoA R–CH2–CH2–CH2–C~S–CoA
CYCLE
Acetyl CoA Acyl CoA
[Shorter by 2c]
CO2 H2O ATP
Fig. 17.3 β Oxidation of fatty acids
Aggregate production is 131 ATP molecules (35 + 96 ATP).

Consumption of ATP in activation of fatty acid = 2 ATP.
Net Gain = 129 ATP (131 − 2 ATP) through beta oxidation of palmitic acid.
17.3 Biosynthesis of Cholesterol 459
Oxidation of Odd Carbon Chain Fatty Acids
• Beta oxidation of odd carbon chain fatty acid occurs through similar steps as
even carbon chain fatty acids.
• In the last cycle of beta oxidation, a three-carbon residue is produced unlike a
two-carbon residue in even carbon chain fatty acids. The three-carbon residue
is called as propionyl CoA.
Fate of Propionyl CoA
• Propionyl CoA undergoes carboxylation to form D-methyl malonyl

CoA. Reaction is catalyzed by propionyl CoA carboxylase in the presence of a
molecule of CO2, ATP, and biotin as coenzyme.
• D-methyl malonyl CoA is changed into L-methyl malonyl CoA by enzyme
methyl malonyl CoA epimerase.
• L-methyl malonyl CoA is converted into succinyl CoA by enzyme methyl malo-
nyl CoA mutase in the presence of vitamin B12. In the deficiency of vitamin
B12, L-methyl malonyl CoA is converted into methyl malonic acid which accu-
mulates in body, and it is excreted in urine. Its detection in urine has a diagnostic
value in the screening of deficiency of vitamin B12.
• Succinyl CoA enters TCA cycle.
Metabolism of Cholesterol
Cholesterol is a steroid and is present in animals. It is called as animal sterol. A
healthy person has nearly 2 g of cholesterol per kg of body weight (140 g in 70 kg
weight).
17.3 Biosynthesis of Cholesterol
Site of Occurrence
• Cholesterol is synthesized in most of the body tissues.

• The liver, adrenal cortex, gonads, and skin are actively involved in the synthesis
of cholesterol.
• Brain tissues of infants synthesize cholesterol, while, in adults, synthesis of cho-
lesterol is absent in brain tissues.
Enzymes
• Enzymes for cholesterol biosynthesis are located in cytosol and microsomes.

17.3.1 Steps in Biosynthesis
Cholesterol biosynthesis can be explained in the following steps:
1. Formation of HMG CoA

• Acetyl CoA (CH3COS.CoA) is the precursor of cholesterol. The Acetate
(CH3COO) from acetyl COA constitute carbon skeleton of cholesterol (27C).
• Two molecules of acetyl CoA (2C) condense together to form acetoacetate
(4C). It further condenses with one molecule of acetyl CoA to form β-hydroxy
β-methylglutaryl CoA (6C).
• Reaction is catalyzed by HMG CoA synthase (cytosolic enzyme) as in
Fig. 17.4.
2. Formation of Mevalonate
• β-Hydroxy β-methylglutaryl CoA (6C) is converted into mevalonate (6C).
• Reaction is catalyzed by HMG CoA Reductase. It is a regulatory enzyme
and it is a rate-limiting step in cholesterol biosynthesis. Enzyme is present
in endoplasmic reticulum.
• NADPH (reducing equivalents) provides hydrogen atom for reduction and it
is oxidized into NADP.
3. Formation of Isoprenoid (5C) Units
• Mevalonate undergoes phosphorylation to form 3-phospho
5-pyrophosphomevalonate.
• Reaction is catalyzed by kinase enzyme in the presence of ATP.
• 3-Phospho 5-pyrophosphomevalonate (6C) is unstable and undergoes decar-
boxylation to form isopentenyl pyrophosphate (5C).
• Isopentenyl pyrophosphate (IPP) isomerizes into dimethyl allyl pyrophos-
phate (6C) (DPP) as in Fig. 17.4.
4. Formation of Squalene
• One molecule of isopentenyl pyrophosphate condenses with one molecule of
dimethyl allyl pyrophosphate to form geranyl pyrophosphate (10C). Reaction
is catalyzed by geranyl pyrophosphate synthase.
• One molecule of isopentenyl pyrophosphate condenses with geranyl pyro-
phosphate to form farnesyl pyrophosphate (15C). Reaction is catalyzed by
farnesyl pyrophosphate synthetase.
• Two molecules of farnesyl pyrophosphate condense together to form squa-
lene (30C). Reaction is catalyzed by squalene synthetase as in Fig. 17.4.
5. Conversion of Squalene into Cholesterol
Squalene is converted into cholesterol in two separate reactions which are
described as follows:
• Squalene conversion into lanosterol
–– Squalene undergoes hydroxylation to form squalene-2,3-epoxide. Reaction
is catalyzed by squalene monoxygenase. Process requires NADPH and
oxygen molecule.
–– Squalene-2,3-epoxide undergoes cyclization into lanosterol. Reaction is
catalyzed by cyclase enzyme.
O O
CH3–C∼S–CoA + CH3–C∼S–CoA
Acetyl CoA (2c) Acetyl CoA (2c)
Thiolase CoA∼SH
O O
CH3–C–CH2–C∼S–CoA
Acetoacetyl-CoA (4c)
H2O HMG-CoA
Acetyl-CoA Synthase
β - Hydroxy - β - Methyl glutaryl CoA

HMG - CoA (6c)
2 NADPH2 HMG - CoA

Reductase
CoA∼SH
2 NADP+
OH H
CH3–C–C–CH2OH
H
H2–C–COOH
Mevalonate (6c)
ATP Kinase
Mg++
ADP
Mevalonate-5-phosphate
ATP Phosphomevalonate
kinase
ADP
Mevalonate-5-pyrophosphate
ATP Kinase
Mg++
CO2 H2O Pi ADP

ISO-pentenyl
pyrophosphate Mevalonate-3-phospho-5pyrophosphate
(5c) Decarboxylase
Fig. 17.4 Biosynthesis of Cholesterol

Isopentenyl 5c Pyrophosphate
Geranyl-Pyrophosphate
Isomerase
Synthetase
3,3 Dimethyl allyl-Pyrophosphate (5c)
PPI
Geranyl-Pyrophosphate (10c)
Isopentenyl
Pyrophosphate (5c) Farnesyl-Pyrophosphate Synthetase
PPI
Farnesyl-Pyrophosphate (15c)
NADPH2
Condensation Squalene Synthetase Mg++
Farnesy NADP+
2PPI
Pyrophosphate (15c)
Squalene (30 C)
NADPH2
Squalene monoxygenase
NADP+ O2
Squalene – 2,3 – epoxide
Cyclase
Lanosterol
(C14) Methyl group
14 – Desmethyl lanosterol
2 CH3 groups (C4)
Mosterol
Cholestadienol
NADPH2
O2
NADP+
Desmosterol (24-Dehydrocholesterol)
NADPH2
Reductase
NADP+
Cholesterol
• Lanosterol conversion into cholesterol

This conversion follows enzyme-controlled series of biochemical reactions.
Important reactions have been mentioned as follows:
–– Demethylation of three methyl groups from lanosterol. This step decreases
carbon atoms from 30C to 27C.
–– Double bond between C8 and C9 position is shifted to new position between
C5 and C6.
–– Double bond between C24 and C25 is saturated.
These reactions are catalyzed by enzymes which are located in endoplasmic

reticulum of cells as in Fig. 17.4.
17.3.2 Regulation of Cholesterol Biosynthesis
HMG CoA Reductase Enzyme
• HMG CoA reductase is the key regulatory enzyme for synthesis of cholesterol in
body tissues. Activity of HMG CoA reductase in turn is controlled at the fol-
lowing stages:
–– Gene expression
Cholesterol controls gene expression of HMG CoA reductase. Normal serum
cholesterol concentration is between 140 and 200 mg/dl. In condition of
hypercholesterolemia, cholesterol itself inhibits gene expression and synthe-
sis of mRNA. Therefore, synthesis of HMG CoA reductase is reduced, and
cholesterol synthesis is minimized.
–– Cholesterol feedback inhibition
↑ cholesterol concentration in serum serves as a negative feedback and inhib-
its HMG CoA reductase.
–– Proteolysis of enzyme
HMG CoA reductase contains sterol-sensing domain (a segment of 180
amino acid residues that bind with sterol group). Cholesterol promotes
proteolysis of HMG CoA reductase enzyme.
–– Phosphorylation-dephosphorylation mechanism
Cyclic AMP-activated protein kinase catalyzes phosphorylation of HMG
CoA reductase and its enzymatic activity is reduced, while dephosphorylation
of HMG CoA reductase increases its activity.
Hormones
• Insulin and thyroxine hormones increase cholesterol synthesis.

• Glucagon and cortisol decrease cholesterol synthesis.
Role of Diet
• Diet rich in saturated fatty acids promotes synthesis of cholesterol.

• Diet rich in PUFA decreases cholesterol synthesis and serum cholesterol level.
PUFA possibly stimulates cholesterol oxidation into bile acids and their excre-
tion through the intestine.
• Fiber-rich diet decreases cholesterol synthesis and controls serum cholesterol level.
• Diet rich in glucose, sucrose, and fructose stimulates cholesterol synthesis.
Heredity
• Heredity is a predisposing factor in hypercholesterolemia and hyperlipidemia.
17.4 Biodegradation of Cholesterol
Cholesterol is converted into bile acids, steroidal hormones, and calcitriol. It cannot
be decomposed into CO2 and water. Biodegradation of cholesterol occurs in the fol-
lowing ways:
Formation of Bile Acids

Bile acids are formed in the liver. They serve as emulsifying agents and help in
digestion of dietary triglycerides. Bile acids contain polar and non-polar groups
(amphipathic). Bile acids are of two types as follows:
Steps in Bile Acid Synthesis
• Cholesterol undergoes hydroxylation in the liver to form 7-hydroxycholesterol.

• Reaction is catalyzed by 7-alpha-hydroxylase enzyme in the presence of
NADPH2 and molecular oxygen.
• The 7-hydroxycholesterol undergoes series of enzymatic reactions to form cho-
lic acid and chenodeoxycholic acid. They are primary bile acids which undergo
the following changes as described below:
17.5 Ketogenesis 465
Primary Bile Acids
• Cholic acid
• Chenodeoxycholic acid
Cholic acid predominates in bile. It undergoes conjugation with glycine and tau-
rine amino acids to form glycocholic acid and taurocholic acid. Conjugated primary
bile acids have better emulsifying activity. These conjugated acids exist in bile in
the form of salts of sodium or potassium as:
Sodium glycocholate and sodium taurocholate are found in bile acids.
Secondary bile acids are formed from deconjugation of primary bile acids by
intestinal bacteria.
Secondary bile acids
• Deoxycholic acid
• Lithocholic acid
Formation of Steroidal Hormones

Cholesterol is a precursor molecule for the synthesis of mineralocorticoids, gluco-
corticoids, androgens, progesterone and estrogens.
Formation of Cholecalciferol and Calcitriol

7-Dehydro-cholesterol is a precursor for synthesis of cholecalciferol which is
hydroxyalted in the liver and kidneys to form calcitriol.
17.5 Ketogenesis
Definition
Ketogenesis is a metabolic process in which a group of organic compounds are
synthesized in body.
Types of Ketone Bodies

Ketone bodies are of three types:
• Acetone
• Acetoacetate
• Β-hydroxy butyrate
Site of Occurrence and Enzymes
• Ketogenesis occurs in the liver.

• Enzymes catalyzing reactions are located in mitochondrial matrix of hepatocytes.
17.5.1 Steps in Ketogenesis
Process occurs through enzyme-controlled biochemical reactions. These are

explained as follows:
• Formation of Acetoacetyl CoA

–– Two molecules of acetyl CoA undergo condensation to form acetoacetyl CoA
–– Reaction is catalyzed by thiolase enzyme. A molecule of CoA-SH is released
in reaction as in Fig. 17.5.
O O
CH3 – C – CH2 – C – S.CoA
Acetoacetyl-CoA
CoA–SH H2O
O HMG-CoA
Synthase
CH3 – C ∼ S.CoA
Acetyl-CoA
OH H O
HO –C –CH2 – C – C ∼C ∼ S – CoA
O CH3 H
β - Hydroxy - β - Methyl glutaryl CoA

[HMG - CoA]
HMG - CoA
Lyase
ACETYL-CoA
Acetoacetate Ketone body

β-hydroxy butyrate
dehydrogenase H
CH3–C–C C O
O H OH
NAD+ NADH+H+
Spontaneous
decarboxylation
OH CO2
CH3–C–CH2–COOH
H
CH3–C–CH3
β-hydroxy butyric Ketone body
acid O
Acetone Ketone body
Fig. 17.5 Ketogenesis

17.5 Ketogenesis 467
• Formation of β-Hydroxy β-Methylglutaryl CoA

–– Acetoacetyl CoA condenses with a molecule of acetyl CoA to form β-hydroxy
β-methylglutaryl CoA.
–– Reaction is catalyzed by HMG CoA synthase (mitochondrial enzyme). A
molecule of CoA-SH is released.
–– HMG CoA synthase is key regulatory enzyme.
• Formation of Acetoacetate
–– β-Hydroxy β-methylglutaryl CoA undergoes cleavage to form a molecule of
acetoacetate with the release of a molecule of acetyl CoA.
–– Reaction is catalyzed by HMG CoA lyase.
• Formation of Acetone
–– Acetoacetate can undergo nonenzymatic spontaneous decarboxylation to
form acetone as in Fig. 17.5.
• Formation of β-Hydroxy Butyric Acid
–– Acetoacetate is converted into β-hydroxy butyric acid by β-hydroxy butyrate
dehydrogenase. Reaction occurs in the presence of NADH2 which supplies
hydrogen atom. It is oxidized into NAD+.
–– β-Hydroxy butyric acid is the dominant ketone body in plasma and urine
during ketosis.
17.5.2 Biological Significance of Ketone Bodies
Source of Energy for Extrahepatic Tissues
• Ketone bodies are transported from the liver to the body tissues by blood circula-
tion. Acetoacetate and β-hydroxy butyric acid are chiefly utilized by heart mus-
cles, skeletal muscles, and renal cortex to generate energy.
• In adverse conditions like starvation, ketone bodies are the source of energy for
extrahepatic tissues due to deficient supply of glucose.
• In diabetes mellitus, glucose uptake by peripheral tissues is reduced due to defi-
ciency of insulin. Peripheral tissues utilize ketone bodies.
• In starvation, brain tissues are primarily dependent on ketone bodies for supply
of energy.
Steps for Ketolysis

Ketone bodies decomposed and are utilized by extrahepatic tissues through the
following reactions:
• β-Hydroxy butyric acid

β-Hydroxy butyric acid undergoes oxidation to form acetoacetate by
β-hydroxy butyrate dehydrogenase. Reaction requires NAD+.
• Acetoacetate
Acetoacetate interacts with succinyl CoA in the presence of enzyme CoA trans-
ferase (thiophorase) to form acetoacetyl CoA and succinic acid.
• Acetone
Acetone is not oxidized in living tissues for energy. Excessive accumulation
of acetone produces a fruity smell in breath and urine.
The liver lacks CoA transferase enzyme. Therefore, the liver cannot utilize
ketone bodies.
17.6 Ketosis
Definition
It is a clinical condition characterized by excessive synthesis and accumulation
of ketone bodies in blood circulation.
Under normal health condition, a state of equilibrium exists between the
synthesis of ketone bodies in the liver and their utilization in extrahepatic tis-
sues of body.
Normal plasma ketone bodies level is nearly 1 mg/100 ml. Ketone bodies are
present in urine in undetectable concentration.
Predisposing Factors for Ketosis

Diabetes Mellitus
• It is an endocrine disorder characterized by decreased synthesis of insulin and

insulin resistance among peripheral tissues of body.
• Insulin regulates the uptake and utilization of glucose by extrahepatic tissues.
In the presence of insulin resistance and insulin deficiency, glucose is not
utilized by body tissues for energy production. Glucose becomes surplus in
blood circulation. Alternately, tissue lipids are mobilized to provide energy to
body. It leads to excessive accumulation of free fatty acids and acetyl CoA. The
vicious cycle terminates into excessive production of ketone bodies
(ketosis).
Starvation
• It is a condition in which an individual is severely deprived from intake of food

for a prolonged period. There is an acute deficiency of calories in the body for the
maintenance of basal metabolic rate. Starvation is an acute and a severe form of
malnutrition. In its initial stage, glycogen store of the body is utilized to provide
energy to the body. In the next stage, tissue lipids from adipose tissues are
decomposed to release fatty acids.
• There is excessive concentration of free fatty acids in circulation. Free fatty acids
are oxidized to provide energy to body, and it leads to the accumulation of acetyl
CoA in blood circulation.
• Excessive acetyl CoA cannot be utilized in citric acid cycle. They are converted
into surplus amount of ketone bodies (ketosis).
17.7 Lipoproteins 469
Pathophysiology
Certain conditions predispose to excessive synthesis of ketone bodies in the liver.
Their utilization does not increase proportionately in extrahepatic tissues.
It results in excessive accumulation of ketone bodies in plasma and it is called
as ketonemia. There is an increase in excretion of ketone bodies by kidneys in
urine and is called as ketonuria.
Ketoacidosis is a metabolic disorder characterized by excessive accumulation of
keto acids in blood circulation owing to excessive synthesis of ketone bodies.
Due to uncontrolled production of ketone bodies and their limited utilization,
there is excessive production of keto acids in body. The buffer systems of body are
unable to neutralize keto acids. There is rise in plasma concentration of keto acids
and it leads to fall in pH of blood. It is called as ketoacidosis.
Ketoacidosis is common consequence of uncontrolled diabetes mellitus and is
labeled as diabetic ketoacidosis. It is manifested as a fruity smell (acetone) from the
mouth of patient.
Ketosis and ketoacidosis are clinical conditions owing to excessive produc-
tion of ketone bodies. However, ketosis leads to ketoacidosis which is a life-
threatening condition that requires immediate medical intervention to save the
life of patient.
17.7 Lipoproteins
Definition
Lipoproteins are conjugated proteins that are composed of protein molecules
complexed with lipid moieties.
Lipoprotein molecules are carriers of lipid molecules. They serve as transport
vehicles and carry lipid molecules from the intestine to the liver and distribute lipids
to body tissues through blood circulation.
17.7.1 Classification of Lipoproteins
Lipoproteins are divided into five categories based on electrophoresis property as

follows:
Chylomicrons
Characteristics
• Chylomicrons are synthesized inside enterocytes.

• They have a density of <0.96.
• They have a diameter of 100–1000 nm.
Composition
• Chylomicrons contain about 2% of proteins.

• Chylomicrons contain approximately 98% of total lipids.
–– Triglycerides constitute 85–88% of total lipids of chylomicron.
–– Free cholesterol and cholesterol ester constitute 4% of the total lipids of
chylomicrons.
–– Phospholipids represent about 7–8% of the total lipids in chylomicrons.
–– Free fatty acids are absent in chylomicrons.
–– Chylomicrons contain least amount of proteins (2%) and the maximum
amount of total lipids (98%).
• Chylomicrons contain B48 apoprotein molecule.
Functions
• Chylomicrons are the lightest in weight (least density) and have the largest size
among lipoproteins.
• Chylomicrons serve to transport exogenous triglycerides (dietary) from the small
intestine to the liver and body tissues.
Very Low Density Lipoproteins (VLDL)

Characteristics
• Very low density lipoproteins VLDL are synthesized in the liver.

• They have a density of 0.96–1.006.
• Normal serum VLDL concentration should be 2–25 mg/100 ml.
Composition
• Proteins constitute 10% of the total weight of VLDL.

• VLDL contain 90% of the total lipids.
–– Triglycerides constitute about 55% of the total lipids in VLDL.
–– Free cholesterol and cholesterol ester constitute 20–24% of the total lipids in
VLDL.
–– Phospholipids represent about 20% of the total lipids in VLDL.
–– Free fatty acids are present in negligible amount (1%) in VLDL.
• VLDL contain B100 apoprotein molecule.
Functions
• Very low density lipoproteins serve to transport endogenous triglycerides from

the liver to body tissues.
17.7 Lipoproteins 471
Low Density Lipoproteins

Characteristics
• Low density lipoproteins are synthesized from VLDL in the blood circulation.
• They have a density of 1.006–1.063.
• They have a diameter of 20 nm.
• Normal serum LDL should be <100 mg/100 ml.
• LDL are considered as bad cholesterol. It is implicated in pathogenesis and
progression of atherosclerosis and coronary artery disease.
Composition
• Proteins constitute about 20% of the weight of LDL.

• LDL contain 80% of total lipids.
–– Triglycerides represent about 10–12% of the total lipids in LDL.
–– Free cholesterol and cholesterol ester constitute 60% of the total lipids in
LDL.
–– Phospholipids represent 25–30% of the total lipids in LDL.
–– Proportion of free fatty acids is negligible (1%) in LDL.
• LDL contain B100 apoprotein molecule.
Functions
• Low density lipoproteins serve to transport cholesterol from the liver to body
tissues.
High Density Lipoproteins

Characteristics
• High density lipoproteins are synthesized in the liver.

• They have a density between 1.063 and 1.20.
• Normal serum HDL concentration is between 40 and 70 mg/100 ml. HDL is
considered as a good cholesterol.
Composition
• Proteins constitute about 40% of the HDL.

• HDL contain about 60% of the total lipids in HDL.
–– Triglycerides constitute 10–12% of the total lipids in HDL.
–– Free cholesterol and cholesterol ester constitute 40% of the total lipids.
–– Phospholipids represent about 45% of the total lipids in HDL.
–– Free fatty acids are present in negligible amount (1%) in HDL.
–– HDL contain the maximum amount of proteins (40%) and least amount
of total lipids (60%).
Functions
• HDL serve to transport endogenous cholesterol from peripheral tissues to the

liver.
Structure of Lipoprotein
• It is composed of a central core of triglycerides and cholesterol ester.

• Central core is surrounded by a layer of apoproteins molecules, cholesterol, and
phospholipid molecules.
• Phospholipids and cholesterol are amphiphilic molecules. Their polar heads are
directed outward toward aqueous medium. Their nonpolar tails are oriented
toward the interior of the lipoprotein molecule.
• Lipoprotein molecules are soluble in aqueous medium in blood circulation and
body tissues.
Suggested Readings
Alberti KGMN (ed) (1978) Recent advances in clinical biochemistry. Churchil Livingstone,
London
Harper HA (1979) Review of physiological chemistry, 17th edn. Lange Medical Publisher,
New York
Latner AL (1975) Cantarow and Trumper. Clinical biochemistry, 7th edn. Saunders, Philadelphia
Publisher, New York
Metabolism of Minerals
18
18.1 Calcium
Calcium is the most significant mineral of hard tissues of the body. It is distributed
in various foods. The human body requires calcium for strength of bones and teeth.
It is also essential for many physiological functions. Calcium is necessary for con-
tractility of muscles and nerve conduction. It is essential cofactor in activity of
enzymes and hormones.
Distribution of Calcium in the Body

An adult person of 70 kg body weight contains around 1000 g calcium. It represents
around 1.5% of the total weight of the body. Endoskeleton of the body contains
around 990 g calcium. It amounts to nearly 99% of the total calcium content of the
body. Calcium in bones exists as complexes of phosphate and carbonate with cal-
cium. Skeletal calcium serves two functions. It is a labile pool of calcium to main-
tain intracellular and extracellular levels of calcium. Skeletal calcium provides
bone strength.
Non-skeletal tissues contain approximately 10g calcium which represents
nearly 1% of the total calcium content of the body. Soft tissues possess 0.5% cal-
cium, and extracellular compartment has 0.1% calcium. Normal plasma calcium
level is between 9 and 11 mg/100 ml.
Dietary Sources
Animal Sources
• A rich source is yogurt (dairy edible obtained by bacterial fermentation of
milk).
• Good sources are milk, cheese, sardine, salmon, and egg yolk.
Plant Sources
• Good sources are cereals, lentils, nuts, turnip, and cabbage.

https://doi.org/10.1007/978-981-13-1035-5_18
474 18 Metabolism of Minerals
Recommended Dietary Allowance

Adults
• RDA for adults is about 800 mg per day.
Children
• RDA for children is 1.2 g per day.
In Pregnancy and Lactation

• RDA in pregnancy and lactation is 1.5 g per day.
Forms of Calcium in Plasma

Calcium in plasma exists in three forms as:
• Ionized Calcium
–– Ionized calcium is about 51% of total non-skeletal calcium (>5 g).
–– Ionized calcium is metabolically active, and it is involved in physiological
and metabolic functions.
• Protein-Bound Calcium
–– Protein-bound calcium is nearly 40% of total non-skeletal calcium
(~ 4 g).
–– Important plasma proteins that bind to calcium are albumin and globulin.
–– Calmodulin (calcium-modulated protein) is present in cytoplasm of cells.
• Calcium Complexes
–– Calcium complexes are nearly 9% of total non-skeletal calcium (~1 g).
–– Important complexes of calcium are calcium phosphate, calcium oxalate, and
calcium carbonate.
Non-skeletal calcium performs multiple functions.
• It is necessary for cell signaling.

• It is necessary for muscle contraction.
• It is responsible for nerve impulse transmission.
• It is important in blood clotting.
Absorption of Calcium
• Dietary calcium is found in complexes like calcium carbonate, calcium
phosphate, and calcium tartrate.
• Calcium absorption occurs by mucosa of the small intestine.
• At low intraluminal calcium level
–– Calcium is absorbed by passive diffusion
–– It occurs in favor of concentration gradient and is the slow process of
absorption.
• At high intraluminal level
–– Calcium absorption occurs by active transport.
–– It is an ATP-dependent process.
–– Active transport is regulated by 1,25-dihydroxy cholecalciferol (calcitriol).
18.1 Calcium 475
–– Calcitriol controls synthesis of calbindin (calcium-binding protein located in

brush borders of duodenum).
–– Calbindin transports calcium across cell membranes of enterocytes.
Factors Influencing Absorption of Calcium

Absorption Promoters
• Calcitriol
–– Calcitriol is a hormone.
–– It governs synthesis of calcium-binding protein on the surface of
enterocytes.
–– It enhances absorption of calcium
• Parathyroid Hormone
–– Parathyroid hormone activates hydroxylase enzyme in kidneys.
–– Synthesis of calcitriol is increased.
–– Parathyroid hormone indirectly enhances calcium absorption.
• Dietary Proteins
–– High proteins in diet enhance calcium absorption.
–– Amino acids like arginine and lysine enhance calcium absorption.
• Dietary Carbohydrates
–– Dietary carbohydrates like lactose enhance calcium absorption.
–– Dietary sugars are fermented by colonic bacteria into organic acids.
–– Organic acids like citric acid enhance calcium absorption.
• Low pH of Intestinal Lumen
–– Calcium complexes like calcium carbonate and calcium phosphate are
readily soluble at low intraluminal pH.
–– Low pH of intestinal lumen enhances calcium absorption.
Absorption Inhibitors
• Dietary Fatty Acids
–– Free fatty acids combine with calcium salts and form insoluble calcium soap.
–– It is excreted in stools.
–– Dietary fatty acids inhibit calcium absorption.
• Dietary Phytic Acids
–– Phytic acids like inositol hexaphosphate are found in cereals.
–– They complex with calcium to form insoluble salts of calcium.
–– Phytic acids inhibit calcium absorption.
• Dietary Oxalates
–– Vegetables like spinach and cabbage possess oxalates.
–– Oxalates complex with calcium to form calcium oxalate.
–– It is poorly absorbed and excreted in stools.
–– Dietary oxalates inhibit calcium absorption.
• Dietary Fibers
–– Dietary fibers inhibit calcium absorption.
• High pH of Intestinal Lumen
–– Alkaline medium of the intestine inhibits calcium absorption.
• Dietary Minerals (Phosphate, Iron, and Magnesium)

–– High level of phosphates in food form calcium phosphate which is
insoluble.
–– A ratio of (1:1) of calcium and phosphate in food is favorable for calcium
absorption.
–– High level of magnesium in food interferes in calcium absorption.
–– High iron in diet interferes in calcium absorption.
• Glucocorticoids
–– Glucocorticoids are steroids synthesized by the adrenal cortex.
–– Glucocorticoids diminish calcium absorption.
18.1.1 Functions of Calcium
Mineralization of Bones and Teeth

• Calcium and phosphate are necessary for mineralization of bones and teeth.
• Calcium is deposited in the form of hydroxyapatite crystals.
• Bones serve as a dynamic store of calcium in the body.
Clotting of Blood
• Ionized calcium represents a blood clotting factor IV.
• Calcium is necessary for formation of extrinsic and intrinsic prothrombin
activator.
• Calcium is helpful in cascade of blood clotting.
Muscle Contraction
• Calcium is necessary for formation of actin-myosin complex in the skeletal
muscle.
• Calcium plays a role in excitation-contraction coupling.
• Calcium is helpful in skeletal muscle contraction.
Myocardial Excitability
• Myocardial contractibility and excitability is dependent on calcium.
• Calcium is necessary for normal systole and diastole.
Conduction of Nerve Impulse

• Influx of calcium at a synapse is helpful in release of a neurotransmitter across
synapse.
• Thus calcium is necessary for nerve impulse conduction.
Enzyme Activity
• Activity of lipase is dependent on calcium as cofactor.
• Activity of neuronal nitric oxide synthase (secreted by specific neurons in the
brain) is dependent on calcium as cofactor.
18.1 Calcium 477
• Activity of calpain (proteolytic enzyme; cysteine protease) is dependent on cal-

cium as cofactor.
• Activity of calcineurin A, B, and B2 (protein serine/threonine phosphatase; role
in T-cell activation) is dependent on calcium as cofactor.
Activity of Calmodulin
• Calmodulin is calcium-modulated protein. It is a cytosolic protein.
• It can attach with four calcium ions and can form calcium-calmodulin complex.
• The complex activates adenylate cyclase and it regulates cell functions.
Intracellular Messenger
• Calcium (intracellular) acts as second messenger (intracellular molecule for cell
signaling).
• Calcium as second messenger is responsible for cell functions like muscle con-
traction, nerve impulse transmission, cell growth, and apoptosis.
• Other second messengers are cAMP, cGMP, inositol triphosphate, and
diglyceride.
• First messengers are peptide hormones like TSH, ACTH, prolactin, and catechol-
amines like adrenaline.
• Calcium (extracellular) acts as tertiary messenger for cell function. In gastric
mucosa, extracellular calcium acts as tertiary messenger for secretion of pepsinogen.
Permeability in Gap Junctions

• Gap junctions are found in almost all tissues of human body except erythrocytes
and sperms.
• It is an intercellular communication. Gap junction has an intercellular space of
2 nm, and it is filled with extracellular fluid.
• Calcium concentration in gap junction is helpful in transmission of electri-
cal impulse, movement of ions, and movement of metabolites between two
cells.
Activity of Neuromuscular Junction

• Transmission of nerve impulse from a neuron to skeletal muscle is regulated by
calcium.
• Influx of calcium at neuromuscular junction brings about release of
acetylcholine.
• This neurotransmitter is responsible for the activity of neuromuscular junction.
18.1.2 Regulation of Plasma Calcium Level
Normal plasma calcium level is between 9 and 11 mg/100 ml. Plasma contains cal-
cium in three forms. Ionized calcium in plasma is a metabolically active form. It
performs various functions.
Calcium homeostasis is regulated by the following factors:
Role of Calcitriol
• Effect on Intestine
–– Calcitriol is 1,25-dihydroxy cholecalciferol. It is an active Vitamin D3.
–– It acts as hormone and induces the synthesis of calbindin from enterocytes.
–– Calbindin (calcium-binding protein) is located on brush border surface (api-
cal surface) of enterocytes.
–– Presence of calbindin increases absorption of dietary calcium from lumen of
gut.
Calcitriol increases plasma calcium level.
• Effect on Bones
–– Calcitriol stimulates osteoblastic activity in bones. Osteoblasts express alka-
line phosphatase enzyme in cell membrane.
–– Alkaline phosphatase increases level of phosphorous in developing
bones.
–– It results in increased mineralization of bones.
Calcitriol enhances mineralization of bones.
Role of Parathyroid Hormone (PTH)

Parathyroid hormone is a peptide hormone. It is secreted by chief cells of two pairs
of parathyroid glands. It binds with receptors located on target tissues. It acts on the
following tissues:
• Effect on Bones
–– Bones are dynamic store house of calcium.
–– PTH enhances osteoclastic activity in bones. The number of osteoclasts is
increased in the bone.
–– Osteoclasts secrete lactic acid and collagenase enzyme.
–– It results in demineralization of the bone.
PTH enhances demineralization of the bones.
• Effect on Kidneys
–– PTH stimulates reabsorption of calcium ions through distal convoluted tubules
and collecting tubules (daily excretion of calcium is 5 mmol/day).
–– It diminishes excretion of calcium ions by renal tubules.
–– PTH decreases reabsorption of phosphates by proximal renal tubules.
–– PTH stimulates hydroxylation of 25-hydroxy cholecalciferol into
1,25-dihydroxy cholecalciferol.
PTH decreases plasma phosphate level.

PTH increases plasma calcium level.
18.1 Calcium 479
• Effect on Intestine
–– PTH enhances absorption of calcium indirectly.
Role of Calcitonin
Calcitonin is a peptide hormone. It is secreted by parafollicular cells of the thyroid
gland.
• Calcitonin is antagonistic to PTH.

• Calcitonin inhibits osteoclastic activity in bones.
• Calcitonin increases mineralization of bones.
• Calcitonin diminishes plasma calcium level.
• Calcitonin increases excretion of phosphates by kidneys.
Role of Kidneys
• Kidneys bring about hydroxylation of 25-hydroxy cholecalciferol.
• Kidneys reabsorb calcium.
• Kidneys excrete phosphate.
Role of Intestine
• The small intestine absorbs calcium and phosphates.
Role of Bones
• Bones are reservoir of calcium.
• Bones undergo mineralization and demineralization (bone remodeling) under the
control of hormones.
18.1.3 Disorders of Calcium Metabolism
Hypercalcemia
It is a clinical condition characterized by an increase in plasma calcium level
more than reference value (>11 mg/100 ml).
Etiology
• Hyperparathyroidism
• It may be caused by any one of the following factors:
–– Parathyroid adenoma (benign tumor)
–– Parathyroid malignancy
• Iatrogenic (drug-induced)
–– Loop diuretics like thiazides
• Granulomatous diseases
–– Tuberculosis
–– Sarcoidosis
Characteristic Features
• Lethargy (weakness)
• Nausea, vomiting, and constipation
• Polyuria and renal calculi
• Increase in cardiac contractility
• Decrease in deep tendon reflex (normal reflex is knee jerk on striking with rubber
hammer)
• Confusion (lack of clarity)
Hypocalcemia
It is a clinical condition characterized by a decrease in plasma calcium level
lower than reference value (<8.5 mg/100 ml).
Etiology
• Decrease plasma albumin level
• Hypoparathyroidism
• Renal disease
• Acute pancreatitis
• Iatrogenic (glucocorticoids)
Characteristic Features
• Convulsions
• Cardiac arrhythmia
–– Irregular heart beat either very slow or high, palpitation, angina pectoris, and
decrease in cardiac contractility
• Tetany
• It is a sudden, forceful, and involuntary contraction of muscles (muscle
spasm). The following events occur in tetany:
–– Low calcium in plasma and ECF.
–– Increase in sensitivity of voltage-gated sodium channels.
–– Low-threshold stimulus can open sodium channels across neuronal
membrane.
–– Influx of sodium ions.
–– Rapid and progressive depolarization of neurons.
–– Muscle spasm.
–– Carpopedal spasm
It is the involuntary contraction of muscles of the hand and feet.
It may be elicited by applying a blood pressure cuff over the upper arm for 3 min.
It is called as Trousseau’s sign.
–– Facial Spasm
It is the involuntary contraction of facial muscles.
It may be elicited by tapping over bones of the cheek.
It is called as Chvostek’s sign.
–– Laryngospasm
Sudden and involuntary contraction of vocal cords.
Difficulty in breathing, inhalation is difficult.
18.2 Phosphorus 481
• Hyperactive deep tendon reflex

• Irritability of muscles and nerves
18.2 Phosphorus
Phosphorus is important mineral of hard tissues of the body. It interacts with the
calcium mineral for the calcification of bones and teeth. Phosphorus has equally
important role in synthesis of nucleic acids in the body. It is essential structural
constituent of all biological membranes. It is necessary in the oxidative phosphory-
lation to produce energy.
Distribution of Phosphorus in Body

The human body contains nearly 700 g of phosphorus. About 80% of total phos-
phorus is found in association with calcium ions in hard tissues of the body.
Muscles and blood contain about 10% phosphorus which exists in bound state
with proteins (phosphoproteins), carbohydrates, and lipids (phospholipids). The
residual 10% phosphorus is disseminated in the form of organic compounds in
tissues.
Sources
Animal Sources
• Animal sources are milk, cheese, and eggs.
Plant Sources
• Plant sources are vegetables and cereals.

Adults
• RDA for adults is 800 mg per day.
Children
• RDA for children varies between 500 mg and 1.2 g per day.
Absorption of Phosphorus
Dietary phosphorus is absorbed through intestinal mucosa (jejunum).
Factors Regulating Absorption of Phosphorus

Promoters
• Parathyroid hormone and calcitriol promote absorption of phosphorus.
• Low pH in intestinal lumen promotes absorption of phosphorus.
• Calcium: Phosphorus ratio governs absorption of phosphorus in the intestine.
Inhibitors
• Organic compound like phytic acid in diet retards absorption of phosphorus.
Excretion
• Phosphorus is excreted by kidneys. Phosphate ions (80%) are reabsorbed in
proximal convolute tubules. Excretion of phosphate is regulated by dietary intake
of phosphorus. Nearly 500 mg of phosphorus is excreted in urine daily.
Biochemical Functions
• Phosphorus is essential for calcification of teeth and bones.
• It is necessary for synthesis of phospholipids, phosphoproteins, and nucleic
acids.
• Phosphorus is essential for synthesis of high-energy phosphate compounds like
ATP and GTP.
• Phosphorus is a structural component of coenzymes like NAD+, FAD, FMN, and
NADP+.
• Phosphate is a component in the phosphate buffer system.
• In renal failure, serum phosphate level is raised.
• In serum phosphate level is affected by disease of parathyroid gland. In hyper-
parathyroidism, serum phosphate level is declined, while in hypoparathyroidism,
it is elevated.
• In renal rickets, serum phosphate level is reduced.
18.3 Selenium
Selenium is a trace mineral. It is an antioxidant element in the human body. In 1957,

Schwartz and Flotz observed that selenium was helpful in the prevention of liver
cell necrosis.
Distribution of Selenium
• Selenium is extensively distributed in body tissues. In the liver and kidneys, con-
centration of selenium is the highest, while less vascular organs like the muscle
and adipose tissues contain low concentration of selenium.
• Selenium exists in association with amino acids as selenomethionine and
selenocysteine.
Dietary Sources
• Cereals are average source of selenium (0.1–0.7 μg).
• Vegetables, fruits, and milk products are poor source of selenium.
Absorption and Transport

• Dietary selenium is absorbed in the duodenum. It enters blood circulation in
selenomethionine.
• It complexes with lipoprotein in blood circulation and is distributed to
tissues.
18.4 Copper 483
• Selenomethionine is taken up by body tissues like adipose tissues, myosin, and

myoglobin.
• Selenium (selenocysteine) is a prosthetic group in glutathione peroxidase
enzyme. This enzyme is located in mitochondria and cytosol. Glutathione per-
oxidase catalyzes reduction of H2O2 into water and oxygen. It is an important
antioxidant enzyme in cells.
• Selenium, ascorbic acid, and vitamin E are antioxidants in cells. Selenium pro-
tects tissues against oxidative damage.
• Selenium has affinity to heavy metals like cadmium, mercury, and silver.
Selenium protects tissues against toxic effects of heavy metals.
Selenium Toxicity
• Excessive intake of selenium results into clinical condition called as selenosis. It
is manifested as:
–– GIT disturbances like nausea, vomiting, and loss of weight
–– Change in behavior
–– Garlic odor from the mouth owing to formation of dimethyl selenide
18.4 Copper
Copper is a trace element. The human body contains around 100 mg of copper in
tissues.
Dietary Sources
• Copper is found in cereals, nuts, egg yolk, and green leafy vegetables.
• Milk is a poor source of copper.

• RDA for children is 0.5 mg per day to 1.5 mg per day.
Absorption
• Dietary copper is absorbed in duodenum.
• Metallothionein is a conjugated protein. It helps in the absorption of copper.
• Zinc, molybdenum, and phytic acid retard absorption of copper in
duodenum.
Transport in Blood Circulation

• About 95% of copper exists as ceruloplasmin in blood circulation. The remain-
ing 5% of copper is bound to albumin in circulation.
–– Ceruloplasmin
Normal serum ceruloplasmin concentration is 25–50 mg/100 ml. It contains

6–8 copper atoms which are present in cuprous state and cupric state. It is a
storage protein.
• Normal serum copper concentration is 100–200 μg/100 ml.
• Copper is a structural component of ALA synthase enzyme. It is necessary for
synthesis of hemoglobin.
• Copper is a component of superoxide dismutase, catalase, cytochrome oxidase
and other copper-dependent enzymes.
• Ceruloplasmin is a copper containing protein. It is essential for oxidation of iron
in blood circulation. Oxidized iron (Fe3+) is distributed in form of transferring in
circulation.
• Copper is necessary for myelination of nerves in the brain.
Menkes’ Disease
• Disease is characterized by impaired absorption of copper in intestinal mucosa.
Probably, copper remains chelated by metallothionein within enterocytes.
• Serum copper concentration is decreased.
• Copper deficiency leads to anemia.
Wilson’s Disease
• It is a disorder of copper metabolism. Excessive deposition of copper occurs in
the liver and brain leading to liver necrosis and brain necrosis.
• Serum copper concentration is reduced.
• Serum ceruloplasmin concentration is decreased.
• Copper is deposited in renal tubules leading to renal necrosis.
18.5 Zinc
Zinc is a trace mineral. The human body contains around 2 g of zinc.
Dietary Sources
• Vegetables, cereals, beans, and milk are good sources of zinc.

• RDA in pregnancy and lactation is about 15 mg per day.
Absorption
• It is absorbed in duodenal mucosa.
• Conjugated protein metallothionein helps in the absorption of zinc through intes-
tinal mucosa.
• Divalent metal ions like copper, iron, and calcium interfere in absorption of zinc.
18.6 Fluorine 485
• Peptides promote zinc absorption.
• Zinc is a structural component of enzymes like alcohol dehydrogenase, carbonic
anhydrase, alkaline phosphatase, and carboxypeptidase.
• Zinc is essential for storage of insulin in the pancreas.
• Zinc has a role in normal reproductive function.
18.6 Fluorine
Fluorine is an electronegative element. It is widely distributed in its oxidized form

(fluoride) in nature. It is found in drinking water in varying proportions.
Dietary Sources
• Drinking water is the exclusive, easily accessible, and most probable source of
fluorides for consumption to humans.
• Alternate sources of fluorides can be salmon, sardine, and tea leaves.

• According to WHO recommendation, safe dose of fluoride in water is between
0.5 and 1.0 mg/L.
• Lethal dose of fluoride is 2.5 g.
Absorption and Transport

• Dietary fluoride enters the stomach. It combines with HCl to form hydrogen
fluoride (HF). It is absorbed from the mucosa of the stomach in two
forms.
• At low pH, hydrofluoric acid remains in undissociated form. It is absorbed by
diffusion through the mucosa.
• At high pH (>2.5), hydrogen fluoride is ionized into hydrogen and fluoride ions.
Diffusibility of fluoride from the stomach is pH-dependent and carrier-
independent. It is owing to the sensitivity of phospholipid layer of plasma mem-
brane hydrogen fluoride. Phospholipid layer is highly permeable to hydrogen
fluoride than fluoride ions. Therefore, HF can rapidly pass through plasma
membrane in favor of pH gradient in the body.
• Large proportion of fluorides is absorbed through intestinal mucosa. Its
absorption in the small intestine is pH-independent and carrier-dependent.
Transport
• Absorbed fluoride rapidly enters blood circulation. It exists in ionic fluoride
and organic fluorocompounds with lipids. From plasma, fluorides are distrib-
uted to hard tissues of the body.
• In bones, fluorides are deposited in bone matrix, as well as fluorides are
incorporated in hydroxyapatite crystal structure.
Excretion
• Dietary fluorides are chiefly excreted by kidneys. Excretion of fluoride in
urine is again pH-dependent. Alkaline urine (owing to intake of fruits and salad)
suppresses excretion of fluorides, while acidic urine (owing to intake of protein
enriched diet) promotes urinary excretion of fluorides.
• Dietary fluorides which are not absorbed from the small intestine are excreted in
feces.
• Dietary fluorides are also secreted in saliva in trace amounts (0.01–0.03 ppm).
Salivary fluorides have biological significance as they are involved in excreting
anticariogenic effect on the teeth.
Tooth Development
• Fluorine has a positive role in normal tooth development. Additionally, fluorine
has anticariogenic effect on teeth.
–– During tooth development stage, fluorine is incorporated into crystal structure
and forms fluoroapatite crystals. These crystals are less soluble in oral acidic
environment than hydroxyapatite crystals. Fluoroapatite crystals have higher
resistance to attack of acids than hydroxyapatite crystals. It also maintains
normal crystal structure of teeth.
Bone Development
• Fluorine has healthy effect on normal bone development.
–– In 1 PPM dose, fluorine promotes deposition of calcium and phosphates in
organic bone matrix. Fluorine helps in retention of Ca++ in the bone, thereby
preventing demineralization of skeletal tissues.
–– Fluorine retards age-dependent osteoporosis in bones.
18.6.1 Fluoride Toxicity
Fluoride toxicity is a clinical condition characterized by excessive intake of

fluoride in water or food leading to a variety of clinical manifestations in the
body.
Fluoride toxicity can be grouped into two categories depending on its manifesta-
tions as:
Dental Fluorosis
Dental fluorosis is a disorder of tooth mineralization owing to intake of exces-
sive amount of fluoride in drinking water during tooth development stage.
Etiology
• High fluorine content in drinking water is the prime cause of dental fluorosis. A
dose of fluorine exceeding 1 PPM in drinking water during stage of tooth
development is detrimental to normal development of teeth.
18.6 Fluorine 487
Pathogenesis
• Ingested fluorides exhibit harmful effects on tooth bud through in situ mode.
High serum fluoride concentration alters normal tooth development via the fol-
lowing probable mechanisms:
–– Inhibitory effect on proteases in maturing enamel
In normal tooth development, ameloblasts lay down enamel organic matrix
rich in amelogenin proteins. These proteins provide nucleation for the growth
of hydroxyapatite crystals.
During enamel maturation stage, enamel matrix proteins are hydrolyzed
and removed from maturing enamel. Proteolysis of matrix proteins provides a
large space for the growth of hydroxyapatite crystals as enamel of the tooth is
95% inorganic by weight.
Enamel matrix proteins proteolytic enzymes
Matrix metalloproteinase-20 (MMP-20) also called as enamelysin.
Kallikrein 4 (KLK-4) also called as enamel matrix serine protease-1.
These enzymes catalyze hydrolysis of amelogenins along with enamel matrix
proteins.
High level of serum fluoride has an inhibitory effect in situ on proteases. As a

result, degradation of amelogenin and enamel matrix proteins does not take place. It
impairs deposition of calcium and phosphates in maturation stage of the tooth and
results into hypomineralization.
• Effect on Hydroxyapatite Crystals

In normal tooth mineralization, calcium and phosphates are mineralized into
hydroxyapatite crystals.
In high level of serum fluoride, excess fluoride ions are incorporated into crystal
structure. Fluoride ions replace hydroxyl ions from hydroxyapatite crystals to form
fluoroapatite crystals. Accordingly, high amount of hydroxyl ions are released in
extracellular matrix space of enamel which maintain alkaline pH.
High pH converts amelogenin into insoluble mass and impairs its removal from
extracellular matrix space. Alkaline pH additionally favors growth of apatite
crystals along the diameter than length which retains enamel proteins.
Overall effect is hypomineralization of tooth enamel.
Critical period in which toxic effects of fluoride intake are manifested is
between 1st year and 6th year of life.
Depending upon the severity of dental fluorosis, hypomineralized enamel exhib-
its structural deformities as follows:
1. Subsurface hypomineralization
• Mineralization of surface enamel is normal. It appears translucent, glossy,
and smooth. However, subsurface enamel shows hypomineralization and
porosity which may extend to dentinoenamel junction.
2. White opaque spots

• With higher level of hypomineralization, enamel shows white opaque areas
over cusps of teeth.
3. White spots with discoloration
• White opaque spots are widespread over enamel surfaces of tooth. Appearance
of brown discoloration is also manifested on tooth surfaces.
4. Enamel discoloration with attrition
• With further rise in severity of hypomineralization, enamel surface shows
brown to black discoloration. Enamel undergoes attrition over cusps and inci-
sal edges of teeth.
• There is formation of pits over enamel surfaces.
Dental fluorosis is also called as enamel mottling.
Skeletal Fluorosis
Excessive fluoride intake is manifested into skeletal fluorosis. It has the follow-
ing manifestations:
• Initial lesion is marked by bone pain, stiffness of joints, and osteosclerosis of the
pelvis and vertebral column.
• Later stages of skeletal fluorosis are marked by chronic joint pain, arthritis, and
ligament calcification.
• Fluorosis causes increase in bone density.
• Terminal stages of skeletal fluorosis are marked by limited joint movement,
deformity of the large joints and spine, wasting of muscles, and neurological
manifestations.
18.7 Iron Metabolism
Iron is the most essential trace element in the human body. It is needed in a dose
(<100 mg) per day. Total body iron represents a small fraction (0.01%) of total body
weight in healthy person. It is around 3.5 g in adult males and 2.5 g in adult females.
Iron has multiple functions in the body. Iron is necessary for gaseous transport in
pulmonary alveoli and body tissues.
Dietary Sources of Iron

Animal Sources
• Rich sources of iron are liver, meat, spleen, and eggs.
• Liver is the richest source containing around 5 mg/100 mg of iron.
Plant Sources
• The richest source is green leafy vegetables containing around 20 mg of iron per
100 mg serving of vegetables.
• Good sources are cereals, pulses, lentils, spinach, molasses, and nuts.
• Jaggery is another good source of iron.
18.7 Iron Metabolism 489

Adults
• RDA of iron for adults is nearly 10 mg per day.
Children
• RDA of iron for children varies between 10 and 15 mg per day.
Pregnancy and lactation

• RDA of iron in pregnancy and lactation varies between 20 and 25 mg per day.
Forms of Iron in Body Tissues

Iron is present in two forms in tissues as:
• Functional Iron
Functional form of iron is essential for iron-dependent enzymatic activity, meta-
bolic functions, transport proteins, and hemopoietic functions in the body.
Functional iron is found in the following compounds:
–– Heme-conjugated proteins
Hemoglobin
Myoglobin
Catalases
Peroxidases
–– Cytochromes
–– Other enzymes
Succinate dehydrogenase
Aconitase
• Storage Iron
• Storage form of iron is the iron reservoir of the body. Iron is stored in two forms as:
–– Ferritin
Ferritin form of iron is made up of ferric form of iron which is coplexed with
apoferritin protein. Ferritin is stored in the liver, spleen, bone marrow, blood,
and intestinal mucosa.
–– Hemosiderin
It is another form of iron storage in the body. Hemosiderin contains larger
amount of iron than ferritin. In the condition of iron overload, iron is stored in
hemosiderin. It is deposited in the liver, pancreas, and spleen.
Absorption of Dietary Iron

In a routine diet, about 10–20 mg of iron is consumed with diet. However, only 2 g
(10% of intake) of iron is absorbed in circulation. About 80–90% of total iron con-
sumed is excreted in stools.
Iron in blood circulation is in dynamic equilibrium with iron store of the body, bone
marrow uptake, uptake for synthesis of iron-dependent enzymes, and iron loss from body.
Garnick postulated mucosal block theory for absorption of dietary iron.
• Dietary iron enters the stomach. Hydrochloric acid liberates ferric form of iron
from dietary non-heme iron.
• Ferric form of iron is reduced into ferrous form in the gastrointestinal tract.
Ascorbic acid in diet helps in reduction of iron and forms iron-ascorbate com-
plex. This complex is highly soluble in intestinal juices. Dietary amino acids also
help in chelation of iron as iron amino acid complex. Heme iron is absorbed
without reduction in GIT.
• Iron from non-heme iron and heme iron is absorbed in ferrous form (Fe2+)
through duodenal mucosa.
• Ferrous iron is transported across the plasma membrane of enterocytes (luminal
surface of enterocytes).
• Within cytoplasm of enterocytes, ferrous iron is oxidized into ferric form.
Reaction is catalyzed by ferroxidase I enzyme. The intracytoplasmic transport of
ferric iron is carried by intracellular iron carrier. It transports ferric iron to
apoferritin within enterocytes to form ferritin. Iron is stored in the form of fer-
ritin in enterocytes.
• Ferritin is a transient iron stored in intestinal mucosa. A fraction of ferric iron
from intracellular carrier is converted into ferrous form. It is exported through
serosal surface of enterocytes. Ferroportin helps in the export of iron.
Regulation of Absorption of Iron

• Mucosal block regulates absorption of dietary iron. In case iron store of the
body is exhausted, mucosal cells enhances absorption of dietary iron. Otherwise,
in condition of adequate iron store of the body, mucosal cells limit the absorption
of iron.
• Total body iron store regulates iron absorption. Whenever body iron store is
depleted, absorption of iron is increased through intestinal mucosal cells.
• Erythropoietin secretion also regulates iron absorption. In case of iron defi-
ciency in the body, juxtaglomerular apparatus of kidneys secretes erythropoietin.
It stimulates mucosal cells to enhance absorption of iron.
Factors Promoting Absorption of Iron

• Presence of ascorbic acid in diet promotes iron absorption.
• Low pH in stomach favors iron absorption.
• Protein-rich diet favors iron absorption.
• Gastroferrin in the stomach favors iron absorption.
Factors Retarding Absorption of Iron

• Presence of phytic acid in diet retards iron absorption.
• Diet rich in phosphates retards iron absorption.
• Achlorhydria retards iron absorption.
Transport of Iron
• In blood circulation, ferrous iron is converted into ferric state. It combines with
apotransferrin to form transferrin.
• Iron is chiefly transported by transferrin from GIT to the bone marrow and other
body tissues.
18.7 Iron Metabolism 491
Excretion of Iron
• Iron is an exclusive element that is primarily stored in the body. A negligible
fraction of iron is excreted from the body in the following ways:
–– Desquamated mucosal cells of GIT containing ferritin
–– Desquamated skin cells
–– Menstrual bleeding
• Iron loss in urine is non-traceable. Appearance of blood in urine is considered a
pathological condition.
• Undigested iron is lost in feces.
18.7.1 Clinical Significance
Iron metabolism is clinically oriented toward two conditions that arise either due to
iron deficiency or overload of iron in the body. These are described as follows:
Iron Deficiency
• This condition is characterized by deficiency of iron in body stores. Iron defi-
ciency is associated with the following biochemical parameters:
–– Concentration of hemoglobin is reduced.
–– RBC count is reduced.
–– Serum ferritin level is declined.
–– Erythropoiesis is retarded.
Iron deficiency is clinically manifested as iron-deficiency anemia.
Iron Overload
Iron overload is the excessive accumulation of iron in body tissues. It is generally
caused by frequent blood transfusions and or a genetic disorder.
Iron overload can be grouped into two categories as:
Hemosiderosis
Hemosiderosis is a localized excessive accumulation of iron without any injury
to body tissues. Hemosiderin is deposited in tissues.
It is of two types as follows:
• Hemosiderosis is associated with repeated blood transfusions. Hemosiderin is

deposited in the liver. It is called as transfusional hemosiderosis. It is commonly
observed in patients suffering from thalassemia, sickle cell anemia, and leukemia.
• Hemosiderosis can be idiopathic. In this condition, hemosiderin is deposited in
lungs. It is called as idiopathic pulmonary hemosiderosis.
Hemochromatosis
Hemochromatosis is generalized excessive accumulation of iron in body tissues
accompanied by irreversible cell injury.
It is of two types as follows:

Primary Hemochromatosis
• It is a hereditary disorder of iron metabolism. P. hemochromatosis is caused by
autosomal recessive genes.
• It is manifested by the following signs:
–– Cirrhosis of the liver is caused by deposition of iron in hepatocytes. Liver
cell undergo irreversible injury.
–– Diabetes mellitus is caused by accumulation of iron in islets of Langerhans
in the pancreas. Beta cells are damaged.
–– Pigmentation of the skin
Skin appears bronze colored. Skin pigmentation is associated with insulin
deficiency and collectively termed as bronze diabetes.
–– Cardiomyopathy
Iron is deposited in cardia muscle fibers. It results in hypertension, arrhyth-
mia, and valvular defects.
Secondary Hemochromatosis
S. hemochromatosis is a consequence of another disorder. It is frequently fol-
lowed by the following conditions:
• Severe hemolysis
• Frequent blood transfusion
• Beta-thalassemia major
• Parental iron therapy
Muscle Spasm
• It is the sudden, forceful, and involuntary contraction of muscles.
Muscle Cramp
• Muscle cramp persisted for prolonged period.
• Quadriceps, hamstrings, and gastrocnemius muscles in thigh, back
thigh, and calve regions may undergo cramp.
Tetany
• It is caused by hypocalcemia.
• Increased neuronal membrane permeability to sodium ions.
Tetanus
• It is a bacterial disease. It is caused by invasion of Clostridium tetani.
• Bacteria release tetanospasmin (toxin) which inhibit the release of
inhibitory neurotransmitter (glycine) from Renshaw cells in the spinal
cord.
• Rapid and progressive depolarization of motor neurons.
• Persistence of skeletal muscle contraction
Suggested Readings
London
Gupta A (2017) Iron metabolism in human body. In: Nutritional anemia in preschool children.
Springer-Nature, Singapore
New York
Publisher, New York
Whitford GM (1994) Effects of plasma fluoride and dietary calcium concentrations on GI absorp-
tion and secretion of fluoride in the rat. Calcif Tissue Int 54:421–425
Biological Oxidation
19
19.1 Definition
Biological oxidation is an enzyme-controlled biochemical reaction in which

organic molecules of food are oxidized in tissues to release energy.
Dietary carbohydrates and lipids are major sources of energy through oxida-
tion. Carbohydrates undergo oxidation in tissues to yield energy along with car-
bon dioxide and water. Fatty acids are oxidized in tissues to yield energy as well
as formation of acetyl CoA which subsequently enter TCA cycle to generate
energy and carbon dioxide. Dietary proteins are minimally utilized for energy
production. They are involved in the synthesis and repair of tissues. However,
protein also undergoes oxidative deamination to form ammonia and α-keto acids
whose carbon skeleton is subjected to oxidation (TCA cycle) to yield energy and
produce CO2 and H2O.
19.2 Methods of Biological Oxidation
Addition of Oxygen to Substrate

• Oxygen is added to substrate. It is catalyzed by oxidase enzyme as shown in the
following reaction:
Oxidase
2AH2 + O2 2H2O + 2A
(Addition of O2) (Oxidized)

https://doi.org/10.1007/978-981-13-1035-5_19
496 19 Biological Oxidation
Removal of Hydrogen from Substrate
• In this reaction, hydrogen atoms are removed from a substrate. Hydrogen atoms
must be accepted by a molecule, called as hydrogen acceptor. Reaction is cata-
lyzed by dehydrogenase enzyme as shown in the following reaction:
Dehydrogenase
BH2 + AB 2B + AH2
(H-acceptor) (Oxidized)
Transfer of Electrons
In living tissues, biological oxidation can be better explained in terms of trans-
fer of electrons, wherein a molecule releases electron (electron donor) and it
becomes oxidized. Other molecule accepts electron (electron acceptor) and becomes
reduced.
This type of biological oxidation is called as oxidation-reduction or redox
reaction.
Electron donor in redox reaction is called as reducing agent or reductant, while
electron acceptor is called as oxidizing agent or oxidant.
In general, a redox reaction can be written as:
Donor electron e– + Acceptor electron
Fe++ e– + Fe+++
(reduced form) (oxidized form)
Therefore, biological oxidation is characterized by simultaneous occurrence

of oxidation and reduction reaction.
In redox reactions, a substance exists into two forms:
• Oxidized form
• Reduced form
Example: (NAD+/NADH), (FMN/FMNH2)

These two forms in redox reaction are called as redox couple or conjugate
redox pair.
Oxidation-reduction reaction is dependent on two factors:
• Standard reduction potential (standard redox potential)

• Oxidoreductase enzymes
19.3 Standard Reduction Potential (Redox Potential) 497
19.3 Standard Reduction Potential (Redox Potential)
Redox potential is a measure of the ability of a redox couple to accept or donate

electrons under standard conditions (0.1 M conc, 25 °C, pH 7.0).
Redox potential is expressed in volts or millivolts and is symbolized by (E0).
• Reduction potential of hydrogen is taken as (–0.41 V) which is the lowest as in

Table 19.1.
• Oxygen atom has a reduction potential (+0.82 volt) which is the highest.
• Reduction potentials of other substances range between (−0.42 and +0.82 volt)
as in Table 19.1.
• A substance with lower reduction potential donates electrons, and a sub-
stance with higher reduction potential accepts electrons. In ETS, electrons
move from hydrogen atom to molecular oxygen through a series of com-
plexes placed with increasing reduction potential.
• Therefore, electron donors have lower reduction potential than electron
acceptor.
• Transfer of electrons from lower reduction potential to higher reduction potential
liberates energy and is called as free energy change.
• The amount of free energy change is dependent on the difference of redox
potential.
• Free energy change marked positive indicates energy consumption, while
energy change marked negative indicates release of energy.
Table 19.1 Showing Redox

redox potential of conjugate Conjugate pair potential (V)
pairs
2H+ + 2e− → H2 −0.41
NAD+ + H+ + 2e− → NADH −0.32
FMN + 2H+ + 2e− → FMNH2 −0.30
Ubiquinone + 2H+ + 2e− → ubiquinol (CoQ.H2) +0.04
Cytochrome b (Fe+3) + e → cytochrome b (Fe+2) +0.07
Cytochrome c1 (Fe+3) + e− → cytochrome c1 (Fe+2)− +0.23
Cytochrome c (Fe+3) + e− → cytochrome c (Fe+2) +0.25
Cytochrome a (Fe+3) + e− → cytochrome a (Fe+2) +0.29
Cytochrome a3 (Fe+3) + e− → cytochrome a3 (Fe+2) +0.55
½O2 + 2H+ + 2e− → H2O +0.82
At standard conditions (25 °C temp, pH = 0.7,
concentration = 1 M)
19.4 Oxidoreductase Enzymes
These enzymes catalyze electron transfer from an electron donor to electron accep-
tor molecule. Oxidoreductases belong to EC-1 as per enzyme classification system.
They are further divided into 22 subclasses. Important groups of oxidoreductases
are mentioned as follows:
Oxidases
Oxidase is a subclass of oxidoreductase enzyme that catalyzes removal of
hydrogen atoms from a substrate by utilizing molecular oxygen as an acceptor
of hydrogen.
Oxidases transfer electrons from substrate to oxygen atom (oxygen atom is
direct acceptor of electrons).
Oxidases catalyze formation of either H2O or H2O2 as in following reactions:
Oxidase
2AH2 + O2 2A + 2H2O
Oxidase
AH2 + O2 A + H2O2
Example:
• Cytochrome oxidase produces H2O in the reaction.

• Xanthine oxidase and L-amino acid oxidase produce H2O2 in the reaction.
Oxygenases
Oxygenase is a subclass of oxidoreductase enzyme that catalyzes direct addi-
tion of oxygen to substrate.
Depending upon the number of oxygen atom, these enzymes are subdivided
into monooxygenase and dioxygenase enzymes.
• Monooxygenase
Catalyzes addition of single oxygen atom to substrate in the form of hydroxyl
group, while second oxygen atom is reduced to a molecule of water
Example: dopamine β-hydroxylase (monooxygenase).
Monooxygenase
BH2 + O2 + XH2 BOH + H2O + X
• Dioxygenase
Catalyzes addition of two oxygen atoms into substrate
Example: homogentisic acid 1,2-dioxygenase
Dioxygenase
A + O2 AO2
19.4 Oxidoreductase Enzymes 499
Dehydrogenases
Dehydrogenase is a subclass of oxidoreductase enzyme that catalyzes the removal
of hydrogen atoms from a substrate and brings about oxidation of substrate.
Dehydrogenases are of two types as follows:
1. Aerobic dehydrogenases
• These enzymes can transfer hydrogen directly to molecular oxygen.
• Aerobic dehydrogenases contain FMN or FAD as prosthetic groups which are
reduced into FMNH2 and FADH2 after accepting (2H+ + 2e−) two hydrogens.
These reduced coenzymes are reoxidized by transferring hydrogens to molec-
ular oxygen.
• There is always formation of H2O2 which in turn is decomposed by catalase
enzyme.
• Synthesis of ATP is always absent.
2. Anaerobic dehydrogenases
• These enzymes cannot directly transfer hydrogen to molecular oxygen.
• They transfer hydrogens (electrons) to intermediate electron acceptors in
ETC which ultimately are accepted by molecular oxygen.
• There is always formation of H2O.
• Synthesis of ATP is always a feature of anaerobic dehydrogenases.
Flavin coenzymes (FMN/FAD) and nicotinamide coenzymes (NAD+/NADP+)

serve as electron acceptors.
Examples:
Flavin coenzyme-dependent dehydrogenases
Example: FMN-dependent L-amino acid oxidase, FAD-dependent D-amino

acid oxidase, and succinate dehydrogenase
Nicotinamide coenzyme-dependent dehydrogenases
Example: NAD+-dependent isocitrate dehydrogenase, malate dehydrogenase,

and NADP+-dependent glucose-6-P dehydrogenase
Cytochromes (Cyt b, Cyt c, Cyt P450) are dehydrogenase enzymes.

Cytochrome oxidase (Cyt a, a3) oxidase enzyme.
Cytochrome P450 (monooxygenase enzyme).
Hydroperoxidases
Hydroperoxidase is a subclass of oxidoreductase enzyme that utilizes H2O2as a
substrate and catalyzes oxidation of another reduced substrate.
Hydroperoxidases are of two types as follows:
1. Peroxidase
Glutathione peroxidase requires reduced glutathione for activity of enzyme.
Peroxidase
2 G-SH + H2O2 G-s-s-G + H2O
Reduced Oxidized
Glutathione Glutathione
Glutathione reductase enzyme brings about reduction of glutathione disulfide

(oxidized) as:
G-s-s-G +NADPH+ H+ → 2 GSH+ NADP+.
2. Catalase
Catalase decomposes two molecules of H2O2 into a molecule of water and
oxygen molecule.
Catalase
2 H2O2 2 H2O + O2
19.5 Electron Transport System (ETS)
Definition
Electron transport system is defined as a series of complexes which are involved
in the transfer of electrons from a donor to an acceptor molecule coupled with
synthesis of ATP.
19.5.1 Structural Components of Electron Transport

System (ETS)
ETS is comprised of two components as follows:
1. Electron carriers (Acceptors)

2. Enzyme complexes in electron transport system
19.5.2 Electron Carriers (Acceptors)
• Electron carriers are complex organic molecules which transfer electrons from
one molecule to another molecule. Electron carriers act as prosthetic groups
(coenzymes) with enzymes.
• Dehydrogenase enzymes catalyze the dehydrogenation reactions in which
electron are transferred from substrates to electron carriers. Substrate mol-
ecules are oxidized and electron carriers are reduced. Subsequently, electron
carriers transfer electrons to successive molecules and are oxidized for
reuse.
19.5 Electron Transport System (ETS) 501
ypes of Electron Acceptors

T
NAD+
• Nicotinamide adenine dinucleotide is a coenzyme.

• Structurally, it contains two nucleotides. Its one nucleotide has adenine as base,
ribose sugar, and phosphate group. Other nucleotide has nicotinamide as base,
ribose sugar, and phosphate group. Nicotinamide ring is found in highly reactive
group of molecules.
• NAD+ acts as electron acceptor.
• Enzyme catalyzed dehydrogenation of substrate releases two hydrogen atoms
and two electrons. NAD+ accepts one hydrogen atom and two electrons. It is
reduced into NADH. Other hydrogen atom enters in aqueous medium.
• Two electrons are added to carbon atom opposite to N+ atom in nicotinamide ring.
Flavoproteins (Fp)
FAD
• Flavin adenine dinucleotide is a coenzyme. It contains adenine, ribose sugar,

riboflavin ring, and two phosphate groups. Riboflavin ring is highly reactive
group in FAD molecule.
• FAD acts as electron acceptor.
• FAD accepts two hydrogen atoms and two electrons. It is reduced into FADH2.
FMN
• Flavin mononucleotide is a coenzyme. It contains riboflavin, ribose sugar, and

phosphate group. It acts as electron acceptor.
• It accepts two hydrogen atoms and two electrons. It is reduced into FMNH2.
Coenzyme Q (Ubiquinone)
• Coenzyme Q is a ubiquitous coenzyme in living tissues. It is composed of qui-

nine ring and isoprenoid units in side chain. The number of isoprenoid units is
variable. Generally, coenzyme Q10 has ten isoprenoid units in molecule.
• It acts as electron acceptor.
• It is not covalently bound to protein and serves as mobile electron carrier.
• Coenzyme Q can accept two hydrogen atoms and is reduced into ubiquinol.
Otherwise, acceptance of one hydrogen atom reduces coenzyme Q into
semiquinone.
Cytochromes
• Cytochromes are red- or brown-colored conjugated proteins called as hemopro-

teins (“heme”-conjugated proteins) or chromoproteins (pigmented proteins) or
metalloproteins (iron-coordinated proteins).
• In 1925, David Keilin coined the name cytochrome.

• Cytochromes contain heme (iron-porphyrin complex) as a prosthetic
group. Heme is an iron-chelated tetrapyrrole compound. Iron atom in
cytochromes exists in ferrous state (Fe ++) and ferric state (Fe +++). Its inter-
convertibility into ferrous and ferric states is helpful in the transfer of
electrons in ETS.
• Types of Cytochromes
• Depending upon the nature of “heme” and its binding to apoprotein
molecule,
• cytochromes are designated as cytochrome a, cytochrome b, and cyto-
chrome c.
• Three groups of cytochromes have been described as follows:
–– Cytochrome a
This cytochrome contains heme as prosthetic group. Heme is a complex of
iron and porphyrin. It is called as heme A.
Heme A contains copper atoms in addition to iron atoms.
Heme moiety is linked to protein through a coordinate bond between iron of
heme and amino acid residue.
Cytochrome a has further two members which are designated by writing sub-
script as cytochrome a and cytochrome a3.
Cytochrome a and cytochrome a3 are terminal component of ETS. They
contain copper atoms and referred as cytochrome oxidase.
Heme in cytochrome a differs from protoheme in cytochrome b and cyto-
chrome c as it contains:
Formyl group that replaces a methyl group
Isoprenoid side chain that replaces vinyl side chain
–– Cytochrome b
This cytochrome contains protoheme as a prosthetic group. Protoheme is a
complex of iron and protoporphyrin IX. It is called as heme B.
Heme moiety is linked to protein through a coordinate bond between iron of
–– Cytochrome c
This cytochrome also contains protoheme as a prosthetic group. Protoheme
is a complex of iron and protoporphyrin IX. It is called as heme C.
Heme moiety is linked to protein through a covalent bond between iron of
Cytochrome c has further two members which are designated by writing sub-
script as cytochrome c and cytochrome c1.
Cytochrome C is a mobile electron carrier.
Cytochromes can also be classified depending upon their absorbance at a

particular wavelength of light band in reduced state as:
• Cytochrome a
Absorbance at wavelength 605 nm
• Cytochrome b
• Cytochrome c
Heme A is a prosthetic group of cytochrome a and cytochrome a3.
Heme B is a prosthetic group of cytochrome b.
Heme C is a prosthetic group of cytochrome c and c1.
Heme moiety in cytochromes is attached to protein molecule by two thio-
ether linkages (covalent bonding).
Iron-Sulfur Complexes
• They are designated as Fe-S complexes. In these complexes, iron is not a
component of heme group. Therefore, Fe-S complex is also called as non-
heme iron complex. These complexes are associated as prosthetic groups in
various metalloproteins like NADH dehydrogenase, ferrodoxin, and coen-
zyme Q.
• Fe-S complexes contain chelated iron and sulfur atoms. The iron atoms are cova-
lently linked to sulfur atoms. Sulfur atoms are provided by cysteine residues of
protein and inorganic sulfides.
• Types of Fe-S Complexes
–– In Fe4S4 complex
This complex has four iron atoms, four cysteine residues, and four inor-
ganic sulfide ions. Each iron atom is linked to two sulfur atoms from cysteine
residues and two sulfur atoms from inorganic sulfides.
–– In Fe2S2 complex
This complex has two iron atoms, two sulfur atoms, and four cysteine
residues. Each iron atom is linked to two sulfur atoms from cysteine residues
and two sulfur atoms from inorganic sulfides.
–– Fe-S complex
This complex has one iron atom and four cysteine residues. Inorganic sulfide
is absent. Single iron atom is linked to four sulfur atoms of cysteine
residues.
• Ferrodoxin (Fe2S2) was the first recognized Fe-S complex. It is involved in
nitrogen fixation in plants.
• In electron transport chain, complex I and complex II contain complexes of
Fe-S.
• Fe-S complexes are involved in electron transfer in ETS. The iron atom in Fe-S
complex undergoes oxidation and reduction, hence catalyzing the transfer
of electrons.
Cytochrome c and coenzyme Q constitute mobile electron carriers in elec-

tron transport chain.
Cytochromes and Fe-S complexes carry one electron in ETS.
NADH, FAD, FMN, and coenzyme Q carry two electrons in ETS.
19.5.3 Enzyme Complexes in Electron Transport System
• Enzyme complexes are highly organized transmembrane proteins. These

complexes are located in the inner mitochondrial membrane.
• Enzyme complexes are interlinked by electron carrier molecules.
Enzyme complexes are described as follows:

Complex I
• Complex I is NADH dehydrogenase (NADH-CoQ reductase).

• NADH dehydrogenase is associated with coenzyme FMN and Fe-S complex.
• Complex I catalyzes electron transfer from NADH to CoQ as in Fig. 19.1.
Complex II
• Complex II is succinate dehydrogenase (succinate-CoQ reductase).

• Succinate dehydrogenase is associated with coenzyme FAD and Fe-S complex.
• Complex II catalyzes electron transfer from succinate to CoQ.
Complex III
• Complex III is ubiquinol-dehydrogenase (CoQ-Cytochrome c reductase).

• This enzyme is associated with cytochrome b and cytochrome c1 as coenzymes
and iron-sulfur clusters (Fe-S) as in Fig. 19.1.
• This complex catalyzes electron transfer from CoQ.H2 to cytochrome c.
Complex IV
• Complex IV is cytochrome c oxidase.

• This enzyme is associated with cytochrome a and cytochrome a3 as coenzymes.
• This complex catalyzes electron transfer from cytochrome c to oxygen mol-
ecule as in Fig. 19.1.
19.5.4 Physiology of Electron Transport System
Hydrogen atoms are produced in the body through oxidation and dehydrogenation
reactions. Hydrogen atoms are accepted by NAD+ and FAD which in turn furnish
hydrogen atoms to molecular oxygen through a series of complexes in ETC. This
exchange is coupled with synthesis of ATP.
The NADH2 and FADH2 are oxidized for reuse.
Sources of NADH2
1. Carbohydrate metabolism
• Oxidative decarboxylation of pyruvate into acetyl CoA-SH by pyruvate
dehydrogenase
• Oxidative decarboxylation of isocitrate by isocitrate dehydrogenase
ATP
ADP + PI
FMN SITE FAD

NADH SUCCINATE
Fe-S I Fe-S
COMPLEX I COMPLEX II
COQ ADP + PI ATP
Cytb - CytC1 SITE

Fe-S II
COMPLEX III
• MOBILE Cytc
ELECTRON CARRIER
• CONNECTING LINK
Between Complex III &
Complex IV
ADP + PI ATP
Cyta, Cyta3 SITE

Cutt III
COMPLEX IV
2Ht
Molecular
oxygen (½O2) H2O
Fig. 19.1 Diagram showing Electron Transport Chain
• Oxidative decarboxylation of alpha-ketoglutarate by alpha-ketoglutarate

dehydrogenase
• Dehydrogenation of malate into oxaloacetate by malate dehydrogenase
2 . Ketogenesis
• Dehydrogenation of beta-hydroxybutyrate into acetoacetate by beta-
hydroxybutyrate dehydrogenase
3. Lipid metabolism
• Dehydrogenation of beta-hydroxyacyl CoA into beta-ketoacyl COA by beta-
hydroxyacyl CoA dehydrogenase
4. Amino acid metabolism
• Oxidative deamination of L-glutamate into alpha-ketoglutarate by L-glutamate
dehydrogenase
Sources of FADH2
1. Lipid metabolism
• Dehydrogenation of acyl CoA into alpha-beta unsaturated acyl COA by acyl
COA dehydrogenase
2. Carbohydrate metabolism
• Dehydrogenation of succinate into fumarate by succinate dehydrogenase
Sequence of steps in electron transport chain is described as follows:
(Complex I)
• NADH2 furnishes two electrons and two H+ ions and it is oxidized into NAD+.
It is reutilized in dehydrogenation reactions in metabolic pathways as in
Fig. 19.1.
• Complex I (NADH-CoQ reductase) catalyzes transport of electrons from NADH
to CoQ (lipid-soluble electron carrier) in ETC through a series of reactions as
follows:
–– Two electrons are accepted by flavoproteins in complex I to form reduced
flavoprotein.
NADH + H+ + FMN FMNH2 + NAD+
–– Reduced flavoproteins transfer electrons to iron clusters in complex I which in

turn transfer electrons to CoQ.
CoQ + e_ CoQ.H (semi-quinone)
e_
CoQ.H2 (quinol)
Transfer of electrons from NADH to CoQ is associated with the release of free
energy (redox potential +0.42 V) which is sufficient to pump protons from mito-
chondrial matrix to intermembrane space.
Transfer of electron from NADH2 to CoQ synthesizes 1 ATP molecule.
It is the Site I.
(Complex II)
• Succinate molecule furnishes two electrons and two hydrogen ions which
are transferred to CoQ through FAD and iron clusters in complex II as in
Fig. 19.1.
Succinate + CoQ Fumarate + CoQ.H2
Transfer of electron from succinate to CoQ is associated with reduction potential

of +0.11 V. The small energy change is unable to pump protons into intermembrane
space.
Complex II is an electron transport pathway parallel to complex 1.

However, complex II is not associated with proton pumping into intermem-
brane space. ETS through complex II generates 2 ATP than complex I which
produces 3 ATP molecules.
Electron transfer from succinate to CoQ lacks synthesis of ATP.
(Complex III)
• CoQ is a mobile electron carrier in ETC. It is lipid soluble. It acts as an acceptor
of electrons from NADH2 and succinate molecules. The CoQ.H2 transfers elec-
trons to complex III.
• In complex III, electrons are initially accepted by Cyt b which transfers electrons
to Cyt c1, and ultimately electrons are passed on to Cyt c.
• CoQ.H2 is oxidized into CoQ for its reuse.
Transfer of electrons from CoQ.H2 to Cyt c is associated with high-energy

change which is sufficient to pump four protons to intermembrane space.
CoQ.H2 + 2 Cyt c [Fe3+) CoQ + 2 Cyt c [Fe2+) + 2H+
• Cytochrome c is a mobile electron carrier.
Electron transfer from CoQ.H2 to Cyt c through Cyt b and Cyto c1 synthe-
sizes 1 ATP molecule.
It is the Site II.
(Complex IV)
• Reduced Cyt c transfers electrons to complex IV.
• In complex IV, electrons are transferred to Cyt a, which in turn transfers elec-
trons to Cu2+ and finally to Cyt a3.
• Cyt a3 transfers electrons to molecular oxygen (terminal electron acceptor).

• Cyt a and Cyt a3 together are called cytochrome oxidase.
4 Cyt c [Fe2+) + 4H+ + O2 4 Cyt c [Fe3+) + 2H2O
Transfer of electrons from Cyt a3 to O2 is associated with high-energy change which

is sufficient to pump two protons into intermembrane space.
Electron transfer from Cyt a3 to O2 synthesizes 1 ATP molecule.
It is the Site III.
19.6 Oxidative Phosphorylation
Definition
Oxidative phosphorylation is a biochemical process in which ATP is formed by
phosphorylation of ADP-utilizing energy generated in oxidation of reduced
coenzymes (NADH/FADH2).
Oxidative phosphorylation converts free energy released during biological oxi-
dation into chemical energy in the form of ATP.
Oxidative phosphorylation is closely coupled with electron transport system in
mitochondria.
Coenzymes like NAD+ and FAD participate in biological oxidation and are
reduced into NADH and FADH2. These reduced forms of coenzymes are oxidized
through the release of electrons and protons in electron transport chain.
Electrons are transported across a series of complexes in ETC. The electrons are
transferred from one redox couple to another in increasing order of redox potential.
The redox potential difference between two redox couples is associated with release
of free energy change.
Free energy during electron flow drives protons from mitochondrial matrix into
intermembrane space through inner membrane.
Proton pump generates proton gradient across inner membrane that drives pro-
tons back into matrix, and it is coupled with phosphorylation of ADP catalyzed by
ATP synthase.
Free Energy Change in Oxidative Phosphorylation

• Redox potential of redox couple NADH/NAD+ is (Eo = −0.32 V).
• Redox potential of terminal redox couple ½ O/H2O is (Eo = +0.82 V).
• Redox potential difference between two redox couples is (Eo = 1.14 V). This
difference in redox potential is equivalent to 52.6 K cal/mol of free energy
change.
• One ATP formation requires 7.3 K cal/mol of free energy. Oxidation of NADH
in ETC is associated with synthesis of 3 ATP molecules which requires 21.9 K cal
of free energy.
19.6 Oxidative Phosphorylation 509
Percentage of Energy Conservation
21.9 × 100
52.6
Percentage of Energy Conservation = 42%
• Oxidation of NADH helps to conserve 42% of energy in the form of 3 ATP

molecules. The efficiency of energy conservation is 42%, while 58% of the
biological energy is dissipated as heat.
Sites of Oxidative Phosphorylation

Oxidative phosphorylation occurs in the inner mitochondrial membrane at three
sites as:
Site-I
• Electron transfer from NADH2 to CoQ synthesizes 1 ATP molecule at

Site I.
Site-II
• Electron transfer from CoQ.H2 to Cyt c through Cyt b synthesizes 1 ATP

molecule at Site II.
Site-III
• Electron transfer from Cyt a3 to O 2 synthesizes 1 ATP molecule at Site

III.
19.6.1 Theories of Oxidative Phosphorylation
Chemiosmotic Hypothesis
This theory was proposed by Peter Mitchell in 1961. It is a widely accepted theory
for understanding oxidative phosphorylation. The theory is also named as Mitchell’s
hypothesis.
Assumptions of Chemiosmotic Theory

1. Electron transport
• Electrons are translocated by enzyme complexes present in ETS. It occurs at
Site I, Site II, and Site III in electron transport chain. Redox couples are
arranged in order of higher redox potential in ETC. The flow of electrons
from a redox couple with lower redox potential to a redox couple with higher
redox potential is exergonic and liberates free energy.
2. Proton Pump
• Electron transport chain carries proton pumps.
• Proton pump is an intrinsic membrane protein located in biological
membrane. It serves to translocate protons across biological membrane.
–– Proton pumps in ETC are as follows:
NADH-CoQ reductase (Site I)
CoQ-cytochrome c reductase (Site II)
Cytochrome c oxidase (Site III)
Proton pump utilizes released energy associated with electron flow in
ETC. Proton pump undergoes conformational change and actively trans-
ports protons.
• Protons are translocated from mitochondrial matrix to intermembrane space
across inner mitochondrial membrane. Protons accumulate in intermembrane
space. It leads to an increase in concentration of protons toward the outer side
of inner membrane than the inner side of inner membrane. Furthermore, pro-
ton translocation produces proton gradient across the inner membrane.
3. Proton Gradient
• Proton gradient is responsible for two conditions as:
–– Low pH on outer side of inner membrane than the inner side.
–– High electrical potential on the outer side of inner membrane than the inner
side.
–– Therefore, proton gradient generates electrochemical gradient across
inner membrane.
4. ATP Synthesis
• In the inner mitochondrial membrane, proton gradient or electrochemical
gradient is neutralized by backflow of electrons from intermembrane
space into mitochondrial matrix across the inner membrane.
ATP synthase (ATP phosphorylase) is also called as ATPase as it can catalyze

hydrolysis of ATP into ADP and Pi.
ATP synthase is a large-sized asymmetrical protein molecule with a shape resem-
bling mushroom. ATP synthase is a molecular motor (molecule that rotates upon
input of energy).
ATP synthase is composed of total eight subunits which are arranged into
two main fractions as:
• F1 fraction
–– It is named as Fraction 1.
–– F1 fraction is composed of alpha, beta, gamma, delta, and epsilon subunits.
–– F1 fraction is hydrophilic.
–– F1 fraction has catalytic activity.
• Fo fraction
–– It is named with subscript “o” owing to the ability of Fo fraction to bind with
oligomycin antibiotic.
–– Fo fraction is composed of a, b, and c subunits.
19.6 Oxidative Phosphorylation 511
–– Fo fraction is hydrophobic in nature.

–– Fo fraction acts a proton-translocating channel or proton pore in inner mito-
chondrial membrane.
• Protons pass through proton pore (Fo fraction) in inner membrane and induces
conformational change in F1 fraction which has catalytic activity.
• F1 fraction utilizes energy and catalyzes phosphorylation of ADP into ATP.
The overall electron chain transport reaction is:

2 H + +2 e + +1 / 2 O2 ® H 2 O + energy

Two hydrogen ions, two electrons, and an oxygen molecule react to form as a
product water with energy released in an exothermic reaction.
hemical Coupling Hypothesis

C
Chemical coupling hypothesis was proposed by Edward Slater in 1953.
Its assumptions are as follows:
• The flow of electrons in ETC is associated with the formation of high-energy

intermediates. They are cleavaged to release energy that is utilized for phos-
phorylation of ADP.
• This hypothesis relies on chemical coupling to synthesize ATP.
• However, high-energy intermediates have not been isolated from
mitochondria.
onformational Coupling Hypothesis

C
Conformational coupling hypothesis was proposed by Paul Boyer in 1964.
Its assumptions are as follows:
• Flow of electrons in electron transport chain brings about release of free energy.
• This free energy induces conformation change in membrane protein which
assumes high-energy conformational state.
• As membrane protein regains low-energy state, it promotes synthesis of ATP by
union of ADP with Pi.
• However, no concrete evidence has been put forward for high-energy con-
formation state of inner membrane.
19.6.2 Inhibitors of Electron Transport System and Oxidative

Phosphorylation
Inhibitors may be classified into two groups as follows:
Inhibitors of ETC
Inhibitor substances combine with complexes of ETC and interfere in the transfer of
electrons at different sites. These inhibitors are subclassified depending on the site
of action as follows:
• Inhibitors of Site 1 (complex I)

–– These inhibitors prevent transfer of electrons from FMNH2 to CoQ via
complex I.
Rotenone (insecticide and fish poisoning)
Chloropromazine (anxiolytic)
Barbiturates (hypnotic).
• Inhibitors of Site II (complex III)
–– These inhibitors prevent electron transfer from reduced Cytb to Cyt c1
through complex III.
Antimycin A (antibiotic)
BAL (British anti-Lewisite)
Dimercaprol
Phenformin (hypoglycemic drug)
Napthoquinone
• Inhibitors of Site III (complex IV)
–– These inhibitors prevent electron transfer from reduced Cytcto molecu-
lar oxygen through complex IV.
Cyanide
Carbon monoxide (CO)
Hydrogen sulfide (H2S)
Inhibitors of ETC and Oxidative Phosphorylation

Inhibitor substances combine with complexes in ETC and enzymes involved in
oxidative phosphorylation. However, these inhibitors do not inhibit ETC which
are not associated with phosphorylation reactions.
• Rutamycin (inhibits ETC and ATP synthase enzyme)

• Oligomycin (inhibits ETC and ATP synthase enzyme)
• Atractyloside (toxic glycoside, herbicide)
Suggested Readings
London
New York
Mazur A, Harrow B (1971) Textbook of biochemistry, 10th edn. Saunders, Philadelphia
McGilvery RW (1983) Biochemistry-a functional approach, 3rd edn. Saunders, Philadelphia
Publisher, New York
Rawn JD (1989) Biochemistry. Neil Patterson Publsihers, Burlington, NC
Streyer L (1975) Biochemistry, 3rd edn. Freeman WH, New York
Swaminathan M (1981) Biochemistry for medical students, 1st edn. Geetha Publishers, Mysore
Thorpe WB, Bray HG, James HP (1970) Biochemistry for medical students, 9th edn. Churchill,
London
Varley H (1969) Practical clinical biochemistry. WH Medical Books, London
Yudkin M, Offord K (1973) Comprehensive biochemistry. Longman, London
Part IV
Medical Biochemistry
Acid-Base Balance
20
20.1 Definition
Acid-base balance is a regulatory mechanism by which pH of extracellular

fluid is stringently maintained.
Intracellular fluid (ICF) has normal pH 6.7 and it fluctuates between pH 6.7 and
7.4. This high variation in pH of intracellular fluid is attributed to metabolic produc-
tion of carbon dioxide in body tissues.
Extracellular fluid (interstitial fluid and plasma) has normal pH of 7.4, and it has
very slight variation between pH 7.35 and 7.45 (7.4 ± 0.05). Despite the intake of
variety of foods per day, acid-base level of plasma and interstitial fluid is thoroughly
maintained within narrow limit of fluctuation. Human body has homeostatic mecha-
nism of constancy of pH.
A sharp change in pH of ECF leads to wide fluctuation in pH of ICF. It in turn
has high detrimental consequences on metabolic functions of cells. Plasma proteins
and enzymes are extremely sensitive to change in pH beyond the acceptable limits.
They undergo denaturation. There is malfunctioning of ion channels and receptors
across the cell membrane of cells.
20.2 Sources of Acids in Body
Routine diet especially protein-enriched food is a source of enormous amount of

acids in human body. Foods like meat, eggs, milk, cheese, alcohol, and animal
proteins liberate high quantity of acids in body tissues.
Two types of acids are produced in body: volatile acid and nonvolatile acids.
Volatile Acid
It is so called as it is excreted by lungs through exhalation. Carbonic acid is the
single volatile acid which is produced in body tissues. Carbon diaoxide is produced

https://doi.org/10.1007/978-981-13-1035-5_20
518 20 Acid-Base Balance
in tissues due to metabolic reactions of various food products. Carbon dioxide is

produced through complete oxidation of carbon chain of nutrients in carbohydrates,
lipids, and proteins as in Fig. 20.1. It becomes the major source of volatile acid
(carbonic acid) in tissues.
Carbonic acid is produced by dissolution of CO2 into H2O. It occurs in plasma
non-enzymatically and within RBC by action of carbonic anhydrase enzyme.
Metabolic activity of body tissues generates nearly 15,000–20,000 mmol of
carbon oxide. Similar quantity of carbonic acid is produced in plasma as in
Fig. 20.1.
Nonvolatile Acids (Metabolic Acids)

They are not excreted by lungs. They are fixed acids. Nonvolatile acids are further
grouped into organic and inorganic acids.
Organic and Inorganic Nonvolatile Acids

These acids are produced by incomplete oxidation of carbohydrates, lipids, and
proteins in body tissues. Important organic nonvolatile acids are pyruvate, lac-
tate, alpha-keto acids, acetoacetate, and beta-OH-butyric acid.
Important inorganic nonvolatile acids are sulfuric acid and phosphoric acid.
Sulfuric acid is produced by oxidation of sulfhydryl group of methionine and cyste-
ine amino acids. Phosphoric acid is produced by oxidation of phospholipids, phos-
phoprotein, and nucleic acids.
Daily production of nonvolatile acids is between 1 and 1.5 mmol H+/kg
weight and a total amount of 70–80 mmol H+/day as in Figs. 20.2, 20.3, 20.4,
and 20.5.
Complete Oxidation of Nutrients

Fig. 20.1 Diagram
showing oxidation of Carbon Chain → CO2 + H2O
carbon chain from foods
H2CO3
Incomplete Oxidation of Carbohydrates

Fig. 20.2 Diagram
showing incomplete Carbohydrates → Glucose
oxidation of carbohydrates
Pyruvate Lactate
H+
20.3 Sources of Bases in Body 519
Incomplete Oxidation of Trigycerides

Fig. 20.3 Showing
incomplete oxidation of
triglycerides Triglycerides
Acetoacetate β-OH-Butyric acid
H+
Fig. 20.4 Showing Oxidation of Phospholipid, Phosphoprotein and Nucleic acid

oxidation of lipids and
nucleic acids
H3PO4
H+
Oxidation of Sulfhydryl Group of Methionine and Cysteine Amino Acids
H2SO4
H+
Fig. 20.5 Showing oxidation of sulfur containing amino acids
20.3 Sources of Bases in Body
Routine diet delivers a negligible quantity of basic compounds in body. Fruits, veg-
etables, leguminous foods, and nuts constitute alkaline foods and provide alkaline
compounds in the body. These foods are rich in bicarbonates and potassium com-
pounds. They help to raise the pH of plasma and ECF.
Foods like raisins, spinach, bananas, and apple have potential renal acid load
(PRAL) as −20, −13, −6, and −2.5, respectively. They have the highest alkalinizing
effect on plasma and ECF.
Vegetarian food provides sodium salts of citrates and lactates. These compounds
utilize hydrogen ions. Ammonia is also generated through deamination of amino
acids. It is transported to blood circulation. It has alkalinizing effect on plasma and
ECF. However, ammonia is converted into urea.
20.4 Acid-Base Homeostasis
Acid-base balance is maintained by three types of regulatory mechanisms in

body. Each one has its characteristic and role in acid-base homeostasis as
shown in Fig. 20.6.
20.4.1 Buffer Systems
• Bicarbonate buffer
• Phosphate buffer
• Protein buffers
20.4.2 Role of Buffer System in Acid-Base Homeostasis
Buffer
Buffer is an aqueous solution comprised of a weak acid and its salt with a
strong base or a weak base and its salt with strong acid.
Buffer can resist a change in pH of the solution after addition of an acid or a base.
Efficacy of buffer is determined by its pKa (dissociation constant of acid).
Efficacy of a buffer is good at its pKa close to pH ± 1 of solution.
20.4.3 Types of Buffer Systems
Three buffer systems are present in body. With mild variation, their compo-
nents are the same in blood, lymph, interstitial fluid, and intracellular fluid.
Mechanism of Acid-Base balance (Homeostasis)

Acid-base homeostasis is maintained by following systems as:
Buffer Systems First-line Defence Mechanism
Respiratory System Short Term Homeostasis
Renal System Second-line Defence Mechanism
Long Term Homeostasis
Fig. 20.6 Showing systems of acid-base homeostasis

20.4 Acid-Base Homeostasis 521
Based on components of a buffer system, they are grouped into the following
categories:
1. Bicarbonate Buffer
2. Phosphate Buffer
3. Protein Buffer
Bicarbonate Buffer
Composition
• Bicarbonate buffer is composed of a weak acid (carbonic acid) and its salt with
strong base (sodium bicarbonate). It is represented as:
NaHCO3 / H 2 CO3

• Bicarbonate/carbonic acid represents the most important buffer system of blood

and ECF.
• Sodium bicarbonate and carbonic acid are present in a fixed ratio (20:1) in
blood.
• Plasma concentration of bicarbonates is 20 times higher than plasma concentra-
tion of carbonic acid. Therefore, bicarbonates constitute alkali reserve.
Mechanism of Action
Neutralization of Nonvolatile Acids
• Bicarbonate buffer system neutralizes nonvolatile acids like lactic acids, hydro-
chloric acid, and sulfuric acid.
• After an acid enters extracellular fluid, it furnishes H+ ions. The bicarbonate buf-
fer becomes active. Sodium bicarbonate (NaHCO3) dissociates into Na+ ion and
HCO3− ion. The bicarbonate ions react with H+ ions to form carbonic acid as in
Fig. 20.7.
• Carbonic acid is a weak acid. It further dissociates into carbon dioxide and
a molecule of water. Carbon dioxide is removed by lungs through
expiration.
• Therefore, a strong nonvolatile acid is buffered by bicarbonate component
into a weak volatile acid.
Neutralization of Alkali
• After an alkali enters blood, carbonic acid dissociates into hydrogen ions and
bicarbonate ions. Hydrogen ions react with hydroxyl ions to form water as in
Fig. 20.8.
Significance
• Bicarbonate buffer is significant buffer of blood and ECF. It helps to neutralize
effectively hydrogen ions produced in plasma and ECF.
Addition of
Fig. 20.7 Acid buffered NaHCO3 | H2CO3
by bicarbonate buffer
Acid in Body
system
HA
H+ Na+ + HCO3–
Dissociation of
Liberation of Sodium Bicorbonate
H+ ions
By Acid
H+ HCO3–
H2CO3
Carbonic acid
Addition of
Fig. 20.8 Bicarbonate NaHCO3 | H2CO3
Buffer System
Alkaline Substance
[NaOH] Bicarbonate Buffer System
Na+ + OH– H+ + HCO3
Dissociation of Dissociation of
Alkaline Substance Carbonic acid
OH– + H+ H2O
Na+ + HCO3–
NaHCO3
• This buffer system generates weak and volatile carbonic acid. It rapidly dissoci-
ates into carbon dioxide which is easily removed by lungs. This is an advantage
of bicarbonate buffer system over other buffer system in body.
• Bicarbonate buffer system is an indicator of acid-base balance of body.
• Bicarbonate buffer system is directly associated with lungs.
Phosphate Buffer
• Phosphate buffer system is comprised of weak acid (monosodium phosphate)
(NaH2PO4) and its salt (disodium phosphate) (Na2HPO4). It is represented as:
Na 2 HPO4 / NaH 2 PO4

• NaH2PO4 acts as weak acid and Na2HPO4 acts as salt of weak acid with strong
base.
• Disodium phosphate and monosodium phosphate are present in fixed ratio (4:1).
• Phosphate buffer system is an important buffer of intracellular fluid (ICF).
• Concentration of disodium phosphate (alkali phosphate) is four times higher
than concentration of monosodium phosphate (acid phosphate) in blood.
Mechanism of Action
Neutralization of Acid
• After an acid enters blood, it furnishes hydrogen ions.
• Disodium phosphate Na2HPO4 becomes active, and it dissociates into Na+ ion
and NaHPO4− ion as in Fig. 20.9.
• Hydrogen ions (H+) from acid are neutralized by NaHPO4− ions. There is forma-
tion of monosodium phosphate. It is excreted by kidneys.
Neutralization of Alkali
• After an alkali enters blood, phosphate buffer system becomes active. Its
monosodium phosphate component participates to neutralize addition of
alkali.
• Monosodium phosphate dissociates into H+ ions and NaHPO4− ions. Latter ions
react with OH group of alkali and neutralize it. NaHPO4− ions are converted into
disodium phosphate, and it is excreted by kidneys as in Fig. 20.10.
Addition of
Fig. 20.9 Acid buffered Na2HPO4 | NaH2PO4
by phosphate buffer
Acid in Body
[HA] Phosphate Buffer
H+ Na HPO4– + Na+
Acid liberates Dissociation of Buffer

Hydrogen ions
H+ + NaHPO4–
Excreted in
Na H2PO4 Urine
Addition of
Fig. 20.10 Alkali Na2HPO4 | NaH2PO4
buffered by phosphate
Alkaline Compound
buffer Phosphate Buffer
[NaOH]
Na+ + OH– Na HPO4– + H+

Dissociation of Dissociation of
alkali Phosphate Buffer
Na+ + NaHPO4–
Excreted in
Na2 HPO4 Urine
Significance
• Dissociation constant of acid (pKa) of phosphate buffer system is 6.8 and it is
close to pH of blood (7.4). Therefore, physiologically, it is a better buffer than
bicarbonate buffer.
• Phosphate buffer is present in low concentration in blood than bicarbonate buf-
fer; therefore, it is a weak buffer system than bicarbonate buffer system.
• It is directly associated with kidneys.
Protein Buffers
• Plasma Protein Buffer
• Hemoglobin Buffer
Plasma Protein Buffer

• Plasma proteins and hemoglobin constitute protein buffer system.
• Plasma protein represents a better and more effective buffer of blood.
• Plasma proteins particularly albumin represent 95% of protein buffer system of
blood and plasma.
• Buffering efficacy of protein is dependent on dissociable group in plasma pro-
teins. These groups are carboxylic group (COOH), amino group (NH2), guani-
dine group (C=NH), and imidazole group (C-NH).
• In acidic medium, amino group (NH2) accepts hydrogen ions from acidic
medium. It is converted into NH3− and acts as base. Plasma protein gets posi-
tively charged.
• In alkaline medium, carboxylic group (COOH) dissociates to release hydrogen
ions. It is converted into COO− ion and acts as acid. Plasma protein becomes
positively charged.
Hemoglobin Buffer
• Hemoglobin is a conjugated protein and is an effective protein buffer.
• Buffering capability of hemoglobin is due to the presence of imidazole (C-NH)
in histidine amino acid.
• Hemoglobin has 38 histidine residues. Imidazole of histidine is the most effec-
tive buffering group of hemoglobin. Its pKa (7.3) is almost similar to pH of
plasma (7.4).
• Imidazole (C-NH) is closely linked with ferrous iron of heme. In oxygenated
state, imidazole is highly dissociable. It releases hydrogen ion and acts as
acid.
• In deoxygenated state, imidazole is least dissociable. It acts as proton acceptor
and base.
Mechanism of Hemoglobin as Buffer

At Body Tissue Level
• Cellular respiration produces carbon dioxide in tissues. Due to high pCO2 at tis-
sues level, it rapidly diffuses into blood. Inside RBCs, CO2 combines with water
to form H2CO3 under the influence of carbonic anhydrase. Carbonic acid is
unstable and dissociates into H+ ions and HCO3 ions.
• Deoxyhemoglobin accepts H+ ions (H.Hb) and acts as base. Therefore,
hemoglobin buffers hydrogen ions and prevents change in pH of blood and
ECF.
• Bicarbonates ions are transported to level of pulmonary alveoli with a least
change in pH of blood. This transport is called isohydric transport.
At Pulmonary Alveoli Level

• Due to high pO2, deoxyhemoglobin (H.Hb) releases hydrogen ions, and they
combine with bicarbonate ions to form carbonic acid. It dissociates into carbon
dioxide and water.
20.4.4 Role of Respiratory System in Acid-Base Homeostasis
Respiratory system acts as second line of defense in maintaining pH of blood and

ECF. Pulmonary alveoli have rich blood supply and help to remove tissue-gener-
ated carbon dioxide from blood circulation and constancy of pH within a narrow
range. Hemoglobin plays an integral role in the transport of CO2 from tissues to
the lung.
Mechanism of Homeostasis
• Respiration is regulated by respiratory center located in medulla oblongata in
the brain. Respiratory center is a cluster of neurons which are sensitive to change
in pH and pCO2 of blood and ECF. These neurons are called central
chemoreceptors.
• At elevated pCO2, carbon dioxide can rapidly cross blood-brain barrier. It dif-
fuses in cerebrospinal fluid and combines with water to form carbonic acid. It
splits immediately to deliver H+ ions and HCO3− ions.
• H+ ions activate respiratory center in the brain. It stimulates rate and force of
respiration (ventilation). A slight rise (0.2%) in pCO2 (1.5 mm of Hg) in blood
causes 100% increase in pulmonary respiration.
• Therefore, respiratory system helps to wash out excess of carbon dioxide from
blood and preserves pH of blood and ECF.
20.4.5 Role of Renal System in Acid-Base Homeostasis
Kidneys are vital excretory organs of human body. They serve the following vital
functions:
• Removal of nitrogenous waste substances like creatinine, urea, uric acid, xan-
thine, and hypoxanthine from the body
• Secretion of erythropoietin and renin
• Maintenance of total body water
• Maintenance of acid-base homeostasis through the following:
–– Excretion of H+ ions from blood and body fluids
–– Reabsorption of Na+ and HCO3+ ions from glomerular filtrate and preserving
alkali reserve
–– Excretion of ammonium ions
–– Excretion of monosodium dihydrogen phosphate in urine
–– To preserve acidification of urine under normal health condition
Renal system operates through following mechanisms as:
enal Mechanism of H+ Ion Excretion (Bicarbonate Mechanism)

R
Kidneys are importantly involved in removal of hydrogen ions from blood and
ECF. Regulation of hydrogen ions is necessary for maintenance of pH of
blood.
Renal Mechanism of H+ Ion Excretion

• Renal mechanism of hydrogen ion excretion occurs in proximal convoluted
tubules (PCT) of nephrons.
• PCT are surrounded by peritubular capillaries. Carbon dioxide diffuses from
capillaries into the cytoplasm of proximal convoluted tubules. CO2 combines
with water in presence of carbonic anhydrase to form carbonic acid. It splits into
hydrogen ions and bicarbonate ions within PCT as in Fig. 20.11.
• Hydrogen ions are secreted into lumen of tubules. Secretion of one H+ ion is
exchanged with reabsorption of one Na+ ions by cells of PCT.
• Hydrogen ions are excreted in urine. Therefore, kidneys are significantly engaged
in acid-base balance.
Blood Proximal tubule cell
Glomerular
Lumen
filtrate
of
CO2 Na+
CO2 renel
Carbonic Anti porter tubule
H2O anhydrase
H+ H+
H2 CO3
HCO3– HCO3–
Reabsorbtion Na+
HCO3
NC+
Excreted in
NaHCO3 urine
Alkali reserve
Fig. 20.11 Renal bicarbonate mechanism of H+ excretion
eabsorption of Bicarbonate Ions

R
Bicarbonates are important ions of blood and ECF. They represent alkali
reserve of body.
Renal Mechanism of Reabsorption of Bicarbonate Ions

• Bicarbonate ions are reabsorbed by proximal convoluted tubules of nephrons.
• Bicarbonate ions are filtered into glomerular filtrate which is present in lumen of
tubules. HCO3 ions combine with hydrogen ions present in lumen of renal tubule
and form carbonic acid. It dissociates into carbon dioxide and water in tubular
lumen.
• Carbon dioxide diffuses inside the cells of PCT in favor of concentration gradi-
ent. Blood around PCT also delivers carbon dioxide to PCT cells.
• Inside PCT, carbon dioxide combines with water to form carbonic acid. It splits
in presence of carbonic anhydrase to form H+ ions and HCO−3 ions as in
Fig. 20.11.
• Bicarbonate ions and Na+ ions diffuse into capillaries from cytoplasm of PCT
cells.
• Therefore, renal system helps to maintain alkali reserve of blood and ECF in
addition to preserving pH.
enal Mechanism of Phosphate Excretion

R
• Renal mechanism of phosphate excretion occurs in distal convoluted tubule of
nephron.
• Plasma contains alkaline phosphate, disodium monohydrogen phosphate
(Na2HPO2), and acidic phosphate, monosodium dihydrogen phosphate
Blood Renal tubule cell
Glomerular
CO2 CO2 +H2O filtrate
Na+
Carbonic
anhydrase HPO–4
H2CO3
H+
HCO–3 HCO–3 + H+
Na+
Na Na+ Anti porter
Na H2 Po4
Excreted in urine
Fig. 20.12 Renal phosphate mechanism of H+ excretion
(NaH2PO4), in a ratio (4:1). This ratio of phosphates is necessary to maintain

normal acidification of urine.
• Alkaline phosphate, disodium monohydrogen phosphate (Na2HPO2), is filtered
in glomerular filtrate.
• In lumen of tubules, hydrogen ions are exchanged with sodium ions from alka-
line phosphate, disodium monohydrogen phosphate. It is converted into monoso-
dium dihydrogen phosphate which is acidic, and it is excreted in urine as in
Fig. 20.12.
• Sodium ions are reabsorbed into DCT cells and enter blood circulation.
enal Mechanism of Ammonium Ion Excretion

R
Ammonia is a toxic compound produced in blood and tissues as a result of deamina-
tion of amino acids. Kidneys are involved in excretion of hydrogen ions in the form
of ammonium ions to preserve pH of blood.
Mechanism of Excretion of Ammonium Ions

• This mechanism operates in distal convoluted tubules (DCT) of nephrons.
• Glutamine is deaminated inside the DCT cells. Reaction is catalyzed by gluta-
minase enzyme. It results in formation of ammonia and glutamic acid.
• Ammonia diffuses into tubular lumen. It combines with secreted H+ ions to form
ammonium ions (NH4+ ions). Ammonium ions are impermeable to cell mem-
brane of tubules, and hence, they cannot enter cells of DCT.
• Ammonium ions are excreted in urine. Therefore, renal system helps to remove
hydrogen ions in form of ammonium ions to preserve pH of blood and ECF. It
20.5 Disorders of Acid-Base Balance 529
has been estimated that nearly 75% of acid load of body is removed by excretion
of ammonium ions in urine.
• In acidosis, generation of ammonium ions is highly increased to counteract the
excess of hydrogen ions in blood and ECF. This declares ammonium excretion
mechanism as an important method of acid-base homeostasis.
Acidification of Urine
Under normal conditions, urine is acidic with a pH (6.5). Acidification of urine owes
to tubular secretion of H+ ions by proximal convoluted tubules of kidneys. Hydrogen
ions represent acid overload of body. Kidneys are the main organs to excrete
hydrogen ions in the form of acidic urine under normal physiological conditions. It
is surprising that acidic urine is formed through glomerular filtration of components
of alkaline blood (pH 7.4). It represents important excretory function of kidneys by
which hydrogen ions are eliminated and acid-base balance is maintained.
20.5 Disorders of Acid-Base Balance
Disturbance in acid-base homeostasis is manifested in the form of acidosis and

alkalosis.
20.5.1 Acidosis
It is a clinical condition characterized by a decrease in pH of blood.
Metabolic Acidosis
It is characterized by a decline in bicarbonate concentration of blood. It represents
a primary alkali deficit.
• Bicarbonate/carbonic acid ratio is decreased (↓20:1).

• Concentration of H+ ions in blood is increased (↓pH).
• As a compensatory mechanism, respiratory center is stimulated. Ventilation is
increased for rapid removal of CO2 from blood.
• The pCO2 is lowered leading to low blood carbonic acid level. It helps to restore
blood bicarbonate level.
• ↑NH3 and ↑H+ ion excretion by kidneys
• ↑ reabsorption of bicarbonates by kidneys.
Conditions Causing Metabolic Acidosis

Diabetes mellitus, lactic acidosis, starvation, hemorrhage, end-stage renal fail-
ure, and pyelonephritis
Respiratory Acidosis
It is characterized by an elevation of carbonic acid level. It represents primary car-
bonic acid overload.
• A decline in removal of CO2 by pulmonary alveoli. It results in ↑pCO2 in

blood.
• Concentration of carbonic acid in blood is increased (↑ carbonic acid).
• Bicarbonate/carbonic acid ratio is decreased (↓20:1).
• Concentration of H+ ions in blood is increased (↓pH).
• As a compensatory mechanism, kidneys are primarily active in respiratory aci-
dosis. Reabsorption of bicarbonates is increased by tubules. It helps to equalize
bicarbonate to carbonic acid ratio.
Conditions Causing Respiratory Acidosis

Asthma, pulmonary edema, emphysema, drug-induced (morphine, barbitu-
rates), and brain lesions
20.5.2 Alkalosis
It is a clinical condition characterized by an increase in pH of blood.
Metabolic Alkalosis
It is characterized by an elevation of bicarbonate concentration. It represents pri-
mary alkali overload.
• It is caused by increase in bicarbonate level in blood.

• Bicarbonate/carbonic acid ratio is increased (↑20:1) with (↑pH).
• As a compensatory mechanism, respiratory center is depressed. Rate of respi-
ration becomes decreased. Ventilation is reduced. It results in retention of carbon
dioxide and ↑pCO2 in blood. It generates more amount of carbonic acid and
helps to restore bicarbonate/carbonic acid ratio of blood.
• Excretion of bicarbonates is increased by kidneys.
• Hydrogen ion excretion is reduced and K+ ion excretion is increased by
kidneys.
• Decrease in NH3 synthesis by kidneys.
Conditions Causing Metabolic Alkalosis

↑ intake of bicarbonates and hypokalemia
Respiratory Alkalosis
It is characterized by a decline in carbonic acid level. It represents primary carbonic
acid deficit.
• The pCO2 in blood is decreased due to hyperventilation.

• Carbonic acid level in blood is increased.
• Bicarbonate/carbonic acid ratio is increased (↑20:1) with increase in (↑pH).
• As a compensatory mechanism, kidneys are primarily involved in restoration
respiratory alkalosis. Excretion of bicarbonates is decreased. Excretion of hydro-
gen ions is increased.
Diseases Causing Respiratory Alkalosis

Meningitis, salicylates poisoning, fever, and high altitude
Suggested Readings
London
New York
Publisher, New York
Rawn JD (1989) Biochemistry. Neil Patterson Publishers, Burlington, NC
Thorpe WB, Bray HG, James HP (1970) Biochemsitry for medical students, 9th edn. Churchil,
London
Varley H (1969) Practical clinical biochemistry. WH medical books, London
Nutrition
21
21.1 Definition
Nutrition is defined as biological process of consumption of food in proportion

to dietary requirement of living organism.
Nutrition is essential for growth and development of human body. Nutrition
comprises intake of macronutrients like proteins, carbohydrates, and lipids and
micronutrients. Determination of quantity of macronutrients per day for an indi-
vidual is most essential. It involves sound knowledge about the calorific value of
foods and calorie requirement of an individual. Intake of sufficient amount of
micronutrients is another dimension of human nutrition. Micronutrients help to sus-
tain normal endocrine and metabolic functions.
21.2 Food
Definition of Food
Food is defined as any edible substance that is used or intended for use by
humans either raw, cooked, or partly cooked excluding drugs.
21.3 Definition of Calorific Value of Food
Calorific value of food is defined as the amount of energy produced by total

combustion of 1 g of a particular food material in the presence of oxygen. It is
expressed in terms of caloric value.

https://doi.org/10.1007/978-981-13-1035-5_21
534 21 Nutrition
21.4 Measurement of Calorific Value
Carbohydrates, proteins, and lipids constitute important foodstuffs. Different food-

stuffs release variable amount of energy by complete combustion in an apparatus
called as bomb calorimeter.
Procedure
• Bomb calorimeter is a thick-walled steel apparatus. It contains sufficient amount
of oxygen to burn foodstuff completely.
• 1 g of foodstuff is burnt in the presence of oxygen within sealed apparatus
(bomb). Air inside the apparatus becomes hot and it escapes via a copper tube. It
is surrounded by water column. Hot air raises water temperature.
• Thermometer displays rise in temperature.
Calculation
Calorific value of food (H) = W (T2-T1)
W mass of water
(T2-T1) change in temperature
M mass of foodstuff
Calorie
Calorie is defined as energy required to raise the temperature of 1 g of water
by 1° C at a given pressure.
Calorie is food energy and is expressed as (Cal). It is also expressed in Kcal in
routine practice, where 1 Kcal is equal to 1000 cal. It should be noteworthy that heat
is expressed in joules.
Calorie and joule are related as shown in:
1 cal = 4.2 J ( SI unit of energy )

1 Kcal = 1000 cal

Calorific Value of Foodstuffs

Within body tissues, dietary carbohydrates and lipids are completely oxidized to
form carbon dioxide and water and release energy. However, dietary proteins are
incompletely oxidized in body tissues. Proteins undergo deamination and trans-
amination to form ammonia and urea which are excreted. It leads to loss of energy.
Similarly, in carbohydrates and fats, some amount of energy is lost in the diges-
tion and excretion of dietary foodstuff.
21.5 Respiratory Quotient of Foods (RQ) 535
Table 21.1 Showing calorific values of foods

Dietary foodstuff Calorific value in the body Calorific value in calorimeter
Carbohydrates 4.1 cal/g 4.3 cal/g
Lipids 9.3 cal/g 9.4 cal/g
Proteins 4.1 cal/g 5.4 cal/g
Therefore, calorific value of carbohydrates and lipids and proteins differ in bomb
calorimeter and body tissues. However, difference in calorific values of carbohydrates
and lipids is minimum, and it is maximum in proteins. It is due to loss of ammonia
and urea from metabolism of proteins in body (Table 21.1).
21.5 Respiratory Quotient of Foods (RQ)
Definition
Respiratory quotient of foods is the ratio of amount of CO2 produced to amount
of O2 consumed in complete combustion of foods.
Respiratory Quotient of Carbohydrates

• RQ of carbohydrates is nearly 1. It is due to complete combustion of carbohy-
drates into carbon dioxide and water.
C6H12O6 + 6O2 6CO2 + 6H2O
RQ of carbohydrates = 6CO2/6O2 = 1
Respiratory Quotient of Lipids

• RQ of lipids is 0.7.
2 C15H110O6 + 163O2 114CO2 + 110H2O
(Tristerin)
• In the above example, the number of oxygen atoms in comparison to carbon and
hydrogen atoms per molecule of tristearin is considerably reduced. Lipids inher-
ently contain lesser number of oxygen atoms in their structure. Lipids require
more amount of oxygen in combustion.
RQ of Lipids = 114/163 = 0.7
Respiratory Quotient of Proteins

• RQ of proteins is 0.8.
536 21 Nutrition
C72H112N18O22S + 72O2 63CO2 + 38H2O + SO3 + 9CO(NH2)2
RQ of Proteins (Alanine) = 63/72= 0.8
• Proteins represent variable chemical structure. So it is difficult to ascertain num-

ber of oxygen atoms in protein molecule.
Respiratory Quotient of Mixed Diet

• RQ of mixed diet depends on relative proportion of dietary components. In routine
practice, RQ is nearly 0.85. However, it can be higher in carbohydrate-rich diet.
Significance of Respiratory Quotient

• RQ is a numerical value. It represents source of energy in human body. For
example, RQ of 0.7 shows that lipids are mainly utilized for energy production
in the body.
• RQ is a convenient way of determination of energy released by oxidation of
foodstuff.
21.6 Basal Metabolic Rate (BMR)
Calorie requirement of individuals is highly variable. It is dependent on various fac-

tors like age, gender, environmental conditions, and occupation. However, mini-
mum calorie requirement to sustain life under standard conditions is constant in all
individuals.
Definition
Basal metabolic rate is defined as the minimum quantity of energy necessary to
sustain physiological functions of the body under basal conditions in postab-
sorptive state.
Characteristics of Basal Conditions

• Individual should be in complete physical and mental.
• Individual should be in postabsorptive state (12 h without intake of solid or
liquid).
• Individual should be placed in reclining position in bed and should be awake and
alert.
In basal conditions, energy is utilized by body to perform basic physiological

functions like respiration, blood circulation, muscle tone, and brain activity. Cellular
metabolism utilizes energy.
21.6.1 Estimation of Basal Metabolic Rate
Depending on the procedure employed, basal metabolic rate can be estimated by the
following two methods:
21.6 Basal Metabolic Rate (BMR) 537
Open-Circuit Method
This method employs measurement of oxygen consumption and production of car-
bon dioxide.
• Advantage
–– BMR can be estimated with accuracy with this method.
• Disadvantage
–– Apparatus used in this method is sophisticated and costly.
–– Procedure requires high technical efficiency and trained lab technicians.
Examples of Open-Circuit Method
1. Douglas method
2. Tissot method
Closed-Circuit Method
This method employs closed-circuit environment. The consumption of oxygen by
patient is measured for 2–6 min in basal conditions.
Examples of Closed-Circuit Method

Benedict-Roth Apparatus
• It is a cylindrical apparatus which is filled with oxygen. Patient under test is

advised to keep nostrils closed and breathe by mouth. Patient utilizes oxygen
from spirometer during inhalation, and carbon dioxide released during exhala-
tion is absorbed by lime water. Respiratory movements are transmitted to respi-
rometer and in turn are recorded on a drum. Consumption of oxygen is obtained
from recordings on drum.
Calculation of BMR
Standard heat production for 1 L of oxygen consumption = 4.825 °C
Oxygen consumed in 6 min = A
Oxygen consumed in 1 h = 10A
Standard heat production for 1 L of oxygen consumption in 1 h = 4.825 × 10A
Since BMR = cal/h/m2, therefore,
4.825 0 C ´ 10A
BMR =
Square meter of body surface area

• In a state of health, BMR is constant for any individual.

• BMR variation between −15% and +20% is accepted normal.
• BMR requires 60–70% of total calorie requirement of the body.
Normal Value of BMR

In Adult Males
538 21 Nutrition
• BMR is 40 cal/h/m2.
• BMR in adult males per day is nearly 1600 cal/day.
• BMR represents 50–60% of total calorie requirement of individual per day.
In Adult Females
• BMR is 37 cal/h/m2.
• BMR per day is 1400 cal/day.
• BMR represents about 50–60% of total calorie requirement of individual per
day.
21.6.2 Factors Regulating BMR
Age
Infants and children have larger body surface area in relation to body weight. They
have higher BMR than adults. BMR at age of 6 years is 57 cal and at age of 16 years
is 50 cal. It is estimated that BMR declines nearly by 12% for every 10 years of life.
Exception is BMR of newborn babies. It is 20–25 cal/h/m2.
Gender
Males have greater mass of muscles than females. Males have nearly 5% higher
BMR than females.
Body Surface Area

Body surface area is directly proportional to BMR. Since surface area of body is
dependent on weight and height, therefore, thin and tall persons have greater surface
area and higher BMR in comparison to obese and short stature individuals.
Exercise
Regular physical exercise as in sportsmen and athletes increases lean muscle
mass and hence increases body surface area. It leads to increase in BMR (lean
muscle mass is metabolically more active and energy demanding than adipose
tissues).
Starvation
In period of starvation, dietary intake is reduced. Consequently, BMR is decreased.
Probably, it is an adaptation to starvation.
Fever
BMR is increased in fever. It is estimated that about 10% BMR rises with every
increase in 1 °C of body temperature.
Diseases
In diseased state of body, BMR is altered.
21.6 Basal Metabolic Rate (BMR) 539
Increase in BMR
In following diseases, BMR is increased as:
• Infectious diseases (sore throat, cellulitis)

• Polycythemia
• Leukemia
• Cushing syndrome
• Acromegaly
• Thyrotoxicosis
• Hypertension
• Chronic obstructive pulmonary disease
Decrease in BMR
In the following diseases, BMR is decreased:
• Myxedema
• Addison’s disease (adrenal insufficiency)
• Parkinson’s disease
In thyrotoxicosis, BMR increases 50–70% higher than normal. However, respi-

ratory quotient does not change owing to rise in oxygen consumption proportionate
to carbon dioxide production.
• In myxedema, BMR declines by 30–40% below normal.
Climate
Environmental conditions determine BMR of an individual. It increases in cold cli-
mate than hot climate.
Hormones
Hormones from thyroid gland (T3 and T4), hormones from adrenal medulla (adren-
aline), and hormones from anterior pituitary gland are responsible for rise in BMR.
Pregnancy
BMR in pregnancy increases after sixth month of pregnancy. Basal metabolic rate
of pregnant women is decided as:
• BMR of women as in nonpregnant state.

• BMR of fetus.
• Conclusively, BMR in pregnancy rises.
Racial Variations
Human race differs in physique and composition of body in different continents.
Racial variations influence BMR. Eskimos have 33% higher BMR than normal.
540 21 Nutrition
Women belonging to Eastern countries living in the USA have 10% lesser BMR
than women of America in the same age group.
Drugs
Alcohol, caffeine, nicotine, and adrenaline elevate BMR.
21.6.3 Clinical Significance of BMR
Planning of Diet
BMR helps in estimation of calorie requirement of an individual. It in turn is neces-
sary for selection of nutrients from basic food groups and planning a balanced diet.
Research Based-Activity
Effect of drugs and nutrients on basal metabolic rate can be ascertained.
Diagnosis
BMR estimation is a useful tool in diagnosis of diseases.
21.7 Specific Dynamic Action (SDA)
Definition
Specific dynamic action is the surplus heat production in the body, which is
above the estimated calorific value, after a given quantity of food is metabo-
lized in the body.
Specific dynamic action is also termed as food-induced thermogenesis.
Specific Dynamic Action of Foods

Dietary foodstuff involves digestion, absorption, and storage of food in body tis-
sues. These cellular activities release heat which is above the heat of cellular metab-
olism during basal conditions. Therefore, basal metabolism and food processing
together produce heat in body tissues, which together constitute food-induced
thermogenesis or SDA.
SDA is dependent on the nature of nutrient in diet; hence, proteins, carbohy-
drates, and lipids have different specific dynamic action.
SDA for Carbohydrates

• Specific dynamic action for carbohydrates is 5% higher than calculated calorific
value.
SDA for Proteins

• Specific dynamic action for proteins is 30% higher than calculated calorific
value.
21.8 Balance Diet 541
SDA for Lipids

• SDA for lipids varies between 5 and 13% higher than calculated calorific value.
SDA for Mixed Diet

• SDA is between 6 and 10%.
• Carbohydrates and lipids in diet reduce SDA of proteins. In mixed diet, SDA is
lowered. It is not the sum of individual SDA of food components. Addition of
carbohydrates in protein reduces SDA by 12%. Addition of lipids in protein diet
reduces SDA by 54%.
• Addition of lipids comparatively reduces SDA more than addition of carbohy-
drates in mixed diet.
Explanation
Suppose a 20 g of proteins are oxidized in bomb calorimeter in laboratory. This
results in heat production.
Heat produced in burning of proteins in bomb calorimeter
= 20 × 4 = 80 cal (calorific value of protein 4 cal/g)
Heat produced in metabolism of 20 g of proteins in body tissues
= calculated calorific value + food induce thermogenesis (SDA)
= 80 cal + 30% higher than calculated calorific value for proteins
= 80 cal + 24 cal
Total heat production in body tissues = 104 cal
Specific dynamic action or food-induced thermogenesis is always higher
than basal metabolic rate.
Mechanism of Specific Dynamic Action

Food processing involves conversion of nondiffusible foodstuff into diffusible form,
absorption of micronutrients into circulation, and storage of food.
In case of dietary proteins, SDA is the highest among all macronutrients. In
amino acids, nitrogenous fraction undergoes oxidative deamination and carbon
skeleton enters intermediate metabolism. The body spends energy in these physio-
logical activities.
In the case of dietary carbohydrates, the body spends energy when glucose is con-
verted into lipids as well as when glucose is metabolized into carbon dioxide and water.
In the case of dietary lipids, oxidation of fats releases energy.
Therefore, food-induced physiological activities generate heat in body. It is
called SDA.
21.8 Balance Diet
Definition
Balanced diet is defined as a diet which comprises proportional amounts of
food stuffs drawn from basic food groups to fulfill energy and nutrient require-
ments of body.
542 21 Nutrition
Characteristics of Balanced Diet
• Balanced diet should contain one part protein, one part lipids, and four parts
carbohydrates (1:1:4).
• It should contain vitamin and minerals in appropriate quantity.
• It should be enriched with appropriate amount of foods selected from basic food
groups so as to provide phytonutrients, vitamins, and trace elements.
• Balanced diet should be prepared keeping in mind the physiological needs of an
individual. The quantity of its ingredients should be adjusted to fulfill increased
calorie requirement in pregnancy, lactation, childhood, and convalescent
periods.
• Balanced diet should be economical and contains local food groups.
• Balanced diet should have adequate amount of fiber foods (salad).
21.8.1 Basic Food Groups
Basic food groups are fundamental groups of foods that deliver wide range of
macro- and micronutrients. They are essential in maintaining health. Each food
group has characteristic nutrients that serve particular function. It is recommended
to select food from each basic food group so as to provide necessary calories and
minerals to match energy requirement of an individual.
21.8.2 Types of Basic Food Groups
In 1943, the US Department of Agriculture introduced a guide for healthy nutri-

tion. USDA promulgated seven basic food groups for general health.
1 . Yellow green vegetables

2. Oranges, grapefruit, and tomatoes
3. Fruits, other vegetables, and potatoes
4. Milk and milk products
5. Meat, fish, poultry, or eggs
6. Bread, flour and cereals
7. Vitamin A fortified butter and fortified margarine
The Australian government Department of Health recommended five basic

food groups as:
1 . Grains (breads, cereals, rice, pasta, noodles, and other grains) (40% of daily diet)
2. Vegetables (30% of daily diet)
3. Fruits (10% of daily diet)
4. Milk (milk, yoghurt, cheese, and/or alternatives) (10% of daily diet)
5. Meat (lean meat, fish, poultry, eggs, and beans) (10% of daily diet)
In 1992, US Department of Agriculture again recommended four basic food

groups as:
1. Green leafy vegetables and fruits

• This food group delivers vitamins A and C. Vegetables and fruits are good
sources of dietary fibers and carbohydrates and minerals. It is recommended
to have 2–3 servings per day from this group.
2. Milk
• Milk is an excellent source of calcium, phosphorous, lipids, and proteins.
Cheese constitutes as good casein protein. Milk should be served 2–3 times a
day in preschool children, adolescents, and adults.
3. Meat
• Meat group represents good source of animal and plant proteins. Meat group
includes meat, fish, eggs, pulses, beans, peas, and nuts. Two servings are rec-
ommended per day.
4. Cereals
• Whole grains as wheat, barley, maize, and rice are good source of carbohy-
drates and proteins. They are rich in minerals and vitamin B complex. Whole
grains also provided fibers. Two to three servings of cereals are recommended
per day.
Food Guide Pyramid

In 1992, the US Department of Agriculture (USDA) recommended the number of
adequate servings which should be consumed from each basic food group per day.
It was called as Food Guide Pyramid.
Food Pyramid
Food pyramid is also called as diet pyramid.
It is a diagrammatic representation indicating the type of nutrient and its
optimum servings which should be selected from each basic food group and
should be consumed per day to meet calorie and mineral requirement of an
individual.
Cereals and bread constitute the base of food pyramid with 6–11 servings per
day to be consumed depending on age, gender, and dietary needs. Further, oils, fats,
and sweets are placed at the top of food pyramid which should be used judiciously
per day as in Fig. 21.1.
MyPyramid
In 2005, the USDA Center for Nutrition Policy and Promotion introduced
updated version of nutrition guidelines. It replaced earlier Food Guide Pyramid.
MyPyramid is a diagrammatic representation in the form of colorful verti-
cal bars without images of foods.
In left side of pyramid, image of stairs and climber represent a need for physical
activity. In extreme left-hand side, orange vertical bar represents proportion of
cereals per day. Vegetables and milk groups are represented by green and blue bars,
544 21 Nutrition
HYDRATES
CARBO-
OIL
2 SERVINGS
GROUP
GROUP
MEAT
MILK
2–4 SERVINGS
2–4 SERVINGS
2–4 SERVINGS
FRUIT GROUP
VEGETABLE
GROUP
BREAD, CEREALS, RICE

6–11 SERVINGS
DEPENDING ON AGE,
GENDER HEALTH
Fig. 21.1 Showing food pyramid
respectively. Both have equal proportions. Red bar represents fruit group which is
smaller than vegetables and milk groups. Another yellow narrow band represents
protein group and thin silver bar gives amount of oils per day to be consumed.
MyPlate
In 2011, the USDA Center for Nutrition Policy and Promotion introduced a lat-
est version of nutrition guidelines. It replaced MyPyramid.
MyPlate is a diagrammatic representation in the form of a pie chart depict-
ing a plate and glass representing five food groups.
Plate is divisible into four zones indicating 30% cereals, 40% vegetables,
10% fruits, and 20% proteins to be consumed per day. Plate is shown to be asso-
ciated with a small circle representing a dairy (glass of milk) as in Fig. 21.2
(Table 21.2).
21.8.3 Types of Balanced Diet
Quantity of ingredients of balanced diet varies according to age group, gender, and
nature of work. Few examples of balanced diet have been provided from the report
of nutrition expert group, ICMR (1968) as in Tables 21.3, 21.4, 21.5 and 21.6.
Fig. 21.2 My Plate
DAIRY
10%
FRUITS 20%
PROTEINS
40%
VEGETABLES
30%
GRAINS
Table 21.2 Calorie Adult male

requirement per day Sedentary work 2400 cal
Light work 2600 cal
Moderate work 3000 cal
Heavy work 4000 cal
Adult female
Sedentary work 1800 cal
Light work 2000 cal
Household women 2400 cal
Table 21.3 Balanced diet Sedentary lifestyle

recommended for adult men Food groups Vegetarians (g) Non-vegetarians (g)
with sedentary lifestyle
Cereals 400 400
Pulses 70 55
Green leafy vegetables 100 100
Other vegetables 75 75
Roots and tubers 75 75
Fruits 30 30
Milk 300 100
Fats/oils 400 35
Meat/fish Nil 30
Eggs Nil 30
Sugars 30 30
Source: Report of nutrition expert group, ICMR (2007, 2010a,
2010b, 2012); Deb 2004
546 21 Nutrition
Table 21.4 Balanced diet Moderate work

recommended for adult men Food groups Vegetarians (g) Non-vegetarians (g)
with moderate work
Cereals 475 475
Pulses 80 65
Green leafy 125 125
vegetables
Fruits 30 30
Milk 300 100
Fats/oils 45 40
Meat/fish Nil 50
Eggs Nil 30
Sugars 40 40
2010b, 2012); Deb 2004
Table 21.5 Balanced diet Moderate work

recommended for adult Food groups Vegetarian (g) Non-vegetarian (g)
women with moderate work
Cereals 350 350
Pulses 70 55
Fruits 30 30
Milk 200 100
Fats/oils 40 35
Meat/fish Nil 30
Eggs Nil 40
Sugars 30 30
2010b, 2012); Deb (2004)
Table 21.6 Balanced diet Food groups Age 1–3 years Age 4–6 years
recommended for preschool Cereals 150 200
children
Pulses 50 60
Fruits 50 50
Milk 500 400
Fats/oils 20 25
Sugars 30 40
2010b, 2012); Deb 2004
21.9 Nutritional Value of Dietary Carbohydrates 547
21.9 Nutritional Value of Dietary Carbohydrates
Carbohydrates are important component of human diet. A healthy adult person

requires around 2800 cal per day. Calorific value of carbohydrates is 4 cal/g. It is
recommended that around 60% of calories should be obtained from carbohydrates.
Ideally, an individual should consume around 450 g of carbohydrates per day.
However, carbohydrates constitute major ingredient of daily diet in economi-
cally weaker section of society. Carbohydrates furnish 90% of total calorie require-
ment per day to poor population.
Carbohydrates can be categorized into two groups depending on their
nutritional value:
Digestible Carbohydrates
These carbohydrates are easily digested and metabolized in the body, for example,
glucose, starch, glycogen, fructose, and sucrose.
Nondigestible Carbohydrates
These carbohydrates are not digested in the body, for example, cellulose, inulin, and
pectin.
Chief Source of Energy

• Carbohydrates fulfill 60–70% of total calorie need of the body per day. Glucose
is an instant source of energy during hypoglycemia. They fulfill 90% of energy
requirement in poor population owing to major proportion of carbohydrates in
daily diet.
Exclusive Source of Energy

• Brain tissues and erythrocytes are exclusively dependent on glucose metabolism
for their total energy demand.
Source of Energy for Muscles

• Skeletal muscles store about 500 g of glycogen. It is an important source of
energy for skeletal muscles. It is converted into glucose which in turn is metabo-
lized to produce energy for muscular contractions.
Roughage Value
• Salads, fruits, and whole grains are rich in cellulose. It is a nondigestible carbo-
hydrate among humans owing to the absence of cellulose enzyme in alimentary
canal. Cellulose accumulates in the intestine and provides bulk to volume to the
intestine. It promotes intestinal peristalsis. Cellulose relieves constipation, and
this effect is termed as roughage effect.
Protein-Sparing Effect
• Body tissues fulfill energy demand from dietary carbohydrates and proteins that
are marginally utilized for energy production. This is protein-sparing effect.
• Dietary proteins utilized for growth and catalysis of metabolic reactions.
548 21 Nutrition
Formation of Pentose
• Dietary carbohydrates are metabolized in hexose monophosphate shunt. This
results in formation of pentoses like ribose and deoxyribose. Pentoses are con-
stituents of nucleic acids.
Role in Lipogenesis
• Dietary carbohydrates are converted into triglycerides. They are stored in adi-
pose tissues.
Role in Fat Oxidation

• Fatty acid oxidation produces acetyl CoA. It is condensed with oxaloacetate to
form citric acid in TCA cycle. Therefore, carbohydrates are essential in proper
utilization of end products of fatty acid oxidation.
21.10 Nutritional Value of Dietary Proteins
Dietary proteins are building elements of living body. Proteins are primarily neces-
sary for generation and regeneration of body tissues. Proteins have higher impact on
growth than lipids and carbohydrates. 1 g of protein provides 4 cal of energy.
A healthy adult person should consume around 1 g of protein per kg of weight of
the body per day. It is around 70 g in adult males and 60 g in adult women. Its daily
requirement increases during pregnancy and lactation. Children in age group between
1 and 5 years require 30–40 g of protein daily. Old-aged persons, convalescents, and
persons suffering from chronic diseases like cirrhosis and renal failure require higher
quantity of protein. The carbohydrates are important component of human diet.
A healthy adult person requires around 2800 cal per day. It has been recom-
mended to consume 0.75 g/kg body weight of proteins for adults per day.
Dietary proteins primarily serve as a source of amino acids to the body pool of
amino acids. They are utilized for synthesis of structural proteins as well as func-
tional proteins. Dietary proteins are necessary for synthesis of hormones. Dietary
proteins help to maintain positive nitrogen balance. They are necessary for repair of
body tissues.
Nutritional value of dietary proteins can be assessed from two stand-
points as:
21.10.1 Quality of Dietary Proteins
There are many procedures that grade dietary proteins on the basis of quality.
iological Value of Dietary Protein

B
Definition
Biological value is the percentage of absorbed protein from diet which becomes
part of tissue proteins.
Or
21.10 Nutritional Value of Dietary Proteins 549
Biological value can also be defined as percentage of absorbed nitrogen which

is retained in body.
It is a scale which determines what percentage of a protein is utilized by the
body. Biological value describes how rapidly the body can utilize the dietary
protein.
Biological value is considerable in the case of dietary proteins owing to lack of
storage of amino acids in the body. Biological value is of little importance in carbo-
hydrates and lipids because sufficient quantity of carbohydrates and lipids is stored
in body tissues. However, dietary proteins should have high biological value to ful-
fill daily requirement of amino acids.
Limiting amino acid in dietary protein determines biological value of protein.
For example, suppose the body requires daily 1 g of methionine. Routine diet sup-
plies 300 g of protein with 500 mg of methionine. It means dietary proteins are
deficient of 500 mg of methionine and proteins have low biological value. It can be
increased by mixing the protein with other dietary protein which has high histidine
content. In this way, biological value of mixture of proteins is higher than individual
dietary proteins.
Estimation of Biological Value (BV)

It is estimated in terms of percentage of absorbed nitrogen which is retained in tis-
sues of the body.
Procedure
• An experiment is performed on weaning albino rats.

• Rats are fed upon protein-free diet for 10 days. Amount of nitrogen is calculated
in urine and feces samples of rats.
• Later on, rats were fed upon 10% protein diet. Amount of nitrogen is estimated
in urine, feces, and diet.
Calculation (BV) = Amount of N2 retained × 100
Amount of N2 absorbed
BV = ((IN – (FN – FC) – (UN – UC)

(
IN – (FN – FC)
IN Ingested nitrogen
FN Nitrogen content in feces with protein diet
FC Nitrogen content in feces without protein in diet
UN Nitrogen content in urine with protein diet
UC Nitrogen content in urine without protein in diet

550 21 Nutrition
Significance of Biological Value

• It is a measure of usefulness of proteins.
• It is an index of nutritive importance of proteins.
• Biological value is a useful index for vegans and vegetarians to select a good
source of dietary protein. Animal proteins, in general, have high biological value
as in Table 21.7.
Egg has the highest biological vale. It is considered to have the best amino
acid composition for humans. In plant-based proteins, soya bean has a compara-
tive composition of amino acids, but it has low biological value in comparison
to egg.
Limitation
• This method does not consider digestibility criteria of proteins.
et Protein Utilization (NPU)

N
Definition
Net protein utilization is defined as a ratio between the proportion of protein
retained in body to the proportion of protein ingested.
This method is designed to assess quality of proteins. It is a better procedure. It
involves digestibility criteria of dietary proteins.
Calculation (NPU) = Amount of nitrogen retained × 100
Amount of nitrogen consumed
Significance
• It is an index of quality of dietary protein for human consumption.
• The value of net protein utilization varies from 0 to 100. A value of 100 indicates
that dietary protein is completely utilized in the body, whereas a value of “0”
indicates that ingested protein is not utilized in the body.
• Egg has NPU score of 100 and is considered as reference.
Limitation
• Net protein utilization is affected by limiting amino acids in dietary proteins.
Table 21.7 Showing Biological value Food source

biological values of food 94 Egg
sources
84 Milk
75 Beef
65 Soya bean
67 Whole cereals
60 Corn
21.10 Nutritional Value of Dietary Proteins 551
Chemical Score
Definition
Chemical score is defined as a ratio between quantity of the most limiting
essential amino acid in protein under test to quantity of similar essential amino
acid in egg protein.
This method determines chemical composition of proteins. It analyzes composi-
tion of amino acids of a given protein. It is compared with a reference composition
of amino acids, usually egg.
Chemical Score = quantity(mg) of limiting essential amino acid per gram of test protein
quantity (mg) of limiting essential amino acid per gram of egg protein ×100
Chemical score of egg protein is considered as 100. Its value is taken as reference
for comparison with same amino acid.
Egg protein contains adequate proportion of essential amino acids. Therefore,
chemical score of egg protein is used as a mark to determine chemical score of other
dietary proteins as in Table 21.8.
rotein Efficiency Ratio

P
Definition
Protein efficiency ratio is defined as a ratio between the weight gain of a subject
under test and quantity of protein consumed by the subject.
PER had been a widely used method for evaluating the quality of protein in food.
Calculation
weight gain ( g )
Protein efficiency ratio =
quantity of protein consumed ( g )

Supplementation of Dietary Proteins

• Egg is the source of all essential amino acids to humans.
• Animal proteins are better in quality than plant proteins. Wheat and rice proteins
are limiting in lysine and threonine amino acids. Therefore, intake of wheat or
rice as staple food results in deficiency of amino acids. It can be overcome by
mixing of one type of food with another food. It helps to supplement deficient
amino acids in one food with other food. Human race is consuming mixed diet.
This activity helps to improve nutritive value of proteins.
Table 21.8 Showing nutritive values of amino acids

Protein source BV NPU Chemical score PER Limiting amino acid
Egg 94 91 100 4.5 None
Milk 84 75 65 3.0 Sulfur amino acids
Meat 75 76 70 2.7 Sulfur amino acids
Wheat 67 47 42 1.5 Lysine, threonine
Soya bean 65 55 55 2.1 Sulfur amino acids
552 21 Nutrition
Example:
Rice (deficient in lysine, low in threonine) is supplemented with a preparation of
red grams.
Wheat (deficient in lysine, high in methionine) is supplemented with potatoes
(sufficient methionine, high in lysine).
Rice is supplemented with meat.
Limiting Amino Acid

• It is the most deficient essential amino acid in a dietary protein. This amino
acid becomes limiting for protein, for example, sulfur-containing amino
acid in wheat.
21.10.2 Quantity of Dietary Proteins
Quantity of dietary proteins signifies the quantity of proteins which is to be

consumed daily to fulfill calorie need of an individual.
It may be called as recommended dietary allowance of proteins.
RDA of dietary proteins is dependent on total calorie requirement of a person,
age of a person, health and disease state of a person, and job profile of a
person.
A healthy adult individual must consume around 0.8–1 g/kg body weight of pro-
tein daily. This value amounts to nearly 560 g in adult with 70 kg of body weight.
However, amount of protein intake is raised during physiological conditions like
pregnancy and lactation. Additionally, preschool children in age group between 1
and 5 years need 30–40 g of protein daily. Aged individuals, convalescents, and
patients affected with diseases like liver cirrhosis and kidney failure need addi-
tional quantity of proteins per day. This group of individuals exhibit negative
nitrogen balance. Additional amount of proteins help to replenish nitrogen
balance.
21.11 Nutritional Value of Dietary Lipids
Lipids are important source of energy for living organisms.

It is recommended that around 20–30% of total calories should be obtained from
dietary lipids. Appropriate calculation of calories from lipids depends on total calo-
ries demand per day for the person. A healthy adult person requires around 2800 cal
per day. Calorific value of fat is (9 cal/g). Therefore, a quantity of lipids between 60
and 90 g per day is recommended.
Triglycerides constitute 80–90% of total dietary lipids consumed per day.
Triglycerides of polyunsaturated, short-chain, and medium-chain fatty acids are
digested more rapidly and easily than saturated fatty acids. Oils from plant seeds
like mustard, sunflower, soya bean, and groundnut are beneficial to saturated and
trans-fatty acids.
21.12 Protein Energy Malnutrition 553
According to American Heart Association, calories from saturated fatty acids

should not be more than 10% of total calories for healthy persons. For individuals
who are at risk of coronary artery disease, it should be less than 7% of total calories
per day. Further, proportion of trans-fatty acids in diet should be minimal to provide
<2% of total calories.
Source of Energy
• Fats are source of energy. Calorific value of 1 g of fat is 9 cal, and it is the highest
among all macronutrients. Oxidation of palmitic acid (C15H31COOH) produces
129 ATP molecules, whereas oxidation of glucose (C6H12O6) produces 38 ATP
molecules.
Transport Fat-Soluble Vitamins

• Dietary lipids help to carry fat-soluble vitamins A, D, E, and K.
Protein-Sparing Effect
• Dietary lipids are good source of energy and fulfill energy demand of body tis-
sues. They exert protein-sparing effect similar to carbohydrates.
Palatability of Food
• Palatability is a pleasure gained through eating a favorable food. Fats in diet
enhance palatable value. Fats also provide a feeling of satiety.
Source of Essential Fatty Acids

• Dietary lipids contain essential fatty acids. Linoleic acid is abundant in oils from
plant seeds. Arachidonic acid is found in animal fats. Proportion of EFA in bal-
anced diet should be 30% of total fat intake per day. Essential fatty acids have
high beneficial effects on health. They are useful in lowering low-density lipo-
protein. They are necessary for health of the liver and transport of cholesterol in
blood circulation. They are essential for growth and cognitive development in
preschool children.
21.12 Protein Energy Malnutrition
Definition
Protein energy malnutrition (PEM) is a type of malnutrition which is associ-
ated with inadequate intake of calories and/or insufficient intake of proteins in
diet.
Causes of Protein Energy Malnutrition

Primary Causes
• Inadequate intake of calories
• Intake of poor quality and inadequate quantity of proteins
554 21 Nutrition
Secondary Causes
• Malabsorption syndrome
• Celiac disease
Types of Protein Energy Malnutrition

PEM is categorized into three types as:
• Kwashiorkor
• Marasmus
• Marasmic-kwashiorkor
21.12.1 Kwashiorkor
Definition
Kwashiorkor is a type of severe protein deficiency disorder.
Etiology
• Kwashiorkor is caused by severe inadequacy of proteins in diet. Protein-
deficient diet supplies adequate calories to children for survival but impairs phys-
iological functions.
Predisposing Factors
• Natural calamity leading to food crisis
• Poverty
• Improper weaning
• Persistent diarrhea
• Acute respiratory infections
• Poor sanitation and hygiene
• Geophagia
Age Predilection
• It is commonly seen in children in 2–3 years age group.
• Retarded growth of children.
• Pitting edema of ankles and feet is the characteristic sign of kwashiorkor.
• Distension of the abdomen.
• Hairs dry and sparse. Brown discoloration of hairs.
• Dermatitis and loss of skin pigmentation.
• Anorexia (loss of appetite).
• Liver enlargement (fatty liver).
• Muscle wasting.
• Physical fatigue.
• Anemia.
21.12 Protein Energy Malnutrition 555
Prognosis
• Malnutrition (weight to height <−3SD) in kwashiorkor is associated with poor
prognosis despite the start of proper treatment.
21.12.2 Marasmus
Definition
Marasmus is primarily an energy (calorie) deficiency disorder.
Etiology
• Inadequate intake of diet (including protein, carbohydrates, lipids, and
minerals) for prolonged period
Predisposing Factors
• Natural calamity leading to food crisis
• Poverty
• Improper weaning
• Persistent diarrhea
• Acute respiratory infections
• Poor sanitation and hygiene
• Geophagia
• Helminthic infestations
• Preterm infants
• Prolonged breast feeding
Age Predilection
• Marasmus is observed in infants (<1 year).
• Retarded growth, pronounced emaciation, and underweight are common
features.
• Persistent diarrhea is another important sign of marasmus.
• Edema is absent.
• The skin becomes flaccid, wrinkled, and pale in color.
• Hairs are dry and thin and have loss of luster.
• Acute dehydration.
• Infant is highly irritable and restless.
• Anemia.
Prognosis
• Marasmus has good prognosis after the start of proper treatment.
556 21 Nutrition
Suggested Readings
Deb AC (2004) Fundamentals of biochemistry, 8th edn. New Central Book Agency, Kolkata
Food Act (2014) New Zealand Legislation, no. 32, 2014, New Zealand. http://www.legislation.
govt.nz/act/public/2014/0032/latest/whole.html#DLM2996074
Gopalan C, Ramasastri BV (1990) Nutritive value of Indian food. National Institute of Nutrition,
ICMR, Hyderabad
Gupta A (2015) Effect of geophagy on the nutritional status of children under five years of age.
Ind Stream Res J 4(12):5859
Gupta A (2017) Assessing stunting and predisposing factors among children. Asian J Pharm Clin
Res 10(10):1–8
ICMR (2007) Diet and diabetes. NIN, ICMR, Hyderabad
ICMR (2010a) Nutrient requirements and recommended dietary allowances for Indians. NIN,
ICMR, Hyderabad
ICMR (2010b) Nutritive value of Indian foods. NIN, ICMR, Hyderabad
ICMR (2011) Dietary guidelines for Indians – a manual. NIN, ICMR, Hyderabad
Khanna K et al (2003) Textbook of nutrition and dietetics. Phoenix Publishing House Pvt. Ltd.,
New Delhi
Passmore R (1986) Eastwood. Human nutrition and dietetics, 8th edn. Churchill Livingstone,
London
Planning Commission (2005) 10th plan (2002–2007) volume II, nutrition. Planning Commission,
GOI, New Delhi
Robinson CH, Lawler MN (1986) Normal and therapeutic nutrition, 17th edn. Macmillan
Publishing Company, New York
Swaminathan M (1990) Essentials of food and nutrition, vol vol 1/vol 2. Bangalore Printing and
Publishing Co Ltd., Bengaluru
Wadhwa A, Sharma S (2003) Nutrition in the community. Elite Publishing House Pvt. Ltd., Darya
Ganj
Serum Enzymes and Organ Function
Tests 22
22.1 Definition
Clinical enzymology is the branch of medical science which deals with study of
enzyme activity for diagnosis and prognosis of diseases.
In 1956, Wroblewski and his colleagues published their work on SGOT and
LDH. It was a new venture in medical science that paved a way to emergence of
clinical enzymology.
22.2 Types of Plasma Enzymes
Plasma enzymes are two types depending on their source and function.
Functional Plasma Enzymes

• Concentration of these enzymes is very high in plasma.
• These enzymes are synthesized by the liver.
• These enzymes have high catalytic activity in plasma.
• They may be called as plasma-derived enzymes.
• Examples are fibrinogen, prothrombin, and lipoprotein lipase.
Nonfunctional Plasma Enzymes

• The concentration of these enzymes has very low plasma.
• These enzymes are synthesized by rough endoplasmic reticulum in cells.
• These enzymes have no specific function in plasma.
• They may be called as cell-derived enzymes.
• Examples are SGPT, SGOT, and creatine kinase.

https://doi.org/10.1007/978-981-13-1035-5_22
558 22 Serum Enzymes and Organ Function Tests
Nonfunctional enzymes are continuously discharged into plasma by the follow-

ing activities:
1 . Normal diffusion through membrane of cells

2. Normal cell death
There exists a balance between rate of influx of an enzyme in plasma and its
catabolism and clearance from plasma.
A disease causes imbalance by either increasing release or decreasing clear-
ance of nonfunctional enzyme from plasma.
Clinical enzymology is based on the estimation and interpretation of plasma
enzymes in disease.
22.3 Significance of Clinical Enzymology
Diagnosis of Disease
• Estimation of plasma enzymes is a noninvasive method. It helps in the detection
of organ dysfunction and diagnosis of disease.
Pattern of Disease
• Plasma enzyme estimation helps to understand the pattern of a disease. For
example, SGPT and SGOT estimations aid in diagnosis of acute viral hepatitis,
alcoholic cirrhosis, fatty liver, and obstructive liver disease.
Prediction of Severity of Disease

• Plasma enzyme is helpful in assessing the severity of a disease. For example,
rising value of SGPT helps to predict severity of acute viral hepatitis.
Prognosis of Disease
• Plasma enzyme estimation helps to assess the prognosis of a disease. Their study
predicts the morbidity and mortality.
Differential Diagnosis
• The study of plasma enzymes helps in differential diagnosis of diseases with
similar clinical manifestations. For example, pulmonary embolism can exhibit
the same clinical manifestation as acute myocardial infarction. Assessment of
LDH, SGOT, and CK is helpful in differential diagnosis.
Interpretation Unit of Serum Enzymes (Plasma Enzymes)

Plasma enzyme activity is expressed in international unit.
It is defined as enzyme activity that converts 1 μmol of substrate per minute
per liter under optimum conditions.
22.4 Serum Enzymes in Heart Diseases 559
22.4 Serum Enzymes in Heart Diseases
22.4.1 Creatine Kinase (CK or CPK)
Creatine kinase is an important enzyme that catalyzes phosphorylation of cre-

atine into creatine phosphate (phosphocreatine, a mobile source of energy in
skeletal muscles and brain tissues which provides high energy phosphate to
ADP to form ATP).
Creatine
• Nitrogenous compound is formed from glycine and arginine in the liver and kid-
neys and released into blood circulation and enters skeletal muscles (95%) and
brain tissues (small fraction).
Occurrence
It is present in high amount in cardiac muscle fibers, skeletal muscle fibers, brain
tissues, and the retina. It is absent in liver cells, erythrocytes, and the kidneys.
Normal Value
Adult males: 10–100 IU/L
Adult females: 10–80 IU/L
Creatine
• It is a nitrogenous compound. It is synthesized from glycine and arginine amino
acids in the liver and kidneys. Creatine enters circulation and transported to the
skeletal muscles.
• It is phosphorylated into phosphocreatine by creatine kinase in the presence of
ATP. Phosphocreatine is energy currency for the muscles and brain. It delivers
high-energy phosphate to ADP to form ATP.
Creatinine
• Creatinine is formed by a nonenzymatic breakdown of phosphocreatine in skel-
etal muscles. It is produced at uniform rate. It is not reabsorbed and excreted
freely by kidneys. Serum creatinine level is an indicator of renal function and is
inversely related to renal function.
Interpretation in Heart Disease

In Acute Myocardial Infarction (AMI)
• In acute myocardial infarction, cardiac muscle fibers are damaged. The enzyme is
released in plasma in large amount. Its detection is helpful in the diagnosis of AMI.
• Value of creatine kinase is elevated in acute myocardial infarction. Its value tends to
rise within 3–6 h, and it attains a maximum value in 24 h. Its value normalizes in 72 h.
Isozyme of creatine kinase (CK)

Creatine kinase is a dimer and its subunits are called as B (brain) and M (muscles).
On the basis of two subunits, creatine kinase has three isoenzymes:
• CK-MM (CK-3)
It is present in high concentration in skeletal muscle fibers (98%). Skeletal
muscles have low concentration (1%) of CK-MB. Its serum concentration is
80%.
• CK-MB (CK-2)
Cardiac fibers have (30%) concentration of CK-MB. Its serum concentration is
5%. Its value rises in serum after onset of AMI owing to its release from dam-
aged cardiac fibers.
• CK-BB (CK-1)
It is present in high concentration in the brain and smooth muscle fibers. Its
serum concentration is 1%.
Clinical Significance in AMI

• Serum creatine kinase is helpful to detect early AMI cases when changes in ECG
do not correlate with onset of AMI.
• Creatine kinase is not able to diagnose congestive cardiac failure and coronary
ischemia.
• Creatine kinase is not increased in hemolysis as in lactate dehydrogenase. Its
estimation is more advantageous in comparison to LDH.
22.4.2 Serum Glutamate Oxaloacetate Transaminase (SGOT)
It is also called as aspartate transaminase (AST). It catalyzes reversible transamina-

tion in between aspartate and glutamate.
Occurrence
This enzyme is present in high concentration in liver cells, cardiac muscle fibers, the
kidneys, and the brain.
Normal Value
In adult males: 5–40 IU/L
In adult female: 5–35 IU/L
Interpretation in Heart Disease

In Acute Myocardial Infarction
Its value increases in 12 h and attains peak value in within 24 h. It normalizes
after 3–5 days.
22.5 Serum Enzymes in Liver Diseases 561
Significance of SGOT in AMI

• Quantity of rise (SGOT value) is related to size of infarct.
• SGOT value is helpful in prognosis of AMI as:
–– SGOT value <50 IU/L indicates good prognosis and low mortality.
–– SGOT value >150 IU/L indicates poor prognosis and high mortality.
–– SGOT value >350 IU/L indicates very high mortality.
22.4.3 Lactate Dehydrogenase (LDH)
It catalyzes reversible conversion in between pyruvate and lactate.
Occurrence
Lactate dehydrogenase is widely distributed in body tissues. It is present in high
concentration in liver cells, cardiac fibers, skeletal fibers, erythrocytes, brain tissues,
and the kidneys. Erythrocytes contain 100 times higher concentration of LDH than
plasma. LDH is liable to false-positive test in hemolysis. Therefore, it is a non-
specific biomarker.
Normal Value
Its normal value varies between 120 and 360 IU/L.
Interpretation in Acute Myocardial Infarction

Its serum value increases 12 h after AMI and attains peak value in 48 h. It normal-
izes between the 8th and 12th day.
Significance in Acute Myocardial Infarction (AMI)
• LDH-2 is the predominant isoenzyme in plasma.

• In normal condition, concentration of LDH-2 in plasma is higher than concen-
tration of LDH-1.
• In acute myocardial infarction, LDH-1 concentration becomes higher than
LDH-2 concentration in plasma. This reversal of concentration of between two
LDH isoenzymes is called flipped pattern.
• Concentration of LDH.
• LDH is not a specific biomarker of AMI.
22.5 Serum Enzymes in Liver Diseases
The liver is the master organ to control metabolism of proteins, carbohydrates, and
lipids. Liver cells contain various enzymes. After injury to hepatocytes, enzymes are
released into plasma. Therefore, estimation of serum enzymes is a valuable clinical
tool that serves multiple functions.
22.5.1 Serum Transaminases
erum Glutamate Pyruvate Transaminase (SGPT)

S
It is also called as alanine transaminase (ALT). It catalyzes reversible transamina-
tion between alanine and glutamate.
Occurrence
SGPT is primarily and abundantly found in liver cells.
Normal Value
Its normal value ranges between 5 and 45 IU/L.
Interpretation
• Elevation of SGPT >500 IU/L is suggestive of viral hepatitis, toxin-induced liver
disease, and ischemic liver disease.
• Value of SGPT rises in biliary cirrhosis (50–350 IU/L).
• Elevation of SGPT between 300 and 500 IU/L is found in Laennec’s cirrhosis.
• Elevation of SGPT between 150 and 300 IU/L is suggestive of obstructive jaun-
dice (posthepatic jaundice).
erum Glutamate Oxaloacetate Transaminase (SGOT)

S
tion between aspartate and glutamate.
Occurrence
SGOT is predominantly found in cardiac muscle fibers. It is also present in liver
cells.
Normal Value
Interpretation
• Elevation of SGOT is seen in cirrhosis patients.
Ratio of SGOT/SGPT has better diagnostic and prognostic significance than
either test performed alone.
Significance
• Elevation in SGPT level starts before clinical appearance of jaundice. Its peak
value is attained between 7 and 10 days. SGPT normalizes within 4 weeks of
onset of acute viral hepatitis.
• SGOT/SGPT ratio is <1in hepatitis excluding viral hepatitis.
• SGOT/SGPT ratio is >1in advanced cirrhosis of the liver and chronic hepatitis C.
• SGOT/SGPT ratio >2 is suggestive of chronic alcoholic cirrhosis.
22.5 Serum Enzymes in Liver Diseases 563
Gamma-Glutamyl Transpeptidase (GGT)

It is a transferase enzyme. It catalyzes the transfer of gamma-glutamyl group from
donor molecule like glutathione to an amino acid.
Occurrence
Gamma-glutamyl transpeptidase is mainly found in the liver. In the liver, it is
necessary for metabolism of drugs. It is also present in the pancreas, kidneys, and
spleen.
Normal Value
Interpretation
• GGT value rises in chronic viral hepatitis, alcoholic cirrhosis, and drug-induced
hepatitis.
Alkaline Phosphatase
It is orthophosphoric-monoester phosphohydrolase (hydrolase) enzyme.
It catalyzes breakdown of phosphate monoesters through addition of water at
alkaline pH. Its exact physiological functions are still obscure. In bones, it is
helpful in bone mineralization. ALP is a biomarker for osteoblastic activity in
bones.
Occurrence
Alkaline phosphatase is ubiquitous in distribution. It is widely found in body tis-
sues. It is abundantly found in the liver, bone, intestine mucosa, kidneys, and
placenta.
Normal Value
Its normal value is 20–140 IU/L.
Interpretation
• Elevation in alkaline phosphatase value is two times its normal value in hepato-
cellular jaundice.
• Elevation in alkaline phosphatase value is nearly ten times its normal in obstruc-
tive jaundice (posthepatic jaundice).
Significance
• GGT to ALP ratio has better diagnostic and prognostic value than either value of
test alone.
• GGT/ALP >1.4 is highly suggestive of alcoholic cirrhosis than other liver
diseases. Therefore, ratio has a value in differential diagnosis of liver
diseases.
22.6 Serum Enzymes in Gastrointestinal Tract Diseases
22.6.1 Serum Amylase
Amylase is a hydrolase. It splits dietary starch into maltose under normal physiolog-
ical condition. In diseases of pancreas (acute pancreatitis), salivary glands (acute
parotitis), kidneys (renal failure) and diabetes mellitus, serum value of amylase is
elevated.
Occurrence
• Amylase is found in the saliva and pancreatic juice.
Normal Value
Interpretation
In Acute Pancreatitis
• Elevation of serum amylase is found in acute pancreatitis. Serum amylase
>1000 IU/L is seen in first 24 h of onset of acute pancreatitis. It normalizes
within 2–3 days of onset.
In Salivary Gland Diseases

• Serum amylase is elevated in mumps, bacterial parotitis, and sialolithiasis (sali-
vary stones). Its value ranges between 500 and1000 IU/L in salivary gland
diseases.
22.6.2 Serum Lipase
Lipase is a hydrolase enzyme. It digests lipids by hydrolyzing the ester linkage in

triglycerides.
Occurrence
Lipase is secreted by Ebner’s gland, gastric glands, pancreatic glands, and intestinal
glands in alimentary canal.
Normal Value
Interpretation
• Serum lipase is elevated to 1000 times the normal value in acute pancreatitis. It
normalizes within 10–14 days after onset of disease.
• Serum lipase level is also elevated in duodenal ulcer, carcinoma of the pancreas,
and liver cirrhosis.
22.8 Liver Function Tests 565
22.7 Serum Enzymes in Bone Diseases
Serum alkaline phosphatase

Alkaline phosphatase activity is associated with bone mineralization. Its activity is
a biomarker for osteoblastic activity in bones.
Occurrence
It is found in tissues like liver, intestine, bone and placenta.
Normal Value
In males: 45–110U/L, In females: 40–100 U/L.
• This enzyme has high clinical significance in detection of bone diseases.
• Serum alkaline phosphatase is elevated in Pagets’ disease, rickets, and
osteomalacia.
• Serum alkaline phosphatase is decreased in hypophosphatasia (rare, hereditary
disorder where bone mineralization is impaired).
• Its value is elevated in malignancy of bones.
Serum Acid Phosphatase in Prostate Cancer

Acid phosphatase is hydrolase enzyme. It catalyzes breakdown of phosphate mono-
ester at low pH.
Occurrence
It is found in the prostate gland, kidneys, spleen, liver, and erythrocytes.
Normal Value
Its normal value is <2 ng/ml.
• Its value is elevated in prostate cancer.
22.8 Liver Function Tests
Liver is a major organ of human body that performs multiple functions. It is a mas-
ter organ that controls metabolism of carbohydrates, lipids, and proteins. It detoxi-
fies ammonia into urea. It metabolizes drugs. It synthesizes physiologically
important compounds. The liver excretes biles.
Definition
Liver function tests comprise a batter of biochemical tests that help in assess-
ment and management of liver diseases.
Indications
Liver function tests (LFT) serve following functions as:
Diagnosis of Liver Disease
Liver function tests are noninvasive biochemical tests. They help to assess function-
ing of the liver. They are helpful in diagnosis of liver disease.
Differential Diagnosis of Liver Diseases
Liver function tests help to differentiate liver diseases. For example, the ratio of
SGOT/SGPT describes pattern of liver diseases and helps in differential diagnosis
between viral hepatitis and alcoholic hepatitis.
Prognosis of Liver Diseases
Liver function tests are useful in detection of severity of liver diseases. They are also
needful in determining outcome of liver diseases.
Long-term Follow-Up
Liver function tests are helpful in long-term management of liver diseases. For
example, chronic hepatitis B and C and alcoholic cirrhosis are associated with com-
plications. It requires periodic evaluation of liver functioning and evaluation of
therapeutic response to disease.
Limitations
Lack sensitivity: Test sensitivity is the ability to detect persons actually suffer-
ing from a disease (true positives).
Liver function tests may have compromised sensitivity in certain liver diseases.
Example:
Normal LFT in non-cirrhotic portal hypertension.
Lack specificity: Test specificity is its ability to detect persons who are actu-
ally healthy (without disease) (true negatives).
Liver function tests have poor specificity in liver diseases.
Example:
Hypoalbuminemia is found in chronic liver diseases and renal failure.
Serum aminotransferases are elevated in liver diseases and acute myocardial
infarction.
22.8.1 Classification of Liver Function Tests
Group I (Tests for Excretory Function of the Liver)

• Serum bilirubin
• Urine bilirubin
• Urine urobilinogen
Group II (Tests for Enzymes of the Liver)

• Serum aminotransferases
• Serum alkaline phosphatase
• Serum γ-glutamyl transpeptidase
Group III (Tests for Synthesizing Function of the Liver)

• Serum total protein, serum albumin, and serum globulin
• Prothrombin time
Group IV (Test for Carbohydrate Metabolism)

• Glucose tolerance test
Group V (Test for Lipid Metabolism)

• Serum cholesterol level
Group VI (Test for Amino acid Metabolism)

• Serum ammonia level test
• Ammonia tolerance test
Group VII (Test for Detoxification Function)

• Hippuric acid synthesis test
22.8.2 Group I (Tests for Excretory Function of the Liver)
• Serum bilirubin
• Urine bilirubin
• Urine urobilinogen
Serum Bilirubin Test

Bilirubin is a metabolic product of heme catabolism in the spleen. It is insoluble in
water and is called as unconjugated bilirubin or indirect bilirubin.
It undergoes conjugation with glucuronic acid in hepatocytes and forms bilirubin
diglucuronide. It becomes water soluble and called as conjugated bilirubin or
direct bilirubin.
Principle of Estimation
Its estimation is based on van den Bergh reaction. Serum bilirubin reacts with
diazo reagent to form a purple-colored compound.
Normal Value
Serum total bilirubin varies between 0.2 and 1.2 mg/100 ml.
Serum direct bilirubin is 0.2 mg/100 ml (represents 15% of total serum
bilirubin).
Serum indirect bilirubin is <1.2 mg/dl (represents 85% of total serum
bilirubin).
Interpretation
In Prehepatic Jaundice (Hemolytic Jaundice)
• Indirect bilirubin level is elevated. VD Bergh reaction is indirect positive (pur-
ple color appears only after addition of alcohol).
In Hepatocellular Jaundice
• Direct and indirect bilirubins are elevated. VD Bergh reaction is biphasic (purple
color appears with diazo reagent, its intensity ↑ with alcohol).
In Posthepatic Jaundice (Obstructive Jaundice)

• Direct bilirubin is elevated. VD Bergh reaction is direct positive (purple color
appears on addition of diazo reagent in sample).
Unconjugated Hyperbilirubinemia
Conditions like hemolytic jaundice and Gilbert syndrome are associated with
increased bilirubin formation and impaired conjugation of bilirubin, respectively.
It results into elevation of unconjugated fraction of bilirubin in body (>85%) and
is called as unconjugated hyperbilirubinemia. Serum unit may rise to <5 times
the normal value of serum unconjugated bilirubin (<6 mg/dl).
Conjugated Hyperbilirubinemia
Condition like obstructive jaundice or cholestasis is associated with obstruction
inflow of the bile from the liver to the duodenum. As a result, conjugated bilirubin
regurgitates into systemic circulation leading to rise in fraction of direct bilirubin in
serum (>50%) and is called as conjugated hyperbilirubinemia.
Urine Bilirubin Test

Principle of Test
Urine bilirubin is estimated by Gmelin’s test. Concentrated nitric acid oxidizes bili-
rubin into biliverdin to form greenish color in test tube.
Direct bilirubin is water soluble. It can pass the glomerular filtration membrane.
It appears in urine and the condition is called bilirubinuria. It is associated with
increase in serum direct bilirubin.
Normal Value
Urine bilirubin is 0.02 mg/100 ml. It is not traceable under normal condition.
Interpretation
• Serum indirect bilirubin is elevated which is insoluble in water. Therefore, bili-
rubin is absent in urine. The condition is called as acholuric jaundice.
• Direct and indirect bilirubins are elevated.

• Direct bilirubin is formed in the liver. However, its excretion is obstructed and it
regurgitates into blood circulation. It is excreted by kidneys and appears in urine.
The condition is called as choluric jaundice.
rine and Fecal Urobilinogen

U
Bilirubin is reduced into urobilinogen by colon bacteria. It is a colorless com-
pound. A small amount of urobilinogen enters hepatic portal vein and reaches
the liver. It is delivered to circulation and excreted by kidneys. On exposure to
air, urobilinogen is oxidized into urobilin which provides yellow color to
urine.
Intestinal urobilinogen is reduced into stercobilin. It is excreted in feces. On
exposure to air, stercobilinogen is oxidized into stercobilin which provides
yellowish brown color to feces.
Principle of Test
Urine urobilinogen is detected by addition of Ehrlich reagent (mixture of paradi-
methyl amino benzaldehyde + Conc HCL) in 2 ml of urine sample. Red color of
solution appears in the presence of urobilinogen.
Normal Value
Urine Urobilinogen
Its value is 0.6 mg/per 24 h. In normal condition, it is untraceable in urine.
Interpretation
• Urine urobilinogen is increased. Urine appears dark yellow colored.
In Posthepatic Jaundice (Obstructive Jaundice) and Hepatocellular Jaundice

• Urobilinogen in urine is not detected.
Fecal Urobilinogen
Principle of Test
Fecal urobilinogen is detected by Edelman’s reagent (alcoholic mercuric chlo-
ride + alcoholic zinc chloride + amyl alcohol) with feces. Appearance of greenish
fluorescence indicates urobilinogen.
Normal Value
Its value ranges between 50 and 200 mg per day.
Interpretation
• Fecal urobilinogen concentration is increased. Feces appear dark brown
colored.
• Fecal urobilinogen is decreased. Feces appear pale colored.

• Urobilinogen in feces becomes absent. Feces appear clay colored.
22.8.3 Group II (Tests for Enzymes of the Liver)
• Serum aminotransferases
• Serum alkaline phosphatase
• Serum γ-glutamyl transpeptidase
Serum Transaminases
Serum Glutamate Pyruvate Transaminase (SGPT)

It is also called as alanine transaminase (ALT). It catalyzes reversible transamina-
tion between alanine and glutamate.
Occurrence
SGPT is primarily and abundantly found in liver cells.
Normal Value
Interpretation
• Elevation of SGPT >500 IU/L is suggestive of viral hepatitis, toxin-induced liver
disease, and ischemic liver disease.
• Value of SGPT rises in biliary cirrhosis (50–350 IU/L).
• Elevation of SGPT between 300 and 500 IU/L is found in Laennec’s cirrhosis.
• Elevation of SGPT between 150 and 300 IU/L is suggestive of obstructive jaun-
dice (posthepatic jaundice).
Serum Glutamate-Oxaloacetate Transaminase (SGOT)

tion between aspartate and glutamate.
Occurrence
SGOT is predominantly found in cardiac muscle fibers. It is also present in liver cells.
Normal Value
Interpretation
• Elevation of SGOT is seen in cirrhosis patients.
Ratio of SGOT/SGPT has better diagnostic and prognostic significance than

either test performed alone.
Significance
• Elevation in SGPT level starts before clinical appearance of jaundice. Its peak
value is attained between 7 and 10 days. SGPT normalizes within 4 weeks of
onset of acute viral hepatitis.
• SGOT/SGPT ratio is <1in hepatitis excluding viral hepatitis.

• SGOT/SGPT ratio is >1in advanced cirrhosis of liver and chronic hepatitis C.
• SGOT/SGPT ratio >2 is suggestive of chronic alcoholic cirrhosis.
Gamma-Glutamyl Transpeptidase (GGT)

It is a transferase enzyme. It catalyzes the transfer of gamma-glutamyl group from
donor molecule like glutathione to an amino acid.
Occurrence
Gamma-glutamyl transpeptidase is mainly found in the liver. In the liver, it is
necessary for metabolism of drugs. It is also present in the pancreas, kidneys, and
spleen.
Normal Value
Interpretation
• GGT value rises in chronic viral hepatitis, alcoholic cirrhosis, and drug-induced
hepatitis.
Alkaline Phosphatase
It is orthophosphoric-monoester phosphohydrolase (hydrolase) enzyme. It cata-
lyzes breakdown of phosphate monoesters through addition of water at alkaline
pH. Its exact physiological functions are still obscure. In bones, it is helpful in bone
mineralization. ALP is a biomarker for osteoblastic activity in bones.
Occurrence
Alkaline phosphatase is ubiquitous in distribution. It is widely found in body tis-
sues. It is abundantly found in the liver, bone, intestine mucosa, kidneys, and
placenta.
Normal Value
Its normal value is 20–140 IU/L.
Interpretation
• Elevation in alkaline phosphatase value is two times its normal value in hepato-
cellular jaundice.
• Elevation in alkaline phosphatase value is nearly ten times its normal in obstruc-
tive jaundice (posthepatic jaundice).
Significance
• GGT to ALP ratio has better diagnostic and prognostic value than either value of
test alone.
• GGT/ALP >1.4 is highly suggestive of alcoholic cirrhosis than other liver
diseases. Therefore, ratio has a value in differential diagnosis of liver
diseases.
22.8.4 Group III (Tests for Synthesizing Function of the Liver)
• Serum total protein, serum albumin, and serum globulin

• Serum prothrombin
erum Total Protein, Serum Albumin, Serum Globulin

S
The liver is the principal site of synthesis of albumin and globulin. Estimation of
serum albumin and globulin provide useful information regarding synthesizing
function of the liver.
Principle of Serum Total Proteins

Serum total protein can be estimated by biuret reaction. Protein in serum reacts
with copper sulfate in alkaline medium to form violet-colored biuret complex.
Serum protein concentration is proportional to intensity violet color.
Principle of Serum Albumin

Albumin in serum has the ability to interact with bromocresol green dye to form
BCG-albumin complex. This complex absorbs light at a specific wavelength.
Normal Value
• Serum albumin varies between 3.5 and 5.5 g/dl.
• Serum globulin varies between 2.5 and 3.5 g/dl.
• Serum total protein varies between 6 and 8 g/dl.
• Normal albumin/globulin ratio is 2:1.
Interpretation
• Serum albumin and globulin ratio remain normal in early stage of viral
hepatitis.
• In later stage, elevation of serum globulin has been found.
In Hepatocellular Disease
• Serum albumin is greatly reduced and serum globulin is elevated. A/G ratio is
reversed. It is highly suggestive of chronic cirrhosis liver.

• Serum albumin and globulin ratio remain normal.
Prothrombin Time
Prothrombin is a clotting factor and plasma protein. It is synthesized by liver.
Prothrombin estimation is performed in the form of prothrombin time.
It is the time needed for clotting of a sample of citrated plasma which con-
tains thromboplastin and calcium.
Normal Value
Normal value of prothrombin time is 14 s (PTcontrol).
Another method of expression of prothrombin time is international normalized
ratio (INR).
INR is the ratio of PT of patient to PT control as:
INR = PTpatient / PTcontrol

Normal value of INR is 1.0.
Interpretation
In Hepatocellular Diseases
• Prothrombin time is elevated depending upon severity of disease. It may rise to
ten times the normal value.
In Obstructive Jaundice
• Prothrombin time is elevated.
22.8.5 Group IV (Test for Carbohydrate Metabolism)
• Glucose tolerance test
lucose Tolerance Test

G
It determines the ability of body tissues to utilize carbohydrate after an intake of a
given amount of glucose.
22.8.6 Types of Glucose Tolerance Test
ral Glucose Tolerance Test

O
Preparation of Patient
• Patient is advised to abstain from eating or drinking at least 8–12 h before test.
• Patient should be alert physically and mentally.
Procedure of Test
• A baseline fasting blood sample is collected.
• Patient is asked to drink a solution of 75 g of glucose (recommended by WHO
for adults) dissolved in 250 ml of water within 5 min.
• After every 30 min, five blood samples are collected.
• All six samples are estimated to determine blood glucose level. A graph is plot-
ted for six values of blood glucose concentration against time and it is called
glucose tolerance curve.
Interpretation
Normal Glucose Tolerance Curve
Characteristics
• Fasting blood glucose level should be <110 mg/dl.
• At 1 h period, blood glucose level should be <180 mg/dl. It is the highest peak
of blood glucose concentration. It should not exceed the renal threshold for glucose.
• At 21/2 h period, fasting blood glucose level (<110 mg/dl) should be obtained.
Diabetic Glucose Tolerance Curve

• Fasting blood glucose level is elevated (>110 mg/dl). Fasting glucose level
between 110 and 125 mg/dl indicates borderline impaired glucose tolerance.
• At 1 h period, blood glucose level rises >180 mg/dl. The highest peak of blood
glucose concentration is obtained after 1 h.
• At 21/2 h period, fasting blood glucose level is not obtained. It confirms
hyperglycemia.
I ntravenous Glucose Tolerance Test

It is indicated in condition of malabsorption of glucose from alimentary canal.
Indications
• Coeliac disease
• Hypothyroidism
Procedure
• A dose of 3 g/kg of weight of glucose is administered intravenously in 50% solu-
tion in 5 min.
• Baseline blood sample and half hourly five blood samples are taken.
Interpretation of glucose tolerance curve is same as in OGTT.
22.8.7 Group V (Test for Lipid Metabolism)
• Serum Cholesterol
Liver regulates synthesis of cholesterol and helps in maintenance of serum cho-

lesterol level.
Normal Value
Normal total serum cholesterol varies between 150 and 250 mg/dl.
Interpretation
• Serum cholesterol level is reduced.

• Serum cholesterol is elevated.
22.8.8 Group VI (Test for Amino Acid Metabolism)
Serum Ammonia Level

Liver is the chief organ for transamination and deamination of surplus amino acids.
These metabolic activities result into formation of ammonia.
Principle
• Serum ammonia level is determined by blood gas analysis.
Ammonia Tolerance Test

Test determines capability of liver to convert portal ammonia from intestine into urea.
Procedure
• Patient is advised to avoid any food overnight before the test.
• Blood sample in fasting state is procured.
• Patient is given 10 g of ammonium citrate in flavored syrup base to drink.
• Successive four blood samples are collected at intervals of ½ h, 1 h, 2 h, and
3 h.
• Ammonia gas is estimated by blood gas analysis.
Normal Value
Normal serum ammonia level varies between 40 and 70 μg/dl.
Interpretation
In Hepatocellular Disease
• Serum ammonia level is elevated. Its value may exceed 300 μg/dl. The liver func-
tions to convert ammonia coming from the intestine via portal vein into the urea.
This activity is impaired in hepatocellular diseases like cirrhosis, chronic hepati-
tis B and C, and alcoholic cirrhosis.
• Serum ammonia level is highly elevated (hyperammonemia) in hepatic
encephalopathy.
22.8.9 Group VII (Test for Detoxification Function)
Hippuric Acid Synthesis Test

Test determines two hepatic functions when benzoic acid in the form of sodium benzoate
is administered in body. Test determines synthesizing ability of liver along with
conjugation capability of the liver. Overall, test assesses detoxification ability of the liver.
Principle
Test is based on the following chemical reaction in hepatocytes:
Benzoic acid + aminoacetic acid ( glycine ) ® hippuric acid

Procedure
• Patient is provided breakfast.
• After 2 h, patient is instructed void the bladder.
• Provides a drink containing 6 g of sodium benzoate in 200 ml of water.
• All urine samples till next 4 h are collected and combined. Amount of hippuric
acid is estimated.
Interpretation
In Healthy Condition
• An amount of 3 g of hippuric acid is excreted.
• Excretion of hippuric acid is decreased below 3 g.
Renal Function Tests

The kidneys are an indispensable excretory organ of the human body. The kidneys
perform multiple functions involving removal of waster substances, homeostasis,
and secretion of important hormones.
Functions of Kidneys
• Excretion of metabolic waste substances (urea, uric acid, xanthine, hypoxan-
thine, and creatinine)
• Excretion of exogenous substances from body (drugs, hormones)
• Homeostasis of acid-base balance
• Homeostasis of body fluid
• Secretion of hormones (renin, erythropoietin)
22.9 Renal Function Tests (RFT)
Renal function tests comprise a batter of biochemical tests that help in assess-
ment and management of renal diseases.
Indications
Renal function tests (LFT) serve the following functions.
Diagnosis of Renal Diseases
Renal function tests are noninvasive biochemical tests. They help to assess func-
tioning of kidneys. They are helpful in early detection of renal diseases.
Prognosis of Renal Diseases
Renal function tests are useful in detection of severity of renal diseases. They are
also needful in determining outcome of renal diseases.
Long-term Follow-Up
Renal function tests are helpful in long-term management of renal diseases.
These tests help to assess efficacy of drugs and need for kidney replacement.
22.9 Renal Function Tests (RFT) 577
Classification of Renal Function Tests

Group I (Tests for Glomerular Filtration)
• Urea clearance test
• Creatinine clearance test
• Inulin clearance test
Group II (Tests for Renal Plasma Flow)

• P-amino hippuric acid test
• Filtration fraction test
Group III (Tests for Tubular Functions)

• Concentration and dilution tests
Glomerular Filtration Tests

Glomerular filtration test estimates the capability of nephrons to form glomerular fil-
trate. In a healthy adult person, 180 L glomerular filtrate is formed in 24 h by kidneys.
Normal Value of Glomerular Filtration Rate (GFR)

Its normal value is 125 ml/min.
Glomerular filtration rate cannot be estimated directly. It is assessed by clear-
ance test.
Definition
Clearance (C) is the volume of plasma which is cleared from an indicator sub-
stance in 1 min.
Clearance of a substance can be calculated by the following formula:
U´V
Clearance of substance ( Z ) =
P
where U = Concentration of substance in urine, V = Volume of urine, P = Concentration
of substance in plasma.
Since
GFR = clearance of an indicator substance subject to certain characteristics,

therefore
GFR = U z ´ V / Pz

where Uz (concentration of substance in urine) and Pz (concentration of substance in
plasma).
Characteristics of Indicator Substance

• In case of endogenous substance, the rate of its formation should be constant.
• In case of exogenous substance, it should be inexpensive, easy to administer, and
physiologically inert.
• Indicator substance should be freely filtered by kidneys.

• It should not be reabsorbed by tubules.
Based on type of indicator substance, clearance tests are the following types:
Urea Clearance Test

Kidneys excrete urea from plasma.
Creatinine Clearance Test

Creatinine is endogenous substance. It is a metabolic waste product formed from
decomposition of creatine phosphate in skeletal muscles.
Characteristics of Creatinine as Indicator Substance
• Creatinine is synthesized at constant rate.
• Its synthesis is proportional to muscle mass.
• It is freely filtered by kidneys.
• It is not reabsorbed by tubules.
• It is secreted by renal tubules.
Procedure
• Urine sample is collected in 24 h.
• Blood sample is taken.
• Measure volume of urine, urine creatinine concentration, and serum creatinine
concentration.
Principle
• Serum creatinine is estimated by Jaffe’s reaction. Creatinine reacts with picric
acid in alkaline medium to form reddish orange color. Its intensity is proportional
to creatinine concentration.
Creatinine clearance = GFR
Creatine clearance = U cr ´ V / Pcr
Normal Value
• Normal value of serum creatinine is 0.7–1.2 mg/dl in males and 0.6–1.1 mg/dl in
females.
• Serum creatinine is inversely proportional to renal function.
• Normal creatinine clearance is nearly 105 ml/min.
Interpretation
• Serum creatinine concentration is elevated in renal diseases. It is inversely pro-
portional to GFR.
• Creatinine clearance is decreased in renal diseases.
22.9 Renal Function Tests (RFT) 579
Remarks
• Creatinine is secreted by renal tubules. In renal failure, GFR decreases. However,
total creatinine clearance increases owing to tubular secretion. So it is not an
ideal and reliable biomarker for renal function. It leads to overestimation of
GFR.
Inulin Clearance Test

Inulin is a homopolysaccharide of fructose. It is employed as exogenous marker for
ascertaining renal dysfunction.
Characteristics of Inulin as Indicator Substance
• It is an exogenous substance.
• It is easy to administer.
• It is economical and chemically inert substance. It is not metabolized in the
body.
• It is freely filtered by the kidneys.
• It is neither reabsorbed nor secreted by renal tubules.
Procedure
• Test is performed after a light breakfast.
• Solution of 10 g of inulin in 100 ml water is prepared. It is administered through
IV route.
• Urine is collected after 2 and 4 h. Urine volume is measured.
• Blood sample is collected after 2 h of inulin administration. Serum inulin is
estimated.
Inulin clearance = U inulin ´ V / Pinulin

Normal Value
Its normal value is 125 ml/min. Inulin clearance is almost comparable to glomerular
filtration rate.
Interpretation
• Inulin clearance is decreased in renal diseases. It indicates decline in glomerular
function of kidneys.
Test for Renal Blood Flow

This test is not performed in routine clinical practice.
Para-aminohippuric acid is utilized as a marker to determine for renal plasma
flow (RPF). RPF is estimated by PAH clearance, since it is filtered by glomeruli and
also secreted by tubules.
Normal Value of RPF
• In males
–– Its value is 650 ml/min/1.73 m2.
• In females
–– Its value is 600 mL/min/1.73 m2.
Tests for Tubular Function

Renal perfusion is important in determining normal tubular functioning. Renal
hypoperfusion due to hyperdynamic changes, drug- and/or toxin-induced, definitely
reduces blood flow to kidneys. It impairs tubular functions.
Concentration Test
Principle
Concentration test determines capability of kidneys to reabsorb water and concen-
trate urine. Test relies on estimation of specific gravity of urine by urinometer.
Procedure
• Patient is advised to have dinner 12 h before the test (8PM if test is scheduled on
next day morning 8AM). Thereafter, patient is advised to keep fasting till next
day morning.
• Any urine passed midnight is discarded.
• In the morning at 8AM, collect sample of urine. Thereafter, two more urine
samples are collected after an interval of 1 h (9AM–10AM).
Normal Value
• Normal specific gravity of urine is 1.025.
Interpretation
• In normal tubular functioning, specific gravity of at least one sample should be
≥1.025.
• In abnormal tubular functioning, specific gravity of urine sample is <1.020.
• In severe renal failure, specific gravity of all urine samples is fixed at 1.010.
Dilution Test
Principle
Test determines capability of the kidneys to remove water. Test relies on estimation
of urine output and its specific gravity after intake of a given volume of water.
Procedure
• Patient is advised to have dinner 12 h before the test (8PM if test is schedule on
next day morning 8AM). Thereafter, patient is advised to keep fasting till next
day morning.
• Patient is asked to empty bladder and discard urine.
• Patient is given 1200 ml of water to drink in 30 min.
• Four urine samples are collected after an interval of 1 h in between each sample.
Interpretation
• Normal Kidney Function
–– About 80% of total water intake (1000 ml) should be evacuated in urine
within 4 h.
–– Specific gravity of at least one urine sample should be ≤1.003.
• Kidney Dysfunction
–– Excretion of water intake is delayed.
–– Specific gravity of samples should not be ≤1.0031. It becomes almost con-
stant at 1.010.
Suggested Readings
Latner AL, Cantarow A (1975) Clinical biochemistry, 7th edn. Saunders, Philadelphia
McGilvery RW (1983) Biochemistry-a functional approach, 3rd edn. Saunders, Phialdelphia
Rawn JD (1989) Biochemistry. Neil Patterson Publishers, Burlington, NC
Thorpe WB, Bray HG, James HP (1970) Biochemsitry for medical students, 9th edn. Churchill,
London
Part V
Immunochemistry
Immunoglobulins
23
Extrinsic substances invade the body. They stimulate the immune system of the
host. It produces endogenous substances which destroy extrinsic substances. The
former substance is the antigen, while the latter is called as antibody.
23.1 Definition
Antigen
Antigen is exogenous organic molecule which can elicit either one or both types
of immune reactions in the body of host.
Generally, antigens can be polypeptides or polysaccharides in nature. For exam-
ple, pneumococcal capsule contains polysaccharide as antigen. The peptidoglycan
is the chief structural component of gram-positive bacteria. It is composed of poly-
mer of disaccharides cross-linked to peptides. Bacterial peptidoglycan is antigenic
in nature.
Lipids and nucleic acids are rarely antigen DNA fragments, or oligoribonucleo-
tides covalently linked to large protein molecule can act as antigen. Lipids or ste-
roids linked to large protein molecule are antigens and are called as “haptene,” for
example, aniline, dinitrophenol, and hydralazine.
Antibody
Antibody is an endogenous glycoprotein which exhibits specificity for a par-
ticular antigen.
In antibody, glycoprotein is made up of oligosaccharides covalently attached to
polypeptides. This is called as glycosylation. The polypeptides belong to immuno-
globulin superfamily of proteins. Immunoglobulin superfamily is constituted by cell
surface proteins and soluble proteins. Antibody and immunoglobulin are synony-
mously used terms.
Immunoglobulins are endogenous glycoproteins belonging to immunoglob-
ulin superfamily of proteins.

https://doi.org/10.1007/978-981-13-1035-5_23
586 23 Immunoglobulins
Immunoglobulin represent gamma-globulin fraction of plasma proteins. They

constitute around 20% of total plasma proteins. An immunoglobulin is composed of
about 80–96% of protein and 4–10% of oligosaccharides.
23.2 Structure of Immunoglobulin
23.2.1 Edelman-Gally Model of Immunoglobulin
• Immunoglobulin molecule is Y shaped.

• Ig molecule is a heterotetramer composed of four polypeptide chains. Two heavy
chains represented as “H”-chain and two light chains represented as “L”-chain
are linked together by disulfide bonds. Immunoglobulin molecule can be written
as (H2 L2).
• Each H-chain is made up of 450 amino acid residues and each L-chain is com-
posed of 212 amino acid residues. Amino acid residues are arranged into specific
domains. Each domain contains around 110 amino acid residues. These domains
are interconnected by intra-chain disulfide bonds.
• The domains are broadly grouped into two categories as variable and constant
domains. In L-chain, the domain located toward N-terminal is called as variable
domain of light chain (VL), whereas the domain located toward C-terminal is
called as constant domain of light chain (CL). The sequence of amino acid resi-
dues is variable in variable domains.
• In H-chain, the domain located toward N-terminal is called as variable domain of
heavy chain (VH), and the domain on the C-terminal is called as constant domain of
heavy chain (CH). There are three constant regions in H-chain as CH1, CH2, and CH3.
The sequence of amino acid residues is comparatively stable in constant domains.
• Heavy chain characteristics.
–– On the basis of structure, five classes of H-chains have been identified among
humans. Their molecular weight is about 55,000. They are labeled as alpha
(α), gamma (γ), mu (μ), epsilon (ε), and delta (δ). Class of H-chain deter-
mines the type of immunoglobulin. Five types of immunoglobulins are named
as IgA, IgG, IgM, IgE, and IgD, respectively.
• Light chain characteristics.
–– On the basis of structure, two classes of L-chains have been identified among
humans. Their molecular weight is about 25,000. They are labeled as kappa
(k) and lambda (λ). A particular Ig molecule always possesses either kappa or
lambda class of light chain.
• Hinge region.
–– It is an inter-domain region on heavy chain. It is a highly variable region of
amino acid residues located in the central portion of H-chain. The hinge
region has predominantly cysteine and proline amino acid residues. It is sensi-
tive to enzymatic proteolysis.
23.2 Structure of Immunoglobulin 587
• Antigen-binding fragment (Fab).

–– It is the region of antibody which has specificity to antigen. It is composed of
two constant domains (CH and CL) and two variable domains (VH and VL)
toward N-terminal. Variable domains possess antigen-binding site called as
“paratope.” The antigen contains a specific site that attaches to the antibody
and is called as “epitope.” It is the antigen determinant.
• Fragment crystallization region (Fc).
–– This region is located toward the tail end of antibody. It is composed of three
constant domains (CH-2-4) in each H-chain. This region interacts with cell sur-
face receptors of immune cell. Fc region is helpful in the activation and regu-
lation of immune response.
• Enzymatic proteolysis of immunoglobulin.
–– Papain enzyme breaks immunoglobulin molecule into three similar-sized
fragments. Two fragments are named as “Fab,” and the third fragment is
named as “Fc.”
–– Pepsin enzyme cleavages Ig molecule below hinge region and yields a large
fragment called as F(ab)2 which can be further cleavaged into two Fab frag-
ments. Pepsin degrades Fc fragment completely.
–– Another enzyme, called as immunoglobulin-degrading enzyme from
Streptococcus pyogenes (IdeS), breaks IgG into F(ab)2 fragment as in
Fig. 23.1.
HT
LIG AIN ANTIBODY
CH BINDING SITE
2
NH
S
2
NH S
S HEAVY
HEAVY S S CHAIN
CHAIN
GIO E
S
RE IABL
N
SS
R
VA
S S O H COO
H
S CO
S
CONSTANT
REGION HINGE S S
REGION S S
S
S COMPLIMENT BINDING SITE
S
S
COOH COOH
Fig. 23.1 Structure of Immunoglobulin

23.3 Characteristics of Individual Immunoglobulins
23.3.1 Immunoglobulin A (IgA)
Features
• It is composed of two H-chains of class alpha (α) and two L-chains of class either
kappa or lambda class.
• IgA exists either as a single Y-shaped monomer or a dimer linked by J chain.
• Its molecular formula is α2k2 or α2λ2.
• Its molecular weight is between 150,000 and 500,000.
• Its carbohydrate content is 8%.
• IgA represents about 10–20% of total immunoglobulins.
• Its normal serum concentration is between 150 and 400 mg/dl.
• IgA is abundantly found in body secretions like saliva, sweat, tears, milk,
and gastrointestinal, nasal, and bronchial secretions. It is a highly predomi-
nant antibody in colostrum (first secretion from human breast after birth
of baby).
Functions
• It provides local immunity against pathogens. It is due to its high concentration
in body fluids. It prevents invasion of pathogens from the skin as well as from
mucosal surfaces. It binds with the antigens and destroys them.
23.3.2 Immunoglobulin G (IgG)
Features
• It is composed of two H-chains of class gamma (γ) and two L-chains of class
either kappa or lambda.
• It exists as a single Y-shaped monomer.
• Its molecular formula is γ2k2 or γ2λ2.
• IgG represents about 70–80% of total immunoglobulins. It is the maximum
abundant immunoglobulin in body.
• IgG is the unique antibody that can cross placental barrier. It provides immunity
to growing fetus. IgG can also cross pass through blood vessels.
• It is the predominant antibody that is produced in secondary immune response.
Functions
• IgG is the most predominant antibody against most of the bacterial and viral
infections.
• It provides humoral immunity against bacterial and viral infections.
23.3 Characteristics of Individual Immunoglobulins 589
23.3.3 Immunoglobulin M (IgM)
Features
• IgM is the largest antibody. It is composed of five Y-shaped monomeric units, so
IgM is a pentameric immunoglobulin.
• Each Y-shaped monomer is composed of two H-chains of class “mu” (μ) and two
L-chains of class either kappa or lambda.
• Its molecular formula is (μ2 k2)5 or (μ 2λ2)5.
• Its molecular weight is about 900,000.
• IgM represents about 7% of total immunoglobulins in the body.
• In IgM, individual monomers are linked together by J-shaped polypeptide chain.
This chain possesses high amount of aspartic acid and glutamic acid residues.
This polypeptide chain is highly elongated.
• Due to large size of IgM, it cannot pass through the blood vessels. It cannot cross
the placental barrier. It is the predominant antibody that circulates in the blood.
• It can bind with five antigenic sites simultaneously.
Functions
• IgM is the first-line antibody. It is the predominant antibody that is produced in
primary immune response.
• IgM constitutes naturally antibodies. They are the immunoglobulins of class IgM
produced by B lymphocytes in the absence of any antigenic exposure. Natural
antibodies are anti-A, anti-B, and anti-Rh antibodies.
23.3.4 Immunoglobulin D (IgD)
Features
• IgD is composed of two H-chains of class delta (δ) and two L-chains of class
• Its molecular formula is δ2k2 or δ2λ2.
• IgD represents about 0.5–2% of total immunoglobulins.
• Its normal serum concentration is around 5 mg/dl.
• It is the predominant antibody that is located on the cell surfaces of B lym-
phocytes along with another antibody IgM.
Functions
• Function of IgD is uncertain.
• IgD is helpful in the differentiation of B lymphocytes. IgD coats the surfaces of
B lymphocytes along with IgM.
• IgD is necessary for activation of B lymphocytes.

• IgD stimulates mast cells and basophils to release antimicrobial substances. So
IgD provides local immunity in respiratory system.
23.3.5 Immunoglobulin E (IgE)
Features
• IgE is composed of two H-chains of class epsilon (ε) and two L-chains of class
• Its molecular formula is ε2k2 or ε2λ2.
• IgE represents about 0.004% of total immunoglobulins.
• Its normal serum concentration is 0.02–0.05 mg/dl. It occurs in least
concentration.
• IgE is bound to Fc cell surface receptors on mast cells through its Fc region. The
IgE coats mast cells and is responsible for allergic response of host body. It is
called as “reaginic antibody.”
Functions
• Dual nature of IgE

–– IgE antibody binds with allergens through its Fab region. But IgE binds to
surface of mast cells through its Fc region.
• Protection against helminthic infection
–– IgE is primarily involved in defense against helminthic infection caused by
Schistosoma mansoni, Fasciola hepatica, and Trichinella spiralis parasites.
• Defense against Plasmodium
–– IgE has a role in immune response in Plasmodium parasite infection.
• Type-1 hypersensitivity
–– IgE is responsible for type-1 hypersensitivity. IgE interacts with allergens on
the surface of mast cells. The allergen-antibody complex induces degranula-
tion of mast cells. These granules release histamine, prostaglandins, and leu-
kotrienes. These substances are responsible for allergic manifestations in
body of the host. IgE is implicated in allergic asthma, allergic rhinitis, sinus-
itis, urticarial, and atopic dermatitis.
Suggested Readings
Charles J (2001) Immunobiology, 5th edn. Garland Publishing, Oxford
Edelman GM, Gally JA (1964) A model for the 7S antibody molecule. Available at: http://www.
pnas.org/content/51/5/846.full.pdf
Kirkham PM, Schroeder HW Jr (1994) Antibody structure and the evolution of immunoglobulin V
gene segments. Semin Immunol 6:347–360
Kleiner IS, Orten JM (1966) Biochemistry, 7th edn. Mosby publisher, St Louis
Pier GB, Lyczak JB, Wetzler LM (2004) Immunology, infection, and immunity. ASM Press,
Washinton DC
Rawn JD (1989) Biochemistry. Neil Patterson Publsihers, Burlington, NC
Thorpe WB, Bray HG, James HP (1970) Biochemsitry for medical students, 9th edn. Churchill,
London
Part VI
Dental Biochemistry
Dental Biochemistry
24
24.1 Introduction
The teeth are the calcified tissues of the oral cavity which serve to masticate foods,
are helpful in speech, and have aesthetic function. A tooth is made up of calcified
tissues and vascular tissues. Calcified tissues cover crown and root portions of the
tooth. The calcified tissues are labeled as follows:
• Enamel (the hardest tissue of body and surrounds crown of tooth)

• Dentin (forms crown and root of tooth)
• Cementum (hard tissue that surrounds root of tooth)
Chemical composition of three layers is described separately as follows:
• Chemical composition of enamel

• Dentin
• Cementum
24.2 Enamel
Enamel is the outermost, avascular, nonliving, calcified and protective layer over the
crown of teeth.
Characteristics of Enamel
• Enamel is developed from the ectodermal layer.
• Enamel is the protective layer of the crown of teeth.
• Enamel is synthesized by ameloblasts.
• It is the hardest tissue of the body.
• It is minimally porous in nature.

https://doi.org/10.1007/978-981-13-1035-5_24
596 24 Dental Biochemistry
• Its color is yellowish white.

• Enamel thickness is maximum at cusps of molars and premolars (2–2.5 mm). Its
thickness is minimum at the neck region of the tooth.
24.2.1 Chemical Composition of Enamel
Enamel is composed of water and solids. These constituents of enamel layer are
explained as follows:
Water
• Water constitutes only 3% of weight of enamel.
• Water is present in loosely bound state in organic matrix and in hydroxyapatite
crystals.
Solids
Solids in the enamel are further sub-divided into organic solids and inorganic solids.
Each type of solid component of enamel is described below:
Organic Solids
• Organic solids form only 1% of the weight of tooth enamel.
• Organic solids are deposited by ameloblasts in developmental stage of tooth.
• In mature tooth, organic solids are present around enamel rods. Enamel rods are
the densely packed mass of hydroxyapatite crystals which are supportive units of
enamel of teeth.
Chemical nature of organic solid in enamel:

• Chemical structure of organic solids in the enamel is variable and it depends
on the stage of tooth development. it is described under two headings as
below:
–– Developmental enamel
Histological sectioning of developmental enamel shows that organic matrix
of enamel resembles to keratin. Nevertheless, analysis of amino acid com-
position of mature enamel does not prove keratin nature of organic matrix.
–– Mature enamel
Analysis of proteins in mature enamel shows high proportion of amino
acids like serine, glycine, and glutamic acid in organic matrix of enamel.
X-ray diffraction studies of mature enamel show the presence of beta-pleated

structure of proteins in enamel matrix. Organic solids in enamel contains predomi-
nantly proteins like amelogenin, and enamelin. The important characteristics of
these proteins are described below:
24.2 Enamel 597
Enamel Proteins
Amelogenin
• It is the chief structural protein of enamel.
• Amelogenin is secreted by ameloblasts in developmental stage. It is the chief
protein of extracellular matrix of enamel. It constitutes around 90–95% of all
enamel proteins.
• Amelogenin is rich in serine, proline, leucine, and histidine amino acid
residues.
• Amelogenin residues aggregate to form nanoaggregates. They provide nucleus
to initiate crystallization. It regulates growth and alignment of apatite crystals.
Enamelin
• Enamelin is another structural protein of enamel.
• It represents only 1–2% of total enamel proteins.
• Enamelin is necessary for normal synthesis of enamel. Enamel synthesis is regu-
lated by ENAM gene. Any mutation in ENAM gene results in a disorder called
as enamel hypoplasia (amelogenesis imperfecta).
• Enamelin controls the formation of amelogenin nanoaggregates.
Inorganic Solids
• Inorganic solids constitute chief structural and supportive components of tooth
enamel. They constitute around 96% of the weight of enamel.
• Calcium and phosphorous are the chief minerals in inorganic components of
enamel. These minerals exist in crystalline form which are called as apatite
crystals. Apatite crystals are associated with hydroxyl ions and are named as
hydroxyapatite crystals.
• These crystals are highly organized and tightly packed to form enamel rods.
• Hydroxyapatite crystals
–– Hydroxyapatite crystals are hexagonal shaped.
–– Hydroxyapatite crystals are made up of calcium phosphate (apatite crystals)
with hydroxyl ions. Their formula is Ca10(PO4)6 X2.
–– Width of crystals is 60 nm and thickness is 30 nm.
–– Calcium ions are arranged to form a hexagon. Inside a hexagon, three calcium
ions are arranged to form a triangle. Two such triangles are placed parallel to
each other inside a hexagon.
–– Phosphate ions are arranged in two tetrahedrons in between two calcium ions.
One tetrahedron is made up of one phosphorous and four oxygen atoms.
–– Two hydroxyl ions are located inside calcium triangles within hexagon.
Fluoride ions can replace hydroxyl ions to form “fluoroapatite crystals.”
These crystals are less soluble in acids and more stable than hydroxyapatite
crystals. Fluoroapatite crystals render enamel resistant to dental caries.
Trace Minerals in Inorganic Solids of Enamel

• Minerals like fluoride, zinc, chloride, and selenium are present in higher concen-
tration on the surface enamel than deeper layers of enamel.
• Minerals like sodium, magnesium, and carbonates are present in higher concen-
tration in deeper layer of enamel than its surface layer.
24.3 Dentine
Dentine is the inner, calcified, avascular, and sensitive layer of teeth that forms
crown and root of teeth.
Characteristics of Dentine
• Dentine is developed from mesodermal layer.
• It is yellow in color.
• Dentine is synthesized by odontoblasts. They are large sized columnar cells and
are packed between dentine and pulp in the tooth. These cells form dentine
through the process of dentinogenesis.
• Hardness property of dentine is higher than the bone, lesser than enamel, and
almost equal to cementum.
• Inner to Dentine, pulp cavity is present in the tooth. This cavity contains highly
vascular soft tissues. It is richly innervated.
24.3.1 Chemical Composition of Dentine
Dentine is made up of water and solids. Solids are further grouped into inorganic
solids and organic solids as follows:
Water
• Water represents nearly 10% of weight of dentine.
• Water is present in bound state in organic matrix and in hydroxyapatite crystals
in dentine.
Organic solids
• Organic solids constitute around 20% of weight of dentine.
• Organic solids are further grouped into two categories as dentine proteins
and ground substance which are explained below as:
–– Dentine proteins are the chief organic elements of dentine. The collagen
and sialophosphoprotein are the predominant dentine proteins. Their
characteristics are described below:
Collagen
Collagen represents about 90% of the total organic solids of dentine. It is an
important structural protein of dentine.
24.4 Cementum 599
Dentine contains type-I collagen protein. It is made up of two alpha-1

chains and one alpha-2 chain. These three chains are right-handed helically
coiled to form a triple helix protein structure.
Each chain contains 1000 amino acids residues. Glycine residues consti-
tute 30% of total amino acid residues in each chain. Proline and lysine together
represent another 30% proportion of amino acid residues in each chain.
Other dentine proteins
Dentin sialophosphoprotein
It is secreted by odontoblasts in pulp of teeth and osteoblasts in bone tissues.
This protein is essential for calcification of organic matrix of dentine.
Dentine sialophosphoprotein is a precursor molecule that produces three
dentine proteins as dentin sialoprotein, dentin glycoprotein, and dentin
phosphoprotein.
Three proteins constitute chief non-collagen proteins of dentine. They con-
trol calcification of dentine.
–– Ground substance
Ground substance constitutes about 10% of the total organic solids of dentine.
It is an amorphous gel-like substance in extracellular matrix in dentine.
It is made up of glycosaminoglycans (GAG) and peptides. The prominent
GAGs are chondroitin sulfate moieties. They are linked with short peptides to
form glycoproteins.
Inorganic Solids
• Inorganic solids represent around 70% of the weight of dentine.
• They are mainly calcium and phosphate ions which are crystalized in form of
hydroxyapatite crystals as in enamel.
• Size of hydroxyapatite crystals in dentine is smaller than enamel. It is 1/10 of the
size in enamel.
24.4 Cementum
It is a calcified and protective layer on root of the teeth.
Characteristics of Cementum
• It is yellowish in color and mildly softer in comparison to dentine.
• Primary cementum is present on coronal 1/3 of root. It is acellular. Secondary
cementum is present on middle 1/3 and apical 1/3 of root. It is cellular in nature.
24.4.1 Chemical Composition of Cementum
Water
• Water represents 10% of weight of cementum.
Organic Solids
• It forms about 25% of weight of cementum.
• It is mainly composed of type-I collagen fibers.
Inorganic Solids
• It forms about 65% of the weight of cementum.
• It is in the form of hydroxyapatite crystals.
24.5 Biochemical Basis of Dental Caries
24.5.1 Dental Caries
Definition
Dental caries is defined as a microbial, invasive, and progressive disorder char-
acterized by demineralization of inorganic components and proteolysis of
organic components of teeth.
24.5.2 Role of Biochemical Compounds Involved in Dental Caries
Role of Sucrose and Lactic Acid

• Streptococcus mutans is a gram-positive bacterium. It is the predominant bacte-
ria of the oral cavity. It is present in dental plaque.
• S. mutans is the acidogenic bacteria. It has glucansucrase enzyme. This enzyme
can split dietary sucrose into glucose and fructose. Glucose is converted into
lactic acid through glycolysis pathway.
• Lactic acid decreases pH at the interface dental plaque and enamel surface. This
activity favors demineralization of hydroxyapatite crystals and initiation of
dental caries.
Role of Extracellular Polysaccharides

• S. mutans have remarkable ability to synthesize extracellular polysaccharides
(EPS) from sucrose.
• Bacteria can decompose sucrose into glucose and fructose by glucansucrase
enzyme. S. mutans has enzyme glucosyltransferase which catalyzes polymeriza-
tion of glucose into glucans. Another enzyme fructosyltransferase catalyzes con-
version of fructose into long chain polysaccharides called as fructans.
• Glucans and fructans are extracellular polysaccharides (mucilaginous carbohy-
drates) which are clearly and significantly implicated in initiation and progres-
sion of dental caries.
• EPS has the following impacts as follows:
–– EPS promotes adherence of bacteria to enamel pellicle and dental plaque.
–– EPS is insoluble in water. It serves as barrier between enamel and oral envi-
ronment. It retains acids in close contact with enamel.
–– EPS helps to raise thickness of plaque.
24.5 Biochemical Basis of Dental Caries 601
The role of sucrose, lactic acid, and EPS has been postulated in acidogenic
theory of dental caries. It was proposed by W. D. Miller in 1890.
Theory states that dental caries involves decalcification of hard tissues of teeth
owing to acids produced by fermentation of dietary sucrose by bacteria.
24.5.3 Fluoride as Anticaries Agent
Fluorine is electronegative element and exists in nature as fluoride in bound form.

Fluoride is present in water, tea, and fish. It is absorbed from intestinal mucosa and
enters circulation. Fluoride is concentrated into calcified tissues of body during
mineralization of bones and teeth.
Fluoride is incorporated into hydroxyapatite crystals during development stage.
It replaces hydroxyl ions, and resultant crystals are termed as fluoroapatite
crystals.
In enamel, fluoride concentration is higher in superficial layer of enamel than
deeper layer.
In dentine and cementum, its concentration is higher in deeper layers. Dentine
and cementum store greater amount of fluoride than enamel.
Mechanism of Action of Fluoride

1. Fluoroapatite Crystals
• Apatite crystals are surrounded by hydration shell (layer of adsorbed water).
Fluoride ions pass through hydration shell and combines with calcium ions to
form a thin layer of calcium fluoride. It protects deeper apatite crystals. Later
on, surface fluoride ions are incorporated into crystal lattice. It replaces
hydroxyl ions to form fluoroapatite crystals.
• Fluoroapatite crystals are resistant to acid attack. Their solubility in acid is
less to that of hydroxyapatite crystals. Fluoroapatite crystals have higher dis-
solution time than hydroxyapatite crystals. Higher dissolution time promotes
remineralization of enamel surface.
2. Inhibition of Enolase Enzyme
• Enolase enzyme catalyzes conversion of phosphoenolpyruvate into pyruvate.
Fluoride ions inhibit enolase enzyme in bacteria. Fluoride ions intercept
glucose metabolism of bacteria. Overall, fluoride ions prevent formation of
lactic acid.
24.5.4 Salivary Proteins in Dental Caries
Dental caries is a progressive disorder caused by multiple factors. Salivary proteins

have protective and diagnostic roles in dental caries.
Saliva contains lactoferrin, immunoglobulins, lysozymes, albumin, and mucin as
important proteins. Salivary mucin and glycoproteins rich in proline are helpful in
formation of a coat over enamel surface. These proteins protect enamel surfaces of
teeth from attrition and abrasion. Salivary proteins protect from demineralization of
enamel surfaces. Proteins retain calcium and phosphate ions in contact with enamel
surfaces and are helpful in remineralization.
Immunoglobulin A is the predominant antibacterial protein of saliva. It is synthe-
sized by plasma cells and secreted in saliva. IgA prevents proliferation of cariogenic
organisms and decreases their colonization in plaque.
Mucin is a glycoprotein and main salivary protein. Mucin plays multiple roles in
oral cavity. Mucin forms a sticking coat on the surfaces of teeth. It protects teeth
from acid attack by minimizing contact with enamel surface. Mucin decreases bac-
terial colonization and adherence on tooth surfaces.
Proline-rich proteins in saliva are basic proteins. They have affinity to hydroxy-
apatite crystals. These proteins help to neutralize acid produced by S. mutans. These
proteins chelate free calcium ions in saliva and help in remineralization of enamel.
C-reactive protein is an acute phase protein. It has a diagnostic role in dental car-
ies and periodontal diseases. It is synthesized in the liver. It is secreted by salivary
glands in the oral cavity. It is an important biomarker in dental diseases.
24.6 Chemical Composition of Saliva
Saliva is a colorless viscous fluid secreted by salivary glands.
Saliva contains 99.5% water and 0.5% solids.
Solids
Organic Solids
Organic solids can further be subdivided into following substances as:
• Enzymes
–– Salivary amylase
Salivary amylase is a calcium-dependent metalloenzyme. It is also called as
alpha-amylase or ptyalin. It splits starch into maltose and dextrin.
–– Maltase
Maltase is a disaccharide-splitting enzyme. It cleavages maltose into two mol-
ecules of glucose.
–– Lingual lipase
Lingual lipase is secreted by Ebner’s gland on the dorsum of the tongue. It is
an acidic lipase. It hydrolyzes triglycerides into mono- and diglycerides with
release of free fatty acids.
–– Lysozymes
Lysozyme is an antibacterial enzyme. It hydrolyzes 1,4-glycosidic bond
between N-acetylmuramic acid and N-acetyl-D-glucosamine in peptidoglycans
of bacterial cell wall. Lysozymes destroy bacteria in saliva.
–– Carbonic anhydrase
Carbonic anhydrase helps to maintain pH homeostasis in the oral cavity.
Saliva has buffering activity.
–– Kallikrein
24.6 Chemical Composition of Saliva 603
Kallikrein is a proteolytic enzyme. It is secreted by acinar cells of salivary

glands. Kallikrein splits kininogens into bradykinin (vasodilator).
–– Lactoperoxidase
Lactoperoxidase belongs to peroxide enzyme. It catalyzes oxidation of thio-
cyanates, bromides, and iodides. It is a strong antibacterial enzyme. It pro-
vides innate immunity against pathogens in oral cavity.
• Proteins
–– Immunoglobulin-A
IgA is an antibody. It is synthesized by plasma cells and secreted into saliva
by acinar cells of salivary glands. IgA provides local immunity in oral cavity.
–– Proline-rich proteins
Proline-rich proteins are intrinsic disordered proteins. These proteins are devoid
of regular three-dimensional structure. These proteins contain multiple short
repeats of proline-rich sequences. Proline-rich proteins in saliva help to bind
calcium to enamel surface and have antimicrobial property, for example, PRB4.
–– Lactoferrin
Lactoferrin is an iron-binding conjugated protein. It belongs to the transferrin
family. Lactoferrin is secreted by salivary glands. It helps to regulate
concentration of free iron in the blood and secretion of the body. It is also a
part of the innate immune system. Lactoferrin sequesters free iron in saliva.
Iron is a growth factor of pathogens. Therefore, lactoferrin helps to kill
pathogens. Its another antibacterial property is that it can attach to receptors
located on microbial cell wall. The ferric iron in lactoferrin catalyzes oxidation
of lipids and polysaccharides in bacterial cell wall.
–– Mucin
Mucin is a glycoprotein. It is the chief organic component of mucus (viscous
secretion that covers oral mucous membranes) of saliva. Mucin helps in the
lubrication of mucosa, prevents from chemical injury, and acts as a barrier
between pathogens and mucosa.
–– Epidermal growth factor
Epidermal growth factor in saliva is a protein. It is made up of 53 amino acid
residues. EGF helps in growth of cells, proliferation, and differentiation.
–– Albumin
Albumin is an important protein in saliva. Salivary albumin functions as pro-
tein buffer in saliva. It also has a diagnostic role in the oral cancerous lesions.
–– Carbohydrates
Saliva does not contain carbohydrates in normal and healthy individuals.
However, in diabetes mellitus, glucose is present in saliva.
–– Nonprotein nitrogenous substances
Saliva excretes nonprotein nitrogenous substances like xanthine, hypoxan-
thine, creatinine, urea, and uric acid.
Inorganic Solids
• Saliva contains cations like Na+, K+, Ca++, Mg++, and anions like Cl−, HCO3−, F−.
Saliva also contains oxygen, carbon dioxide, and nitrogen gases.

24.7 Periodontal Diseases and Immunity
Oral cavity harbors hundreds of microbial species. Streptococcus species are impli-
cated in the formation of plaque on teeth surfaces. Poor oral hygiene causes a shift
in the nature of microbes in dental plaque with preponderance of facultative anaer-
obes and gram-negative microbes. These organisms release endotoxins in gingival
sulcus which activates acquired immune system. Antigens are recognized and pre-
sented by antigen-presenting cells to lymphocytes. Antigen-presenting cells release
pro-inflammatory cytokines like interleukin-1, IL-6, and alpha-tumor necrosis fac-
tor to activate cytotoxic T cells. These lymphocytes phagocytose the pathogens.
Endotoxins and pro-inflammatory cytokines induce vascular changes in the blood
circulation of periodontium. There is an increase in capillary permeability in the
vasculature of periodontium. It leads to emigration of neutrophils, macrophages,
and monocytes to the site of junctional epithelium and crevicular fluid in gingival
sulcus.
There is formation of inflammatory exudate in gingival sulcus. These changes
are associated with breakdown of collagen fibers, junctional epithelium, and con-
nective tissues surrounding the root of the teeth. As the inflammation progresses, B
lymphocytes appear at the site of inflammation. The junctional epithelium migrates
apically, and it leads to formation of periodontal pocket.
Poor oral hygiene associated with bacterial invasion of periodontal tissues trig-
gers immunogenic inflammation of host oral tissues which damage periodontium.
Suggested Readings
Bhasker SN (ed) (2005) Orban’s oral histology and embryolog. Elsevier, New Delhi
Van Nieuw Amerongen A, Bolscher JG, Veerman EC (2004) Salivary proteins: protective and
diagnostic value in cariology? Caries Res 38:247–253

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Anil

ISBN 978-981-13-1034-8 ISBN 978-981-13-1035-5 (eBook)

Library of Congress Control Number: 2018950138

© Springer Nature Singapore Pte Ltd. 2019

It gives me a great satisfaction to write a brief note on Comprehensive Biochemistry

It is my privilege to review and comment on the book Comprehensive Biochemistry

I am glad to know about the publication of Comprehensive Biochemistry for

It is a pleasure to write a foreword for Comprehensive Biochemistry for Dentistry,

Biochemistry is a vast and challenging field for dental students. Comprehensive

I wish great success and my heartiest congratulations to my senior colleague, Dr.

P. K. Garg, MBBS, MD (Pathology)

Puneet Garg, MBBS, MD (Pathology)

Hasan Kamal, PhD (Biochemistry)

Biochemistry is an essential subject in the medical, dental, nursing, paramedical,

Kotputli, India Anil Gupta

1 Introduction to Biochemistry�������������������������������������������������������������������� 1

Part I Cellular Biochemistry

2 Cell and Organelles������������������������������������������������������������������������������������ 5

Part II Structural Biochemistry

3 Protein and Amino Acids�������������������������������������������������������������������������� 35

4.7 β-Globulins����������������������������������������������������������������������������������������� 72

6.3 Characteristics of Monosaccharides �������������������������������������������������� 103

9.6.5 Effect of pH���������������������������������������������������������������������������� 195

10.11 Thyroid Hormones���������������������������������������������������������������������������� 243

12 Digestion and Absorption of Proteins������������������������������������������������������ 367

12.4 Mechanism of Absorption���������������������������������������������������������������� 372

15.6 Hexose Monophosphate Shunt (HMP) �������������������������������������������� 420

18.3 Selenium ������������������������������������������������������������������������������������������ 482

Part IV Medical Biochemistry

20 Acid-Base Balance�������������������������������������������������������������������������������������� 517

21.4 Measurement of Calorific Value������������������������������������������������������� 534

23 Immunoglobulins �������������������������������������������������������������������������������������� 585

Part VI Dental Biochemistry

Anil Gupta is currently working as Professor and Head of the Department of

Biochemistry is a branch of life science (medical science) which deals with

1.2 Historical Development of Biochemistry

• Roots of modern biochemistry are traceable to the science of Indian medicine

© Springer Nature Singapore Pte Ltd. 2019 1

2.2 Landmark Discoveries

In 1665, Robert Hooke investigated a piece of cork under microscope. He found

2.3 Cell Theory Postulates

• Schleiden (1838) and Schwann (1839) together proposed Cell theory.

© Springer Nature Singapore Pte Ltd. 2019 5

2.4 Modern Concept of Cell

• All living organisms are made up of one or more cells.

2.5 Prokaryotic Cell

Pro- means “primitive,” and karyon means “nucleus.”

• Prokaryotic cell has primitive nucleus. It is not enclosed by nuclear membrane.

2.6 Eukaryotic Cell

The word Eu means “true” and karyon means “nucleus.”

• Eukaryotic cells have properly defined nucleus. It is surrounded by nuclear

Fig. 2.1 Diagram of prokaryotic cell

Golgi body Nuclear pore

Fig. 2.2 Diagram of eukaryotic cell

• Cell contains 80S ribosomes attached to endoplasmic reticulum.

2.7 Plasma Membrane

Plasma membrane is a lipoprotein aceous semipermeable structure that sur-

2.7.3 Chemical Composition

Plasma membrane is essentially composed of carbohydrates, lipids, and proteins in

• Example: Lipid contents vary between 20% and 75%

Lipids in Plasma Membrane

hydrophobic (nonpolar) tail. Hydrophilic head is oriented outward towards the

Proteins in Plasma Membrane

Kotputli, India Anil Gupta

1 Introduction to Biochemistry�� 1

Part I Cellular Biochemistry

2 Cell and Organelles�� 5

Part II Structural Biochemistry

3 Protein and Amino Acids�� 35

4.7 β-Globulins�� 72

6.3 Characteristics of Monosaccharides �� 103

9.6.5 Effect of pH�� 195

10.11 Thyroid Hormones�� 243

12 Digestion and Absorption of Proteins�� 367

12.4 Mechanism of Absorption�� 372

15.6 Hexose Monophosphate Shunt (HMP) �� 420

18.3 Selenium �� 482

Part IV Medical Biochemistry

20 Acid-Base Balance�� 517

21.4 Measurement of Calorific Value�� 534

23 Immunoglobulins �� 585

Part VI Dental Biochemistry

1.2 Historical Development of Biochemistry

2.2 Landmark Discoveries

2.3 Cell Theory Postulates

2.4 Modern Concept of Cell

2.5 Prokaryotic Cell

2.6 Eukaryotic Cell

2.7 Plasma Membrane

2.7.3 Chemical Composition

2.8 Models of Plasma Membrane Structure

2.8.1 Lipid Bilayer Model

2.8.2 Sandwich Model

2.8.3 Unit Membrane Model

2.8.4 Fluid Mosaic Model

2.8.5 Functions of Plasma Membrane

2.9 Cytoplasmic Organelles

2.10 Golgi Body

2.10.1 Functions of Golgi Body

• Proteins are glycosylated at asparagine residues by addition of mannose-6-