Textbook of Biochemistry for Medical Students PDF, 6th Edition

 TEXTBOOK OF BIOCHEMISTRY For Medical Students  TEXTBOOK OF BIOCHEMISTRY For Medical Students Sixth Edition DM VASUDEVAN MBBS MD FAMS FRCPath...

 TEXTBOOK OF BIOCHEMISTRY For Medical Students  TEXTBOOK OF BIOCHEMISTRY For Medical Students Sixth Edition DM VASUDEVAN MBBS MD FAMS FRCPath Distinguished Professor of Biochemistry College of Medicine, Amrita Institute of Medical Sciences, Cochin, Kerala (Formerly Principal, College of Medicine, Amrita, Kerala) (Formerly, Dean, Sikkim Manipal Institute of Medical Sciences, Gangtok, Sikkim) E-mail: [email protected] SREEKUMARI S MBBS MD Professor, Department of Biochemistry Sree Gokulam Medical College and Research Foundation Thiruvananthapuram, Kerala E-mail: [email protected] KANNAN VAIDYANATHAN MBBS MD Clinical Associate Professor, Department of Biochemistry and Head, Metabolic Disorders Laboratory Amrita Institute of Medical Sciences, Kochi, Kerala Email: [email protected] ® JAYPEE BROTHERS MEDICAL PUBLISHERS (P) LTD Kochi St Louis (USA) Panama City (Panama) London (UK) New Delhi Ahmedabad Bengaluru Chennai Hyderabad Kolkata Lucknow Mumbai Nagpur Published by Jitendar P Vij Jaypee Brothers Medical Publishers (P) Ltd Corporate Office 4838/24 Ansari Road, Daryaganj, New Delhi - 110002, India, Phone: +91-11-43574357, Fax: +91-11-43574314 Registered Office B-3 EMCA House, 23/23B Ansari Road, Daryaganj, New Delhi - 110 002, India Phones: +91-11-23272143, +91-11-23272703, +91-11-23282021 +91-11-23245672, Rel: +91-11-32558559, Fax: +91-11-23276490, +91-11-23245683 e-mail: [email protected], Website: www.jaypeebrothers.com Offices in India Ahmedabad, Phone: Rel: +91-79-32988717, e-mail: [email protected] Bengaluru, Phone: Rel: +91-80-32714073, e-mail: [email protected] Chennai, Phone: Rel: +91-44-32972089, e-mail: [email protected] Hyderabad, Phone: Rel:+91-40-32940929, e-mail: [email protected] Kochi, Phone: +91-484-2395740, e-mail: [email protected] Kolkata, Phone: +91-33-22276415, e-mail: [email protected] Lucknow, Phone: +91-522-3040554, e-mail: [email protected] Mumbai, Phone: Rel: +91-22-32926896, e-mail: [email protected] Nagpur, Phone: Rel: +91-712-3245220, e-mail: [email protected] Overseas Offices North America Office, USA, Ph: 001-636-6279734, e-mail: [email protected], [email protected] Central America Office, Panama City, Panama, Ph: 001-507-317-0160, e-mail: [email protected] Website: www.jphmedical.com Europe Office, UK, Ph: +44 (0) 2031708910, e-mail: [email protected] Textbook of Biochemistry for Medical Students © 2011, DM Vasudevan, Sreekumari S, Kannan Vaidyanathan All rights reserved. No part of this publication should be reproduced, stored in a retrieval system, or transmitted in any form or by any means: electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the authors and the publisher. This book has been published in good faith that the material provided by authors is original. Every effort is made to ensure accuracy of material, but the publisher, printer and authors will not be held responsible for any inadvertent error (s). In case of any dispute, all legal matters are to be settled under Delhi jurisdiction only. First Edition : 1995 Second Edition: 1998 Third Edition: 2001 Fourth Edition: 2005 Fifth Edition: 2007 Reprint: 2008 Sixth Edition: 2011 ISBN: 978-93-5025-016-7 Typeset at JPBMP typesetting unit Printed at.........  With humility and reverence, this book is dedicated at the lotus feet of the Holy Mother Sri Mata Amritanandamayi Devi “Today's world needs people who express goodness in their words and deeds. If such noble role models set the example for their fellow beings, the darkness prevailing in today's society will be dispelled, and the light of peace and non-violence will once again illumine this earth. Let us work together towards this goal.” —Mata Amritanandamayi Devi  Preface to the Sixth Edition We are glad to present the sixth edition of the Textbook of Biochemistry for Medical Students. With this sixth edition, the textbook is entering the 16th year of existence. With humility, we may state that the medical community of India has warmly received the previous editions of this book. Many medical colleges and universities in India have accepted it as one of the standard textbooks. We are happy to note that this book has also reached in the hands of medical students of neighboring countries of Nepal, Pakistan, Bangladesh, Sri Lanka, etc. and also to distant countries in Africa and Europe. Apart from the medical community, this book has also become popular to other biology group of students in India. In retrospect, it gives immense satisfaction to note that this book served the students and faculty for the past one and half decades. At this time, a revision of the textbook has become absolutely necessary for two reasons. Firstly, the Medical Council of India has revised the syllabus for biochemistry, especially enhancing the topics on Clinical Biochemistry. Accordingly, we have made elaborate changes in the order of chapters, old chapters on clinical chemistry have been extensively updated and clinically relevant points were further added. Secondly, rapid progress has been made in the area of molecular biology during past few years, and these advances are to be reflected in this book also. The major change in this sixth edition is that advanced knowledge has been added in almost all pages, a few sentences were added here and there in almost all pages; sometimes, a few pages are newly incorporated; while it became necessary to include a few new chapters also. From the first edition onwards, our policy was to provide not only basic essentials but also some of the advanced knowledge. About 30% contents of the previous editions were not required for a student aiming for a minimum pass. A lot of students have appreciated this approach, as it helped them to pass the PG entrance examinations at a later stage. However, this asset has paved the way for a general criticism that the extra details are a burden to the average students. Especially when read for the first time, the student may find it difficult to sort out the essential minimum from the desirable bulk. So, in the fifth edition, we have promised that we shall make two different books, one for MBBS and another one for postgraduate courses in Biochemistry. Thus, the content has been reduced substantially in the last edition. But, due to various reasons, most of which beyond the control of the authors, the postgraduate book could not be published. This led to the criticism that the content is sub-optimal. Many PG students were enquiring about the advanced book. The advanced students felt that they were neglected. This 6th edition is a compromise. Advanced topics are given in small prints. In essence, this book has three components; rather this book is composed of three books. The bold printed areas will be useful for the students at the time of revision just before the examinations; regular printed pages are meant for an average first year MBBS student (must-know areas) and the fine printed paragraphs are targeted to the advanced students preparing for the PG entrance (desirable to know areas). The readability has been markedly improved by increasing the font size in the regular areas. Essay and short notes questions, problem solving exercises, viva voce, quick look, multiple choice questions (MCQs) are given as a separate book, but free of cost. These questions are compiled from the question papers of various universities during the last decade. These questions will be ideal for students for last-minute preparation for examinations. A textbook will mature only by successive revisions. In the preface for the first edition, we expressed our desire to revise the textbook every 3 years. We were fortunate to keep that promise. This book has undergone metamorphosis during each edition. Chemical structures with computer technology were introduced in the second edition. Color printing has been launched in the third edition. The fourth edition came out with multicolor printing. In the fifth edition, the facts were presented in small paragraphs and that too with numbers, so as to aid memorization. In this sixth edition, figures are drastically increased; there are now about 1,100 figures, 230 tables and 200 boxes (perhaps we could call it as Illustrated Textbook of Biochemistry), altogether making the book more student-friendly. The quality of paper is also improved during successive editions. We were pleasantly surprised to receive many letters giving constructive criticisms and positive suggestions to improve the textbook. These responses were from all parts of the country (we got a few such letters from African students also). Such contributors include Heads of Departments, very senior professors, middle level teachers and mostly postgraduate students. We have tried to incorporate most of those suggestions, within the constraints of page viii Textbook of Biochemistry limitations. In a way, this book thus became multi-authored, and truly national in character. This is to place on record, our deep gratitude for all those “pen-friends” who have helped us to improve this book. The first author desires more interaction with faculty and students who are using this textbook. All are welcome to communicate at his e-mail address The first author is in the process of retirement from active service, and would like to reduce the burden in due course. A successful textbook is something like a growing institution; individuals may come and go, but the institution will march ahead. Therefore, we felt the need to induce younger blood into the editorial board. Thus, a third author has been added in this sixth edition, so that the torch can be handed over smoothly at an appropriate time later on. In this connection, I would like to introduce the young author, Dr Kannan Vaidyanathan. He has teaching experi- ence of 15 years. He took MD in Biochemistry from Kerala, and done extensive research at the Indian Institute of Science, Bengaluru, Karnataka. He has also visited many advanced laboratories world over, and presented papers in different international conferences. He has many publications to his credit. He is now Clinical Associate Professor, Department of Biochemistry, and Head, Metabolic Disorders Laboratory, Amrita Institute of Medical Sciences, Kochi, Kerala. The help and assistance rendered by our students in preparing this book are enormous; the reviews collected by Dr Sukhes Mukherjee is specially acknowledged. The official website of Nobel Academy has been used for pictures and biographies of Nobel laureates. Web pictures, without copyright protection, were also used in some figures. The remarkable success of the book was due to the active support of the publishers. This is to record our appreciation for the co-operation extended by Shri Jitendar P Vij, and his associates. We hope that this sixth edition will be friendlier to the students and be more attractive to the teachers. Now this is in your hands to judge. “End of all knowledge must be building up of character.”—Gandhiji DM Vasudevan Sreekumari S Kannan Vaidyanathan  Preface to the First Edition There are many textbooks of biochemistry written by Western and Indian authors. Then what is the need for yet another textbook? Putting this question to ourselves, we have waited for many years before embarking on this project. Most western textbooks do not emphasise nutrition and such other topics which are very vital to an Indian student. While Indian authors do cover these portions, they sometimes neglect the expanding fields such as molecular biology and immunochemistry. Thus during our experience of more than 25 years in teaching, the students have been seen compelled to depend on different textbooks during their study of biochemistry. We have tried to keep a balance between the basic essentials and the advanced knowledge. This book is mainly based on the MBBS curriculum. However, some advanced portions have also been given in almost all chapters. These areas will be very beneficial to the readers preparing for their postgraduate entrance examinations. Chapters on diabetes, cancer and AIDS are included in this book. During their clinical years, the students are going to see such cases quite more often, hence knowledge of applied biochemistry of these diseases will be very helpful. The authors, themselves medical graduates, have tried to emphasise medical applications of the theoretical knowledge in biochemistry in almost all the chapters. A few questions have been given at the end of most of the chapters. These are not comprehensive to cover all the topics, but have been included only to give emphasis to certain points which may otherwise be left unnoticed by some students. We are indebted to many persons in compiling this textbook. We are highly obliged to Dr ANP Ummerkutty, Vice- Chancellor, University of Calicut, for his kind gesture of providing an introduction. Dr M Krishnan Nair, Research Director, Veterinary College, Trichur, has provided his unpublished electron micrographs for this book. Dr MV Muraleedharan, Professor of Medicine, and Dr TS Hariharan, Professor of Pharmacology, Medical College, Trichur, have gone through the contents of this book. Their valuable suggestions on the applied aspects of biochemistry have been incorporated. Two of our respected teachers in biochemistry, Prof R Raghunandana Rao and Prof GYN lyer (both retired) have encouraged this venture. Prof PNK Menon, Dr S Gopinathan Nair, Assistant Professor, Dr Shyam Sundar, Dr PS Vasudevan and Mr K Ramesh Kumar, postgraduate students of this department, have helped in collecting the literature and compiling the materials. Mr. Joby Abraham, student of this college has contributed the sketch for some of the figures. Prof CPK Tharakan, retired professor of English, has taken great pains to go through the entire text and correct the usage of English. The secretarial work has been excellently performed by Mrs Lizy Joseph. Many of our innumerable graduate and postgraduate students have indirectly contributed by compelling us to read more widely and thoroughly. “A lamp that does not glow itself cannot light another lamp” —Tagore Our expectation is to bring out new editions every 3 years. Suggestions to improve the contents are welcome from the teachers. November 1994 DM Vasudevan Sreekumari S  Contents SECTION A : CHEMICAL BASIS OF LIFE 1. Biochemical Perspective to Medicine............................................................................................... 1 Historical background; Stabilizing forces in Biomolecules; Electrostatic bonds; Hydrophobic interactions; van der Waals force; Hydrogen bond; Properties of water; Principles of Thermodynamics; Donnan Membrane Equilibrium 2. Subcellular Organelles and Cell Membranes.................................................................................. 7 Cell Composition, Subcellular organelles, Nucleus, Endoplasmic reticulum, Golgi apparatus, Lysosomes, Peroxisomes, Mitochondria. Fluid Mosaic Model, Lipid rafts, Caveolae, Tight junction, Cytoskeleton, Transport mechanisms, Facilitated diffusion, Ion channels, Ligand gated channels, Voltage gated channels, Ionophores, Active transport, Sodium pump, Uniport, Symport, Antiport, Exocytosis, Endocytosis, Pinocytosis, Phagocytosis. 3. Amino Acids: Structure and Properties........................................................................................... 19 Classification based on structure, Classification based on side chain characters, Classification based on metabolic fate, Classification based on nutritional requirement, properties and reactions, Iso-electric point, Decarboxylation, Amide formation, Transamination, Oxidative deamination, Amino acid derivatives of importance, Peptide bond, Color reactions of amino acids and proteins 4. Proteins: Structure and Function..................................................................................................... 27 Structure of proteins, primary, secondary, tertiary and quaternary structures, Primary structure of insulin, Structure-function relationship, Sequence analysis, iso-electric pH, Precipitation reactions, Denaturation of proteins, Heat coagulation, Classification of proteins, Quantitative estimation of proteins. 5. Enzymology: General Concepts and Enzyme Kinetics.................................................................. 40 Classification, Co-enzymes, Mode of action of enzymes, Active center, Kinetics, Michaelis constant, Activation, Competitive inhibition, Noncompetitive inhibition, Allosteric inhibition, Key enzymes, Feedback inhibition, Covalent modification, Repression, Induction, Specificity of enzymes, Enzyme engineering, Enzyme units, Iso-enzymes. 6. Chemistry of Carbohydrates............................................................................................................. 60 Monosaccharides, Glucose, Fructose, Mannose, Galactose, Stereoisomers, Epimers, Reactions, Benedict’s reaction, Osazone, Glycosides, Amino sugars, Deoxy sugars, Disaccharides, Sucrose, Lactose, Maltose, Polysaccharides, Starch, Glycogen, Cellulose, Mucopolysaccharides 7. Chemistry of Lipids............................................................................................................................ 73 Classification of lipids, Classification of fatty acids, Saturated fatty acids, Unsaturated fatty acids, Polyunsaturated fatty acids, Triglycerides, Classification of compound lipids, Phospholipids, Liposomes, Lecithin, Phospholipases, Lung surfactants, Cephalin, Plasmalogens, Sphingolipids, Nonphosphorylated lipids, Compound lipids, Glycerophosphatides, Sphingolipids, Sphingomyelin, Cerebrosides, Gangliosides. SECTION B : GENERAL METABOLISM 8. Overview of Metabolism................................................................................................................... 83 Experimental approach to study of metabolism, Tissue culture, Radioisotope tracers, Metabolic profile in organs, Brain, Skeletal muscle, Cardiac muscle, Adipose tissue, Liver, Metabolic adaptations during starvation. xii Textbook of Biochemistry 9. Major Metabolic Pathways of Glucose............................................................................................ 90 Digestion and absorption of carbohydrates, Glucose transporters, Regulation of blood sugar, Embden- Meyerhof pathway, Glycolysis, Regulation, Cori’s cycle, BPG shunt, Fate of pyruvate, Gluconeogenesis, Glucose-alanine cycle, Glycogenolysis, Glycogen synthesis, Glycogen storage diseases 10. Minor Metabolic Pathways of Carbohydrates............................................................................... 113 Hexose monophosphate shunt pathway, Glucose-6-phosphate dehydrogenase deficiency, Glucuronic acid pathway, Essential pentosuria, Polyol pathway, Fructose metabolism, Hereditary fructose intolerance, Fructosuria, Galactose metabolism, Galactosemia, Metabolism of alcohol, Amino sugars, Glycoproteins, Blood group substances, Mucopolysaccharidoses, Inborn errors associated with carbohydrate metabolism. 11. Metabolism of Fatty Acids............................................................................................................... 127 Digestion and absorption of fat, Beta oxidation, Energetics, Oxidation of odd chain fatty acids, Alpha oxidation, Omega oxidation, Organic acidurias, De novo synthesis of fatty acids, Elongation, Synthesis of triglycerides, Metabolism of adipose tissue, Hormone sensitive lipase, Liver adipose tissue axis, Obesity, Fatty liver, Lipotropic factors, Ketone bodies, Ketogenesis, Ketolysis, Ketosis. 12. Cholesterol and Lipoproteins......................................................................................................... 146 Steroids, Structure of cholesterol, Biosynthesis of cholesterol, Plasma lipids, Transport of lipids, Lipoproteins, Apolipoproteins, Chylomicrons, VLDL, LDL, HDL, Lp(a), Free fatty acid, Non-esterified fatty acids, Bile salts, Steroid hormones. 13. MCFA, PUFA, Prostaglandins and Compound Lipids................................................................... 160 Digestion of medium chain fatty acids, Monounsaturated fatty acids, Beta oxidation of unsaturated fatty acids, Polyunsaturated fatty acids, Desaturation of fatty acids, Essential fatty acids, Eicosanoids, Prostaglandins, Leukotrienes, Very long chain fatty acids, Synthesis of Compound Lipids, Phosphatidyl choline, Sphingomyelin, Lipid storage diseases. 14. General Amino Acid Metabolism (Urea Cycle, One Carbon Metabolism).................................. 170 Digestion of proteins, Absorption of amino acids, Meister cycle, Intracellular protein degradation, Cathepsins, Ubiquitin pathway, Proteasomes, Inter-organ transport of amino acids, Glucose Alanine cycle; Catabolism of amino acids, Formation of ammonia, Transamination, Oxidative deamination, Nonoxidative deamination, Disposal of ammonia, Urea cycle, Disorders of urea cycle, Hepatic coma, Blood urea. One carbon compounds, Generation and utilization of one carbon groups. 15. Simple, Hydroxy and Sulfur Containing Amino Acids (Glycine, Serine, Methionine, Cysteine)........................................................................................ 183 Glycine, Creatine, Creatinine, Primary hyperoxaluria, Serine, Serine choline glycine cycle, Selenocysteine, Alanine, Glucose alanine cycle, Beta alanine, Threonine, Methionine, Transmethylation reactions, Cysteine, Glutathione, Sulphur, Cystinuria, Homocystinurias, Cystathioninuria. 16. Acidic, Basic and Branched Chain Amino Acids (Glutamic Acids, Aspartic Acid, Lysine, Arginine, Nitric Oxide, Histidine, Valine, Leucine, Isoleucine)................................................................... 194 Glutamic acid, GABA, Glutamine, Aspartic acid, Asparagine, dicarboxylic amino aciduria, Lysine, Arginine, Nitric Oxide, Ornithine, Polyamine synthesis, Valine, Leucine, Isoleucine, Maple syrup urine disease, Isovaleric aciduria, Histidine, Histamine 17. Aromatic Amino Acids and Amino Acidurias (Phenylalanine, Tyrosine, Tryptophan, Proline)........................................................................... 203 Phenylalanine, Tyrosine, Melanin, Catecholamines, Phenylketonuria, Alcaptonuria, Albinism, Tryptophan, Nicotinic acid pathway, Serotonin, Melatonin, Indican, Hartnup’s disease, Proline, Inter-relation of amino acids; Amino acidurias 18. Citric Acid Cycle................................................................................................................................ 216 Citric acid cycle reactions, Significance of TCA cycle, Amphibolic role, Regulation, Integration of metabolism,  Contents xiii 19. Biological Oxidation and Electron Transport Chain.................................................................... 223 Primary, secondary and tertiary metabolism, Redox potential, Biological oxidation, Oxidases, Cytochrome oxidase, Dehydrogenases, NAD+, FAD, Cytochromes, Oxygenases, High energy compounds, Organization of electron transport chain, NADH shuttle, Malate aspartate shuttle, Flow of electrons, Oxidative phosphorylation, Chemi-osmotic theory, ATP synthase, Inhibitors of ATP synthesis, Uncouplers of oxidative phosphorylation, lonophores. 20. Free Radicals and Anti-Oxidants.................................................................................................... 236 Free radicals, Reactive oxygen species, Generation, Damage, Free radical scavenger systems, Inflammation, Respiratory diseases, Retrolental fibroplasia, Reperfusion injury, Atherosclerosis, Skin diseases, Age-related diseases, Lipid peroxidation, Initiation, propagation and termination phases, Preventive anti-oxidants, Chain breaking anti-oxidants. 21. Heme Synthesis and Breakdown................................................................................................... 242 Structure of heme, Biosynthesis of heme, Porphyria, Shunt bilirubin, Catabolism of heme, Plasma bilirubin, Hyperbilirubinemias, Congenital hyperbilirubinemia, Hemolytic jaundice, Hepatocellular jaundice, Obstructive jaundice, Differential diagnosis of jaundice. 22. Hemoglobin (Structure, Oxygen and Carbon Dioxide, Transport, Abnormal Hemoglobins)... 254 Structure of hemoglobin, Transport of gases, Oxygen dissociation curve, Hemoglobin interaction, Effect of 2,3-BPG, Isohydric transport of carbon dioxide, Chloride shift, Fetal hemoglobin (HbF), Hemoglobin derivatives, Carboxy hemoglobin, Met-hemoglobin, Hemoglobin variants, Sickle cell hemoglobin (HbS), Thalassemias, Myoglobin, Anemias. SECTION C : CLINICAL AND APPLIED BIOCHEMISTRY 23. Clinical Enzymology and Biomarkers............................................................................................ 266 Creatine kinase, Cardiac troponins, Lactate dehydrogenase, Markers of Cardiac diseases; Aspartate amino transferase, Alanine amino transferase, Alkaline phosphatase, Nucleotide phosphatase, Gamma glutamyl transferase, Markers of liver diseases, Acid phosphatase, Cholinesterase, Glucose-6-phosphate dehydrogenase, Amylase, Lipase, Aldolase, Enolase, Enzymes as therapeutic agents, Enzymes used for diagnosis, Immobilized enzymes. 24. Regulation of Blood Glucose, Insulin and Diabetes Mellitus...................................................... 274 Regulation of blood glucose, Determination of glucose, Glucose tolerance test, Impaired glucose tolerance, Impaired fasting glycemia, Gestational diabetes mellitus, Alimentary glucosuria, Renal glucosuria, Reducing substances in urine, Glycosuria, Diabetes mellitus, Clinical presentation, Diabetic keto acidosis, Hyperosmolar nonketotic coma, Lactic acidosis, Chronic complications, Glycated hemoglobin. 25. Cardiovascular Diseases and Hyperlipidemias............................................................................ 292 Lipid profile, Atherosclerosis, Coronary artery disease, Relation of cholesterol with myocardial infarction, Risk factors of atherosclerosis, Prevention of atherosclerosis, Hypolipoproteinemias, hyperlipoproteinemias. 26. Liver and Gastric Function Tests.................................................................................................... 301 Tests for liver function, Serum bilirubin, Classification of jaundice, Bile acids and bile salts, Tests based on the metabolic capacity of the liver, Test based on synthetic function, Serum enzymes as markers of hepatobiliary diseases, Gastric function, Hydrochloric acid secretion, Assessment of free and total acidity, Pancreatic function tests. 27. Kidney Function Tests.................................................................................................................... 314 Formation of urine, Functions of the tubules, Renal threshold, Tubular maximum, Abnormal constituents of urine, Proteinuria, Reducing sugars, Clearance tests, Inulin clearance, Creatinine clearance test, Cystatin C, Urea clearance test, Tests for tubular function, Osmolality, Acidification test. xiv Textbook of Biochemistry 28. Plasma Proteins............................................................................................................................... 329 Serum electrophoretic pattern in normal and abnormal states, Albumin, Transport proteins, Polymorphism, Acute phase proteins, Ceruloplasmin, Alpha-1-anti-trypsin, Alpha-2- macroglobulin, Negative acute phase proteins, Clotting factors, Anticoagulants, Fibrinolysis, Hemophilia. 29. Acid-Base Balance and pH............................................................................................................. 339 Acids and bases, Henderson-Hasselbalch equation, Buffers, Buffer capacity, Buffers of body fluids, Respiratory regulation of pH, Renal regulation of pH, Titratable acid, Cellular buffers, Disturbances in acid- base balance, Anion gap, Metabolic acidosis, Metabolic alkalosis, Respiratory acidosis, Respiratory alkalosis. 30. Electrolyte and Water Balance...................................................................................................... 355 Body water compartments, Donnan membrane equilibrium, Osmolality, Electrolyte concentration of body fluid compartments, Regulation of sodium and water balance, Renin-angiotensin system, Assessment, Disturbances, Isotonic contraction, Hypotonic contraction, Hypertonic contraction, Isotonic expansion, Hypotonic expansion, Hypertonic expansion; Clinical applications of Sodium, Potassium, Chloride, Hypernatremia, Hyponatremia, Hypokalemia, Hyperkalemia, Hyperchloremia, Hypochloremia. 31. Body Fluids (Milk, CSF, Amniotic Fluid)........................................................................................ 365 Milk, Colostrum, Aqueous humor, Cerebrospinal fluid, Amniotic fluid, Assessment of fetal maturity. 32. Clinical Laboratory: Screening of Metabolic Diseases; Quality Control.................................... 368 Prenatal diagnosis; AFP, hCG, uE3, DIA, PAPP-A; Newborn screening; Investigations for Metabolic disorders; Reference values, Pre-analytical variables, Collection of blood, Anticoagulants, Preservatives for blood, Urine collection, Quality control, Accuracy, Precision, Specificity, Sensitivity, Limits of errors allowable in the laboratory, Percentage error. SECTION D : NUTRITION 33. Fat Soluble Vitamins (A, D, E, K).................................................................................................... 379 Vitamin A, Role in vision, Wald’s visual cycle, Vitamin D, Calcitriol, Vitamin E, Vitamin K. 34. Water Soluble Vitamins (Thiamine, Riboflavin, Niacin, Pyridoxine, Pantothenic acid, Biotin, Folic acid, Vitamin B12 and Ascorbic acid).................................................................................... 391 Thiamine (B1), Beriberi, Riboflavin (B2), Niacin, Pyridoxine (B6 ), Pantothenic acid, Acetyl CoA, Succinyl CoA, Biotin, Folic acid, Folate antagonists, Folate trap, Vitamin B12, Choline, Inositol, Ascorbic acid (Vitamin C), Scurvy. 35. Mineral Metabolism and Abnormalities........................................................................................ 411 Calcium, Homeostasis, Parathyroid hormone, Calcitonin, Hypercalcemia, Hypocalcemia, Bone metabolism; Markers of bone metabolism; Phosphorus, Magnesium, Sulphur, Iron, Absorption, Iron deficiency, Hemochromatosis, Copper, Ceruloplasmin, Iodine, Zinc, Fluoride, Selenium, Manganese, Molybdenum, Cobalt, Nickel, Chromium, Lithium. 36. Energy Metabolism and Nutrition................................................................................................... 432 Calorific value, Respiratory quotient, Resting metabolic rate (RMR), Specific dynamic action, Requirements, Dietary carbohydrates, Dietary fiber, Nutritional importance of lipids, Nutritional importance of proteins, Essential amino acids, Nitrogen balance, Biological value of proteins, Protein energy malnutrition (PEM), Marasmus, Kwashiorkor, Obesity, Prescription of diet, Special diets, Glycemic index, Total parenteral nutrition. 37. Detoxification and Biotransformation of Xenobiotics.................................................................. 446 Phase one reactions, Oxidative reactions, Reductive reactions, Hydrolysis, Phase two reactions, Conjugation, Phase three reactions.  Contents xv 38. Environmental Pollution and Heavy Metal Poisons..................................................................... 451 Corrosives and irritants, Organic irritant poisons, Neurotoxins, Heavy metal poisons, Lead, Mercury, Aluminium, Arsenic, Pesticides and insecticides, Organophosphorus compounds, Industrial hazards, Air pollutants, Sulphur dioxide, Toxic substances in foodstuffs, Lathyrism. SECTION E : MOLECULAR BIOLOGY 39. Nucleotides; Chemistry and Metabolism....................................................................................... 457 Purine bases, Pyrimidine bases, Nucleosides, Nucleotides, Biosynthesis of purine nucleotides, Salvage pathway, Regulation of synthesis, Degradation of purines, Uric acid, Gout, Secondary hyperuricemia, Lesch-Nyhan syndrome, Synthesis of pyrimidine nucleotide, Regulation, Orotic aciduria, Deoxyribonucleotide formation, Degradation of pyrimidine. 40. Deoxyribo Nucleic Acid (DNA): Structure and Replication.......................................................... 469 Structure of DNA, Watson-Crick model, Supercoiling of DNA, Nucleoproteins, Chromosomes, Replication of DNA, Meselson-Stahl experiment, DNA polymerases, Replisome, Primosome, Okazaki fragments, Repair mechanisms, Diseases associated with repair mechanisms, Xeroderma pigmentosum, Telomeres, Telomerase, Inhibitors of DNA replication. 41. Transcription and Translation....................................................................................................... 481 Ribonucleic acid, mRNA, RNA polymerase, Transcription, Transcription signals, Initiation of transcription, Elongation of transcription, Termination of transcription, Post-transcriptional processing, Spliceosomes, Ribozymes, Introns and exons, Reverse transcriptase, tRNA, rRNA, Ribosomes, snRNA, Protein biosynthesis, Genetic code, Translation, Initiation of translation, Elongation of translation, Termination of translation, Protein targetting, Post-translational processing, Protein folding, Chaperones, Heat shock proteins, Inhibitors of protein synthesis, Antibiotics, Mitochondrial DNA and RNA, Oxphos diseases; Genomics and proteomics; Micro RNA, interfering RNA, RNA silencing; Antisense therapy; Fusion proteins. 42. Inheritance, Mutations and Control of Gene Expression............................................................. 498 Principles of heredity, Dominant inheritance, Recessive inheritance, X-linked inheritance, Population genetics, Chromosomal recombination, Genetic loci on chromosomes, Mutations, Point mutation, Termination codon mutation, Frame shift mutation, Conditional mutation, Ame’s test, Mutagens, Site directed mutagenesis, Cell cycle, Check points, Oncosuppressor proteins, Rb protein, p53, Apoptosis, Caspase activation cascade, Regulation of gene expression, Operon concept, Repression, Derepression, Lac operon, Hormone response elements; Gene amplifications, Gene switching, Viruses, Antiviral agents; Lysogeny; Transduction; Epigenetic modifications. 43. Recombinant DNA Technology and Gene Therapy..................................................................... 512 Application of recombinant DNA technology, Restriction endonucleases, Restriction map, cDNA, Vectors, Plasmids, Cosmids, Homopolymer tailing, Chimeric molecules, Cloning, Transfection, Selection, Expression vectors, Gene therapy, Vectors for gene therapy, Retroviruses, Adenoviruses, Plasmid liposome complex; Stem cells. SECTION F : HORMONES 44. Mechanisms of Action of Hormones............................................................................................... 520 G proteins, Cyclic AMP, Protein kinases, Phosphatidyl inositol biphosphate, Inositol triphosphate, Diacyl glycerol, Cyclic GMP, Steroid receptors, Insulin Signaling pathway, mTOR, Jak-STAT pathway, NFkB 45. Hypothalamic and Pituitary Hormones.......................................................................................... 528 Anti-diuretic hormone, Oxytocin, Hypothalamic releasing factors, Growth hormone, Adrenocorticotropic hormone, Endorphins, Glycoprotein hormones, Thyroid stimulating hormone, Gonadotropins. xvi Textbook of Biochemistry 46. Steroid Hormones............................................................................................................................ 532 Adrenal cortical hormones, Synthesis of steroid hormones, 17-ketosteroids, Assessment of glucocorticoid secretion, Assessment of mineralocorticoid function, Adrenal hyperfunction, Adrenal hypofunction, Primary hyperaldosteronism, Adrenogenital syndrome, Ovarian hormones, Testicular hormones. 47. Thyroid Hormones........................................................................................................................... 538 Thyroid hormones, Synthesis, Secretion, Mechanism of action, Metabolic effects, Assessment of thyroid function, Hyperthyroidism, Hypothyroidism., 48. Signal Molecules and Growth Factors........................................................................................... 543 Adiponectin, Cadherins, CKK, C-Jun, C-Kit, EGFR, Erythropoietin, ERK, FGF, GIP, Gastrin, Gaunylin, Ghrelin, GLP, GSK, GCSF, GMCSF, HSP, HGF, HGFR, HER2/neu, HIF, ICAM, IGF, IGFR, IR, IRS, Interferons, JNKs, MCSF, MIP, MMP, MAPKK, MCP, Neuropeptide Y, Osteocalcin, Osteonectin, Osteopontin, Osteoprotogerin, p38, p53, p70S6, Pancreatic polypeptide, PIGF, PDGF, PARP, Protein C, RANTES, Rb, Resistin, SSA, SSAP, Secretin, Selectins, Somatostatin, STAT, Tau, TGF, Thrombopoietin, Thrombomodulin, TIMP, TNF, VCAM, VEGF, VIP SECTION G : ADVANCED BIOCHEMISTRY 49. Immunochemistry............................................................................................................................ 553 Immune response, Effector mechanisms, Cell mediated immunity, Humoral immunity, Structure of immunoglobulins, Variability, Classes of immunoglobulins, IgG, IgM, IgA, IgE, Isotopes, allotypes, ideotypes, Multiple myeloma, Plasmacytoma, Bence-Jones Proteinuria, Macroglobulinemia, Hypergamma-globulinemia, Complement system, Hereditary angio neurotic edema, Immunodeficiency states, Molecular mechanisms of antibody production, Transposition of genes, Somatic recombination, Molecular structure of antigens, HLA antigens, Cytokines, Lymphokines. 50. Biochemistry of AIDS and HIV........................................................................................................ 563 Transmission, Natural course of the disease, Laboratory analysis, Virus, Replication, HIV genes and gene products, Immunology of AIDS, Anti-HIV drugs, Prevention. 51. Biochemistry of Cancer................................................................................................................... 567 Etiology, Chemical carcinogens, Antimutagens, Oncogenic viruses, Oncogenes, Proto oncogene, Antioncogenes, Oncosuppressor genes, Growth factors, Tumour kinetics, Doubling time, Contact inhibition, Anchorage dependence, Apoptosis, Oncofetal antigens, Tumor markers, Alpha fetoprotein, Carcinoembryonic antigen, Tissue polypeptide antigen, Prostate specific antigen, Other tumor markers, Anticancer drugs, Drug resistance. 52. Tissue Proteins in Health and Disease.......................................................................................... 581 Collagen, Elastin, Keratins, Contractile proteins, Actin, Myosin, Troponin, Muscle contraction, Calmodulin, Micro filaments, Micro tubules, Lens proteins; Prions, Human prion diseases, Biochemistry of aging, Alzheimer’s disease. 53. Applications of Isotopes in Medicine............................................................................................. 592 Subatomic particles, Valency, Isotopes, Radioactive decay, Alpha, Beta, Gamma radiations, Half-life, Units of radioactivity, Research applications, Diagnostic applications, Teletherapy, Radiosensitivity, Fractionation of doses, Biological effects of radiation, Radiation protection. 54. General Techniques for Separation, Purification and Quantitation........................................... 599 Electrophoresis, PAGE, Immuno-electrophoresis, High voltage electrophoresis, Capillary electrophoresis, Chromatography (adsorption, partition, ion exchange, gel filtration, affinity), HPLC, Ultracentrifugation, Determination of molecular weight of proteins, Radioimmunoassay, ELISA test, pH meter, Colourimeter, Spectrophotometer, Flame photometer, Autoanalysers, Dry chemistry systems, Ion selective electrodes, Tandom mass spectroscopy, Fluorescent activated cell sorter.  Contents xvii 55. Molecular Diagnostics..................................................................................................................... 612 Gene library, Linkage analysis, Microsatellite markers, Human Genome Project, Southern blot, In-situ hybridisation, Northern blotting, Western blot, Animal cloning, Molecular cloning in clinical diagnosis and management, DNA finger printing. Restriction fragment length polymorphism (RFLP), Polymerase chain reaction (PCR), Reverse PCR, Hybridoma technology and Monoclonal antibodies, Single Strand Conformation Polymorphism (SSCP), Heteroduplex Analysis, Conformation Sensitive Gel Electrophoresis (CSGE), Protein Truncation Test (PTT), Denaturing High Performance Liquid Chromatography (DHPLC), Transgenesis, DNA sequencing, Bio-informatics, Nanotechnology. APPENDICES I. Abbreviations Used in this book......................................................................................................... 625 II. Normal values (Reference values)...................................................................................................... 629 III. Conversion Chart................................................................................................................................ 631 IV. Greek Alphabet (Commonly Used letters as Symbols)....................................................................... 631 V. Recommended Daily Allowance (RDA) of Essential Nutrients............................................................ 632 VI. Composition of Nutrients in Selected Common Food Materials.......................................................... 633 VlI. Table Showing Surface Area for Different Heights and Weights.......................................................... 635 VIII. Ideal Body Weight and Height of Different Age Groups....................................................................... 635 Index............................................................................................................................................637  CHAPTER Biochemical 1 Perspective to Medicine The word chemistry is derived from the Greek word "chemi" CHAPTER AT A GLANCE (the black land), the ancient name of Egypt. Indian medical The reader will be able to answer questions on science, even from ancient times, had identified the metabolic and genetic basis of diseases. Charaka, the great the following topics: master of Indian Medicine, in his treatise (circa 400 BC) 1. History of biochemistry observed that madhumeha (diabetes mellitus) is produced 2. Biomolecules and metabolism by the alterations in the metabolism of carbohydrates and fats; 3. Ionic bonds the statement still holds good. 4. Hydrogen bonding Biochemistry has developed as an offshoot of organic chemistry, and this branch was often referred as "physiological 5. Hydrophobic interactions chemistry". The term "Biochemistry" was coined by Neuberg 6. Principles of thermodynamics in 1903 from Greek words, bios (= life) and chymos (= juice). 7. Donnan membrane equilibrium One of the earliest treatises in biochemistry was the "Book of Organic Chemistry and its Applications to Physiology and Pathology", published in 1842 by Justus von Liebig (1803- 73), who introduced the concept of metabolism. The "Textbook Biochemistry is the language of biology. The tools of Physiological Chemistry" was published in 1877 by Felix for research in all the branches of medical science Hoppe-Seyler (1825-95), who was professor of physiological are based on principles of biochemistry. The study chemistry at Strausbourge University, France. Some of the of biochemistry is essential to understand basic milestones in the development of science of biochemistry are functions of the body. This will give information given in Table 1.1. The practice of medicine is both an art and a science. regarding the functioning of cells at the molecular The word "doctor" is derived from the Latin root, "docere", level. How the food that we eat is digested, absor- which means "to teach". Knowledge devoid of ethical back- bed, and used to make ingredients of the body? ground may sometimes be disastrous! Hippocrates (460 BC How does the body derive energy for the normal to 377 BC), the father of modern medicine articulated "the day to day work? How are the various metabolic Oath". About one century earlier, Sushrutha (500 BC), the processes interrelated? What is the function of great Indian surgeon, enunciated a code of conduct to the medical practitioners, which is still valid. He proclaims: "You genes? What is the molecular basis for immuno- must speak only truth; care for the good of all living beings; logical resistance against invading organisms? devote yourself to the healing of the sick even if your life be Answer for such basic questions can only be derived lost by your work; be simply clothed and drink no intoxicant; by a systematic study of medical biochemistry. always seek to grow in knowledge; in face of God, you can Modern day medical practice is highly dependent take upon yourself these vows." Biochemistry is perhaps the most rapidly developing on the laboratory analysis of body fluids, especially subject in medicine. No wonder, the major share of Nobel the blood. The disease manifestations are reflected prizes in medicine has gone to research workers engaged in the composition of blood and other tissues. in biochemistry. Thanks to the advent of DNA-recombination Hence, the demarcation of abnormal from normal technology, genes can now be transferred from one person constituents of the body is another aim of the study to another, so that many of the genetically determined of clinical biochemistry. diseases are now amenable to gene therapy. Many genes, (e.g. human insulin gene) have already been transferred to microorganisms for large scale production of human proteins. Advances in genomics like RNA interference for silencing of genes and creation of transgenic animals by gene targeting of embryonic stem cells are opening up new vistas in therapy of diseases like cancer and AIDS. It is hoped that in future, physician will be able to treat the patient, understanding his genetic basis, so that very efficient "designer medicine" could cure the diseases. The large amount of data, especially with regard to single 2 Textbook of Biochemistry; Section A: Chemical Basis of Life Table 1.1. Milestones in history of biochemistry Scientists Year Landmark discoveries Rouell 1773 Isolated urea from urine Lavoisier 1785 Oxidation of food stuffs Wohler 1828 Synthesis of urea Berzelius 1835 Enzyme catalysis theory Louis Pasteur 1860 Fermentation process Edward Buchner 1897 Extracted enzymes Fiske & Subbarow 1926 Isolated ATP from muscle Lohmann 1932 Creatine phosphate Hans Krebs 1937 Citric acid cycle Avery & Macleod 1944 DNA is genetic material Lehninger 1950 TCA cycle in mitochondria Watson & Crick 1953 Structure of DNA Nirenberg 1961 Genetic code in mRNA Holley 1963 Sequenced gene for tRNA linked to each other to form macromolecules of the Khorana 1965 Synthesized the gene cell, e.g. glucose to glycogen, amino acids to Paul Berg 1972 Recombinant DNA technology proteins, etc. Major complex biomolecules are Kary Mullis 1985 Polymerase chain reaction Proteins, Polysaccharides, Lipids and Nucleic acids. 1990 Human genome project started The macromolecules associate with each other by 2003 Human gene mapping completed noncovalent forces to form supramolecular systems, e.g. ribosomes, lipoproteins. nucleotide polymorphisms (SNPs) that are available, could Finally, at the highest level of organisation in the be harnessed by "Bioinformatics". Computers are already hierarchy of cell structure, various supramolecular helping in drug designing process. Studies on oncogenes complexes are further assembled into cell organelle. have identified molecular mechanisms of control of normal In prokaryotes (e.g. bacteria; Greek word "pro" = and abnormal cells. Medical practice is now taking more and more help from the field of biochemistry. With the help before; karyon = nucleus), these macromolecules of human genome project (HGP) the sequences of the whole are seen in a homogeneous matrix; but in eukaryotic human genes are now available; it has already made great cells (e.g. higher organisms; Greek word "eu" = true), impact on medicine and related health sciences. the cytoplasm contains various subcellular organelles. Comparison of prokaryotes and BIOMOLECULES eukaryotes are shown in Table 1.2. More than 99% of the human body is composed of 6 elements, i.e. oxygen, carbon, hydrogen, nitrogen, STUDY OF METABOLIC PROCESSES calcium and phosphorus. Human body is composed Our food contains carbohydrates, fats and proteins of about 60% water, 15% proteins, 15% lipids, 2% as principal ingredients. These macromolecules are carbohydrates and 8% minerals. Molecular structures in organisms are built from 30 small Table 1.2. Bacterial and mammalian cells precursors, sometimes called the alphabet of Prokaryotic cell Eukaryotic cell biochemistry. These are 20 amino acids, 2 purines, 3 pyrimidines, sugars (glucose and ribose), Size Small Large; 1000 to 10,000 times palmitate, glycerol and choline. Cell wall Rigid Membrane of lipid bilayer In living organisms the biomolecules are Nucleus Not defined Well defined ordered into a hierarchy of increasing molecular Organelles Nil Several; including mito- complexity. These biomolecules are covalently chondria and lysosomes  Chapter 1; Biochemical Perspective to Medicine 3 Fig. 1.3. Ionic bonds used in protein interactions Fig. 1.1. Covalent bond Fig. 1.2. Ionic bond group of histidine. Negative charges are provided by beta and gamma carboxyl groups of aspartic acid to be first broken down to small units; carbohydrates and glutamic acid (Fig. 1.3). to monosaccharides and proteins to amino acids. 3. Hydrogen Bonds This process is taking place in the gastrointestinal These are formed by sharing of a hydrogen tract and is called digestion or primary metabolism. between two electron donors. Hydrogen bonds After absorption, the small molecules are further result from electrostatic attraction between an broken down and oxidised to carbon dioxide. In this electro-negative atom and a hydrogen atom that is process, NADH or FADH2 are generated. This is bonded covalently to a second electronegative named as secondary or intermediary metabolism. atom. Normally, a hydrogen atom forms a covalent Finally, these reducing equivalents enter the electron bond with only one other atom. However, a transport chain in the mitochondria, where they are hydrogen atom covalently bonded to a donor atom, oxidised to water; in this process energy is trapped may form an additional weak association, the as ATP. This is termed tertiary metabolism. hydrogen bond with an acceptor atom. In biological Metabolism is the sum of all chemical changes of a systems, both donors and acceptors are usually compound inside the body, which includes synthesis nitrogen or oxygen atoms, especially those atoms (anabolism) and breakdown (catabolism). (Greek in amino (NH2) and hydroxyl (OH) groups. word, kata = down; ballein = change). With regard to protein chemistry, hydrogen releasing groups are -NH (imidazole, indole, STABILIZING FORCES IN MOLECULES peptide); -OH (serine, threonine) and -NH2 (arginine 1. Covalent Bonds lysine). Hydrogen accepting groups are COO –, Molecules are formed by sharing of electrons (aspartic, glutamic) C=O (peptide); and S–S between atoms (Fig. 1.1). (disulphide). The DNA structure is maintained by hydrogen bonding between the purine and 2. Ionic Bonds or Electrostatic Bonds pyrimidine residues. Ionic bonds result from the electrostatic attraction between two ionized groups of opposite charges (Fig. 1.2). They are formed by transfer of one or more electrons from the outermost orbit of an electropositive atom to the outermost orbit of an electronegative atom. This transfer results in the formation of a ‘cation’ and an ‘anion’, which get consequently bound by an ionic bond. Common examples of such compounds include NaCl, KBr and NaF. With regard to protein chemistry, positive charges are produced by epsilon amino group of lysine, guanidium group of arginine and imidazolium Fig. 1.4. Hydrophobic interaction 4 Textbook of Biochemistry; Section A: Chemical Basis of Life 4. Hydrophobic Interactions Non-polar groups have a tendency to associate with each other in an aqueous environment; this is referred to as hydrophobic interaction. These are formed by interactions between nonpolar hydrophobic side chains by eliminating water molecules. The force that causes hydrophobic molecules or nonpolar portions of molecules to aggregate together rather than to dissolve in water is called the ‘hydrophobic bond’ (Fig. 1.4). This serves to hold lipophilic side chains of amino acids together. Thus, nonpolar molecules will have minimum exposure to water molecules. Fig. 1.5: Water molecules hydrogen bonded toward 100°C, the kinetic energy of its molecules 5. Van Der Waals Forces becomes greater than the energy of the hydrogen These are very weak forces of attraction between bonds connecting them, and the gaseous form of all atoms, due to oscillating dipoles, described water appears. A few gifted properties of water by the Dutch physicist Johannes van der Waals make it the most preferred medium for all cellular (1837-1923). He was awarded Nobel prize in 1910. reactions and interactions. These are short range attractive forces between a. Water is a polar molecule. Molecules with polar chemical groups in contact. Van der Waals bonds that can easily form hydrogen bonds with interactions occur in all types of molecules, both water can dissolve in water and are termed polar and nonpolar. The energy of the van der “hydrophilic”. Waals interaction is about 1 kcal/mol and are b. It has immense hydrogen bonding capacity both unaffected by changes in pH. This force will with other molecules and also the adjacent water drastically reduce, when the distance between molecules. This contributes to cohesiveness of atoms is increased. Although very weak, van der water. Waals forces collectively contribute maximum c. Water favors hydrophobic interactions and provides towards the stability of protein structure, especially a basis for metabolism of insoluble substances. in preserving the nonpolar interior structure of Water expands when it is cooled from 4oC to o 0 C, while normally liquids are expected to contract proteins. due to cooling. As water is heated from 0oC to WATER: THE UNIVERSAL SOLVENT 4 oC, the hydrogen bonds begin to break. This results in a decrease in volume or in other words, Water constitutes about 70 to 80 percent of the increase in density. Hence, water attains high weight of most cells. The hydrogen atom in one density at 4oC. However, above 4oC the effect of water molecule is attracted to a pair of electrons in temperature predominates. the outer shell of an oxygen atom in an adjacent molecule. The structure of liquid water contains PRINCIPLES OF THERMODYNAMICS hydrogen-bonded networks (Fig. 1.5). Thermodynamics is concerned with the flow of heat The crystal structure of ice depicts a tetrahedral and it deals with the relationship between heat and arrangement of water molecules. Four others bound work. Bioenergetics, or biochemical thermo- by hydrogen bonds surround each oxygen atom. dynamics, is the study of the energy changes On melting, the molecules get much closer and this accompanying biochemical reactions. Biological results in the increase in density of water. Hence, systems use chemical energy to power living liquid water is denser than solid ice. This also processes. explains why ice floats on water. Water molecules are in rapid motion, constantly 1. First Law of Thermodynamics making and breaking hydrogen bonds with adjacent The total energy of a system, including its molecules. As the temperature of water increases surroundings, remains constant.  Chapter 1; Biochemical Perspective to Medicine 5 Or, ΔE = Q – W, where Q is the heat absorbed by conditions are defined for biochemical reactions the system and W is the work done. This is also at a pH of 7 and 1 M concentration, and differen- called the law of conservation of energy. If heat tiated by a priming sign ΔG0. It is directly related is transformed into work, there is proportionality to the equilibrium constant. Actual free energy between the work obtained and the heat dissipated. changes depend on reactant and product. A system is an object or a quantity of matter, chosen Most of the reversible metabolic reactions are for observation. All other parts of the universe, near equilibrium reactions and therefore their ΔG outside the boundary of the system, are called the is nearly zero. The net rate of near equilibrium surroundings. reactions are effectively regulated by the relative concentration of substrates and products. The 2. Second Law of Thermodynamics metabolic reactions that function far from The total entropy of a system must increase if a equilibrium are irreversible. The velocity of these process is to occur spontaneously. A reaction reactions are altered by changes in enzyme occurs spontaneously if ΔE is negative, or if the activity. A highly exergonic reaction is irreversible entropy of the system increases. Entropy (S) is a and goes to completion. Such a reaction that is measure of the degree of randomness or disorder part of a metabolic pathway, confers direction to of a system. Entropy becomes maximum in a the pathway and makes the entire pathway system as it approaches true equilibrium. Enthalpy irreversible. is the heat content of a system and entropy is that fraction of enthalpy which is not available to do Three Types of Reactions useful work. A. A reaction can occur spontaneously when ΔG is A closed system approaches a state of negative. Then the reaction is exergonic. If ΔG equilibrium. Any system can spontaneously is of great magnitude, the reaction goes to proceed from a state of low probability (ordered completion and is essentially irreversible. state) to a state of high probability (disordered B. When ΔG is zero, the system is at equilibrium. state). The entropy of a system may decrease C. For reactions where the ΔG is positive, an input with an increase in that of the surroundings. The of energy is required to drive the reaction. The second law may be expressed in simple terms reaction is termed as endergonic.and those with as Q = T x ΔS, where Q is the heat absorbed, T a negative ΔG as exergonic. (Examples given is the absolute temperature and ΔS is the change below). Similarly a reaction may be in entropy. exothermic (ΔH is negative), isothermic (ΔH is zero) or endothermic (ΔH is positive). 3. Gibb's Free Energy Concept Energetically unfavorable reaction may be driven The term free energy is used to get an equation forward by coupling it with a favorable reaction. combining the first and second laws of Glucose + Pi → Glucose-6-phosphate (reaction1) thermodynamics. Thus, ΔG = ΔH – TΔS , where ATP + H2O → ADP + Pi (reaction 2) ΔG is the change in free energy, ΔH is the change Glucose+ATP→Glucose-6-phosphate+ADP (3) in enthalpy or heat content of the system and ΔS is the change in entropy. The term free energy Reaction 1 cannot proceed spontaneously. denotes a portion of the total energy change in But the 2nd reaction is coupled in the body, so a system that is available for doing work. that the reaction becomes possible. For the first For most biochemical reactions, it is seen that reaction, ΔG0 is +13.8 kJ/mole; for the second ΔH is nearly equal to ΔE. So, ΔG = ΔE – TΔS. Hence, reaction, ΔG0 is –30.5 kJ/mole. When the two ΔG or free energy of a system depends on the reactions are coupled in the reaction 3, the ΔG 0 change in internal energy and change in entropy of becomes –16.7 kJ/mole, and hence the reaction a system. becomes possible. Details on ATP and other high-energy phosphate bonds are described in 4. Standard Free Energy Change Chapter 19. It is the free energy change under standard Reactions of catabolic pathways (degradation conditions. It is designated as ΔG0. The standard or oxidation of fuel molecules) are usually exergonic, 6 Textbook of Biochemistry; Section A: Chemical Basis of Life Donnan's equation also states that the electrical neutrality in each compartment should be maintained. In other words the number of cations should be equal to the number of anions, such that In left : Na+ = R¯+ Cl¯; substituting: 9 = 5 + 4 In right : Na+ = Cl¯; substituting: 6 = 6 Figs 1.6A and B. Donnan membrane equilibrium The equation should also satisfy that the number of sodium ions before and after the equilibrium are whereas anabolic pathways (synthetic reactions or the same; in our example, initial Na+ in the two building up of compounds) are endergonic. compartments together is 5 + 10 = 15; after Metabolism constitutes anabolic and catabolic equilibrium also, the value is 9 + 6 = 15. In the case processes that are well co-ordinated. of chloride ions, initial value is 10 and final value is also 4 + 6 = 10. DONNAN MEMBRANE EQUILIBRIUM In summary, Donnan's equations satisfy the When two solutions are separated by a membrane following results: permeable to both water and small ions, but when 1. The products of diffusible electrolytes in both one of the compartments contains impermeable compartments are equal. ions like proteins, distribution of permeable ions 2. The electrical neutrality of each compartment is occurs according to the calculations of Donnan. maintained. In Fig. 1.6, the left compartment contains NaR, 3. The total number of a particular type of ions which will dissociate into Na+ and R¯. Then Na+ before and after the equilibrium is the same. can diffuse freely, but R¯ having high molecular 4. As a result, when there is non-diffusible anion weight cannot diffuse. The right compartment on one side of a membrane, the diffusible cations are more, and diffusible anions are less, contains NaCl, which dissociates into Na+ and Cl¯. on that side. Both ions can diffuse freely. Thus, if a salt of NaR is placed in one side of a Clinical Applications of the Equation membrane, at equilibrium 1. The total concentration of solutes in plasma Na+ x R¯ x H+ x OH¯ = Na+ x OH¯ x H+ will be more than that of a solution of same ionic To convey the meaning of the mathematical strength containing only diffusible ions; this provides the net osmotic gradient (see under values, a hypothetical quantity of each of the ion is Albumin, in Chapter 28). also incorporated in brackets. Initially 5 molecules 2. The lower pH values within tissue cells than of NaR are added to the left compartment and 10 in the surrounding fluids are partly due to the molecules of NaCl in the right compartment and concentrations of negative protein ions within the both of them are ionized (Fig. 1.6A). When cells being higher than in surrounding fluids. equilibrium is reached, the distributions of ions are 3. The pH within red cells is lower than that of shown in Figure 1.6B. According to Donnan's the surrounding plasma is due, in part, to the equilibrium, the products of diffusible electrolytes very high concentration of negative non-diffusible in both the compartments will be equal, so that hemoglobin ions. This will cause unequal [Na+] L x [Cl¯ ] L = [Na+] Rx [Cl¯ ] R distribution of H+ ions with a higher concentration within the cell. If we substitute the actual numbers of ions, the 4. The chloride shift in erythrocytes as well as formula becomes higher concentration of chloride in CSF are also 9 x 4 in left = 6 x 6 in right due to Donnan's effect (Chapter 22).  CHAPTER Subcellular Organelles 2 and Cell Membranes CHAPTER AT A GLANCE SUBCELLULAR ORGANELLES Cells contain various organized structures, The reader will be able to answer questions on collectively called as cell organelles (Fig. 2.1). the following topics: When the cell membrane is disrupted, either by 1. Nucleus mechanical means or by lysing the membrane by 2. Endoplasmic reticulum Tween-20 (a lipid solvent), the organized particles 3. Golgi apparatus inside the cell are homogenised. This is usually 4. Lysosomes carried out in 0.25 M sucrose at pH 7.4. The 5. Mitochondria organelles could then be separated by applying 6. Plasma membrane differential centrifugal forces (Table 2.1). Albert 7. Transport mechanisms Claude got Nobel prize in 1974 for fractionating 8. Simple and facilitated diffusion subcellular organelles. 9. Ion channels 10. Active transport Marker Enzymes 11. Uniport, symport and antiport Some enzymes are present in certain organelles only; such specific enzymes are called as marker enzymes (Table 2.1). After centrifugation, the separated organelles are identified by detection of marker enzymes in the sample. NUCLEUS 1. It is the most prominent organelle of the cell. All cells in the body contain nucleus, except mature RBCs in circulation. The uppermost layer of skin also may not possess a readily identifiable nucleus. In some cells, nucleus occupies most of the available space, e.g. small lymphocytes and spermatozoa. 2. Nucleus is surrounded by two membranes: the inner one is called perinuclear membrane with Fig. 2.1. Typical cell 1= Nuclear membrane; 2= Nuclear pore; 3= Nucleolus; 4= endoplasmic reticulum; 5= Golgi body; 6= Mitochondria; 7= Microtubule; 8= Lysosome; 9= Vacuole; 10= Plasma membrane Fig. 2.2. Nucleus 8 Textbook of Biochemistry; Section A: Chemical Basis of Life 2. This will be very prominent in cells actively syn- thesizing proteins, e.g. immunoglobulin secreting plasma cells. The proteins, glycoproteins and lipoproteins are synthesized in the ER. 3. Detoxification of various drugs is an important function of ER. Microsomal cytochrome P-450 hydroxylates drugs such as benzpyrine, amino- pyrine, aniline, morphine, phenobarbitone, etc. 4. According to the electron microscopic appear- numerous pores (Fig. 2.2). The outer membrane ance, the ER is generally classified into rough is continuous with membrane of endoplasmic and smooth varieties. The rough appearance reticulum. is due to ribosomes attached to cytoplasmic side 3. Nucleus contains the DNA, the chemical basis of of membrane where the proteins are being genes which governs all the functions of the cell synthesized.. The very long DNA molecules are complexed 5. When cells are fractionated, the complex ER is with proteins to form chromatin and are further disrupted in many places. They are automatically organized into chromosomes. re-assembled to form microsomes. 4. DNA replication and RNA synthesis (trans- cription) are taking place inside the nucleus. GOLGI APPARATUS 5. In some cells, a portion of the nucleus may be 1. Camillo Golgi described the structure in 1898 seen as lighter shaded area; this is called (Nobel prize 1906). The Golgi organelle is a nucleolus (Fig. 2.2). This is the area for RNA network of flattened smooth membranes and processing and ribosome synthesis. The vesicles. It may be considered as the converging nucleolus is very prominent in cells actively area of endoplasmic reticulum (Fig. 2.1). synthesizing proteins. Gabriel Valentine in 1836 2. While moving through ER, carbohydrate groups described the nucleolus. are successively added to the nascent proteins. These glycoproteins reach the Golgi area. ENDOPLASMIC RETICULUM (ER) 3. Golgi apparatus is composed of cis, medial and trans 1. It is a network of interconnecting membranes cisternae. Glycoproteins are generally transported from ER enclosing channels or cisternae, that are to cis Golgi (proximal cisterna), then to medial Golgi continuous from outer nuclear envelope to outer (intermediate cisterna) and finally to trans Golgi (distal plasma membrane. Under electron microscope, cisterna) for temporary storage. Trans Golgi are particularly abundant with vesicles containing glycoproteins. Newly the reticular arrangements will have railway track synthesized proteins are sorted first according to the sorting appearance (Fig. 2.1). George Palade was signals available in the proteins. Then they are packed into awarded Nobel prize in 1974, who identified the transport vesicles having different types of coat proteins. ER. Finally, they are transported into various destinations; this is an energy dependent process. Table 2.1. Separation of subcellular organelles 4. Main function of Golgi apparatus is protein Subcellular Pellet formed at the Marker enzyme sorting, packaging and secretion. organelle centrifugal force of 5. The finished products may have any one of the Nucleus 600–750 x g, 10 min following destinations: Mitochondria 10,000–15,000 x g, Inner membrane: a. They may pass through plasma membrane 10 min ATP Synthase to the surrounding medium. This forms Lysosome 18,000–25,000 x g, Cathepsin continuous secretion, e.g. secretion of 10 min immunoglobulins by plasma cells. Golgi complex 35,000–40,000 x g, Galactosyl transferase b. They reach plasma membrane and form an 30 min integral part of it, but not secreted. Microsomes 75,000–100,000 x g, Glucose-6- c. They are formed into a secretory vesicle, where 100 min phosphatase these products are stored for a longer time. Cytoplasm Supernatant Lactate Under appropriate stimuli, the contents are dehydrogenase secreted. Release of trypsinogen by pancreatic  Chapter 2; Subcellular Organelles and Cell Membranes 9 Table 2.2. Metabolic functions of subcellular d. Lipid hydrolysing enzymes (fatty acyl esterase, organelles phospholipases) Organelle Function PEROXISOMES Nucleus DNA replication, transcription 1. The peroxisomes have a granular matrix. They Endoplasmic Biosynthesis of proteins, glycoproteins, are of 0.3–1.5 μm in diameter. They contain reticulum lipoproteins, drug metabolism, ethanol peroxidases and catalase. They are prominent oxidation, synthesis of cholesterol (partial) in leukocytes and platelets. Golgi body Maturation of synthesized protein 2. Peroxidation of polyunsaturated fatty acids Lysosome Degradation of proteins, carbohydrates, in vivo may lead to hydroperoxide formation, R- lipids and nucleotides OOH → R- OO. The free radicals damage Mitochondria Electron transport chain, ATP generation, molecules, cell membranes, tissues and genes. TCA cycle, beta oxidation of fatty acids, (Chapter 20). ketone body production, urea synthesis 3. Catalase and peroxidase are the enzymes (part), heme synthesis (part), gluconeo- present in peroxisomes which will destroy the genesis (part), pyrimidine synthesis (part) unwanted peroxides and other free radicals. Cytosol Protein synthesis, glycolysis, glycogen Clinical applications of peroxisomes are shown metabolism, HMP shunt pathway, in Box 2.2. transaminations, fatty acid synthesis, cholesterol synthesis, heme synthesis (part), urea synthesis (part), pyrimidine MITOCHONDRIA synthesis (part), purine synthesis 1. They are spherical, oval or rod-like bodies, about 0.5–1 μm in diameter and up to 7 μm in length acinar cells and release of insulin by beta cells of Langerhans are cited as examples. Box 2.1. Clinical Applications of Lysosomes d. The synthesized materials may be collected into lysosome packets. 1. In gout, urate crystals are deposited around knee joints (Chapter 39). These crystals when phagocytosed, cause physical damage to lysosomes and release of enzymes. LYSOSOMES Inflammation and arthritis result. 1. Discovered in 1950 by Rene de Duve (Nobel 2. Following cell death, the lysosomes rupture releasing prize 1974), lysosomes are tiny organelles. Solid the hydrolytic enzymes which bring about postmortem wastes of a township are usually decomposed autolysis. in incinerators. Inside a cell, such a process is 3. Lysosomal proteases, cathepsins are implicated in tumor metastasis. Cathepsins are normally restricted to the interior taking place within the lysosomes. They are bags of lysosomes, but certain cancer cells liberate the cathepsins of enzymes. Clinical applications of lysosomes out of the cells. Then cathepsins degrade the basal lamina by are shown in Box 2.1. hydrolysing collagen and elastin, so that other tumor cells 2. Endocytic vesicles and phagosomes are fused with can travel out to form distant metastasis. lysosome (primary) to form the secondary lysosome or 4. There are a few genetic diseases, where lysosomal digestive vacuole. Foreign particles are progressively enzymes are deficient or absent. This leads to accu- digested inside these vacuoles. Completely hydrolysed mulation of lipids or polysaccharides (Chapters 10 and products are utilized by the cell. As long as the lysosomal 13). membrane is intact, the encapsulated enzymes can act 5. Silicosis results from inhalation of silica particles into only locally. But when the membrane is disrupted, the the lungs which are taken up by phagocytes. Lysosomal released enzymes can hydrolyse external substrates, membrane ruptures, releasing the enzymes. This stimulates leading to tissue damage. fibroblast to proliferate and deposit collagen fibers, resulting 3. The lysosomal enzymes have an optimum pH around 5. in fibrosis and decreased lungs elasticity. These enzymes are 6. Inclusion cell (I- cell) disease is a rare condition in which a. Polysaccharide hydrolysing enzymes (alpha-gluco- lysosomes lack in enzymes, but they are seen in blood. sidase, alpha-fucosidase, beta-galactosidase, alpha- This means that the enzymes are synthesized, but are not mannosidase, beta-glucuronidase, hyaluronidase, aryl able to reach the correct site. It is shown that mannose-6- sulfatase, lysozyme) phosphate is the marker to target the nascent enzymes b Protein hydrolysing enzymes (cathepsins, colla- to lysosomes. In these persons, the carbohydrate units are not added to the enzyme. Mannose-6-phosphate- genase, elastase, peptidases) deficient enzymes cannot reach their destination (protein c. Nucleic acid hydrolysing enzymes (ribonuclease, deo- targetting defect). xyribonuclease) 10 Textbook of Biochemistry; Section A: Chemical Basis of Life Fig. 2.3. Mitochondria Fig. 2.4A. The fluid mosaic model of membrane (Fig. 2.1). Erythrocytes do not contain mito- chondria. The tail of spermatozoa is fully packed with mitochondria. 2. Mitochondria are the powerhouse of the cell, where energy released from oxidation of food stuffs is trapped as chemical energy in the form of ATP (Chapter 19). Metabolic functions of mitochondria are shown in Table 2.2. 3. Mitochondria have two membranes. The inner membrane convolutes into folds or cristae (Fig. 2.3). The inner mitochondrial membrane contains the enzymes of electron transport chain (Chapter 19). The fluid matrix contains the enzymes of citric acid cycle, urea cycle and heme synthesis. Fig. 2.4B. Proteins are anchored in the 4. Cytochrome P-450 system present in mito- membrane by different mechanisms chondrial inner membrane is involved in steroidogenesis (Chapter 46). Superoxide 5. Mitochondria also contain specific DNA. The integral inner membrane proteins, are made by mitochondrial protein dismutase is present in cytosol and mito- synthesising machinery. However the majority of proteins, chondria (Chapter 20). especially of outer membrane are synthesised under the control of cellular DNA. The division of mitochondria is under the command of mitochondrial DNA. Mitochondrial Box 2.2. Peroxisomal Deficiency Diseases ribosomes are different from cellular ribosomes. 1. Deficiency of peroxisomal matrix proteins can lead to Antibiotics inhibiting bacterial protein synthesis do not adreno leuko dystrophy (ALD) (Brown-Schilder’s disease) affect cellular processes, but do inhibit mitochondrial characterized by progressive degeneration of liver, kidney protein biosynthesis (Chapter 41). and brain. It is a rare autosomal recessive condition. The 6. Taking into consideration such evidences, it is assumed defect is due to insufficient oxidation of very long chain fatty that mitochondria are parasites which entered into cells acids (VLCFA) by peroxisomes (see Chapter 13). at a time when multicellular organisms were being 2. In Zellweger syndrome, proteins are not transported evolved. These parasites provided energy in large quanti- into the peroxisomes. This leads to formation of empty ties giving an evolutionary advantage to the cell; the cell peroxisomes or peroxisomal ghosts inside the cells. Protein gave protection to these parasites. This perfect targetting defects are described in Chapter 41. symbiosis, in turn, evolved into a cellular organelle of 3. Primary hyperoxaluria is due to the defective peroxisomal mitochondria. metabolism of glyoxalate derived from glycine (Chapter 15). 7. A summary of functions of organelles is given in Table 2.2 and Box 2.3.  Chapter 2; Subcellular Organelles and Cell Membranes 11 Box 2.3. Comparison of Cell with a Factory 4-C. The lipid bilayer shows free lateral movement Plasma Fence with gates; gates open of its components, hence the membrane is said membrane when message is received to be fluid in nature. Fluidity enables the membrane to perform endocytosis and Nucleus Manager’s office exocytosis. Endoreticulum Conveyer belt of production units 4-D. However, the components do not freely move Golgi apparatus Packing units from inner to outer layer, or outer to inner layer Lysosomes Incinerators (flip-flop movement is restricted). During apoptosis (programmed cell death), flip-flop Vacuoles Lorries carrying finished products movement occurs. Mitochondria Power generating units This Flip-flop movement is catalyzed by enzymes. Flippases catalyse the transfer of amino phospholipids across the membrane. Floppases catalyse the outward PLASMA MEMBRANE directed movement which is ATP dependent. This is 1. The plasma membrane separates the cell from mainly seen in the role of ABC proteins mediating the the external environment. It has highly selective efflux of cholesterol and the extrusion of drugs from cells. The MDR (multi drug resistance) associated permeability properties so that the entry and exit p-glycoprotein is a floppase. Ernst Ruska designed the of compounds are regulated. The cellular first electron microscope in 1939. Gerd Binning and metabolism is in turn influenced and probably Heinrich Rohrer introduced the scanning electron regulated by the membrane. The membrane is microscopy in 1981 by which the outer and inner layers of membranes could be visualized separately. All the metabolically very active. three workers were awarded Nobel prize in 1986. 2. The enzyme, nucleotide phosphatase (5' 4-E. The cholesterol content of the membrane nucleotidase) and alkaline phosphatase are seen alters the fluidity of the membrane. When on the outer part of cell membrane; they are cholesterol concentration increases, the therefore called ecto-enzymes. membrane becomes less fluid on the outer 3. Membranes are mainly made up of lipids, surface, but more fluid in the hydrophobic core. proteins and small amount of carbohydrates. The The effect of cholesterol on membrane fluidity contents of these compounds vary according to is different at different temperatures. At the nature of the membrane. The carbohydrates temperature below the Tm cholesterol in- are present as glycoproteins and glycolipids. creases fluidity and there by permeability of Phospholipids are the most common lipids the membrane. At temperatures above the Tm, present and they are amphipathic in nature. Cell cholesterol decreases fluidity. membranes also contain cholesterol. In spur cell anemia and alcoholic cirrhosis membrane studies have revealed the role of excess cholesterol. 4. Fluid Mosaic Model The decrease in membrane fluidity may affect the activities of receptors and ion channels. This has The lipid bilayer was originally proposed by Davson been implicated in conditions like LCAT deficiency, and Danielle in 1935. Later, the structure of the Alzheimer’s disease and hypertension. biomembranes was described as a fluid mosaic Fluidity of cellular membranes responds to variations model (Singer and Nicolson, 1972). in diet and physiological states. Increased release of 4-A. The phospholipids are arranged in bilayers with reactive oxygen species (ROS), increase in cytosolic calcium and lipid peroxidation have been found to the polar head groups oriented towards the adversely affect membrane fluidity. Anesthetics may act extracellular side and the cytoplasmic side with by changing membrane fluidity. a hydrophobic core (Fig. 2.4A). The distribution 4-F. The nature of the fatty acids also affects the of the phospholipids is such that choline fluidity of the membrane, the more unsaturated containing phospholipids are mainly in the cis fatty acids increase the fluidity. external layer and ethanolamine and serine The fluidity of the membrane is maintained by the length of containing phospholipids in the inner layer. the hydrocarbon chain, degree of unsaturation and nature of the polar head groups. Trans fatty acids (TFA) decrease the 4-B. Each leaflet is 25 Å thick, with the head portion fluidity of membranes due to close packing of hydrocarbon 10 Å and tail 15 Å thick. The total thickness is chains. Cis double bonds create a kink in the hydrocarbon chain about 50 to 80 Å. and have a marked effect on fluidity. Second OH group of 12 Textbook of Biochemistry; Section A: Chemical Basis of Life glycerol in membrane phospholipids is often esterified to an by caveolae mediated transcytosis. The endocytosis of unsaturated fatty acid, mono un