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    VK Malhotra - Biochemistry for Students, 12th Edition VK Malhotra - Biochemistry for Students, 12th Edition Document Transcript

    • BIOCHEMISTRY for Students
    • BIOCHEMISTRY for Students 12th Edition VK Malhotra PhD (Gold Medalist) Department of Biochemistry Maulana Azad Medical College (MAMC) New Delhi, India Foreword Nancy Kaul ® JAYPEE BROTHERS MEDICAL PUBLISHERS (P) LTD New Delhi • Panama City • London
    • Published by 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 Website: www.jaypeebrothers.com Offices in India • Ahmedabad, e-mail: ahmedabad@jaypeebrothers.com • Bengaluru, e-mail: bangalore@jaypeebrothers.com • Chennai, e-mail: chennai@jaypeebrothers.com • Delhi, e-mail: jaypee@jaypeebrothers.com • Hyderabad, e-mail: hyderabad@jaypeebrothers.com • Kochi, e-mail: kochi@jaypeebrothers.com • Kolkata, e-mail: kolkata@jaypeebrothers.com • Lucknow, e-mail: lucknow@jaypeebrothers.com • Mumbai, e-mail: mumbai@jaypeebrothers.com • Nagpur, e-mail: nagpur@jaypeebrothers.com Overseas Offices • Central America Office, Panama City, Panama, Ph: 001-507-317-0160 e-mail: cservice@jphmedical.com, Website: www.jphmedical.com • Europe Office, UK, Ph: +44 (0) 2031708910 e-mail: info@jpmedpub.com Biochemistry for Students © 2012, Jaypee Brothers Medical Publishers All rights reserved. No part of this publication should be reproduced, stored in a retrieval system, or transmitted in any form or by any means: electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the author and the publisher. This book has been published in good faith that the material provided by the author is original. Every effort is made to ensure accuracy of material, but the publisher, printer and author 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. Previous Editions: 1978, 1980, 1982, 1984, 1985, 1987, 1989, 1991 (Reprint 1993), 1996, 1998, 2003 (Reprint 2006, 2008) Twelfth Edition: 2012 ISBN 978-93-5025-504-9 Typeset at JPBMP typesetting unit Printed at
    • Foreword Biochemistry has been playing a very important role in dayto-day life of medical students. The book Biochemistry for Students written by Dr VK Malhotra, Gold Medalist, serves as a quick reading material being purposefully written in clear, lucid and precise manner. This book will certainly serve the needs of medical students. Dr (Mrs) Nancy Kaul Ex-Head, Department of Biochemistry Lady Hardinge Medical College New Delhi, India
    • Preface to the Twelfth Edition This book is revised keeping in view all categories of students and it addresses their needs in a simple and practical manner as biochemistry tries to explain the mystery of life in the language of chemistry. I hope the book will be received warmly by the students as well as teachers for both desire maximum benefits out of it. All the chapters are revised to gain understanding and clarity. Suggestions to improve the future editions are most welcome and will be highly appreciated. I would like to thank Shri Jitendar P Vij (Chairman and Managing Director) and Mr Tarun Duneja (Director Publishing) of M/s Jaypee Brothers Medical Publishers (P) Ltd, New Delhi for the publication of this book. Mr Subrata Adhikary (Author Coordinator) deserves special praise for this venture. VK Malhotra
    • Preface to the First Edition Biochemistry currently occupies an eminent position particularly among medical subjects. However, there are few texts in the market at present which enable the students to acquire a working knowledge of the subject. Having been connected with the teaching profession for the past few years, I am well acquainted with the difficulties encountered by the students while trying to master the subject. The present book is the result of my humble attempt to overcome these handicaps and present the subject in a simple and easily comprehensible form. Attempts have been made to illustrate the subject matter with diagrams and chemical formulae wherever necessary. Special thanks to my publisher Shri Jitendar P Vij, without whose help, this book could not have seen the light of the day. VK Malhotra
    • Contents 1. Biophysics ......................................................................... 1 • • • • • • 2. Chemistry of Carbohydrates ..................................... 19 • • • • • • 3. Hydrogen Ion Concentration, pH ................................................. 1 Osmosis and Osmotic Pressure .................................................... 12 Colloids ............................................................................................ 16 Surface Tension ............................................................................... 17 Absorption ....................................................................................... 18 Viscosity ........................................................................................... 18 Carbohydrates ................................................................................ 19 Functions of Carbohydrates ......................................................... 19 Classification of Carbohydrates ................................................... 19 Oligosaccharides ............................................................................. 40 Polysaccharides ............................................................................... 45 Heteropolysaccharides .................................................................. 49 Chemistry of Lipids .................................................... 53 • Simple Lipids ................................................................................... 54 • Compound Lipids ........................................................................... 62 • Derived Lipid ................................................................................... 68 4. Chemistry of Amino Acids and Proteins ............ 74 • Chemistry of Amino Acids ........................................................... 74 • Proteins ............................................................................................ 85 5. Hemoglobin .................................................................. 102 • Porphins ......................................................................................... 102 • Porphyrins ..................................................................................... 103
    • xii BIOCHEMISTRY FOR STUDENTS • Hemoglobin .................................................................................. 103 • Porphyria ....................................................................................... 114 6. Enzymes ........................................................................ 120 • • • • • • • 7. Biological Oxidation ................................................. 140 • • • • 8. Biological Oxidation ...................................................................... 140 Mixed Function Oxidases ............................................................. 142 High Energy Compounds ........................................................... 143 Respiratory Chain ........................................................................ 144 Metabolism of Carbohydrates ............................... 151 • • • • • • • • • • 9. Enzymes ......................................................................................... 120 Factors Influencing the Rate of Enzymatic Reactions ............. 124 Enzyme Activity ........................................................................... 129 Enzyme Inhibitions ...................................................................... 130 Catalytic Site or the Active Sites of the Enzymes .................... 134 Enzyme Induction ........................................................................ 135 Diagnostic Value of Plasma Enzymes ....................................... 137 Glycolysis ....................................................................................... 151 Citric Acid Cycle ........................................................................... 155 Energetics ....................................................................................... 158 Glycogenesis .................................................................................. 163 Gluconeogenesis ........................................................................... 168 Galactose Metabolism .................................................................. 169 Fructose Metabolism .................................................................... 172 Lactose Synthesis .......................................................................... 173 Uronic Acid Pathway ................................................................... 174 Regulation of Blood Glucose ....................................................... 175 Metabolism of Lipids ............................................... 184 • Plasma Lipoproteins ..................................................................... 184
    • CONTENTS xiii 10. Metabolism of Proteins ........................................... 210 • • • • • • • • Digestion and Absorption ........................................................... 210 Urea Cycle (Krebs-Henseleit Cycle) .......................................... 214 Glycine ............................................................................................ 221 Methionine ..................................................................................... 226 Cysteine and Cystine ................................................................... 227 Phenylalanine and Tyrosine ........................................................ 229 Tryptophan .................................................................................... 237 Leucine, Isoleucine and Valine .................................................... 240 11. Nucleic Acid—Chemistry and Metabolism ...... 241 • Nucleic Acids ................................................................................. 244 12. Vitamins ....................................................................... 259 • • • • • • • • • • • • • • • • Fat Soluble Vitamins .................................................................... 261 Vitamin A ....................................................................................... 261 Vitamin D ....................................................................................... 264 Vitamin E ....................................................................................... 265 Vitamin K ....................................................................................... 266 Water Soluble Vitamins ............................................................... 268 Vitamin C ....................................................................................... 268 Thiamine ........................................................................................ 270 Riboflavin ....................................................................................... 272 Niacin .............................................................................................. 273 Pantothenic Acid ........................................................................... 275 Pyridoxine ...................................................................................... 275 Biotin ............................................................................................... 277 Folic Acid ........................................................................................ 279 Cyanocobalamin ........................................................................... 281 Antivitamins .................................................................................. 283 13. Acid-base Balance ..................................................... 284 • Acid-base Balance ......................................................................... 284
    • xiv BIOCHEMISTRY FOR STUDENTS 14. Water and Mineral Metabolism ........................... 292 • Biological Importance of Water .................................................. 292 • Minerals .......................................................................................... 295 15. Xenobiotics .................................................................... 306 16. Nutrition ....................................................................... 310 • Food Values ................................................................................... 319 • 1500 Calories Diabetic Diet Chart .............................................. 322 17. Organ Function Tests ............................................... 326 • • • • Liver Function Tests ..................................................................... 326 Renal Function Tests .................................................................... 330 Pancreatic Function Test .............................................................. 335 Gastrointestinal (Git) Function Test ........................................... 338 18. Immunology ................................................................. 339 • Introduction ................................................................................... 339 • Functions of T Cells ...................................................................... 343 19. Cancer ............................................................................ 356 20. Hormones...................................................................... 360 • • • • • • Insulin ............................................................................................. 364 Glucagon ........................................................................................ 367 Triiodothyronine (T3) and Thyroxine (T4) ................................. 367 Calcitonin ....................................................................................... 368 Parathormone ............................................................................... 369 Thyroid Gland ............................................................................... 370
    • CONTENTS xv 21. Protein Biosynthesis ................................................. 371 • • • • • • Activation Step .............................................................................. 372 Initiation of Polypeptide Chain (In Ribosomes) ...................... 374 Elongation ...................................................................................... 376 Termination ................................................................................... 378 Codon ............................................................................................. 380 Regulation of Gene Expression .................................................. 381 22. Instrumentation .......................................................... 385 • • • • • • Colorimetry ................................................................................... 385 Electrophoresis .............................................................................. 386 Isotopes and their Application .................................................... 387 Electrometric Determination of pH ........................................... 388 Estimation of Nitrogen Content by Micro-Kjeldahl Method ..... 390 Chromatography ......................................................................... 393 Index ........................................................................................ 397
    • CHAPTER Biophysics 1 HYDROGEN ION CONCENTRATION, pH Acids are substances which furnish hydrogen ions (H+) in the solution, whereas bases are substances that furnish hydroxide ions (OH–) in the solution. Substances that dissociate in water into a cation (positively charged ion) and an anion (negatively charged ion) are classified as electrolytes. Whereas sugar or alcohols which dissolve in water but do not carry a charge or dissociate into species with a positive and negative charge are classified as nonelectrolytes. Strong electrolytes are completely ionized in aqueous solutions whereas weak electrolytes are partially ionized in aqueous solutions. pH of a solution is defined as the negative logarithm of its hydrogen ion concentration. pH = – log10 [H+] = 1 log 10 [H + ] Pure water has equal concentration of H+ and OH– ions, the concentrations of each is very small and each being equal to 10–7 moles/liter at room temperature. Water dissociates into: H2O H+ + OH– From the Law of Mass action, the dissociation of water can be represented as: Kw = [H+ ] [OH – ] [H2 O]
    • 2 BIOCHEMISTRY FOR STUDENTS The bracket indicates the concentration of each component in moles per liter. The concentration of undissociated water is so large as compared to the concentration of H+ and OH– ions, so that for all the practical purposes it is fairly constant. This simplifies the above equation to: [H+] [OH–] = K [H2O] [H+] [OH–] = Kw Where Kw is ionic product of water or the dissociation constant of water. Electrical conductivity measurements have shown that dissociation constant of water is constant at a given temperature and changes with the change in temperature. Ionic product of water is usually taken as 10–14 at the room temperature (25ºC). Then [H+] [OH–] = 10–14 Taking logarithm of both sides log [H+] + log [OH–] = –14 By rearrangement –log [H+] –log [OH–] = 14 According to the definition of pH, the above equation simplifies to: pH + pOH = 14 At neutrality, both hydrogen and hydroxide ions have equal concentration, i.e. pH = 7 pOH = 7 There exists an inverse relationship between [H+] and [OH–] ions in solution. As hydrogen ion concentration increases, the hydroxide ion concentration decreases and vice versa. The acidity or alkalinity of a solution is determined by the amount of [H+] and [OH–] ions present.
    • BIOPHYSICS 3 A solution having hydrogen ions concentration of one normality (1 N) will have a pH 0, and other having hydroxide concentration of one normality (1 N) will have pH 14. It should also be kept in mind that a change of one pH unit brings a ten-fold change in acidity or alkalinity, i.e. a solution of pH 5 has ten times more the hydrogen ion concentration than that of a solution of pH 6 and a hundred times more than that of a solution of pH 7. If hydrogen ion concentration is doubled, the pH falls by 0.3 units. The average pH values of some of body fluids are: Gastric juice 1.4 Saliva 6.8 Urine 6.0 Milk 7.1 Tears 7.2 Blood 7.4 Pancreatic juice 8.0 Q. Calculate the pH of a solution of which hydrogen ion concentration is 4.6 × 10–9 M. Ans. pH = –log10 [CH+] = –log10 [4.6 × 10–9] = –log10 4.6 + 9 log10 10 = –0.66 + 9 = 8.34. Q. Calculate the hydrogen ion concentration of a solution, the pH of which is 4.50. Ans. pH = –log10 [CH+]
    • 4 BIOCHEMISTRY FOR STUDENTS log 10 [CH+] = – pH = – 4.50 = 5.50 [CH+] = Antilog 5.50 [CH+] = Antilog 0.5 × antilog 5.00 = 3.16 × 10–5 M. Buffers Buffers are the solutions, which resist changes in pH, when small amount of acid or alkali is added to them. The best buffer is the one which gives the smallest change in pH. Buffers act like shock absorber against the sudden changes of pH. Acetic acid: sodium acetate (CH3COOH; CH3COONa) and carbonic acid: sodium carbonate (H2CO3; NaHCO3) are examples of buffer systems. Physiologic buffers include bicarbonate, orthophosphate and proteins. A buffer is a pair of weak acid and its salt with a strong base or a pair of weak base and its salt with a strong acid. If either free H+ or free OH– are added to a solution of such a pair they will be partially converted to the unionized form. Thus or B– + H+ HB + OH– BH H2O + B– Where HB denotes a weak acid and B– its conjugate base. The combination of a weak acid and the base that is formed on dissociation is referred to as a conjugate pair. Ammonium ion NH+ is an acid because it dissociates to yield a H+ and 4 NH3 which is conjugate base. Phosphoric acid (H3PO4) is an acid and PO4–3 is a base. NH4+ (acid) H3PO4 H+ + NH3 (conjugate base) 3¯ 3H+ + PO4 The ability to buffer hydrogen ions is more important to the body than the buffering of hydroxyl ions. The most commonly used buffers in the laboratory are: Acetate buffer (Sodium acetate/acetic acid). Phosphate buffer (Na2HPO4/KH2PO4). Citrate buffer (Sodium citrate/citric acid).
    • BIOPHYSICS 5 Barbitone buffer (Sodium diethyl barbiturate/diethyl barbituric acid). The pH of a buffer solution is calculated by the HendersonHasselbalch equation. Suppose the solution is composed of a weak acid [HA] and its salt with a strong base [BA]. The dissociation of weak acid [HA] and salt [BA] can be represented as follows: HA H+ + A– Weak acid Proton + Conjugate base Conjugate base (A–) is the ionized form of a weak acid [H+] + [A+] [HA] + [BA] – [B ] + [A ] ...(1) ...(2) [HA] dissociates less because it is a weak acid, whereas [BA] dissociates completely because it is a salt of a strong base. Larger the ka, the stronger the acid, because most of the HA will be converted into H+ and A–. Conservely, smaller the ka, less acid will be dissociated and hence weaker the acid. The dissociation constant of equation (1) is represented as: Ka = [H+ ] [A - ] [HA] By rearrangement [H+] [A–] = Ka [HA] [H+] = K a [HA] [A + ] As the acid [HA] is weak acid, it will be very slightly ionized, and most of it will be present as [HA], whereas the
    • 6 BIOCHEMISTRY FOR STUDENTS salt [BA] will be highly ionized, the concentration of [A–] can be taken as the total concentration of [BA]. [H+] = K a [HA] [BA] Taking logarithm of both sides [HA] [BA] log [H+] = log Ka + log –log [H+] = –log Ka + log [BA] [HA] pH = pK + log [BA] [HA] pH = pK + log [Salt] [Acid] This equation is called Henderson-Hasselbalch equation. If the value of K (the dissociation constant) is known, the pH of a buffer solution of a given composition can be readily calculated. The above equation indicates that the pH of the buffer solution depends on the ratio of the concentrations of the salt and the acid. The buffering power of a mixture of a weak acid and its salt is greatest when the two substances are present in equivalent proportions. Then the buffer has its maximum capacity to absorb either H+ or OH– ions. So that pH is approximately equal to the pK of the acid, i.e. when the acid is half neutralized. [salt] For example [salt] acid log Therefore = [acid] = 1 [salt] = log 1 = 0 acid pH = pK
    • BIOPHYSICS 7 The effective range of a buffer is 1 pH unit higher or lower than the pKa. The pKa value of most of the acids produced in the body is well below the physiological pH, hence, they ionizes, immediately and add H+ to the medium. The effect of dilution on the pH of a buffer mixture and on the apparent pK of the acid is slight. The pH depends upon the ratio of salt: acid and this ratio is not much affected by dilution. The pH of the buffer solution is determined by the pK and the ratio of salt to acid concentration. Lower the pK value, lower is the pH of the solution; whereas the ratio of salt to acid concentration may vary with no change in pH as long as the ratio remains the same. When the ratio between the salt and the acid is 10:1, the pH will one unit higher than the pKa whereas when the ratio between salt and acid is 1:10 the pH will be one unit lower than the pKa. Maximum buffering capacity occurs ± 1 pH unit on either side of pKa. Buffers are of main importance in regulating the pH of the body fluids and tissues within limits consistent with life and normal function. Many biochemical reactions, including those catalysed by enzymes, require pH control which is provided by buffers. Dissociation constant and pK of acids of importance in biochemistry. Compound Dissociation constant Acetic acid Citric acid Lactic acid Pyruvic acid Water Succinic acid 1.74 8.12 1.38 3.16 1 6.46 × × × × × × 10–5 10–4 10–4 10–3 10–14 10–5 pK 4.76 3.09 3.86 2.50 14 4.19 Q. A mixture of equal volumes of 0.1 M NaHCO3 and 0.1 M H2CO3 shows a pH of 6.1. Calculate the pKa of H2CO3. Ans. Concentration of H2CO3, i.e. acid = 0.1 M. Concentration of NaHCO3, i.e. salt = 0.1 M. Applying Henderson-Hasselbalch equation pH = pK acid + log [NaHCO 3 ] [H 2 CO 3 ]
    • 8 BIOCHEMISTRY FOR STUDENTS 0.1 0.1 6.1 = pK acid + log 1 6.1 = pK acid + log pK acid = 6.1 [log 1 = 0] Q. Phosphate buffers are prepared by mixing together 0.1 M Na2HPO4 and 0.1 M KH2PO4 in different ratios. Calculate the expected pH of the buffer solution prepared by mixing the salt and the acid in the above system in the ratio 2:1 (Given log 2 = 0.30 and pK2 of phosphoric acid 6.7) Ans. Concentration of Na2HPO4 (i.e. salt) = 2 × 0.1 M. Concentration of KH2PO4 (i.e. acid) = 1 × 0.1 M. Applying Henderson-Hasselbalch equation pH = pK phosphoric acid + log = 6.7 + log [Na 2 HPO 4 ] [KH 2 PO 4 ] 2 × 0.1 1 × 0.1 = 6.7 + log 2 = 6.7 + 0.3 = 7 So the e×pected pH of the buffer solution is 7. Q. You are provided with ample supply of carbonic acid and sodium bicarbonate. How would you prepare a buffer solution of pH 6.1. Give the theoretical basis of the procedure to be followed (pKa of carbonic acid = 6.1). Ans. Applying Henderson-Hasselbalch equation: pH = pK + log [Salt] [Acid] pKa of carbonic acid = 6.1 The buffer solution to be prepared should have a pH of 6.1. This can be achieved if the concentration of sodium carbonate and carbonic acid is the same.
    • BIOPHYSICS 9 So buffer solution of pH 6.1 can be made by mixing equal volume of sodium carbonate and carbonic acid of same concentration. Q. What would be the pH of 100 cm3 of a 0.2 M acetic acid solution to which has been added 10 cm3 of 1.5 M sodium hydroxide. (Given the pK for acetic acid 4.74.). Ans. Before the addition of NaOH, The number of moles of acetic acid present is: 100 = 0.02 M 1000 Also the number of moles of sodium hydroxide present in 10 cm3 of 1.5 M NaOH solution are: 0.2 × 100 = 0.015 M 1000 Before the start of reaction, the concentration of acetic acid is 0.02 M and that of sodium hydroxide is 0.015 M. When the reaction takes place, i.e. 0.015 M NaOH neutralizes 0.015 M of CH3COOH to form 0.015 M of sodium acetate. After the reaction is over, the concentration of CH3COOH left behind 0.02 M – 0.015 M = 0.005 M. Reaction CH3COOH + NaOH ↔ CH3COONa + H2O Now, applying Henderson-Hasselbalch equation 1.5 × pH = pK + log 10 [Acetate] [Acetic acid] = 4.74 + log 10 0.015 0.005 = 4.74 + log103 = 4.74 + 0.48 = 5.22 Blood Buffers The buffer systems of blood are: 1. Bicarbonate-carbonic acid (BHCO3 : H2CO3)
    • 10 BIOCHEMISTRY FOR STUDENTS 2. 3. 4. 5. Hemoglobinate-hemoglobin (BHb : HHb) Oxyhemoglobinate-oxyhemoglobin (BHbO2 : HHbO2) Phosphate buffer (B2HPO4 : BH2PO4) Protein buffer (B Protein : H Protein). The most important buffer of plasma is bicarbonate-carbonic acid system. It is present in high concentration. It is of great importance in the acid-base balance of the extracellular fluid and in the maintenance of the blood pH within normal limits. The bicarbonate system is of prime physiological importance, and acts cooperatively with other buffers. The hemoglobinate-hemoglobin and oxyhemoglobinateoxyhemoglobin buffer, i.e. hemoglobin buffers are of prime importance in the erythrocytes. Hemoglobin actually absorbs 60 percent of the hydrogen ions produced by H2CO3. Hemoglobin is a better buffer than most proteins at pH 7.4 because of relatively high concentration of imidazole group (pKa approximately 7) of the constituent histidine molecules. Deoxyhemoglobin is a better buffer than oxyhemoglobin. The converse is also true, i.e. the hydrogen ions decrease the affinity of hemoglobin for oxygen. Protein and phosphate buffers are of little importance in the blood, i.e. they are the minor buffering systems in the blood. Proteins are present in much higher concentrations in cells than in plasma. They are probably important in buffering H+ ions before their release from cells. But phosphate buffer is of importance in raising the plasma pH through excretion of H2PO¯ by kidney. It is an important urinary buffer and 4 works cooperatively with the bicarbonate system. Approximate contribution of individual buffers to total buffering in whole blood is given below. Individual buffers Hemoglobin and oxyhemoglobin Organic phosphates Inorganic phosphates Plasma proteins Plasma bicarbonate Erythrocyte bicarbonate Percent buffering in whole blood 35 3 2 7 35 18
    • BIOPHYSICS 11 Indicators Indicators are substances which change in color with change in the pH of the solution in which they are present. Indicators are dyes which are weak acids or weak bases and have the property of dissociating in solution. Their ionized form have one color and their unionized form have another color. The color of an indicator solution depends on the relative amounts of its acid and base form present in the solution. An indicator which is a weak acid, is undissociated in acid solution and gives the acid color. In the presence of alkali, it forms a salt which dissociates and displays alkali color. Indicators are used in: 1. Determining the end point in acid-base titrations. 2. Determining pH of solutions. Universal Indicator It is a mixture of a number of indicators which gives a variety of color changes over a wide-range of pH. Some common indicators useful for biological pH range are: Indicators 1. Thymol blue (acid range) pK pH range solution Color In acid In alkaline solution 1.65 1.2–2.8 Red Yellow – 2.9–4.0 Red Yellow 3. Methyl orange 3.46 3.1–4.4 Red Orange Yellow 4. Methyl red 5.00 4.3–6.1 Red Yellow 5. Phenol red 7.81 6.7–8.3 Yellow Red 6. Thymol blue (alkaline range) 8.90 8.0–9.6 Yellow Blue 7. Phenolphthalein 9.70 8.2–10 Colorless Pink 2. Methyl yellow (Topfer’s reagent)
    • 12 BIOCHEMISTRY FOR STUDENTS OSMOSIS AND OSMOTIC PRESSURE Osmotic flow occurs whenever a semipermeable membrane separates a solution and its pure solvent or between two solutions differing in concentrations. Water molecules pass through the membrane until the concentration on both sides becomes same. Such a movement of solvent molecules from a pure solvent or dilute solution through a semipermeable membrane is called osmosis. Osmotic Pressure Osmotic pressure is the pressure that must be applied on a solution to keep it in equilibrium with the pure solvent when the two are separated by semipermeable membrane or osmotic pressure is the force required to oppose the osmotic flow. Hypertonic solutions: If the osmotic pressure of the surrounding solution is high, water passes from the cell to the stronger solution outside, this causes the cell to shrink away. Isotonic solutions: If external solution has the same osmotic pressure, no flow of water takes place and hence no effect upon the cell protoplasm is observed. Hypotonic solutions: If the osmotic pressure of the surrounding solution is low, water passes into the cell from the surrounding, the cells become turgid and rupture. Van’t Hoff’s law of osmotic pressure: 1. The osmotic pressure of a solution is directly proportional to the concentration of the solute in the solution. 2. The osmotic pressure of a solution is directly proportional to the absolute temperature. Thus indirectly they follow Boyle’s and Charle’s Law. Osmotic pressure is given by the formula. π = CRT where π = Osmotic pressure C = Concentration in moles per liter R = Gas constant T = Absolute temperature
    • BIOPHYSICS 13 Osmotic pressure is dependent upon the number of dissolved particles (i.e. on concentration) and is independent of the size or weight of the particle. According to the law of osmotic pressure, 1 molar solution exerts an osmotic pressure of 22.4 liters at 0ºC. The osmotic pressure of substances which ionizes is given by the formula. π = i CRT where i the isotonic coefficient is given by: i = 1 + α (n–1) α = degree of ionization n = number of ions obtained on ionization The value of i, depends upon the degree of dissociation of the electrolyte, which varies from one electrolyte to another. It increases as the dilution increases and depends upon the number of ions formed. Since osmotic pressure is proportional to the total number of solute particles in solution, the substances which ionize, will have the higher osmotic pressure as compared to those substances which do not ionize. The osmotic pressure exerted by colloidal solutions is always less as compared to that of crystalloids of similar concentrations in gram per liter because the magnitude of osmotic pressure depends upon number of particles present in unit volume of the solution. Solutions that exert the same osmotic pressure are called isomotic. The osmotic pressure of 1 M NaCl will be double, as compared to the osmotic pressure of 1 M sucrose or glucose solution because each molecule of NaCl on ionization gives two ions, i.e. Na+ and Cl– ions and each ion will exert the respective osmotic pressure. The unit of osmotic pressure is osmol or milliosmol. An osmolar solution is defined as one exerting the osmotic pressure of a molar solution of a nondissociated solute in one liter of solution. Thus the number of osmoles of a undissociated substance in a liter of solution would be the weight in grams divided by its molecular weight. The milliosmolar concenwhere
    • 14 BIOCHEMISTRY FOR STUDENTS tration of glucose in a sample of plasma containing 90 mg per 100 ml therefore would be: 90 mg per 100 ml × 10 = 5 milliosmol per liter 180 (Mol. wt of glucos e) For nonelectrolytes such as glucose or sucrose, 1 millimol is equal to 1 milliosmol. For electrolytes such as NaCl, one millimol of NaCl is equivalent to 2 milliosmol (Na+ and Cl¯). Q. 1 Molar solution of glucose has an osmotic pressure of 22.4 atmosphere at 0ºC. Calculate the osmotic pressure of 0.1 M sucrose and 0.1 M NaCl at the same temperature. Assume 100% dissociation of NaCl. Ans. 1 molar solution exerts an osmotic pressure of 22.4 atmosphere. So 0.1 Molar solution will exert an osmotic pressure of 2.24 atmosphere. So 0.1 M sucrose will have an osmotic pressure of 2.24 atmosphere. In case of sodium chloride, each molecule of NaCl on dissociation gives Na+ ions and Cl– ions. Each ion, i.e. Na+ and Cl– will exert an independent osmotic pressure. Also the dissociation of sodium chloride is 100%. So 0.1 M solution of NaCl will exert an osmotic pressure of 2 × 2.24, i.e. 4.48 atmospheres. Q. Calculate the osmolarities of: i. 0.1 M NaCl solution ii. 0.1 M sucrose solution Ans. The term milliosmol is used in connection with osmotic pressure. 0.1 M solution of NaCl will have an osmotic pressure of 0.1 × 2 = 0.2 milliosmol (because each molecule of sodium chloride on ionization gives two ions). Whereas 0.1 M sucrose will have an osmotic pressure of 0.1 milliosmol. Milliequivalent One milliequivalent is one thousandth of an equivalent and is the same as millimol as long as the valency is one.
    • BIOPHYSICS 15 For valence 1; 1 millimol = 1 milliequivalent For valence 2; 1 millimol = 2 milliequivalent For valence 3; 1 millimol = 3 milliequivalent How to calculate millimols? millimol = milligrams per liter Formula weight Example: 78 mg of K+ ions per liter = 78/39, i.e. 2 millimols = 2 milliequivalent = 2 milliosmols Whereas 100 mg of Ca++ per liter = 100/40, i.e. 2.5 millimols = 2.5 milliosmols = 5 milliequivalent 222 mg of CaCl2 per liter Ca = 40, 2Cl – = 222/111, i.e. 2 millimols of CaCl2 = 6 milliosmols. = 2 × 35.5 = 71 Gibbs Donnan Equilibrium Gibbs Donnan equilibrium is concerned with the distribution of electrolytes in systems separated by membranes which are impermeable to certain components. This resultant unequal distribution of diffusible ions due to the presence of nondiffusible ions on one side of the membrane is called Gibbs Donnan Effect. Example: Consider a semipermeable membrane separating a solution of NaCl and Protein (NaR). The membrane is permeable to Na+ and Cl– but not to R–. Na+ R– Na+ Cl– In the beginning (A) Na+ Na+ – R Cl– Cl – At equilibrium (B)
    • 16 BIOCHEMISTRY FOR STUDENTS When the equilibrium is attained, the product of concentrations of diffusible ions (Na+ and Cl–) on one side of membrane is equal to the product of concentrations of same ions on the other side, i.e. (Na+)(Cl–) > (Na+)(Cl–) The concentration of diffusible positive ion is greater on the side of membrane containing nondiffusible ion, i.e. [Na+]1 > [Na+]2 Donnan effect is of physiological significance in biological systems involving ion exchanges across permeable membranes when the fluid on one side of the membrane contains a nondiffusible component. This results in difference of concentration of diffusible ions which leads to junction potential across the membrane, which is a driving force for most of the body reaction. Donnan effect is also involved in absorption, secretion and maintenance of different electrolyte concentrations between various compartments of the body. COLLOIDS Graham classified substances into: 1. Crystalloids: Substances which pass through parchment or animal membrane. 2. Colloids: Substances which do not pass through parchment or animal membrane. But nowadays, the size of the molecule or particle determines whether they will form crystalloidal or colloidal solutions. According to modern concept. True solution Colloidal solution Suspension solution where the size (diameter) of the particle is less than 1 mμ where the size is between 1-20 mμ where the size is more than 200 mμ Properties of Colloidal Solutions 1. Dialysis: The process of separation of crystalloids from colloids by diffusion through a membrane by osmotic force
    • BIOPHYSICS 2. 3. 4. 5. 17 is called dialysis. Dialysis has an important application in medicine in the artificial kidney. This device is inserted into the patient’s circulation and diffusible material particularly urea passes out from the blood substituting for the action of the faulty kidneys. As the size of the colloidal particle is large, few particles are present in small concentration, the osmotic pressure of the colloidal solution will be very small. This is of prime importance in driving the passage of water and other substances through cell membranes. Precipitation: Colloids possess net charge at the surface which arises from ionisable groups on the particle surface and also from absorption of ions and can be precipitated by neutralizing the charge. Brownian motion. Tyndall effect. SURFACE TENSION The force with which the surface molecules are held in a solution is called surface tension. Some substances such as bile salts have the property of lowering the surface tension of the medium in which they are present. This effect is used in the absorption of fats from the intestine. Other properties of surface tension are formation of drops of liquids falling through air; rise of liquid in a capillary tube and formation of meniscus at the surface of liquids. Surface tension decreases with increase in temperature. Role of Surface Tension Substance which lower the surface tension becomes concentrated in the surface layer whereas substances which increase surface tension are distributed in the interior of the liquid. Soaps, oils, proteins and bile acids reduce the surface tension of water, while sodium chloride and inorganic salts increase the surface tension. Surface tension leads to better adsorption.
    • 18 BIOCHEMISTRY FOR STUDENTS ABSORPTION Certain substances have the power of making water insoluble substances soluble in water without any apparent chemical alteration of the dissolved substance. The substances having such quality are called hydrotropic substances. Among the insoluble substances which are brought into the solution are fats, phospholipids, sterols, calcium carbonate, magnesium phosphate, etc. Substance which bring about the solubility are cholic acids, benzoic acid, hippuric acid, soaps of higher fatty acids, etc. The biological importance of the solution of an insoluble substance in hydrotropic substances lie in the fact that the substances so dissolved are diffusible through membranes. VISCOSITY Viscosity of a liquid is the resistance to flow. Viscosity of blood is 4.5 times more than water. Viscosity of blood is lowered in anemia, nephritis, leukemia, malaria, diabetes mellitus, jaundice, whereas excessive sweating and shock leads to increase of blood viscocity.
    • CHAPTER 2 Chemistry of Carbohydrates CARBOHYDRATES Carbohydrates are defined as the aldehydic or ketonic derivatives of polyhydroxy alcohols and their polymers having hemiacetal glycosidic linkages. The general formula for carbohydrates is Cn(H2O)n. Carbohydrates are the main source of energy in the body. Brain cells and RBCs are exclusively depend on carbohydrates (glucose) as the energy source. The sugar is a carbohydrate and is sweet to taste, soluble in water and chars on heating. Glucose (Grape sugar), fructose (fruit sugar), sucrose (cane sugar), lactose (milk sugar), and maltose (malt sugar) are few examples of sugar. All sugars are carbohydrates but all carbohydrates are not sugars. Glycogen and inulin are carbohydrates but not sugars. FUNCTIONS OF CARBOHYDRATES 1. Provides energy, i.e. as major source of energy to the body. Their calorific value is 4 kcal per gm. 2. As structural components of membranes. 3. As structural basis for DNA and RNA (Ribose/Deoxyribose). 4. As structural basis for nucleosides and nucleotides. 5. As source of carbon skeltons for some amino acids. 6. As basis of some intracellular messenger systems. CLASSIFICATION OF CARBOHYDRATES Monosaccharides Monosaccharides consists of single polyhydroxy aldehyde or ketone unit which cannot be broken down to simpler substances on acid hydrolysis. They are also called simple sugars. Monosaccharides are further divided into: i. Aldoses, i.e. Aldo sugars ii. Ketoses, i.e. Keto sugars.
    • 20 BIOCHEMISTRY FOR STUDENTS Aldoses Monosaccharides containing aldehydic group as the functional group are called aldoses. They are classified according to the number of carbon atoms present. Monosaccharides containing three to seven carbon atoms are called trioses, tetroses, pentoses, hexoses and heptoses respectively. Trioses : D-glyceraldehyde (aldotriose) Dihydroxy acetone (ketotriose) Tetroses : D-Erythrose (aldotetrose) Pentoses : D-Xylulose (ketopentose) : D-Ribose (aldopentose) : D-Deoxyribose (aldopentose) : D-Xylose (aldopentose) : D-xylulose (aldopentose) Hexoses : D-Glucose, D-Galactose, D-Mannose (aldohexose) : D-Fructose (ketohexose) Structures of Erythrose, Ribose, Glucose, Galactose, Mannose are:
    • CHEMISTRY OF CARBOHYDRATES 21 Ketoses Monosaccharides containing ketonic group as the functional group are called ketoses. Examples: Xylulose, Ribulose, Fructose, etc. Stereochemistry The presence of asymmetric carbon atoms (an asymmetric carbon atom is one to which four different atoms or groups are attached) in the compound results in the formation of isomers of that compound. The number of isomers of a compound depends on the number of asymmetric carbon atoms and is given by 2n, where n indicates the number of asymmetric carbon atoms in that compound. If the hydroxyl group on the highest asymmetric carbon atom or on the penultimate carbon atom is on the right hand side, than the compound will belong to D-Series. If the hydroxyl group is on the left side, than the compound will belong to LSeries.
    • 22 BIOCHEMISTRY FOR STUDENTS The D-and L-forms of glucose are given below: Two compounds that resemble each other but are different because their carbons are asymmetric. The relationship exhibited by each compound is called stereoisomerism and the two compounds are called stereoisomers or enantiomorphs. Stereoisomers are those compounds which have the same composition but differ in spatial arrangements. Carbohydrates exhibit the property of optical activity and exist as optical isomers. Glucose with four asymmetric carbon atom will have 24, i.e., 16 isomers. 8 of these isomers will belong to D-series and other 8 to L-series. (Where X denotes that particular carbon atom is asymmetric).
    • CHEMISTRY OF CARBOHYDRATES 23 In the open chain structure of D-glucose, C2, C3, C4, and C5 are the asymmetric carbon atoms. But in nature, D-glucose exists in 32 stereoisomers, i.e. 32 isomers of D-glucose has been isolated. The 32 isomers can be best explained if there is one more asymmetric center in the D-glucose. This is possible if glucose exists in ring or cyclic structure. The cyclic structure involves the formation of hemiacetal linkage between aldehyde group (i.e. C1) and hydroxyl group at C4. In the process, a new asymmetric centre C1 is created at glucose. In the ring form of D-glucose, C1, C2, C3, C4, and C5 are asymmetric and will have 25, i.e., 32 stereoisomers. During the process of cyclization a six membered ring consisting of five carbon atoms and an oxygen atom is formed in case of glucose. This ring structure is also called pyranose structure. Similarly a five membered ring consisting of four carbon atoms and an oxygen atom is formed in case of fructose. This ring structure is also called furanose structure.
    • 24 BIOCHEMISTRY FOR STUDENTS The planar formula of sugars is also called Fischer formula and the ring formula is called Haworth formula. Epimers: Carbohydrates that differ in their configuration about a specific carbon atom other than the carbonyl carbon atom are called epimers. Glucose and galactose are epimers as they differ in their configuration at C-4 carbon atom. Similarly, glucose and mannose are epimers as they differ at C-2 carbon atom. The process of interconversion of glucose and galactose is known as epimerization. In glucose, the hydroxyl group at C-4 is on the right hand side whereas in galactose, the hydroxyl group at C-4 is on the left hand side.
    • CHEMISTRY OF CARBOHYDRATES 25 Anomers: Carbohydrates that differ only in their configuration around the carbonyl carbon atom are called anomers. The carbonyl carbon atom is called the anomeric carbon atom. α-D-glucose and β-D-glucose are the anomeric forms of D-glucose. In α-D-glucose, the hydroxyl group at C-1 (i.e. carbonyl carbon atom) is on the right hand side whereas in β-D-glucose, the hydroxyl group at C-1 is on the left hand side.
    • 26 BIOCHEMISTRY FOR STUDENTS Anomeric form arises as a result of cyclization or ring formation. During the process of cyclization, the C-1 carbon atom which is symmetrical in the open chain formula of glucose is converted into asymmetric carbon atom. The presence of asymmetrical carbon atom give rise to optical activity. When a beam of plane polarized light is passed through a solution of carbohydrates, it will rotate the light either to left or to the right. Depending upon rotation, molecules are called dextrorotatory (+) or (d), levorotatory (–) or (l). A compound that rotates the plane of polarized light in a clockwise direction is said to be dextrorotatory (+), whereas that which rotates the plane of light in a anticlockwise direction is said to be levorotatory (–). Amino Sugars The amino sugars occurring most frequently are glucosamine and galactosamine. They occur as N-acetyl compounds. Glucosamine is present in chitin, shells of insects and mammalian polysaccharides whereas galactosamine is present in polysaccharides of cartilage and chondroitin. Reactions of Monosaccharides 1. 2. 3. 4. Action of acids Mutarotation Reducing property Osazone formation
    • CHEMISTRY OF CARBOHYDRATES 5. 6. 7. 8. 27 Action of dilute alkali Oxidation Reduction Glycoside formation. Action of Acids This is a general test for carbohydrates. Monosaccharides on treatment with concentrated sulphuric acid undergoes dehydration to give furfural or furfural derivatives which on condensation with α-naphthol yield a violet or purple colored complex. Pentoses yield furfural whereas hexoses yield 5-hydroxy furfural.
    • 28 BIOCHEMISTRY FOR STUDENTS Mutarotation Mutarotation is defined as the change in specific rotation of optically active solution without any change in other properties. When glucose is dissolved in water, the optical rotation of the solution gradually changes and attains an equilibrium value. This change in optical rotation is called mutarotation. Mutarotation occurs due to the cyclization of open chain form of glucose into α or β form with equal probability. This α and β cyclic form of glucose have different optical rotations. This is because, the α and β form are not mirror images of each other. They differ in configuration about the anomeric carbon (C1) but have the same configuration at C2, C3, C4, and C5 asymmetric carbons. These cyclic forms are in equilibrium with open chain structure in aqueous solution. Such a change from a single form to an equilibrium mixture that includes its other form is called mutarotation. +112o α-D-glucose +52-5o Equilibrium mixture contains α, β and open chain forms +19o β-D-glucose α-form 36%, β-form 63% and open chain form 1%. The predominance of the β-form in aqueous solution is due to its more stable conformation relative to the α-form. Biologically this change is catalyzed by the enzyme, mutarotase.
    • CHEMISTRY OF CARBOHYDRATES 29 In aqueous solution, many monosaccharides behave as if they have one more asymmetric center than is given by open chain structure. Ring formation involves the formation of internal hemiacetal linkage between the aldehyde group, i.e. C-1 and the hydroxyl group at C-5 and a new asymmetric carbon at C1 is created in glucose. In this cyclic form, there are now five asymmetric carbon atoms (i.e. C-1, C-2, C-3, C-4, C-5) which best explains about the existence of 25, i.e. 32 isomers of glucose. Reducing Property Monosaccharides by virtue of free aldehydic or ketonic group in their structure, i.e. presence of free anomeric carbon atom, reduces certain heavy metallic cation, e.g. Cu++ ions in alkaline solution at high temperature. So all the reducing sugars will give Benedict’s qualitative test and Fehling test positive. The reaction is as follows: The color of the solution or precipitate gives an approximate (rough) amount of reducing sugars present in the solution. Green color......up to 0.5% (+) Green precipitate.....0.5-1% (++) Green to yellow precipitate.....1.0-1.5% (+++) Yellow to orange precipitate.....1.5-2.0% (++++) Brick red precipitate....more than 2%
    • 30 BIOCHEMISTRY FOR STUDENTS Benedict’s qualitative reagent contains cupric sulfate, sodium carbonate and sodium citrate whereas Fehling solution contains cupric sulfate, sodium hydroxide and sodium potassium tartrate (Rochelle salt). Sodium citrate in Benedict’s reagent and sodium potassium tartrate (Rochelle’s Salt) in Fehling solution prevent the precipitation of cupric hydroxide or cupric carbonate, by forming a deep blue soluble slightly dissociated complexes with the cupric ions. These complexes dissociate sufficiently to provide a continuous supply of readily available cupric ions available for oxidation. Benedict’s qualitative reagent is preferred above Fehling solution because it is more stable. Also traces of sugar which is destroyed by the strong alkali of Fehling solution is not destroyed by Benedict’s reagent. Osazone Formation Reducing sugars can be distinguished from one another by phenylhydrazine test when characteristic osazones are formed. These osazones have characteristic crystal structures, melting point, precipitation time and show different crystalline forms under a microscope and hence, are valuable in the identification of reducing sugar. Glucose, fructose and mannose give the same osazones and hence, they cannot be differentiated from each other by this test. In the osazone formation only first two carbon atoms, i.e. C-1 and C-2, take part in the reaction. So reducing sugars which differ in their configuration at C-1 and C-2 and have rest of the structure same, i.e. C-3, C-4, C-5 and C-6 have the same configuration, give the same osazones because during osazone formation, the structural dissimilarity at C-1 and C-2 disappears and the rest of the molecule structure is the same. Three molecules of phenylhydrazine are required to produce one molecule of osazone.
    • CHEMISTRY OF CARBOHYDRATES 31 The formation of osazones of glucose is explained below.
    • 32 BIOCHEMISTRY FOR STUDENTS Fructose reacts with phenylhydrazine in a similar manner.
    • CHEMISTRY OF CARBOHYDRATES 33 Glucose osazone, fructose osazone and mannose osazone are identical with respect to its crystal structure and chemical structure. Glucose, fructose and mannose give the needle shape osazones whereas maltose gives sunflower and lactose gives cotton ball shape osazones.
    • 34 BIOCHEMISTRY FOR STUDENTS Appearance of yellow crystals takes place. Observe the shape of crystals under microscope. Lactose (Cotton Ball) Maltose (Sunflower) Osazone of maltose and lactose Action of Dilute Alkali Monosaccharides on treatment with dilute alkali undergo a variety of molecular transformation through enediol formation. The enediols of sugars are good reducing agents and form the basis of reducing action of sugars in alkaline medium. When glucose is treated with dilute alkali for several hours, the resulting mixture obtained contains both fructose and mannose in addition to glucose. A similar mixture of same sugars is obtained with any of the other two sugars. This interconversion of related sugars by the action of dilute alkali is termed as Lobry de Bruyn-van Ekenstein rearrangement (see page 36 for reaction). Whereas sugars on boiling with strong alkalis are caramelized to give yellow to brown resinous product. That is the reason why Benedict’s reagent containing sodium carbonate is preferred to Fehling solution containing sodium hydroxide. Oxidation Aldoses are oxidized under variety of conditions to the following: i. Aldonic acid: Whereby the first carbon atom (C-1) is oxidized to carboxyl group only. The rest of the molecule structure remains unaffected. ii. Uronic acid: Whereby the terminal carbon atom is oxidized to carboxyl group only. The first carbon atom, i.e. aldehydic group and the rest of the molecular structure
    • CHEMISTRY OF CARBOHYDRATES 35 remains unaffected. Uronic acid derivatives are particularly important in detoxification process, i.e., bilirubin is excreted as bilirubin diglucuronide. Besides this, D-glucuronic acid, D-galactouronic acid, D-mannouronic acid, L-induronic acid are important components of polysaccharides. iii. Aldaric or saccharic acid: Whereby both the first carbon atom, i.e. aldehydic group and the terminal carbon atom, i.e. primary alcoholic group are oxidized to carboxyl group. Galactose undergoes oxidation to form a dicarboxylic acid, mucic acid. This reaction is often important in the identification of galactose. Example: The oxidation products of glucose under different conditions are given on Page 37. Glucose Oxidase: The substrate for glucose oxidase is βD-glucopyranose. Blood glucose which is an equilibrium mixture of α- and β-anomers of D-glucose is qualitatively determined by the formation of hydrogen peroxide by the reaction (P-39).
    • 36 BIOCHEMISTRY FOR STUDENTS
    • CHEMISTRY OF CARBOHYDRATES 37 Two very important uronic acids occuring in carbohydrates are D-glucuronates and L-iduronate (from hexose idose). The only difference between these two molecule is that the carboxyl group is above the ring for D-glucuronate and below the ring for L-iduronate.
    • 38 BIOCHEMISTRY FOR STUDENTS This requires that the α-D-glucopyranose be rapidly isomerized by mutarotation into the β-D-isomer. This reaction is fast without catalyst. Q. A reducing carbohydrate gives a positive reaction with Barford’s test and mucic acid crystals on oxidation. Give the structure of that carbohydrate. Would it exhibit property of mutarotation. If so, what products are formed at equilibrium. Ans. Since Barford’s test is positive. It indicates that reducing carbohydrate is monosaccharide. Also mucic acid crystals are obtained on oxidation suggesting that the given reducing carbohydrate is galactose as it is the galactose which on oxidation gives mucic acid crystals. D-galactose will show mutarotation due to the cyclization of open chain form of D-galactose into α- and β- form with equal probability. The products at the equilibrium are:
    • CHEMISTRY OF CARBOHYDRATES 39 Reduction Glucose on reduction gives sorbitol. Whereas fructose on reduction gives a mixture of sorbitol and mannitol. Mannose gives mannitol, galactose is reduced to dulcitol and ribose to ribotol.
    • 40 BIOCHEMISTRY FOR STUDENTS Fermentation: Fermentation is the process of breakdown of complex organic substances into smaller substances with the help of enzymes. Glucose is fermented to ethyl alcohol and carbon dioxide by yeast. Hence this process is called alcoholic fermentation as alcohol is produced. Glycosides Formation Glycosides are sugar derivatives in which hydrogen of the hydroxyl group of hemiacetal or hemiketal form of the sugar is replaced by an organic moiety. A molecule of water is eliminated when this reaction takes place. Glycosides are not reducing sugars and do not show mutarotation. If the organic moiety is derived from another monosaccharide, the product formed is disaccharide. If the organic moiety is a noncarbohydrate, then it is called aglycone. Aglycone: The noncarbohydrate portion of the glycoside is called the aglycone or aglucone. Glycosides do not reduce alkaline copper sulphate because sugar group is combined, i.e. aldehyde group is converted to an acetal group. Glycosides = Carbohydrate + Carbohydrate part or noncarbohydrate part (aglycone) Examples Cardiac glycosides = Carbohydrate + Digoxin or digitoxin (aglycone) Indican = Carbohydrate + Indoxyl (aglycone) Amygdalin = Carbohydrate + Benzaldehyde (aglycone) OLIGOSACCHARIDES Oligosaccharides are arbitrarily defined as carbohydrates that contains two to ten monosaccharide units per molecule joined by glycosidic linkages. On hydrolysis they yield monosaccharides. Depending upon the number of constituent monosaccharide units, the oligosaccharides are called disaccharides, trisaccharides, etc.
    • CHEMISTRY OF CARBOHYDRATES 41 Oligosaccharides are reducing sugars if one of the carbonyl group is free (not involved in glycosidic linkage). The reducing power of carbohydrate decreases as the number of their sugar components increases. Disaccharides Disaccharides consist of two monosaccharides joined by a glycosidic linkage. The most common and important disaccharides are maltose, Lactose and Sucrose. Maltose and lactose are reducing disaccharides whereas sucrose is nonreducing disaccharide. In general, the properties of disaccharides are similar to those of monosaccharides. Reducing disaccharide sugars are not as reducing agents as monosaccharide because of the lower ratio of reducing groups to carbon atoms. Maltose Maltose consists of two molecules of D-glucose joined by α (1,4)-glycosidic linkage. The anomeric carbon of one glucose molecule is joined to the C-4 carbon of the second glucose molecule. The anomeric carbon of the second glucose molecule is free. So maltose is a reducing disaccharide.
    • 42 BIOCHEMISTRY FOR STUDENTS Maltose or malt sugar does not occur in free state but is formed as an important transitory intermediate product of the digestion of starch and glycogen. Maltose reduces heavy metallic ions in alkaline solution (e.g. Benedict’s reagent), undergoes mutarotation and forms sunflower crystals of maltosazone with phenylhydrazine. Lactose Lactose consists of galactose and glucose joined by β (1,4)glycosidic linkage. The anomeric carbon of D-galactose is joined to 4-carbon of D-glucose. The anomeric carbon of Dglucose is free, so lactose is a reducing disaccharide. Lactose is glucose galactoside. Lactose or milk sugar is an animal disaccharide and is present to the extent of 5% in milk only. It is synthesized in mammary gland and during lactation may appear in the urine.
    • CHEMISTRY OF CARBOHYDRATES 43 Lactose on treatment with concentrated nitric acid gives mucic acid crystals. Lactose reduces Benedict’s reagent, undergoes mutarotation and forms cotton ball lactosazone crystals with phenylhydrazine. Sucrose Sucrose is a non-reducing disaccharide. Sucrose consists of glucose and fructose joined by α(1) →β(2) glycosidic linkage. The anomeric carbon (C-1) of glucose molecule in α configuration is linked to anomeric carbon (C-2) of fructose in β configuration. So sucrose is a nonreducing disaccharide as both the reducing groups of glucose and fructose are linked together and hence not available for reduction. Sucrose or sugar cane is a plant disaccharide and is present in high concentration in sugar cane and sugar beet. Sucrose is used for sweetening purpose.
    • 44 BIOCHEMISTRY FOR STUDENTS Sucrose does not reduce Benedict’s reagent, does not show mutarotation and does not form osazone with phenylhydrazine. Invert Sugar Sucrose on hydrolysis yields equimolecular amounts of glucose and fructose. Since this mixture is levorotatory whereas the original sucrose is dextrorotatory, the process is known as inversion because of the inversion of the sign of rotation, and the mixture of glucose and fructose obtained is called as invert sugar. H+ Sucrose Glucose + Fructose (+65.5) (+52.7) (–92) Honey contains large amount of invert sugar. Isomaltose Isomaltose, a disaccharide is derived from the branch point of starch. Isomaltose has α (1→ 6)-D-glucosidic linkage to a second D-glucose residue.
    • CHEMISTRY OF CARBOHYDRATES 45 POLYSACCHARIDES Polysaccharides are the polymers of monosaccharide units which are joined in linear or branched chain fashion by glycosidic linkages. Polysaccharides contain a large number of sugar components per free carbonyl group. In a branched polysaccharides, there is only one reducing end and multiple nonreducing ends. Thus these free carbonyl groups are not sufficiently potential to reduce the Benedict’s Reagent, etc. By convention polysaccharides are given names ending in— an attached to the particular monosaccharide that make up the polymer. Thus a name for a polysaccharide in general is glycans from glucose. Examples are mannans, xylans and arabans which are polymers of mannose, galactose, xylose and arabinose. Polysaccharides have two important biological functions. 1. As storage form of fuel (i.e., glycogen of animal origin and starch of plant origin). Glycogen and starch are both storage form of glucose; glycogen is used by animals to store glucose and starch is used by plants. 2. As structural components, e.g. Cellulose. The structural polysaccharides have β-linkage and the storage polysaccharides have an α-linkage. The β-linkage keeps the molecular linear whereas α-linkage tends to fold the molecule, forming a gloublar structure then linear one. Polysaccharides can be divided into two groups: a. Homopolysaccharides b. Heteropolysaccharides. Homopolysaccharides They contain only one type of monosaccharides as the repeating unit and on hydrolysis gives only one type of sugar. Example: Starch, cellulose, glycogen, dextrins, etc. Starch Native starch is a mixture of two polysaccharides. a. Amylose b. Amylopectins.
    • 46 BIOCHEMISTRY FOR STUDENTS Amylose Amylose is a linear unbranched molecule in which D-glucose units are linked by α–(1→4) glycosidic linkages. It is water soluble and gives blue color with iodine. Amylopectin Amylopectin is a branched chain molecule in which D-glucose units in addition to α-(1,4) linkages are branched by α-(1,6) glycosidic linkages. This branching occurs on an average of 24 to 30 D-glucose units. It is water insoluble and gives violet color with iodine.
    • CHEMISTRY OF CARBOHYDRATES 47 Starch is a nonreducing polysaccharide, tasteless substance and gives blue color with iodine. Starch on hydrolysis with dilute mineral acids, i.e. with hydrochloric acid gives glucose only. Action of amylases on starch: Amylases are hydrolytic enzymes which hydrolyze polymers of glucose containing α-(1 → 4) glycosidic linkages, Amylases are of two types: 1. α-Amylases. 2. β-Amylases. α-amylases are present in saliva and pancreatic juice. They act on starch, hydrolyzing α-(1,4) glycosidic linkages in a random manner to yield glucose, free maltose and smaller units of starch called starch dextrins. These starch dextrins contain the original α-(1,6) glycosidic linkages. α-amylase cannot hydrolyze the α-(1,6) linkages at the branched point of amylopectins. The α-amylases are activated by chloride ions. β-amylases present in barley malt, cleave successive maltose units beginning from nonreducing ends of starch to give maltose. β-amylase yield only maltose with amylose and smaller branched polysaccharides, known as limit dextrins, as well as maltose with amylopectin. β-amylases also cannot hydrolyze α-(1,6) linkages at the branched point of amylopectin. Cellulose Cellulose is a linear polymer of D-glucose units joined together by β–(1,4) glycosidic linkages. On partial hydrolysis, cellulose yields β-1,4 disaccharide cellobiose instead of maltose. Cell-ulose is water insoluble, nonreducing and gives no color with iodine. Unlike starch and glycogen which are readily digested, cellulose cannot be utilized for energy purposes by human beings, because the enzyme which cleavage β-(1,4) linkage is missing in the gastrointenstinal tract and hence, merely provide bulk to the diet. Cellulose is present in plant leaves, stems, and outer coverings of fruits and vegetables. Cellulose
    • 48 BIOCHEMISTRY FOR STUDENTS is a component of fiber (nondigestible carbohydrate) in the diet. Cellulose is present in plant leaves stems and outer coverings of fruits and vegetables. Cellulose aids intestinal mobility and acts as an stool softener and reduces bowel cancer. The nutrition value of cellulose is nil. Celluloses are the most abundant organic compound on earth. Celluloses are the major components of plants comprising 20 to 45% of this cell wall mass. Glycogen Glycogen is the carbohydrate reserve of the body. Glycogen is also called animal starch, because it serves as nutritional reservoir in animal tissues. Glycogen is a highly branched chain molecule in which glucose unit in addition to linear α-(1,4) linkages are also linked by α-(1,6) at the branched point. This branching repeats after every 8-10 glucose units. Glycogen is water soluble and has no reducing property. It gives red color with iodine. Glycogen is stored in liver and muscle. About three-fourth of all the glycogen in the body is stored in muscle. Difference between starch and glycogen. 1. Starch is of plant origin whereas glycogen is of animal origin. 2. Glycogen is much more branched than the starch. In starch, the branching is after every 24 to 30 glucose units, whereas in glycogen, the branching is after every 8 to 10 glucose units. 3. Starch gives blue color with iodine solution whereas glycogen gives red color. Dextrins They are the partial hydrolytic products of starch by α-amylase, β-amylase and acids. Dextrins formed from amylases have
    • CHEMISTRY OF CARBOHYDRATES 49 unbranched chains while those formed from amylopectins are branched. All dextrins have free sugar group and accordingly reduce alkaline copper sulphate solution. HETEROPOLYSACCHARIDES Heteropolysaccharides are made up of mixed disaccharides repeating units and on hydrolysis gives a mixture of more than one product of monosaccharides and their derivatives of amino sugars and sugar acids. They are the essential components of the tissues where they are present in combination with proteins as mucoproteins. They are also called mucopolysaccharides or glycosamino glycans (CAG). The other suitable name for such heteropolysaccharides is Glycosaminoglycan or CAG. Glycosaminoglycans are unbranched polysaccharides consisting of repeating dissaccharide units comprising a sugar linked to either N-acetylglucosamine or N-acetylgalactosamine. They can be divided into: 1. Neutral mucopolysaccharides 2. Acidic mucopolysaccharides. Acid mucopolysaccharides are present in connective tissues. They contain hexosamine as the repeating disaccharide unit. The repeating structure of each disaccharide contains alternate 1,4 and 1,3 linkages. The most common CAGs are: Hyaluronic Acid Hyaluronic acid is present in the connective tissues, synovial fluid and vitreous fluid in combination with proteins. It is an unbranched polymer. The repeating disaccharide is made up of D-glucuronic acid and N-acetyl D-glucosamine. The monosaccharide subunits are linked by β-(1,4) and β-(1,3) glycosidic linkages. Glc UA-β(1 → 3) – Glu NAc connected by β(1 → 4) linkages.
    • 50 BIOCHEMISTRY FOR STUDENTS On acid hydrolysis it gives an equimolar quantities of glucuronic acid, glucosamine and acetic acid. Hyaluronates form viscous lubricants of joints and gel like substance inside the eyes-vitreous humor. Heparin Heparin is glucosaminoglycans. Heparin is an acidic mucopolysaccharide in which both the amino and the hydroxyl groups are combined with sulphuric acid, which causes it to be slightly acidic substance. Heparin is present in liver, lungs, thymus, spleen and blood. Heparin is blood anticoagulant. Heparin contains D-glucosamine, D-glucuronic acid or L-iduronic acid as the repeating disaccharide units. The glucosidic linkage is α(1,4) involving the glucuronic acid anomeric carbon hydroxyl with hydroxyl group at C-4 of glucosamine.
    • CHEMISTRY OF CARBOHYDRATES 51 Chondroitin Sulfates They are present in connective tissues and serve as a structural material such as cartilage, tendons and bones. Chondroitin sulfates are sulfated polysaccharides. Chondroitin sulfate is galacto aminoglycans. The acid hydrolysis of chondroitin sulfate yield D-galactose, D-glucuronic acid, acetic acid and sulfuric acid. Sialic Acids Sialic acids are N-acetyl derivatives of neuraminic acid and are widely distributed in tissues such as mucins are present in blood group substances.
    • 52 BIOCHEMISTRY FOR STUDENTS Neuraminic acid is a condensation product of pyruvic acid and mannosamine. Examples Repeating units Hyaluronic acid Chondroitin Chondroitin-4sulfate (Chondroitin sulfate A) Heparin Glucuronic acid; N-Acetyl glucosamine Glucuronic acid; N-Acetyl galactosamine Glucuronic acid; N-Acetyl galactose-4-sulphate Glucosamine-6-SO4; glucuronic acid-SO4; iduronic acid Other CAGs 1. Chondroistin sulfate and dermatan sulfate are galactosamine glycam. 2. Heparin sulfate, heparin and keratan sulfate are glucosamine glycam. Mucoproteins and Glycoproteins If the carbohydrate associated with protein is greater than 4%, then the complex protein is called mucoprotein. If the carbohydrate content is less than 4%, then is called glycoprotein. Plasma α1 and α2 globulins are glycoproteins. Blood Group Substances They are water soluble, high molecular weight substances, made up of polysaccharides and proteins. They are present in saliva, gastric mucin, erythrocyte membranes, etc. The immunological specificity resides in oligosaccharide part. The residues present in the oligosaccharides are L-fucose, D-galactose, N-acetyl-D-galactosamine and N-acetyl glucosamine.
    • CHAPTER 3 Chemistry of Lipids According to Bloor, lipids are defined as a group of naturally occurring substances consisting of the higher fatty acids, their naturally occurring compounds and substances found naturally in association with them. It includes a wide variety of substances with different structures. They are insoluble in water but are soluble in so-called fat solvents such as ether, acetone, chloroform, benzene, etc. Associated with them are various fat soluble, non-lipid substances which includes carotenoid pigments and certain vitamins, i.e. vitamins A, D, E and K. Lipids are widely distributed throughout both plant and animal kingdom and are essential constituents of cell membrane. Fats are said to be protein sparing because their availability in the diet reduces the need to burn proteins for energy. Lipids have several important biological functions. 1. They serve as the reservoir of energy because of their: a. High energy content. The calorific value is 9 kcal/gm as compared to carbohydrates which have calorific value of 4 kcal/gm. b. Storage in concentrated form in water free state (anhydrous) in the tissues as compared to carbohydrates which are highly hydrated and cannot be stored in such concentrated form. 2. As structural components of cell membranes. 3. As transport forms of various metabolic fuel. 4. As protective coating on the surface of many organs such as kidney, against injury. 5. To facilitate the absorption of the fat soluble vitamins A, D, E and K.
    • 54 BIOCHEMISTRY FOR STUDENTS Dietary fat can be divided into two types: a. Visible fat or fat consumed as such, e.g. butter, oils, ghee. b. Invisible fat or fat present as part of other foods items, e.g. egg, fish, meat, cereal, nuts, etc. Classification and Functions of Lipids Classification 1. Fatty lipids 2. Triglycerides 3. Phospholipids 4. Sphingolipids 5. Ketone bodies Functions Metabolic fuel, building block for other lipids Fatty acid storage, transport Membrane structure, storage of arachidonic acid Membrane structure Fuel SIMPLE LIPIDS They are esters of fatty acids with various alcohols. If the alcohol is glycerol, then they are called fats or neutral fats and are also called triglycerides as all the three hydroxyl groups of the glycerol are esterified. If the fat is liquid at ordinary temperature it is called an oil. Triglycerides are given by the formula R = Same or different All of the three fatty acids can be same or different. If all the three fatty acids are same, then they are called simple triglycerides. If the fatty acids are different, then they are called mixed triglycerides. In nature, mixed triglycerides are more abundant than the simple triglycerides.
    • CHEMISTRY OF LIPIDS 55 If the alcohol is high molecular weight instead of glycerol then they are called waxes. Comparison of simple and compound lipids is terms of their composition. Lipid Components Simple lipids 1. Triglycerides 2. Waxes Compound lipids 1. 2. 3. 4. Glycerol + Fatty acids Alcohol + Fatty acids (Both long chain) Phospholipids Glycerol + Fatty acids + Phosphate Sphingomyelins Sphingosine + Fatty acid + Phosphate + Choline Cerebrosedes (glycolipids) Sphingosine + Fatty acid + Simple sugar(s) Gangliosides (glycolipids) Sphingosine + Fatty acid + 2-6 simple sugars one of which is sialic acid Fatty Acids Fatty acids in nature as such are not very abundant but are present as ester. Fatty acids are represented by general formula R—COOH. A fatty acid is a long chain aliphatic carboxylic acid. General points about them. 1. They are monocarboxylic acids. 2. Number of carbon atoms are even, though odd number fatty acids exist but are very rare. 3. They may be saturated or may be unsaturated. If unsaturated they can be monounsaturated acid or poly-unsaturated acid. Mammals and plants contain both monosaturated and polyunsaturated fatty acids whereas all the fatty acids containing double bonds that are present in bacteria are monounsaturated. Plant and fish fats contain more polyunsaturated fatty acids than animal fats. The double bonds in a polyunsaturated fatty acid are neither adjacent nor conjugated since this would
    • 56 BIOCHEMISTRY FOR STUDENTS make the structure to easily oxidisable when exposed to environment oxygen. Rather the double bonds are three carbon apart; this provide somewhat greather protection against oxidations. Fats obtained from animals are generally saturated and those from plants are commonly polyunsaturated. However, these are some exceptions: coconut, palm oils are highly saturated. The most common among the saturated fatty acids are palmitic acid (C16), stearic acid (C18) and among the unsaturated fatty acid, oleic acid (C18). Unsaturated fatty acids have lower melting point than saturated fatty acids of same chain length. Fatty acids with odd number of carbon atoms occur in trace amounts in terrestrial and marine animals. Fatty acids with one to eight carbons are liquids at room temperature while those with more carbon atoms are solids. The most common fatty acids in neutral fats are: No. of atoms Butyric acid Caproic acid Lauric acid Palmitic acid Stearic acid Oleic acid 4 6 12 16 18 18 Formula CH3—(CH2)2—COOH CH3—(CH2)4—COOH CH3—(CH2)10—COOH CH3—(CH2)14—COOH CH3—(CH2)16—COOH CH2—(CH2)7—CH=CH —(CH2)7 —COOH Fats as an Energy Source Fats/oils are tremendous source of energy and 40% of total calories are provided by fatty acids that come from triglycerides and phospholipids. Naturally occurring straight chain saturated fatty acid No. of Common name C atoms 2 3 4 Acetic acid Propionic acid Butyric acid Type ⎫ ⎬ ⎭ Systematic name Short chain n-Ethanoic acid n-Propanoic acid n-Butanoic acid Contd...
    • CHEMISTRY OF LIPIDS 57 Contd... 8 10 12 14 16 18 20 Caprylic acid Capric acid Lauric acid Myristic acid Palmitic acid Stearic acid Arachidic acid ⎫ ⎬ ⎭ Medium chain ⎫ ⎬ ⎭ Long chain n-Octanoic acid n-Decanoic acid n-Dodecanoic acid n-Tetradecanoic acid n-Hexadecanoic acid n-Octadecanoic acid n-Eicosanoic acid The presence of double bond in the molecule gives rise to geometric isomerism. All naturally occurring unsaturated long chain fatty acids are found in cis isomer. Most plant fats are liquid since they contain a large proportions of unsaturated fatty acids with melting points. Animal fats, on the other hand, contain a high proportion of palmitic and stearic acids, and are solid or semi-solid at room temperature. Milk fat is unusual in containing a high proportion of shorter chain (C4-C14) fatty acids. Essential Fatty Acids They are also called polyunsaturated fatty acids. They are not synthesized in the body and hence, have to be provided in the diet. Although linolenic acid and arachidonic acid are synthesized by the body from linoeic acid, but they are synthesized in insufficient quantity for our needs. The deficiency of essential fatty acids in humans gives rise to dry, scaly skin, hair loss, poor wound healing, failure of growth and increase in metabolic rate. These essential fatty acids requirement is about 1% of the caloric intake be in the form of essential fatty acids. Essential fatty acids are needed for proper cell membrane formation and for synthesis of prostaglandins prostacyclins, thromboxanes and leukotrienes. Essential fatty acids are: No. of carbon atoms 1. 2. 3. 4. Linoleic acid Linolenic acid Arachidonic acid Timnodonic acid No. of double bonds 18 18 20 20 2 3 4 5 Position of double bonds from carboxyl end Dietary source 9, 9, 5, 5, Vegetable oils Vegetable oils Vegetable oils Fish oils 12 12, 15 8, 11, 14 8, 11, 14, 17
    • 58 BIOCHEMISTRY FOR STUDENTS Vegetable oils are oils and have many double bonds hence polyunsaturated appears on the label of must vegetable oils. Butter, on the other hand, is a fat and hence would be expected to have saturated fatty acids, i.e. no double bonds. Two of the essential fatty acids, linoleic and linolenic acids are not synthesized by the mammal but are synthesized by plants. As long as adequate amounts of linoleic acids are available mammals can synthesize other essential acids. Structures Linoleic acid CH 3(CH2)4CH=CHCH2CH=CH=(CH2)7COOH Linolenic acid CH3CH2CH=CHCH2CH=CHCH2CH =CH(CH2) 7COOH Arachidonic acid CH3(CH2)4(CH=CHCH2)4(CH2)2COOH Essential fatty acids are necessary in the biosynthesis of prostaglandins and for proper cell membrane formation. Prostaglandins are hormone-like compounds which in small amounts have profound effect. Important fatty acids in mammalian tissues: Common name No. of carbon atoms Double bonds Acetic acid Lauric acid Myristic acid Palmitic acid Stearic acid Oleic acid Linoleic acid Linolenic acid Arachidonic acid 2 12 14 16 18 18 18 18 20 0 0 0 1 0 1 2 3 4 Position of double bonds 9 9 9, 12 9, 12, 15 5, 8, 11, 14 Prostaglandins Prostaglandins are the derivatives of prostanoic acid which are the cyclic derivatives of unsaturated fatty acids having twenty carbon atoms.
    • CHEMISTRY OF LIPIDS 59 Prostaglandins are synthesized from essential fatty acids such as linoleic acid, linolenic acid and arachidonic acid. Five type of rings are found in the naturally occurring prostaglandins giving rise to prostaglandins of A, B, E, F and G or H series. The prostaglandins which are widely distributed in the body are PGE1, PGE2, PGE3, PGF1α, PGF2α and PGF3α. Linolenic acid is the precursor to PGE3 and PGF1α, Arachidonic acid is the precursor to PGF2 and PGF2α. Prostaglandins are synthesized and released by all mammalian cells and tissues except RBC. Also prostaglandins are not stored in cells but are synthesized and released immediately. Biological function of prostaglandins: 1. They lower blood pressure (PGE, PGA, PGI2). 2. They are used in the induction of labor, termination of pregnancy and prevention of conception (PGE2). 3. They are used in treatment of gastric ulcer (PGE). 4. They are used to prevent inflammation. 5. They are used in asthma. 6. They are used in congenital heart disease. 7. They inhibit platelet aggregation (PGI2) whereas PGE2 promote clotting process. Eicosanoids: Fatty Acid Derivatives Eicosanoids are all derived from C-20 carbon arachidonic acid. Prostaglandins, Thromboxanes and Leukotrienes are collectively referred as eicosanoids. They have a variety of extreme potent hormone like action on various tissues. These compounds are involved in the regulation of blood pressure, diuresis, blood platelet aggregation, effects on immune and nervous systems, gastric acid secretion and muscle contraction.
    • 60 BIOCHEMISTRY FOR STUDENTS Properties of Fats 1. Acrolein formation: When glycerol is heated in the presence of a dehydrating agent such as potassium bisulphate, acrolein is produced. Acrolein has a characteristic unpleasant odor and is easily identified on the basis of this smell. This reaction occurs whether glycerol is in free or esterified form as occurs in the triglycerides. 2. Hydrogenation: Unsaturated fats can be hydrogenated by the addition of hydrogen across the double bonds of the fatty acids in the presence of nickel as catalyst to give fully saturated fats. The above process is called Hardening of oils whereby vegetable oils are hydrogenated to produce commercial cooking fats. 3. Saponification: Hydrolysis of a fat by alkali is called Saponification. The products of hydrolysis are glycerol and alkali salts of fatty acids, which are called soaps. Since the common fats contain palmitic acid, stearic acid and oleic acid predominantly, the soaps used for washing consist largely of sodium salts of these acids. While these fatty acids are insoluble in water their sodium and potassium salts are water soluble. 4. Rancidity: Rancidity is a chemical change resulting in unpleasant odor and taste on storage when fats are exposed to light, heat, air and moisture. Rancidity is more rapid at high temp-
    • CHEMISTRY OF LIPIDS 61 erature. Rancidity may be due to hydrolytic or oxidative change taking place at the double bonds of the unsaturated fatty acids resulting in short chain aldehydes or ketones which have unpleasant odor. The addition of certain substances, called antioxidants such as ascorbic acid and vitamin E prevents rancidity whereas addition of proxidants like copper, lead and nickel quickens rancidity. The oxidation of unsaturated bonds in fatty acids when the are exposed to oxygen in the environment is referred to as either auto oxidation or peroxidation. Rancid fats are those that contain an appreciable amount of peroxidized fatty acid. Antioxidants are generally added to many food fats to improve their storage quantities. Characterization of Fats Saponification number: Saponification number is defined as the “milligrams of KOH required to saponify 1 gm of fat”. Since fats are mixtures of triglycerides largely of mixed type so the saponification number of a fat indicates the average molecular weight (average chain length) of the fatty acids constituting or comprising the fat. Saponification number is inversely proportional to the average chain length of the fatty acids. Higher the saponification number, the shorter will be the chain lengths of the fatty acids and vice versa. The saponification number of some of the fats is given below: Fat Butter fat Human fat Olive oil Cottonseed oil Linseed oil Coconut oil Castor oil Saponification number 210-230 195-200 185-195 194-196 188-195 250-260 175-185 Iodine number: Iodine number of a fat is defined as the number of gm of iodine absorbed by 100 gm of the fat. Halogens, e.g. iodine or bromine are taken up by the fats because of the presence of double bonds present in the fatty acid part of the fat.
    • 62 BIOCHEMISTRY FOR STUDENTS Iodine number is a measure of the degree of unsaturation of fat. Iodine number of some of the fats is given below: Fat Butter fat Human fat Olive oil Peanut oil Corn oil Soyabean oil Linseed oil Iodine number 26 - 28 65 - 70 80 - 90 85 - 100 105 - 115 135 - 145 170 - 200 Acid number: Acid number is defined as the milligrams of KOH required to neutralize the free fatty acids present in 1 gm. of fat. This is used in determining the rancidity due to free fatty acids. Acetyl number: The acetyl number is defined as the milligrams of KOH required to neutralize acetic acid liberated by the saponification of 1 gm of fat after it has been acetylated. Since acetylation takes place at the hydroxy groups of the hydroxy fatty acid residues in the fat, so acetyl number is a measure of the hydroxy fatty acids in the fat content. Polenske number: The ml of N/10 KOH required to neutralize the insoluble fatty acids from 5 gm. of fat which are not steam volatile. Reichert Meissel number: This represents the ml of N/10 KOH required to neutralize the volatile acid obtained from 5 gm of fat which has been saponified then acidified to liberate the fatty acids and then steam distilled. Butter fat, which contains shorter chain fatty acids has a Reichert Meissel number of 26 to 30. COMPOUND LIPIDS They are the esters of fatty acids containing nitrogen base in addition to an alcohol and fatty acids.
    • CHEMISTRY OF LIPIDS 63 A molecule which has changed and an unchanged portion is called an amphipathic molecule. Phospholipids They are also known as phosphatides. Phospholipids act as a detergent and increase the solubility of other lipids. They are present in all cells as well as in the plasma. Phospholipids include the following groups: Phosphatidic Acid The general structure of phosphatidic acid. They are important intermediates in triglyceride synthesis. Phosphatidic acid on hydrolysis yield glycerol, fatty acid and phosphoric acid. Lecithins The structure of lecithins are: Lecithin contains saturated fatty acid residue at the α-position and unsaturated fatty acid residue at the β-position of the glycerol. Lecithins on hydrolysis give glycerol, fatty acid, phosphoric acid and choline.
    • 64 BIOCHEMISTRY FOR STUDENTS Cephalins The structure of cephalins are: Cephalins differ from lecithins with respect to base attached to phosphoric acid. If the base is ethanol amine then it is called phosphatidyl ethanolamine or ethanolamine cephalin. If the base is amino acid serine then it is called phosphatidyl serine which is also called serine cephalin. Cephalins on hydrolysis yield glycerol, fatty acids, phosphoric acid, ethanol amine or serine. Phosphatidyl Inositol The structure of phosphatidyl inositol is: It contains inositol in place of base.
    • CHEMISTRY OF LIPIDS 65 Cardiolipin An important phospholipid of mitochondrial membrane is cardiolipin. It is a diphosphatidyl glycerol in which two phosphatidic acids are joined by a molecule of glycerol. These phospholipids are particularly rich in the polyunsaturated fatty acids especially linoleic acid. Plasmalogens These compounds possess fatty aldehyde in place of fatty acid at the α-position, with the result the normal ester linkage is replaced by the ether linkage on the C1 carbon. In some cases, bases like choline, serine or ethanol amine are also found. They are found in brain and heart.
    • 66 BIOCHEMISTRY FOR STUDENTS Sphingomyelins Phospholipids containing sphingosine are called sphingomyelins. They contain, a complex base sphingosine in addition to phosphoryl choline. A fatty acid is attached to the amino group of the sphingosine. No glycerol is present. Structure of Sphingomyelins Sphingomyelins are present in all tissues especially in brain and other nervous tissues. Sphingomyelins on hydrolysis yield sphingosine, fatty acid, phosphoric acid and choline. Increased concentration of sphingomyelins occur in liver, spleen, etc. in a condition known as Niemann-Picks disease. Cerebrosides or Glycolipids Glycolipids are carbohydrate-glyceride derivatives containing sugar, sphingosine and a fatty acid. These compounds do not contain phosphoric acid. If the sugar component is galactose, the lipid is termed galactolipid. The term cerebroside is used because it is found in large quantities in brain tissues particularly in white matter.
    • CHEMISTRY OF LIPIDS 67 Cerebrosides On hydrolysis cerebrosides give sphingosine, a fatty acid and galactose. Cerebrosides are differentiated on the basis of fatty acid present. Examples: Kerasin: It contains Lignoceric acid Cerebron: It contains Hydroxy Lignoceric acid Nervon: It contains Nervonic acid Oxynervon: It contains Hydroxy Nervonic acid Cerebrosides occur in large amounts in the white matter of brain and in the myelin sheaths of nerves. In Gaucher’s disease, large amount of cerebroside accumulates in the liver and spleen. Gangliosides They are found in nerve tissues. They contain carbohydrates, N-acetyl galactosamine and N-acetyl neuraminic acid.
    • 68 BIOCHEMISTRY FOR STUDENTS Sulfatides (Sulpholipids) They are cerebrosides having a sulfate group attached to the galactosyl residue. DERIVED LIPID Those substances which are derived from the above two groups by hydrolysis. These include fatty acids of various series, steroids, bile acids and substances associated with lipids in nature such as carotenes, vitamin A, D, E and K. Lecithins are hydrolyzed by certain enzymes, phospholipases or lecithinases. The nature of hydrolysis depends upon the type of phospholipase used. Phospholipase A: Present in snake venom (cobra) hydrolyzes fatty acid in α or 1-position of glycerol in the lecithin to form lysolecithins. In the similar manner it acts on cephalin. Phospholipase B: Hydrolyzes the remaining fatty acid of lysolecithin present at β or 2-position to form glyceryl phosphorylcholine. Phospholipase C: Hydrolyzes phosphorylcholine from lecithins to form diglycerides. Phospholipase C catalyses the hydrolysis at the glycerol side of the phosphate group. Phospholipase D catalyses the hydrolysis on the phosphate side of the phosphate group.
    • CHEMISTRY OF LIPIDS 69 Phospholipase D: Hydrolyzes choline from phosphatidyl ethanolamine (cephalin) form phosphatidyl serines. There are two classes of nonsaponificable lipids. Terpenes They are linear or cyclic compounds formed by condensation of two or more isoprene units. Other important terpenoid compounds are: a. Tocopherol (vitamin E) b. Coenzyme Q (also called ubiquinone) c. Vitamin K (a naphthaquinone) They include vitamins A, E, K and carotenes, etc. Cyclopentano-perhydro-phenanthrene (Steroid nucleus) ring Steroids The term steroids includes many compounds which have however one feature in common, the steroid skeleton. Steroids are the derivatives of cyclopentano-perhydro-phenanthrene ring (consists of four fused rings). This is a saturated (perhydro) pheranthrene ring with a cyclopentane ring attached. Steroids are steroidal alcohol. The most important member of this group is cholesterol. The four rings that make up
    • 70 BIOCHEMISTRY FOR STUDENTS perhydro-cyclopentano-phenanthrene are named alphabetically from left to right. Despite popular belief, cholesterol is not a poison but a very necessary part of our cell membranes and the basis of sexual hormones (androgens, estrogens, etc). Cholesterol is only a problem if it is in excess and in this respect we do not need cholesterol in our diets because body can synthesis it. Steroids belong to the class of important biological compounds with diverse physiological activities. Some of the biologically important steroids are: a. Ergosterol: b. Bile acids: c. Adrenal cortex steroids: d. Female hormones: e. Male sex hormones: UV radiation causes rupture of ring B to produce vitamin D. In lipid metabolism. Corticosterone and cortisol. Progesterone and estrogen. Testosterone and androsterone. Cholesterol is an animal fat and it does not occur in plants. Cholesterol contains hydrogen group at C-3, methyl groups at C-10 and C-13, a double bond at C-5 and an 8C branched alkyl group attached to C-17. This marks a total of 27C. This ring structures are lipid soluble and hydroxyl group of C-3 is hydrophilic.
    • CHEMISTRY OF LIPIDS 71 Plants have stigmasterol and β-sitosterol which differ only in the alkyl group side chain attached at C-17. The Antioxidant System In healthy individuals, the antioxidant system defends tissues against free radical attack. Antioxidants are known to prevent cellular damage and enhance repair. Three classes of antioxidants have been identified. a. Primary antioxidants: They prevent the formation of new free radical species, e.g. superoxide dimutase, glutathione peroxidase, ceruloplasmin, transferrin, ferritin. b. Secondary antioxidants: They remove newly formed free radicals before they can initiate chain reactions. These chain reactions can lead to cell damage and further free radical formations, e.g. vitamin E, vitamin C, β-carotene, uric acid, bilirubin, albumin. c. Tertiary antioxidants: They repair cell structures damaged by free radicals attack, e.g. DNA repair enzymes, methionine sulphoxide reductase. Deficiency in the antioxidant system can develop for a number of reasons: a. Low intake of dietary antioxidants b. Total parenteral nutrition c. Decreases that reduce the absorption of antioxidant nutrients from food, e.g. Crohn’s disease d. Renal dialysis In these situations the antioxidant system struggles to protect the body from free radical attack and as a result the risk of free radical-mediated disease increases. Increased antioxidant status by supplementation may indeed reduce the risk of certain diseases. i. High intake of vitamin E has been associated with reduced risk of mortality from ischemic heart disease. ii. High incidence of vitamin C and β-carotene have been associated with a reduced incidence of some cancers. iii. Dietary supplementation of vitamin E, β-carotene and selenium significantly reduces mortality from esophageal cancer. iv. Within one week on antioxidant rich, low fat diet reduces lipid peroxide levels and increased aborrhic acid level in patient in the acute myocardial infarction.
    • 72 BIOCHEMISTRY FOR STUDENTS Free Radicals A free radicals is defined as any atom or molecule that possesses an unpaired elactron. It can be anionic, cationic, or neutral. Free radicals are highly reactive molecules generated by the biochemical redox reactions that occur as part of normal cell metabolism and by exposure to environmental factors such as UV light, cigarette smoking, environmental pollutions and gamma radiations. Human body is constantly under attack from free radicals. Some toxic compounds can result in the production of free radicals which include anticancer drugs, anaesthetics, analgesics, etc. The free radicals species which occur in the human body are: a. Superoxide radical (•O2¯) b. Hydroxyl radical (OH•) c. Nitric oxide radical (NO•) d. Peroxyl radical (ROO•). Once formed, free radicals attack cell structures within the body. As a result, free radicals have been implicated in numerous diseases such as atherosclerosis, cancer, AIDS, liver damage, rheumatoid arthritis, Parkinson’s disease, etc. Process of Lipid Peroxidation This process is responsible for randicity of food. This process involves: i. Initiation ii. Propagation iii. Termination. Initiation + ROOH + Metaln+ → ROO• + Metal(n-1) + H+ X• + RH → R• + HX Propagation R + O2 → ROO• ROO• + RH → ROOH + R•
    • CHEMISTRY OF LIPIDS 73 Termination 2ROO• → ROOR + O2 ROO• + R•→ ROOR R• + R• → RR Eicosanoids Eicosanoids are formed from C20 polyunsaturated fatty acid. Arachidonate and some other C20 fatty acids give rise to eicosanoids which includes prostaglandins, thromboxanes, leukotrienes, lipoxins. There are two pathways of their formation: 1. Cyclooxygenase pathway 2. Lipooxygenase pathway.
    • 74 BIOCHEMISTRY FOR STUDENTS CHAPTER 4 Chemistry of Amino Acids and Proteins CHEMISTRY OF AMINO ACIDS Naturally occurring amino acids are amino acids containing amino group and carboxyl group on the same alpha carbon atom and are represented by the general formula: All amino acids found in living systems, plant and animal proteins are L-α-amino acids. Glycine is the only amino acid, which is optically inactive and cannot be resolved into D-or L-form because of symmetry on the α-carbon atom. All other amino acids are optically active. The configuration of L-α-amino acid is: A variety of classification of amino acids are possible. Either they can be classified according to the presence of acidic, basic or neutral groups or upon their chemical structures, i.e., presence of polar groups, nonpolar groups, sulphur containing groups, aromatic groups, heterocyclic ring, branched chain and so on.
    • CHEMISTRY OF AMINO ACIDS AND PROTEINS 75 Classification of Amino Acids 1. Aliphatic amino acids 2. Aromatic amino acids 3. Heterocyclic amino acids.
    • 76 BIOCHEMISTRY FOR STUDENTS
    • CHEMISTRY OF AMINO ACIDS AND PROTEINS 77
    • 78 BIOCHEMISTRY FOR STUDENTS Tryptophan Besides these there are number of amino acids which are obtained in free or combined form but do not occur in protein molecules, e.g. thyroxine, triiodothyronine, ornithine, citruline, α-aminobutyric acid, β-alanine, etc. Their structures are given here.
    • CHEMISTRY OF AMINO ACIDS AND PROTEINS 79 A dipeptide has two amino acids joined by a single peptide bond; a tripeptide is composed of three amino acids joined by two peptide bonds: a polypeptide is one in which any number (n) of amino acids or (AA)n are linked together by (n-1) peptide bonds. Examples of relatively smaller peptides that possess biological activity are glutathione, oxytocin, vasopressin, hypertensin, etc. Glutathione is a tripeptide consisting of glutamic acid, cystine and glycine and is found in red blood cells. Oxytocin and vasopressin are produced by the posterior of the pituitary gland. Each is made up of nine amino acids. Oxytocin causes contraction of smooth muscle and it is used in obstetrics to initiate labor whereas vasopressin raises blood pressure and reduces the secretion of urine. Angiotensin I has 10 amino acids and angiotensin II has 8 amino acids. They cause hypertension. Functions of Amino Acids Amino acids serve as: 1. Building block of proteins 2. Precursors of: a. Hormones. (peptide and thyroid) b. Purines c. Pyrimidines d. Porphyrins e. Vitamins 3. Neurotransmitter such as tryptophan (sertonin). 4. Transport of nitrogen: Alanine, glutamine. 5.Substrates for protein synthesis: Those for which there is a codon.
    • 80 BIOCHEMISTRY FOR STUDENTS Essential Amino Acids Those amino acids which are not synthesized in the body and hence have to be provided in the diet. They are also called indispensible amino acids. There are eight essential amino acids. They are leucine, isoleucine, threonine, tryptophan, phenylalanine, valine, methionine and lysine. Adequate amounts of essential amino acids are required to maintain the proper nitrogen balance. Deficiency of one or more essential amino acids in the diet gives rise to decrease in protein synthesis resulting in failure in growth of the child, negative nitrogen balance in adults and fall in plasma proteins and hemoglobin levels. Semiessential Amino Acids Those amino acids which are synthesized partially by the body but not at a rate to meet the requirement of the body are called semiessential amino acids. Semiessential amino acids are arginine and histidine. Nonessential Amino Acids Those amino acids which are synthesized by the body. These amino acids are derived from carbon skeletons of lipids and carbohydrates during their metabolism or from the transformation of essential amino acids. Nonessential amino acids are alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine and tyrosine. Nitrogen Balance The ratio of: Intake N = 1, i.e. nitrogen equilibrium. Normal adults Output N are in nitrogen equilibrium. > 1, i.e. positive nitrogen balance, e.g. during pregnancy, convulsions and growth. < 1, i.e. negative nitrogen balance, e.g. in malnutrituion and in certain wasting diseases where, there is tissue breakdown.
    • CHEMISTRY OF AMINO ACIDS AND PROTEINS 81
    • 82 BIOCHEMISTRY FOR STUDENTS Ninhydrin Reaction All amino acids (except proline and hydroxyproline), proteins or protein derivatives containing free amino group and a free carboxyl group react with ninhydrin to give a blue-violet colored compound called Rheumann’s purple, whereas amino acids, proline and hydroxyproline, give a yellow color with ninhydrin. Reaction with Nitrous Acid α-amino acids are deaminated to the corresponding α-hydroxy acids with nitrous acid. Each amino group yields one molecule of nitrogen which can be measured accurately. Hence, this reaction is used for the estimation of free amino groups in amino acids, peptides and proteins. Formal Titration Sorensen’s formal titration method is used for the estimation of free carboxyl group in amino acid and mixtures of amino acids. By this method one can determine the rate of digestion of proteins by determining the increase in carboxyl groups which accompanies during enzymatic hydrolysis. Amino acids by virtue of Zwitter ion formation are neutral in solution. If formaldehyde is added to a solution of amino acid, an adduct is formed at the amino group, leaving the carboxyl group free and the molecule acidic in reaction. In other words the presence of formaldehyde decreases the basicity of the amino group, permitting free carboxyl group to exert its maximum acidity. Free carboxyl group thus can be titrated.
    • CHEMISTRY OF AMINO ACIDS AND PROTEINS 83 Isoelectric Point of Amino Acids (pl) pl is defined as that pH at which the amino acid does not migrate in an electric field. At this pH, the amino acid molecule exists in the Zwitter ion form, in which the sum of the positive charges are equal to the sum of the negative charges and the net charge on the molecule is zero. pl is calculated as: where pK1 is the pH at which the carboxyl group is halftitrated and pK2 is the pH at which the N+H3 group is halftitrated. Amino acids are amphoteric electrolytes, i.e. they exhibit properties of both an acid and a base. The acidic groups of amino acids are carboxylic group (–COOH → –COO¯+ H+) and protonated α-amino group (–N+H3 → NH2 + H+). Basic groups of amino acids are dissociated carboxyl group (–COO¯ + H+ → –COOH) and α-amino group (–NH2 + H+ → –N+H3). Amino acids in aqueous solutions have been shown to occur as a dipolar species or zwitter ion (Molecules which have both a negative and a positive change). As every amino acid has at least two ionizable groups, it can exist in different ionic forms depending on the pH of the medium. In aqueous solution a neutral amino acid is in the zwitter ion form which is dipolar. It is therefore an amphoteric electrolyte. Ampholytes are those molecules that act as both an acid and a base. In strongly acid pH, it is in cationic form while in strongly alkaline pH, it is in anionic form. At isoelectric pH, the solubility and buffering capacity is minimum.
    • 84 BIOCHEMISTRY FOR STUDENTS Similarly, protons exist as cations in the acid media and anion in the alkaline media of the isoelectric pH. Hence, protein acts as buffers on both sides of isoelectric pH. So a proton is an anion at pH values above the pl and is a cation at pH values below the pl. At the isoelectric pH, glycine exists as Zwitter ion. Addition of acid converts it into cation and addition of alkali converts it into anion. Therefore amino acids depending on the medium pH carry net zero, positive or negative charges. Q. Show the formula of isoelectric glycine. Indicate by formulae what happens on the addition of (a) acid and (b) base to the isoelectric molecule. Ans. At isoelectric point the glycine exists as:
    • CHEMISTRY OF AMINO ACIDS AND PROTEINS 85 PROTEINS Proteins are defined as compounds of high molecular weight made up of α-amino acids linked to one another by peptide linkages. Proteins contain 20 odd individual amino acids present in characteristic proportions and linked in a specific sequence in each protein. Proteins are linear polymers consisting of L-α-amino acids. The amino acids are joined together by peptide bonds. The peptide bond is formed by the union of carboxyl group of one amino acids with amino group of other amino acid with an elimination of water molecule. Classification of Proteins Proteins are classified on the basis of their composition. Simple Proteins Simple proteins are made up of amino acids only and on hydrolysis yield constituent amino acid’s mixture only. Example: 1. Fibrous proteins: These are animal proteins which are highly resistant to digestion by proteolytic enzymes. They are water insoluble. a. Collagens It contains high proportion of hydroxy proline and hydroxylysine. It is a major protein of connective tissues. On boiling with water it forms gelatin. b. Elastins It is present in tendons and arteries. c. Keratins It contains large amount of sulphur as cystine. It is present in hair, wool, nails, etc. 2. Globular proteins: a. Albumins Serum albumin and ovalbumin of egg white. It is water soluble. It is precipitated from solution by full saturation of ammonium sulfate. It is coagulated by heat.
    • 86 BIOCHEMISTRY FOR STUDENTS b. Globulins c. Glutelins d. Gliadins (Prolamines) e. Protamines f. Histones Serum globulins, fibrinogens and muscle myosin. It is soluble in dilute salt solutions. It is precipitated from solution by half saturation of ammonium sulphate. It is coagulated by heat. Cereal proteins such as glutelins of wheat, oxyzenin from rice and zein of maize. It is soluble in weak acids or bases but insoluble in neutral aqueous solutions. Gliadin from wheat and zein from corn. It is water insoluble but soluble in ethanol. Salmine from salmon sperm cells contains high proportion of arginine. Globulin in hemoglobin. It contains high proportion of basic amino acid. It is water soluble. Conjugated Proteins They are proteins which contain nonprotein group (also called prosthetic group) attached to the protein part. On hydrolysis they give nonprotein component and amino acid mixture. Conjugated Protein = Protein part + Prosthetic group. Conjugated proteins are classified according to the nature of the nonprotein group attached to the protein part. Derived Proteins They are formed from simple and conjugated proteins by physical and chemical means. The products of partial hydrolysis of proteins are often classified as derived proteins. 1. Primary derived protein a. Protein These are formed as a result of slight change in structure with little or no hydrolytic cleavage of peptide bonds. Fibrin from fibrinogen.
    • CHEMISTRY OF AMINO ACIDS AND PROTEINS 87 Proteins Prosthetic group 1. Nucleoproteins 2. Phosphoproteins = = Nucleic acid Phosphoric acid 3. Glycoproteins = 4. Lipoproteins = 5. Flavoproteins = Carbohydrate or a derivative of carbohydrate Lipids (Lecithin, Cephalin, cholesterol, etc). Riboflavin 6. Metalloproteins = b. Metaprotein c. Conjugated proteins 2. Secondary derived protein a. Proteoses b. Peptones c. Peptides. Metals (Zinc, iron and copper) Example Virus proteins Casein of milk (Serine residues are phosphorylated), ovovitellin of egg yolk. Mucin of saliva Serum lipoproteins Biological oxidation reduction reactions Carbonic anhydrases, catalase, cyctochrome oxidase They are soluble in dilute acids and bases but insoluble in neutral solvents. Formed by the action of heat, alcohol, UV light, X-rays. Example, cooked egg white and egg albumin. These are formed by the progressive hydrolytic cleavage of the peptide bonds of protein molecules. They are water soluble and are not coagulated by heat. These proteins are formed as a result of various deep seated changes in the structure or composition of the proteins. Separations of proteins by ion exchange resins in a chromatography is also an important technique for the separation and characterization of proteins by change. Ion exchange resins
    • 88 BIOCHEMISTRY FOR STUDENTS are prepared of insoluble materials such as agarose, polyacrylamide, cellulose, etc. that contains negatively changed ligands (such as –CH2COO¯, –C3H6SO3¯) or positively charged legands such as diethyl amino. The degree of retardation of a protein or amino acid by a resin will depend on the magnitude of the charge on the protein at a particular pH of the experiment. Molecule of the same charge as the resin are eluded first in a single band, followed by proteins with an opposite charge to that of the resin. Electrophoresis If a solution of a mixture of proteins is placed between two electrodes, the charged particle will migrate to one electrode or the other at a rate that depends on the net change and, depending on the supporting medium used, on the molecular weight. Structure of Proteins Proteins exhibit four levels of organization: Primary structure Secondary structure Tertiary structure Quaternary structure Refers to amino acid sequence. Refers to folding of polypeptide chain into specific coiled structure which is repititive in one direction. Refers to arrangement and interrelationship of twisted chain into a three dimensional structure. Refers to the association of different monomeric subunit into a composite polymeric protein. Primary Structure It determines the sequence of amino acids in the protein molecule. It indicates the number of amino acids, type of amino acids and in which fashion they are linked up. The sequence of amino acids in proteins can be found out by Sanger’s and Edman’s degradation method.
    • CHEMISTRY OF AMINO ACIDS AND PROTEINS 89 Sanger’s Method This method is used to determine the N-terminal amino acid of proteins. The reagent used is 2, 4-dinitrofluorobenzene (DNFB). DNFB reacts with free amino group of the terminal amino acid of proteins to give a yellow colored 2, 4-dinitrofluorobenzene derivative which on hydrolysis, give the terminal amino acid as the yellow 2,4-dinitro derivative and all the other amino acids of protein are obtained as free amino acids. The yellow derivative is separated and identified by paper chromatography, by comparison with known 2,4-DNP amino acid. Edman’s Method N-terminal amino acid residue of proteins can also be identified by Edman’s method. The reagent used is Phenylisothiocyanate (PITC). It reacts with free alpha amino group of the N-terminal amino acid of proteins to give the phenylisothiocarbamate derivative of the protein which cyclizes in acid medium giving N-terminal amino acid as phenylthiocarbamyl amino acid (PTCA), leaving the rest of the protein chain intact, but shorter
    • 90 BIOCHEMISTRY FOR STUDENTS by one amino acid. PTCA then cyclizes to give the corresponding phenylthiohydration derivative, which is separated and identified by chromatography. The reaction with phenylisothiocyanate is then repeated on the shortened peptide. The amino acid sequence is thus determined from the N-terminal end of the peptide one by one. Phenylthiohydantoin Derivative Edman’s method is superior over Sanger’s method. Edman’s degradation involves the removal of one amino acid at a time from the amino end of a peptide or protein chain, leaving the remaining peptide chain intact. The process can be repeated and the sequence of amino acid from N-terminal end is obtained. Whereas in Sanger’s method, only the N-terminal amino acid is identified because after the removal of N-terminal acid with DNFB, the remaining peptide chain breaks into amino acid mixture. Another reagent often used is Dansyl chloride (Dimethyl aminonaphthalene-5-sulphonyl chloride).
    • CHEMISTRY OF AMINO ACIDS AND PROTEINS 91 The procedure with this reagent is the same as that used with DNFB. A covalent bond is formed with the free N-terminal amino group. The dansylated protein is hydrolyzed with acid and dansylated amino acid is separated and identified by chromatography. C-terminal residues are usually identified with enzyme carboxypeptidase. This enzyme attack only the peptide bond joining the last residue with a free α-carbonyl group of the peptide chain. Amino acids released are identified by chromatography. Also the polypeptide is treated with the anhydrous hydrazine, which breaks peptide bonds forming hydrazides with the carbonyl carbons. The C-terminal residue does not form a hydrazide because its carboxyl group is free. After the removal of the hydrazides, this amino acid is then identified chromatographically.
    • 92 BIOCHEMISTRY FOR STUDENTS The repetition of Edman reactions under favorable conditions can be carried out for 30 to 40 amino acids into the polypeptide chain from the NH2-terminal end. Since most polypeptide chains in proteins contain more than 30 to 40 amino acids, they have to be hydrolyzed into smaller fragments and sequenced in sections. Both enzymatic and chemical methods are used to break polypeptide chains into smaller polypeptide fragments. Trypsin and chymotrypsin are proteolytic enzymes that are used for partial hydrolysis of polypeptide chains in sequencing. Enzyme trypsin catalyse the hydrolysis of peptide bond on the α-COOH side of the basic amino acid residues of lysine and arginine with the polypeptide chains. Chymotrypsin hydrolyzes peptide bonds on the α-COOH side of amino acid residues with larger apolar side chains. The chemical reagent cyanogen bromide cleaves peptide bonds on the carboxyl side of methionine residue with polypeptide chains. R1 Phenylalanine Tyrosine Tryptophan Anginine, Lysine Methionine Tryptophan Reagent Chymotrypsin Trypsin Cyanogen bromide O-Iodosobenzoic acid Secondary Structure The polypeptide back-bone does not assume a random threedimensional structure, but instead generally forms regular arrangements of amino acids that are located near to each other in linear sequence. These arrangements are called as secondary structure of proteins. The durameter of helix is 10Å. This is of following types: 1. α-helix: This is most common type of secondary structure, it is spiral structure. α-helix is stabilized by extensive hydrogen bonding and it consists of 3-6 amino acid per turn. Proline disrupts the α-helical structure because it
    • CHEMISTRY OF AMINO ACIDS AND PROTEINS 93 is imino acid and geometricallly not compatible with helical structure. 2. β-sheet: In this surface appears pleated. So also known as α-pleated sheet. The two or more chains may be parallel or antiparallel. Amyloid protein deposited in brains of individuals with Alzheimer’s disease is composed of β-pleated sheet. 3. β-bends: β-bends reverse the direction of a polypeptide chain, helping it to form a compact, globular shape. These are usually found on the surface of protein molecules.
    • 94 BIOCHEMISTRY FOR STUDENTS 4.Nonrepetitive secondary structure: About half of an average globular protein is organized into repetitive structures. These are not random but have a less regular structure. 5.Supersecondary structures: These mainly form core, i.e. interior to molecule. These are also known as motifs. The common ones are β-α-β unit, greek key and β meander. Tertiary Structure Tertiary structure refers to the coiling of several helical portion of single helix into a three-dimensional structure. The tertiary structure of proteins is stabilized by: 1. Hydrogen bonding: It is formed by sharing of hydrogen atom between electronegative oxygen atoms, nitrogen atoms or combination of two. 2. Disulphide bonding: This results from electrostatic attraction between positively and negatively charged spacies. 3. Ionic interactions or salt bridges: These are nonpolar bonds between hydrocarbon containing compounds. 4. Ester bonding. 5. Hydrophobic interactions: These are the result of mutual interaction of electron and nuclei of molecules. 6. van der Waal’s forces. Quaternary Structure Proteins containing more than one polypeptide chain display fourth level of structural organization called quaternary structure. In quaternary structure of proteins, the individual polypeptide chains are arranged in relation to each other so as to give a single three dimensional structure of the overall protein molecule. Each polypeptide chain in such a protein is called a subunit. Depending upon the number of subunits such proteins are called dimers, tetramers or polymers, etc. The various examples are hemoglobin, ferritin, etc. Reactions of Proteins 1. They give biuret test positive. 2. They give blue color with ninhydrin.
    • CHEMISTRY OF AMINO ACIDS AND PROTEINS 95 Biuret Reaction The name of the reaction is derived from the organic compound, a biuret, obtained by heating urea at high temperature which gives this test positive. The compound biuret contains two peptide linkage. Biuret test is given by those compounds which contain two or more peptide bonds. Since proteins are polypeptides hence, it is a general test for proteins. When proteins are treated with alkali and minute quantities of cupric ions, a pink or purple color is obtained. Precipitation Reactions Proteins are precipitated from the solution by a large number of reagents and the process is called deproteinization. Such precipitation reactions are important in the isolation of proteins, in the deproteinization of blood and other biological fluids. a. Effect of salt concentration: Proteins are precipitated from the solution by the addition of (NH4)2SO4 and Na2SO4. Addition of large amounts of ionic salts results in increase in protein: protein interaction and decrease in protein: water interaction, the process is called salting out.
    • 96 BIOCHEMISTRY FOR STUDENTS b. Effect of positive ions: The positive ions most commonly used for protein preci- pitations are heavy metal cations such as Cu++, Zn++, Fe+++ etc. These cations precipitate proteins from alkaline solution by combining with the negatively charged protein to form an insoluble precipitate of metal proteinate. c. Effect of negative ions: Addition of tungstic acid, phosphotungstic acid, trichloroacetic acid, picric acid, sulphosalicylic acid results in precipitation of protein in acidic solution. Denaturation Denaturation is the unfolding of the characteristic native folded structure of the polypeptide chain of protein. Comparatively weak forces responsible for maintaining the secondary, tertiary and quaternary structure of proteins are rapidly disrupted during the denaturation. The primary structure held by covalent peptide bonds however is not disrupted. After denaturation such proteins acquire the random coil structure which may renaturate into native form under favorable conditions. Denaturation of oligomeric protein involves the (i) dissociation of subunits peptide chains from each other with or without (ii) the unfolding of individual chains into random coils. Such proteins usually are unable to renaturate or refold into the natural form. There are two conspicuous changes that often result from denaturation. 1. Loss of (Partial or Complete) biological activity of the protein. 2. The solubility decrease drastically, i.e. almost the precipitation takes place. Denaturation results in loss of biological activity caused by heat, pH changes, by organic solvents, effect of radiation, etc. In electrophoresis, an ampholyte such as protein, peptide or amino acid in a solution buffered at a particular pH is placed in an electric field. Depending on the relationship of the buffer pH to the pI of the molecule, the molecule will either move toward the cathode (–) or the anode (+) or remain stationary (pH = pI).
    • CHEMISTRY OF AMINO ACIDS AND PROTEINS 97 For plasma protein separation the solution is buffered at pH 8.6 which is at a pH substantially above the pI of the major plasma proteins. The proteins are negatively charged and move toward the positive pole. The peaks are obtained according to their rate of migration in order of their pI values are these of albumin, α1-, α2- and β-globulins, fibrinogen and α1- and α2-globulins. The different major proteins are designated underneath the peaks. The direction of migration is from right to left. Electrophoretic pattern of normal serum Functions of Proteins in the Body 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Catalytic proteins: Enzymes Structural proteins: Collagen Contractile proteins: Actin, myosin Natural defence proteins (Immunity): Antibodies Transport proteins: Albumin, Globulin, Hemoglobin, ceruloplasmin, apolipoprotein Blood proteins: Fibrinogen Hormonal proteins: Insulin Respiratory proteins: Cytochromes Repressor proteins: Regulate expression of genes of chromosomes Rece ptor proteins: Transport information to cell interior after interacting with proteins on the outside. Ribosomal proteins: Associated in the proteins synthesis Toxin proteins: Venoms
    • 98 BIOCHEMISTRY FOR STUDENTS 13. Vision proteins: Rhodopsin 14. Storage: Ferritin. Plasma Proteins Normal value of plasma proteins is 6 to 8 gm per 100 ml of blood. Plasma protein include albumin, globulin and fibrinogen. They can be separated. 1. By precipitation method using sodium sulfate, ammonium sulfate, etc. 2. By electrophoresis. In normal human plasma, 6 fractions have been separated by electrophoresis. They are: i. Albumin ii. α1-globulin iii. α2-globulin iv. β1-globulin v. γ2-globulin vi. Fibrinogen. Functions of Plasma Proteins 1. Osmotic pressure: Plasma proteins are important in regulating water between blood and tissues. Small molecules of plasma and tissue fluid such as glucose, amino acids, urea, electrolytes, freely diffuse back and forth and hence, exert the same osmotic pressure in both fluids, i.e. on both sides of capillary. However, plasma and lymph protein do not freely diffuse through the capillary walls and since the prot ein concentration of plasma is much higher than of lymph by difference in the protein osmotic pressure of the two fluids. This difference in the osmotic pressure of lymph and plasma is estimated to average about 22 mm Hg and represents effective osmotic pressure of plasma.
    • CHEMISTRY OF AMINO ACIDS AND PROTEINS 99 2. 3. 4. 5. As buffers: Proteins are amphoteric in nature and thus help in maintaining pH of the body. Reserve proteins: Proteins serve as source of proteins for the tissues when the need arises. As carrier of certain metabolites: The transport of certain insoluble substances such as bilirubin, free fatty acids, steroid hormones and lipids is carried out by various fraction of serum proteins. As immunoglobulins: The property of antibodies formation resides in γ-globulin fraction of the proteins. Immunoglobulin Immunoglobulins or antibodies, make up the γ-globulins fraction of the plasma. These defensive proteins are synthesized in response to exposure to a foreign material usually a protein or complex carbohydrate. The foreign material is called antigen. The formation of antibodies affords immunity against the antigen and this response is protective. Immunoglobulins are composed of four polypeptide chains, two light chains (L-chains) and two heavy chains (H-chains) per molecule. These chains are linked by disulphide bonds. There are two classes of light chains, κ and λ thus creating two series of immunoglobulin molecules. Each class of immunoglobulin contains a unique type of heavy chain. These are designated as ρ, α, μ, δ and ε chains. These immunoglobulins and their chemical formulae are represented as follows: Immunoglobulins IgG IgA IgM IgD IgE H-chains K-type ρ α μ δ ε K2r 2 K2α 2 K 2 μ2 K2δ2 K2ε2 λ-type λ2r2 λ2α2 λ2μ2 λ2δ2 λ2ε2 Immunoglobulins also called Antibodies, comprises the gamma-globulin fraction of the plasma. They are synthesized in the body in response to the exposure (or administration)
    • 100 BIOCHEMISTRY FOR STUDENTS to a foreign moiety called Antigen. The foreign material or antigens are usually proteins or carbohydrates. The formation of antibodies give rise to immunity against the antigen and this response is protective. The immunoglobulins are glycoproteins containings 3% to 12% carbohydrates including D-mannose, D-galactose, Lfucose, D-glucosamine and a sialic acid. On ultracentrifugation the immunoglobulins are separated into three major fractions IgM, IgG and IgA. Two other immunoglobulins IgD and IgE occur in plasma in small amount. Most of the antibodies are in IgG fraction which represents 70% of the total r-globulins. Immunoglobulins are made up of subunit peptide chains called heavy chain (mol wt 40,000) and a light chain (mol wt 20,000). The three types of heavy chain are μ, r and α. Two types of lighter chain are K and λ. Both types of light chains contain a segment with a constant sequence of amino acids comprising about half the chains and a variable portion of other half. Heavy chains also contain a variable portion of amino acid sequence (about 110 amino acids) and 330 amino acids forming the constant portion of the chains. The variable portion of the light and heavy chains of immunoglobulins contains the active sites of the molecule. Light chains and heavy chains are linked together in the whole immunoglobulin molecule by means of disulphide linkages. Type Subunit composition Mol wt Carbohydrate content (%) Serum level mg/100 ml IgG γ2k2, γ2λ2 153,00 3 0.81.6 IgA (α2k2)n, n(α2γ2) n = 1 to 4 180,0005000,000 5-10 0.2-0.4 lgM (μ2k2)n, (μ2γ2)n n = 5,6 900,000 10-10 0.2-0.5
    • CHEMISTRY OF AMINO ACIDS AND PROTEINS 101 Each heavy chain has four interchain disulfide bonds; two between the pair of μ-chains in the monomer one to the light chain, and one intersusunit bridge between the monomers. The light chains are denoted by smaller lines and may be of the κ or γ type. The solid circles attached to the heavy chains of one of the monomers represent complex oligosaccharides.
    • 102 BIOCHEMISTRY FOR STUDENTS CHAPTER 5 Hemoglobin PORPHINS Porphins are cyclic compounds formed by the linking of four pyrrole rings through methane bridges (—CH=). The four pyrrole rings are labeled as I, II, III and IV and the bridges as α, β, γ and δ. Substituents on the rings are labeled as 1, 2, 3, 4, 5, 6, 7, and 8. Porphins have hydrogens at all 8 substituent positions. In short, the molecule can be represented as:
    • HEMOGLOBIN 103 PORPHYRINS Substituted porphins are called porphyrins. Porphyrins are of two types, i.e. type I and type III. A porphyrin with completely symmetrical arrangements of substituents is called type I porphyrins whereas if the arrangement of substituents is not symmetric then it is called type III porphyrins. In nature both type I and type IlI porphyrins are found but type III porphyrins are more abundant. Porphyrins are colored compounds and show characteristic absorption spectra in both UV and visible regions. Some of the important porphyrins are: Porphyrins 1. 2. 3. 4. Nature of the substituents at the following positions Mesoporphyrin Uroporphyrin Coproporphyrin Protoporphyrin where M E A P V = = = = = 1,2 — ME AP MP MV 3,4 — ME AP MP MV 5,6 — MP AP MP MP 7,8 — PM PA PM PM Methyl group (—CH3) Ethyl group (—C2H5) Acetate group (—CH2COOH) Propionate group (—CH2CH2COOH) Vinyl group (—CH = CH2) Porphyrins can form complexes with metal ions. This property is very important in their functioning in biological system. Examples: Heme is iron porphyrin, chlorophyll is a magnesium porphyrin, Vitamin B12 is a cobalt porphyrin. HEMOGLOBIN The red coloring material of blood is because of hemoglobin. It is present in RBC. Hb is globular in shape. Hemoglobin belongs to class of conjugated proteins whereas heme is the prosthetic group and globin, the protein part: Hemoglobin = Heme + Globin
    • 104 BIOCHEMISTRY FOR STUDENTS Normal adult blood contains 97% HbA1, 2% HbA2 and 1% HbF. Both alpha and beta chains have 75 percent alpha helical structure. The α-chains has 7 and β-chains has 8 helical structure. Functions of Hemoglobin 1. In the transport of oxygen from lungs to the tissues and the transport of carbon dioxide from tissues to the lungs. Hemoglobin forms a dissociable hemoglobin-oxygen complex. Hb + O2 ↔ Oxyhemoglobin 2. As buffers. The buffering action of hemoglobin is due to the amino acid histidine present in the globin part of hemoglobin. Histidine comprises 8 percent of the total amino acid make up of the globin. 3. Hemoglobin is required for both carbon dioxide and oxygen transport because these gases are only sparingly soluble in water. The presence of hemoglobin increases the oxygen transporting capacity of a liter of blood from 5 to 250 ml of oxygen. Hemoglobin plays a vital role in the transport of carbon dioxide and hydrogen ion. Myoglobin which is located in muscles, serves as a reserve supply of oxygen and also facilitates the movement of oxygen within muscle. Significance of 2,3-Diphosphoglycerate (2,3-DPG) The stability of deoxy conformation is inceased by 2,3diphosphoglycerate in mammals. It binds electrostatically to 143rd histidine and 82th lysine in β-chains of deoxy-Hb and stabilizes T-conformation. During oxygenation 2,3-diphospho glycerate is released and T form reverts to R-conformation. Mountain sickness: When an unclemetized subject goes to higher attitudes (Hill areas/mountains) than the level of 2,3-DPG increases in the blood. This reduces the affinity of oxygen to hemoglobin liberating more and more of oxygen to peripheral tissues.
    • HEMOGLOBIN 105 Carbon Monoxide Poisoning Carbon monoxide has the tendency to form coordination compounds with metals, in particular with hemoglobin iron. It combines with hemoglobin to form carboxyhemoglobin (Hb CO). Hemoglobin in this form does not carry oxygen efficiently since by competiting specifically and effectively with oxygen for ferrous site0s of hemoglobin, CO can displace oxygen from hemoglobin in arterial blood. The affinity of hemoglobin for CO is approximately 210 times greater than for O2. In the lungs, hemoglobin combines with O2 to form oxyhemoglobin (HbO2) which is carried in this blood stream. O2 is released at the tissue capillary level. Since there are four heme groups in hemoglobin which can combine reversibly with 4CO or 4 O2 molecule in any combination. At the physiological pH and temperature, the combination of CO with human hemoglobin is about 10 times slower than O2. However, once formed the dissociation of carboxyhemoglobin is 210 times slower than oxyhemoglobin, which explains why the affinity of CO for hemoglobin is 210 times more than that of O2. In lead, poisoning the RBC refer to as Howell’s Jolly bodies and Cabot ring. Heme Ferrous protoporphyrin is called heme. Heme is a chelate of ferrous iron with protoporphyrin. Heme is also called protoheme. Synthesis of Heme The starting materials of hemoglobin synthesis are glycine and succinyl CoA.
    • 106 BIOCHEMISTRY FOR STUDENTS Structure of Hemoglobin In hemoglobin, iron is in ferrous form. When hemoglobin is converted to oxyhemoglobin, one of the linkage of iron with imidazole group of histidine in globin is replaced by oxygen. In oxyhemoglobin, iron remains in the ferrous form.
    • HEMOGLOBIN 107
    • 108 BIOCHEMISTRY FOR STUDENTS About 85 percent of the heme thus formed is used for hemoglobin synthesis. About 10% is used for myoglobin synthesis and the remaining 5% for cytochromes and other heme proteins. There are about 250,000 hemoglobin molecules in a single RBC. Hemoglobin molecule contains 4 heme groups combined with a globin molecule, i.e. 2 α-chains and 2 β-chains, i.e. globin part and four heme groups as prosthetic groups (one with each chain). The total molecular weight is 64, 540. globin (ferroheme)4 + 4O2 = globin (ferroheme-O2)4 Deoxyhemoglobin Oxyhemoglobin Each chain has one-heme group. One hemoglobin molecule contains four heme groups as subunits.
    • HEMOGLOBIN 109 Globin Globin contains 4 polypeptide chains. Two are α-chains and other two are β-chains. These four chains are arranged in tetrahedron configuration. α-chain contains 141 amino acids, whereas β-chain contains 146 amino acids. In all there are 574 amino acids in the globin molecule. The globin moiety is formed from amino acid pool in amount of 8 gm per day in the normal adult. Thus, about 14% of the amino acids from the average daily protein intake are used for globin formation. Each α-chain has 141 amino acids whereas β-chain (also gamma and delta chains) have 146 amino acids. There are 38 histidine molecules in hemoglobin molecule. The 58th residue in α-chain is called distal histidine because it is far away from the iron atom, whereas 87th residue in alpha chain is called proximal histidine because it lies near to iron atom. The α- and β-subunits of Hb are connected by weak noncovalent bonds like vander Walls forces and hydrogen bonds. Each of the four polypeptide chains of hemoglobin has its own heme prosthetic group and iron atom. Iron contained in the heme is coordinately linked with each chain by 2 histidine residues at two imidazole nitrogens of histidine at position 58 and 87 in α-chains and 63 and 97 in β-chain of globin. The structure of oxyhemoglobin is described as R (relaxed) form and that of deoxyhemoglobin is T (tight) form. The Tconformation of deoxy Hb is maintained by electrostatic forces between carboxyl and amine groups.
    • 110 BIOCHEMISTRY FOR STUDENTS Methemoglobin Methemoglobin is a hemoglobin derivative in which iron is in the ferric form. It is also called ferrihemoglobin. Methemoglobin is dark brown in color. Conversion of ferrous to ferric iron in hemoglobin destroys its capacity to combine with oxygen and to transport oxygen. Hence, methemoglobin is useless in the transport of oxygen. Normally the conversion of hemoglobin to methemoglobin takes place in the blood but reducing substances present in red cells tend to prevent the accumulation of any appreciable amount of methemoglobin. The amount of methemoglobin present in blood is 0.3 g per 100 ml of blood. Increased amount of methemoglobin in blood gives rise to a condition called methemoglobinemia. It is caused by the failure in the normal reconversion of methemoglobin to hemoglobin or by production of methemoglobin by certain drugs. The symptoms observed in methemoglobinemia are cyanosis (blue skin) and dyspnea (labored breathing). Hemoglobin Cooperativity The oxygenation process of hemoglobin and myoglobin is very peculiar. This can be understood in terms of a graph of fractional saturation of hemoglobin and myoglobin molecules plotted against the partial pressure of oxygen. As shown in figure, myoglobin oxygenation curve is hyperbolic whereas for hemoglobin it is sigmoidal. For myoglobin the half saturation pressure is quite low which tells us that it is a better oxygen storage molecule then oxygen carrier. The difference in the oxygenation curves between hemoglobin and myoglobin is related to their structural difference. In hemoglobin the presence of four subunits alter the nature of the oxygenation curve. As a consequence of the interplay between four subunits the binding of oxygen is cooperative. The affinity of a given heme for oxygen increases as the other heme in the hemoglobin molecule are oxygenated. Consequently the degree of saturation at first does not respond much to the pressure, then begins to rise abruptly and finally the curve levels off at high pressure. This phenomenon is called
    • HEMOGLOBIN 111 cooperative or allosteric effect. There is an advantage of the sigmoidal curve. The structure of hemoglobin differs in the oxygenated and deoxygenated states. The quaternary structure of oxygenated state is called the R state (for released), and the conformation of the deoxygenated state is called the T state (for tense). The ability of hemoglobin to bind oxygen decreases with an increase in acidity protons make hemoglobin dump oxygen. Oxygenation curves for hemoglobin and myoglobin Hemoglobin Variants Hemoglobin A1 HbA1 contains two α-chains and two β2-chains. HbA1 constituteover 98% of the total hemoglobin of the normal adult hemoglobin and is designated as α2A β2-A, or more simply α2β2. Hemoglobin A2 HbA2 contains two α2-chains and two δ2-chains. HbA2 constitute about 2 percent of the total hemoglobin in the normal adult and is designated as α2 δ2. Hemoglobin F Human fetal hemoglobin is designated as HbF and is represented as α2 γ2.
    • 112 BIOCHEMISTRY FOR STUDENTS HbF contains two α-chains and two γ-chains. HbF is predominant form present at birth but is almost totally replaced by HbA1 within few months after birth. Hemoglobin S (Sickle Cell Hemoglobin) HbS contains two α-chains and two β-chains in which glutamic acid at 6 position from the N-terminal end of the β-chain is replaced by valine. HbS is also called sickle hemoglobin due to the fact the red cells assume the shape of sickle on deoxygenation. HbS gives rise to sickle cell anemia. Hemoglobin Gun Hill It contains only two heme groups instead of four. Five amino acids are missing from the β-chain and this leads to the interference with the heme binding. In short hemoglobin variants are represented as: Chains HbA1 HbA2 HbF HbS 2α 2α 2α 2α 2β 2δ 2γ 2β glu →val at position 6 Myoglobin Myoglobin is a single polypeptide chain. Human myoglobin contains 152 amino acids with a molecular weight of 17,500. The heme is attached to 92nd histidine residue. One molecule of myoglobin can combine with one molecule of oxygen. Myoglobin has higher affinity to oxygen than that of Hb. Myoglobin has high oxygen affinity while Bohr effect, cooperative effect and 2,3-diphosphoglycerate effect can absent. The isoelectric point of myoglobin is 6.5. Bohr Effect The increase in acidity of hemoglobin as it binds oxygen is known as Bohr effect; or Bohr effect is the increase in basicity of hemoglobin as it releases oxygen.
    • HEMOGLOBIN 113 The effect is expressed by the equation. HHb+ + O2↔ HbO2 + H+ The above equation indicates that increase in hydrogen ion concentration will favour the formation of free oxygen from hemoglobin and conversely that oxygenation of hemoglobin will lower the pH of the solution. This reversible uptake and release of protons is responsible for the isohydric transport of carbon dioxide. The term isohydric refers to a lack of change of pH in the process. Breakdown of Hemoglobin Bilirubin in combination with albumin reaches the liver, where it undergoes conjugation to form bilirubin diglucuronide which passes with the bile into the intestines. In the intestines, bilirubin diglucuronide is hydrolyzed, bilirubin is converted to urobilinogen. A portion of urobilinogen is absorbed from the intestines into the blood and some of it is excreted in the urine (4 mg/day). The remainder is re-excreted in the bile. The unabsorbed urobilinogen is excreted in the stool as fecal urobilinogen which is oxidized to urobilin.
    • 114 BIOCHEMISTRY FOR STUDENTS PORPHYRIA When the blood levels of coproporphyrins and uroporphyrins are increased above normal level and excreted in urine or faeces the condition is known as porphyria. Additionally reduced catalase activity has been reported in cases or porphyria. Classification Inherited Erythropoietic Porphyria It is rare inherited disorder and is due to autosomal recessive pattern. Preponderance of type and prophyrias, both uroporphyrin type and coproporphyrin type. This is due to increased deaminase activity with isomerase deficiency. Affected individuals exhibit abnormal sensitivity to lightphoto sensitivity and develop skin lesion. Urine is usually red colored. Explanation: As uroporphyrinogen III is less formed or absent, heme formation surffers. Relative deficiency of heme produces induction of δ. ALA synthetase leading to massive production of type I.
    • HEMOGLOBIN 115 Hepatic Porphyria In this, there occurs abnormal and excessive production of prophyrins (chiefly type III), their precursors δ ALA and porphobilinogen. There is three types of hepatic porphyrias. 1. Acute intermittent porphyria or paroxysmal porphyria: It is autosomal dominant partial deficiency of uroporphyrinogen and synthetase. Patients present with acute attacks of abdominal pain, nausea and vomiting, constipation, CV abnormalities and neuropsychiatric signs. This is due to increased production of porphyrinogen and d-ALA. The patients do not have photosensitivity. Freshly passed urine is often normal in color but on standing in sunlight turns to red urine color. Both colorless compounds porphobilinogen and d-ALA in sunlight. Polymerases to form two colored red compounds porphobilin and porphyrin. Note: Drugs and steroids requiring cyt P-450 can precipitate acute case. Reason is excessive utilisation of cyst P-450 for which heme is utilised. This decrease in heme is associated with depression of δ-ALA synthetase. 2. Porphyria cutanea tarda: It is autosomal dominant: This is due to partial deficiency of uroporphyrinogen decarboxylase and patients are characterized by photosensitivity. Urine contains increased quantities of uroporphyrins and coproporphyrins of both types and also elevated urinary excretion of d-ALA and PBG occurs and is associated with use in serum iron. 3. Varicyate porphyria or mixed (combined) porphyria: In this neurological as well as cutaneous symptoms are seen. This is autosomal dominant. There is deficiency of portoporphyrinogen oxidase and ferrochelatase. Clinically there is vomiting, acute attacks of abdominal pain and neuropsychiatric signs and cutaneous photosensitivity.
    • 116 BIOCHEMISTRY FOR STUDENTS Bilirubin is of two types: 1. Direct bilirubin: Direct bilirubin is bilirubin diglucuronide. It is water soluble. It is expressed as conjugated bilirubin because it can be coupled readily with Diazo Reagent (diazotized sulphanilic acid). This is the direct van den Bergh reaction. 2. Indirect bilirubin: Albumin bound bilirubin is called indirect bilirubin. It is water insoluble. It is expressed as unconjugated bilirubin as it will not react until it is released by the addition of alcohol. The reaction with Diazo reagent after the addition of alcohol is called the indirect van den Bergh reaction. Normal serum bilirubin level is 0.2-0.6 mg %. Jaundice Jaundice is due to increase in the concentration of bilirubin in the blood which imparts yellow color to the skin and conjunctive. Jaundice may be either due to over production of bilirubin than what the liver can normally excrete or a damage in liver, fails to excrete bilirubin in normal amounts. Jaundice is of three types: Hemolytic or Pre-hepatic Jaundice In hemolytic jaundice, there is an increased breakdown of hemoglobin, the liver cells are unable to conjugate all the increased bilirubin formed. Increased production of bilirubin leads to increased production of urobilinogen which appears in urine in large amounts. Bilirubin will be absent in urine. Hepatocellular or Hepatic Jaundice This type of jaundice results from liver damage which cannot conjugate bilirubin. The indirect serum bilirubin level will be high. Urine will show the presence of bilirubin and increased amount of urobilinogen. Stool is light in color.
    • Serum bilirubin Conjugated fraction (Direct) Unconjugated fraction (Indirect) van den Bergh Reaction Urine bilirubin Urine urobilinogen 2. 3. Fecal stercobilinogen Serum cholesterol –Free form –Esterrified form Causes 1. Biochemical investigation Much more than the normal Increased Normal Normal Normal Usually diminished Decreased Decreased More than normal Delayed direct positive Present (but in low amount) Normal Increased More than direct fraction Indirect positive Usually absent Increased Increased Disease of parenchymal cells of liver Increased Increased Hepatocellular (hepatic) Due to excessiv hemolysis Hemolytic (pre-hepatic) Biochemical changes in jaundice Usually diminished Increased Normal Normal Absent Contd. Increased (Direct form is more) Direct positive Increased Increased Increased Due to obstruction of biliary tract Obstructive (post-hepatic) HEMOGLOBIN 117
    • Prothrombin time Serum alkaline Phosphatase Serum transaminases Protein floculation test (Thymol turbidity test) Color of stool 5. 6. 7. 8. *King Armstrong units. 9. Serum proteins A:G ratio 4. Biochemical investigation Contd. Dark colored Normal or weakly Normal Normal Normal Normal Normal Hemolytic (prehepatic) Pale colored usually positive Normal or moderately increased (value below 35 KA units) Very high in first week Decreased Decreased (Reversal of A:G ratio) Increased Hepatocellular (hepatic) Negative except in severe obstruction Clay colored Moderately raised Normal after the parenteral. Injection of vitamin K Increased (value above 5 KA* units Normal Obstructive (posthepatic) 118 BIOCHEMISTRY FOR STUDENTS
    • HEMOGLOBIN 119 Obstructive or Post-hepatic Jaundice This type of jaundice results from the obstruction of common bile duct. As a result of obstruction, bilirubin does not pass into the intestine, so no urobilinogen is found in the urine. Direct serum bilirubin level will be high, urine will show the presence of bilirubin. Stool is clay colored. Physiological Jaundice or Neonatal Jaundice Usually mild form of jaundice appears in some newborn children on the 2nd and 3rd day of life called neonatal jaundice. Causes 1. Excessive destruction of RBCs after birth causing increased in serum bilirubin. 2. Due to hepatic immaturity During IU life, bilirubin formed is mainly eliminiated by placenta immediately after birth where has to eliminate all the bilirubin but it is unable to deal adquately during first 10 days. Note: 1. In infants, when serum bilirubin rises beyond 5% clinical jaundice appears. 2. Jaundice is more common and more severe is premature babies. Phototherapy Exposure of skin to white light converts bilirubin to a compound which has shorter life than bilirubin called lumirubin. Phototherapy is used to treat infants with hemolysis.
    • 120 BIOCHEMISTRY FOR STUDENTS CHAPTER 6 Enzymes ENZYMES Enzymes are biological catalysts which bring about chemical reaction in living cells. They are produced by the living organism and are usually present in only very small amounts in various cells. They can also exhibit their activity when they have been extracted from the source. Enzymes are all organic compounds and a number of them have been obtained in crystalline form. General properties of enzymes are: 1. All enzymes are proteins with exception of ribosomes. 2. Enzymes accelerate the rate of reaction by: a. Not altering the reaction equilibrium b. Being required in a very small amount c. By being not consumed in the overall reaction. 3. They have the enormous power for catalysis. 4. Enzymes are highly specific for their substrate. 5. Enzymes possess active sites at which interaction with substrate takes place. 6. Enzymes catalysis involves the transformation of enzymesubstrate complex as an important intermediate in their action. 7. Enzymes lower the activation energy. 8. Some enzymes are regulatory in function. Some enzymes are purely protein in nature and depend for activity only on their structure while certain enzymes require for their function one or more nonprotein component. They are termed as coenzymes, cofactors or prosthetic groups. If such a compound is firmly attached to enzyme proteins then
    • ENZYMES 121 it is called a prosthetic group. If its attachment to protein is not very firm then it is called coenzyme. Certain coenzymes exist in free state in solution and contact enzyme protein only at the times of reaction. The term apoenzyme refers to the protein part of the enzyme. The apoenzyme in combination with its prosthetic group (or coenzyme) constitute a complete enzyme or holoenzyme system. Holoenzyme = Apoenzyme + Coenzyme = Protein part + Nonprotein part Coenzymes Many enzymes in order to perform their catalytic activity require the presence of small nonprotein molecules. Coenzymes are low molecular weight, organic compounds, nonprotein, thermostable and can be separated by dialysis. Characteristics of Coenzymes 1. 2. 3. 4. They are stable towards heat. Generally derived from vitamins. Function as cosubstrates. They participates in: a. Hydride (H¯) and electron transfer reactions, e.g. NAD+, NADH, FMN, FAD, etc. b. Group transfer reactions, e.g. CoA, TPP, pyridoxal phosphate, tetrahydrofolic acid, etc. Coenzymes NAD+, NADP+ FAD, FMN Thiamine pyrophosphate Pyridoxal phosphate Biotin Coenzyme A Functions performed Hydrogen transfer Hydrogen transfer Acetyl group transfer Amino group transfer Carboxyl group transfer Acyl group transfer Most of the coenzymes are the members of water soluble B-complex group of vitamins. Coenzymes function as the
    • 122 BIOCHEMISTRY FOR STUDENTS intermediate carrier of functional groups of specific atoms or of electrons that are transferred in the overall enzymatic reactions. Classification of Enzymes According to the International Union of Biochemist, the enzymes are classified into six major classes. 1. Oxidoreductases: They catalyze oxidation and reduction reactions. These enzymes are divided into three groups. a. Oxidases: Those which use oxygen as hydrogen acceptor, e.g. tyrosinase, uricase. b. Anaerobic dehydrogenases: Those which use some other substances as hydrogen acceptor, e.g. lactic dehydrogenase, malic dehydrogenase. c. Hydroperoxidases: Those which use hydrogen peroxide as substrate, e.g. catalase, peroxidase. 2. Transferases: They catalyze the transfer of some group from one molecule to another molecule. These enzymes are important in biological synthesis, e.g. transaminases, hexokinases, transacylase, transaldolase, ketolase, phosphomutases. 3. Hydrolases: They catalyze the hydrolysis of substrate by addition of water molecule across the bond which is split, e.g. esterases, peptidases, phosphatases, deamidases. 4. Lyases: They catalyze the addition or removal of groups from the substrate without hydrolysis, oxidation or reduction, e.g. decarboxylases, carboxylase, carbonic anhydrase, aldolase, enolase, etc. 5. Isomerases: They catalyze the conversion of a compound into an isomer, e.g. racemases, epimerases, isomerases, mutases. 6. Ligases: They catalyze the linking together of molecules coupled with the breaking of pyrophosphate bound in ATP, e.g. glutamine synthetase, succinic thiokinases. Enzyme Specificity Enzyme specificity is determined by how well the reactant fit into the enzyme surface. Some enzymes are very specific and show activity with only one substrate. However, some other enzymes are much less particular and will catalyze reaction with similar compounds.
    • ENZYMES 123 Generally two types of enzymatic specificities are observed in different reactions. Zymogens: Several proteins are synthesized in inactive forms. These are called zymogens eq. proteins digesting enzymes and blood clotting proteins. To activate zymogens, a small amount of protein is cleared from one end. This causes the protein to change shape and activate it. These changes are not reversible. Stereospecificity Some enzymes show specificities only with a specific group of a substrate, e.g. Urease catalyzes the hydrolysis of urea. Alteration in the structure of urea results in the loss of activity. For example, N-methyl urea and thiourea are not the substrate for enzyme urease. Also some enzymes show specificity towards D- and Lform of the same substrate, e.g. D-amino acid oxidase acts only on the D-form of amino acid and not on L-form. Substrate Specificity Some enzymes catalyzes similar type of reactions but differ in their action due to absolute substrate specificity, e.g. Pepsin hydrolyzes peptide bond involving amino group of aromatic amino acids as phenylalanine or tyrosine. Similarly trypsin hydrolyzes peptide bond involving the carboxyl group of basic amino acids such as lysine or arginine.
    • 124 BIOCHEMISTRY FOR STUDENTS FACTORS INFLUENCING THE RATE OF ENZYMATIC REACTIONS Effect of Substrate Concentration At a low substrate concentration, the initial velocity of an enzyme catalyzed reaction is proportional to the substrate concentration. However, as the substrate concentration is increased, the initial velocity increases less as it is no longer proportional to the substrate concentration. With a further increase in the substrate concentration the reaction rate becomes independent of the substrate concentration and assumes a constant rate as a result of enzyme being saturated with its substrate. It was Michaelis and Menten who suggested an explanation of these findings by postulating that at low substrate concentrations, the enzyme is not saturated with the substrate and the reaction is not proceeding at maximum velocity whereas when the enzyme is saturated with substrate, maximum velocity is observed. They further visualized the combination of enzyme with the substrate to form an enzyme-substrate complex and assumed that the rate of decomposition of the substrate being proportional to the concentration of enzymesubstrate complex. The velocity of the reaction at this high
    • ENZYMES 125 substrate concentration is termed as maximum velocity. The substrate concentration at which the velocity is half of the maximum velocity is called the Michaelis constant and is termed as Km. Km indicates the affinity of the substrate towards the enzyme and is inversely proportional to the affinity. 1 Km ∝ Affinity Higher the affinity the smaller will be the Km and lower the affinity, the higher will be the Km. The Michaelis-Menten equation is given by the expression V0 Vmax [S] = K + [S] m where V0 = Initial velocity Vmax = Maximum velocity Km = Michaelis constant [S] = Substrate concentration The Michaelis-Menten equation relates the initial velocity, the maximum velocity and the initial substrate concentration through Michaelis-Menten constant. When the initial velocity is exactly half of the maximum velocity the Michaelis-Menten equation assumes the form V [S] 1 Vmax = max 2 K m + [S] Km + [S] = 2 [S] i.e. Km = [S] Thus Michaelis-Menten constant is equal to the substrate concentration at which the initial velocity is half of the maximum velocity. Determination of important physical constants of an enzyme such as V and Km would be difficult from the curve that would be obtained by plotting [V] against [S]. So the MichaelisMenten equation can be transformed into the form which is useful in plotting experimental data. Taking the reciprocals of both the sides of Michaelis-Menten equation.
    • 126 BIOCHEMISTRY FOR STUDENTS K + [S] 1 = m Vmax [S] V0 1 Km [S] = + Vmax [S] Vmax [S] V0 or 1 Km 1 = + Vmax [S] Vmax V0 This equation is called Line-weaver Burk equation and is the equation for a straight line y = mx + c, where m is the slope of the straight line, c is the intercept on the y-axis and x is the intercept on x-axis. When 1 1 is plotted against a straight line is obtained, [V0 ] [S]' the slope of which is the Km 1 and has an intercept of on Vmax Vmax 1 1 1 axis and intercept of on the axis. Km [V0 ] [S]
    • ENZYMES 127 Since, Line-weaver-Burk equation is in the form of a straight line, so it requires few points to define, Km. By using small concentrations of substrate it is possible by this double reciprocal plot to determine Km. Significance of Km and Vmax Values The Michaelis constant [Km] has two meanings: One is that it is equal to that substrate concentration at which half of the active sites are filled and so once the Km is shown, the fractions of sites filled (fs) at any substrate concentration can be calculated by: fs = [S] [S] + K m V = Vmax Second, Km is related to rate constant of the individual steps K + K2 Km = 1 K1 Now, when the K1 is much more than K2, the K2 becomes negligible and Km is then equal to K −1 − K1 , which is the disso- ciation constant of the ES complex, a reversible reaction, i.e. K1 E + S ⇔ ES K−1 when or R1 R2 R1 K1 [E] [S] = = = = K1 [E][S] K–1 [ES] R2 (at equilibrium) K–1 [ES] [E] [S] K −1 = = KSE K1 [ES] (the equilibrium constant of ES) K −1 Km = K1
    • 128 BIOCHEMISTRY FOR STUDENTS So when this condition is met, Km indicates the strength of ES complex and at such conditions a high Km indicates weak binding and a low Km indicates strong binding. But this is true only when the K2 is much less then K-1. Vmax Vmax indicates the turn over number of the enzyme if the concentration of active sites, i.e. the total enzyme (Et) is known since Vmax = K2[Et]. Here, in this relation K2 is called the turn over number of an enzyme which is defined as number of substrate molecules converted into product per unit time when the enzyme is fully saturated with the substrate and the time required for each round of catalysis is thus given by 1/K2. Method of Determining Km Km can be determined by double reciprocal Line-weaver-Burk method. In this the velocity of reaction is noted with different 1 1 and from the graph, the value of Km is determined. [S] [V ] Another advantage of this equation is that it is used to differentiate certain type of inhibitors of enzyme system. Effect of Enzyme Concentration The rate of an enzyme catalyzed reaction is directly proportional to the concentration of the enzyme. The greater the concentration of enzyme, the faster will be reaction taking place. Effect of pH Most enzymes have a characteristic pH at which their activity is maximum. Above or below that pH, the enzyme activity decreases. If a curve is drawn between the activity of an enzyme on a given substrate with the pH of the reaction mixture, it will reveal a maximum activity at a definite pH. This value is known as optimum pH. See Diagram on Page No. 129.
    • ENZYMES 129 This is probably due to the changes in the net charge on enzymes, (as they are protein in nature) resulting from changes in pH. Excessive changes of pH brought on by the addition of strong acids or bases may completely denature and inactivate enzymes. Effect of Temperature The rate of an enzyme catalyzed reaction generally increases with temperature, within the temperature range in which the enzyme is stable and retains its full or maximum activity. Enzyme catalyzed reactions have an optimum temperature at which the reaction is most rapid. Above this temperature the reaction rate decreases as enzymes being protein in nature are denatured by heat and becomes inactive. The increase in rate below optimal temperature results from increased kinetic energy of the reacting molecules. ENZYME ACTIVITY Activity: Amout of substrante converted to products by the enzyme per unit time (e.g. micromoles/minutes) Specific activity: Activity per quality of protein (e.g. micromoles/ minute/mg protein) Catalytic constant: Proportionality constant between the reaction velocity and the concentration of enzyme catalyzing the reaction. Unit: Activity/mole enzyme. Turnover number: Catalytic constant/number of active sites/ mole enzyme.
    • 130 BIOCHEMISTRY FOR STUDENTS International unit (IU): Quality of enzyme needed to transform 1.0 micromole of the substrite to product per minute at 30°C of optimal pH. The activity of an enzyme is expressed in standard units U = the amount of activity of an enzyme which catalyzes the transformation of one micromole of substrate per minute. The specific activity of an enzyme is the number of units of enzyme activity per mg of protein. The reason for needing this is that often the enzyme is not pure and there is contamination protein in the sample. The catalytic constant is units of enzyme activity per mol of protein (mmol/min/mol enzyme). Katab (kat) are the conversion of 1 mol/sec (International units). Turnover Number The number of molecules of substrate converted to products per enzyme molecule per minute is called turnover number. ENZYME INHIBITIONS Since, enzymes are proteins, any agent which denatures proteins will inactivate the enzyme. Inhibitors are the substances which lower down the rate of enzyme reactions. They exert their effect by acting on the apoenzyme, coenzyme, prosthetic group or activator present in the enzyme system or by interfering with the binding of the substrate to the enzyme. Reversible inhibitors bind the enzymes through noncovalent bonds and dilution of the enzyme-inhibitor complex results in dissociation of the reversibly bound inhibitor where as irreversible inhibitors occurs when an inhibited enzyme does not regain activity on dilution of the enzyme-inhibitor complex. Substances that inhibit enzymatic reactions are classified into three groups: 1. Competitive inhibition 2. Noncompetitive inhibition 3. Uncompetitive inhibition.
    • ENZYMES 131 This classification depends upon the manner of combination of the inhibitor with the enzyme. Competitive Inhibition As the name implies, the competition is between normal substrate and the inhibitor molecules for binding at the active site of the enzyme to form enzyme-substrate or enzyme inhibitor complex. As a result of structural similarity between the substrate molecules and inhibitor molecules, they compete both for active sites of the enzyme molecule and tie up to the active sites. These sites are then not available to the normal
    • 132 BIOCHEMISTRY FOR STUDENTS substrate molecules. The overall rate of inhibition is governed by the affinities of inhibitor molecules and normal substrate molecules for the enzyme binding site and by the concentrations of the reactants. The presence of competitive inhibitor thus increases the apparent Km of the enzyme for the substrate, i.e. causes it to require a higher substrate concentration to achieve the maximum velocity. On the other hand, a competitive inhibitor does not affect the Vmax indicating that it does not interfere with the rate of breakdown of enzyme substrate complex. Competitive inhibitors are frequently called antagonists or antimetabolites of the substrate with which they compete. The example of competitive inhibitions are: i. The inhibition of enzyme succinate dehydrogenase by malonate for succinate. Both have similar structural resemblance and hence, both compete for the active site of enzyme succinate dehydrogenase. ii. Sulphanilamide has structural resemblance with paraminobenzoic acid and blocks the folic acid synthesis which results in the deficiency of the vitamin to microorganisms.
    • ENZYMES 133 In case of competitive inhibition. Affinity — Decreases Efficiency — Remains same — Decreases as Km increases 1/Km 1/Vmax — Remains same Noncompetitive Inhibition As the name implies there is no competition between the sub strate and the inhibitor molecules. There is little or no structural resemblance between the substrate and the inhibitor molecules and hence they bind to the different sites of the enzyme. Inhibitors combine with the allosteric site of the enzyme, this combination results in the distortion of the active site. In noncompetitive inhibition, the affinity of enzyme remains same but its efficiency decreases. This inhibition is also known as allosteric inhibition. The inhibitory action cannot be overcome by increasing the substrate concentration. The complex formation between the inhibitor and enzyme is reversible. Noncompetitive inhibitors lowers the Vmax but does not effect the Km. Examples of noncompetitive inhibitions are: There are many enzymes which require free sulphydryl group (i.e.—SH group) for activity, are noncompetitively inhibited by heavy metal ions such as Pb++, Hg++, etc. Urease is an example of an enzyme which experiences heavy metal
    • 134 BIOCHEMISTRY FOR STUDENTS inhibition. The action of nerve gas poisons on acetylcholinesterase, is an example of noncompetitive inhibition. In case of noncompetitive inhibition. Affinity — Remains same Efficiency — Decreases Remains same because the substrate 1/Km concentration has no effect on the inhibitory action. — Increases as V has decreased. 1/Vmax Uncompetitive Inhibition These inhibitors combine only with the enzyme-substrate forming an irreversible complex. The inhibition is dependent only on the concentration of the inhibitor. In case of uncompetitive inhibition Vmax is lower Slope is same Apparent Km < Km CATALYTIC SITE OR THE ACTIVE SITES OF THE ENZYMES The portion of the enzyme protein molecule which actually takes part in catalysis is called active site or the catalytic site of the enzyme. Although the enzymes differ widely in structure specifically and catalysis, there are certain common features about the active sites.
    • ENZYMES 135 1. Normally the active sites makes up a small volume of the total portion of an enzyme. 2. The active site is a three dimensional activity. 3. It is made up of groups that come from the different parts of the linear amino acid chain. Indeed the residues are far a part in the linear sequence but may come together to bring about catalysis. 4. The specificity of the substrate binding depends upon the precisely defined arrangement of the atoms or groups at the active site. Emil Fisher postulated that substrate and enzyme reacted in a well defined clear cut lock-key fashion signifying the predominated structure of the active fit complementary to the substrate molecule structure with which it will bind. This model implied therefore the rigidity of the catalytic site. But this hypothesis was soon found unable to explain the possibility of such a catalytic site reacting with the product to reform substrate in a reversible manner. Then Koshland proposed a more flexible hypothesis called “induced fit model” regarding the structure of the active site. According to this hypothesis, enzymes in the inactive state in the absence of substrate and that various groups in the active site are not correctly oriented to interact with the complimentary groups on the substrate. Binding of the specific substrate however, results in a conformational change in the enzyme and thus to the active site and shifting of those groups or atoms in the site into the correct position for proper binding with the substrate and catalysis. Feedback inhibition: In many multienzyme systems, the end product of the reaction sequence may act as a specific inhibitor of an enzyme at or near the beginning of the sequence, with the result that the rate of entire sequence of reactions is determined by the steady state concentration of the end product. This type of inhibition is called feed back inhibition. For example, cholesterol synthesis is regulated, by feedback inhibition. ENZYME INDUCTION Enzymes are classified according to the condition under which they are present in a cell. They are of two types.
    • 136 BIOCHEMISTRY FOR STUDENTS a. Constitutive enzymes. b. Inducible enzymes. Constitutive Enzymes These enzymes are formed at constant rates and in constant amounts. Their presence in a cell is not related to the presence or absence of their substrates. They are considered to be part of the permanent enzymatic make of the cell. For example, enzymes of glycolytic pathway. Inducible Enzymes Also called adaptive enzymes. They are always present in trace amounts but their concentrations vary in proportions of their substrates. Isoenzymes They are multiple forms of a given enzyme having different mobilities on electrophoresis, differently depressed by inhibitors towards different substrates. Isoenzymes catalyze the same reaction but differ in Km, Vmax or both. The relative amounts of the isoenzymes of a particular enzyme differ in different organs so that in disease, different isoenzyme patterns are found according to the organs from which they have come. These forms are the characteristics of different organs and tissues of the human body. Example 1. Lactate dehydrogenase (LDH): This enzyme catalyzes the dehydrogenation of lactate to pyruvate. This occurs in five different isoenzymes. This enzyme is a tetramer having two types of units, i.e. L and M units. Depending upon the various combination, five isoenzymes are known, i.e. thest two subunits can combine in five different ways. Test Composition Location LD-1 LD-2 LD-3 LD-4 LD-5 HHHH HHHM HHMM HMMM MMMM Heart and RBC Heart and RBC Brain and kidney Liver and skeletal muscle
    • ENZYMES 137 LD-1 is the predominant form in heart and LD-5 in muscles. LDH is elevated from 12 to 48 hours after initial attack. 2. Alkaline phosphatase: It occurs in two forms. 3. Isocitrate dehydrogenase: It occurs in two forms. 4. Creatine phosphokinase: It occurs in three forms. CPK is a dimer consisting of one subunit found in the brain (B) and other in muscle (M). CPK is found in three isoenzymes, as CPK1 (BB), CPK2 (MB) and CPK3 (MM). In normal serum 95% of the CPK activity is in CPK3. CPK is, found in three isoenzymes as: i. CPK (MM), largely found in skeletal muscle tissue. ii. CPK (BB), predominately found in brain tissue. iii. CPK (MB), exclusively found in heart tissue. Blood level of both CPK (total) and CPK (MB) usually markedly increases following acute myocardial infarction. Only CPK (MB) elevation is highly specific for the diagnosis of MI. CPK (MM) increases rapidly following exercise or muscle trauma. CPK (BB) is heat labile and rarely detected in serum. The activitiy of CPK2 is the cornerstone for the diagnosis of myocardial infarction because of its abundance in heart and absence from other cells. It may be elevated after 4 hours and its activity may increase from two to ten folds after 16-24 weeks. DIAGNOSTIC VALUE OF PLASMA ENZYMES A determination of enzyme levels in the serum is often helpful in pinpointing which, if any, body tissue or organ has been damaged or malfunctioning. When a tissue is injured some cells of that tissue are destroyed and their contents, enzymes included, are released into the blood stream. Therefore, if an enzyme is normally found predominately in a tissue other than blood, an increase in its level in the blood indicates that tissue has been damaged. Serum acid phosphatase is increased in Paget’s disease of bone, hyperparathyroidism, metastases of bone, Gaucher’s diseases, chronic renal failure and prostatic carcinoma.
    • 138 BIOCHEMISTRY FOR STUDENTS Serum alkaline phosphatase is increased in rickets, hyperparathyroidism, obstructive jaundice, osteomalacia. Serum glutamate oxaloacetate transaminase SGOT/AST (Aspartate transaminase) is increased in myocardial infarction and skeletal muscle dystrophies. Serum glutamate pyruvate transaminase SGPT/ALT (Alanine transaminase) is increased in viral hepatitis, toxic hepatitis and other forms of liver diseases associated with some degree of hepatic necrosis. Typical profiles of serum enzymes following a myocardial infarction. SGOT catalyzes the reversible transfer of the amino group from glutamate to oxaloacetate to form α-ketoglutarate and aspartate. GOT is released from many diseased cells into serum as SGOT. SGOT is elevated in liver disease and following a myocardial infarction. The serum level has diagnostic value. It can be moderately elevated (5-fold) in people with cirrhosis and obstructive liver disease (a stone blocking bile duct). It can become very high (25-fold) in viral hepatitis. Serum lactate dehydrogenase (LDH) is increased in myocardial infarction, acute liver disease, pernicious anemia, progressive muscular dystrophies. Serum creatine phosphokinase (CPK) is increased in muscular dystrophy, myocardial infarction. Serum amylase is increased in various forms of pancreatic disturbances (Pancreatitis). Serum isocitrate dehydrogenase is increased in liver diseases, severe pulmonary infarction. Serum lipase is increased in acute pancreatitis and carcinoma. Enzyme Creatine kinase (CK-MB) Aspartate transaminase (AST) Lactate dehydrogenase (LDH) Evidence of rise 3-6 hr 6-8 hr 12 hr
    • ENZYMES 139 Typical profiles of serum enzymes following a myocardial infarction.
    • 140 BIOCHEMISTRY FOR STUDENTS CHAPTER 7 Biological Oxidation BIOLOGICAL OXIDATION All body reactions require energy which is obtained from chemical reactions carried out in the living cells. The stepwise oxidation of various metabolites is the principal mechanism for the liberation of energy. The utilization of oxygen and production of carbon dioxide by the tissues in the process of cellular respiration is the final phase of biological oxidation. Transfer of electrons are involved in all oxidation-reduction reactions. Every oxidation must be accompanied by simultaneous reduction and the energy required for the removal of electrons in oxidation is supplied by the reduction. The electron transport is important for the following reasons: 1. It explains how oxygen finally enter the metabolism. 2. It provides the mechanism for the regeneration of oxidation-reduction coenzymes. 3. It provides the majority of the energy derived from metabolic processes. The energy transfers involved in the oxidation-reduction systems are measured by difference in potential of various systems. Oxygen has the highest oxidation potential of the systems in the living cells and hydrogen atom the lowest. Biological oxidation is catalyzed by enzymes which functions in combination with coenzymes or electron carriers. Oxidases These enzymes catalyze the removal of hydrogen from the substrate directly to the molecular oxygen, e.g. cytochrome a3 (cytochrome oxidase), tyrosinase, uricase, etc. 2H + ½ O2———→ H2O
    • BIOLOGICAL OXIDATION 141 Dehydrogenases They are further divided into: a. Aerobic dehydrogenases b. Anaerobic dehydrogenases. a. Aerobic dehydrogenases: These enzymes remove hydrogen from the substrate using either O2 or artificial substance as hydrogen acceptor. These dehydrogenases are flavoproteins, e.g. xanthine oxidase, D-amino acid oxidase, catalase, peroxidases. b. Anaerobic dehydrogenases: These enzymes use substances other than oxygen as hydrogen acceptor. These dehydrogenases are classified as: i. Pyridine nucleotides: Under this group comes nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+). The effective part which participates in the reaction is nicotinamide. ii. Flavonucleotides: They are flavin mono nucleotide (FMN) and flavin adenine dinucleotide (FAD). The effective part which participates in the reaction is riboflavin. FAD + 2H+ + 2 e¯ —→ FADH2+ iii. Cytochromes: The cytochromes are iron containing hemoproteins in which iron oscillates between Fe++ and Fe+++ during oxidation-reduction. Various cytochromes are cytochromes b, c1, c, a.
    • 142 BIOCHEMISTRY FOR STUDENTS Oxygenases The enzymes incorporate oxygen into the substrate molecule. They are divided into two groups: a. Dioxygenases: These enzymes catalyze the incorporation of both atoms of oxygen into the substrate. A + O2 → AO2 For example, tryptophan dioxygenase and homogentisic acid dioxygenase. b. Monoxygenases: They add only one atom of oxygen into the substrate. MIXED FUNCTION OXIDASES These oxidases cause reduction of one atom of O2 and utilization of other atom for specific oxygenation of hydroxylation of the substrate. Two catalytic functions are performed by these enzymes: i. Reduction of an atom of oxygen to O¯ ii. Transfer of oxygen to the substrate. These enzymes not only require O2 but also a source of electrons, i.e. reducing agent to reduce an atom of O2 to O¯ Mixed function oxidases are metalloproteins with prosthetic group containing Fe, Cu. Examples of mixed function oxidases are: i. Phenylalanine hydroxylase ii. p-Hydroxy phenylpyruvate oxidase iii. Imidazole acetic acid oxidase iv. Phenolase complex (this enzyme is involved in the formation of melanins from tyrosine). Hydroperoxidases or Peroxidases These peroxidases catalyze the transfer of electrons from donars (substrates) to H2O2, reducing it to water. The peroxidases are specific in requiring H2O2 as electron acceptor (oxidizing agent) but various substrates may act as substrates or electron donars.
    • BIOLOGICAL OXIDATION 143 Catalases Catalases are specific type of hemin-containing hydroperoxidases which have the property of rapidly catalyzing the decomposition of H2O2. 2H2O2 → 2H2O + O2 Catalase enzymes are simply a specific type of peroxidase enzymes possessing very high activity toward H2O2 as a substrate but also capable of catalysing regular peroxidate reactions. Superoxide anion O¯ is highly reactive. It is generated 2 by a number of biological reactions including the antioxidation of quinones, thiols and reactions catalyzed by xanthine oxidase and flavoprotein dehydrogenases. Superoxides are very toxic to the cells and consequently the enzyme superoxide dismutase, present in all the cells, is responsible for protecting the cell from the harmful effect of superoxide anion, the cell in turn is protected from H2O2 by catalase Superoxide dismutase 2H++ 2O¯ ———————————→ H2O2 + O2 2 Catalase 2H2O 2 ————————————→ 2H2O + O2 HIGH ENERGY COMPOUNDS High energy compounds are: 1. Acid phosphates: They are acid anhydrides of an organic acid (RCOOH) and phosphoric acid. Their general formula is: For example 1, 3, diphosphoglyceric acid, acetyl phosphate. 2. Enol phosphates: For example, 2-phosphoenol pyruvic acid. 3. Guanidinophosphates: Two important compounds in this are creatine phosphate in vertebrate and arginine phosphate in invertebrate.
    • 144 BIOCHEMISTRY FOR STUDENTS 4. Organic pyrophosphates: These compounds are ADP and ATP, GTP, IDP, etc. 5. Coenzyme A: Derivatives of coenzyme A are high energy compounds. 6. Methionione: As S-adenosyl methionine. RESPIRATORY CHAIN Transfer of electrons from substrate to molecular oxygen through a chain of electronic carriers is called electron transport chain or respiratory chain. Mitochondria contains series of catalysts called respiratory chain which are involved in the transfer of hydrogen and electrons and their final reaction with oxygen to form water. The components of respiratory chain are arranged sequentially in the order of increasing redox potential. Electrons flow through the chain in a stepwise manner from lower redox potential to higher redox potential. The amount of energy liberated in transfering electrons from one system to another is determined by difference in redox potential of the two systems. The respiratory chain is given as: A redox potential of 0.2 volt between the components of respiratory chain results in the formation of 1 mole of ATP. The three sites of ATP formation in the respiratory chain are: 1. Between NAD+ and flavoprotein 2. Between cytochrome b and c 3. Between cytochrome a and cytochrome a3. If the substrate enters the respiratory chain through NAD+ than the ATP yield is 3. If the substrate enters the respiratory chain through flavoproteins than the ATP yield is 2.
    • BIOLOGICAL OXIDATION 145 Coenzyme (Ubiquinone) It is called ubiquinone because of its ubiquitous occurrence in nature. It is a lipid soluble hydrogen (electron) carrier found in mitochondrial membranes and is a benzoquinone derivative. It contains an isoprene side chain which varies from source to source. Human coenzyme Q contains 10 isoprene units. It is lipid soluble electron carrying protein and is reversibly reduced by 2H+ from FADH2. Reduced coenzyme Q is the final stage at which oxidation reaction occurs as a process of transfer of hydrogen atoms. Thereafter it is only the electrons of the hydrogen atoms which are carried down the electron transport and the 2H+ ions liberated into the medium. Other homologous of coenzyme Q contains 6 to 10 isoprene units and have been isolated from various microorganisms, e.g. chloroplasts of green plants and mitochondria of beef and other animal tissues. Cytochromes Cytochromes are electron carrier proteins containing heme. They contain protein part to which heme is attached as prosthetic group. The cytochromes undergo oxidation and reduction as a result of oscillation of iron atom with Fe++ and Fe+++ from which donates the reduced and oxidized form respectively. The five different cytochromes that has been identified in the inner mitochondrial membrane are cytochromes a1, a3, b, c, and c1. It has been found that cytochrome a and cytochrome a3 are combined with the same protein molecule to form cytochrome aa3 complex which is also called cytochrome c oxidase or respiratory enzyme. It contains 2 atoms of copper.
    • 146 BIOCHEMISTRY FOR STUDENTS Oxidative Phosphorylation Oxidative phosphorylation means that oxidation is accompanied by phosphorylation. The energy released as a result of biological oxidation is trapped in the form of high energy phosphate bonds in ATP by phosphorylation of ADP. It is divided into two groups: 1. Substrate level phosphorylation: In the substrate level phosphorylation the formation of high energy phosphate takes place on the substrate, without undergoing into the respiratory chain. The characteristics of substrate level phosphorylation are: i. Formation of ATP does not require oxygen ii. Respiratory chain does not participate iii. It is dinitrophenol insensitive. Examples Substrate level phosphorylation is best described by two examples: NAD+ (1) D-glyceraldehyde-3-PO4 + Pi + ADP ——→ PhosphoATP glyceric acid (2) Succinyl CoA + Pi + GDP → Succinic acid + GTP + CoA GTP + ADP → GDP + ATP 2. Respiratory chain phosphorylation: In this phosphorylation the formation of high energy phosphate bonds takes place as a result of transfer of hydrogen and electrons through the respiratory chain to oxygen. The characteristics of respiratory chain or electron-oxygen transport chain are: 1. It is completely inhibited by trace amounts of dinitrophenol (DNP) or by antimycin A. 2. Oxygen uptake however, is not inhibited by DNP. When a substrate is oxidized via NAD linked dehydrogenases, 3 moles of inorganic phosphates are incorporated into 3 moles of ADP to form 3 moles of ATP per atom of oxygen consumed. Similarly when a substrate is oxidized via flavin linked dehydrogenases only 2 moles of ATP is formed.
    • BIOLOGICAL OXIDATION 147 Type of phosphorylation Reactions 1. Substrate level D-Glyceraldehyde -3-PO4 Phosphoenol Pyruvate Succinyl CoA 2. Respiratory chain Phosphorylation isocitrate (electron transport chain) α-ketoglutarate Succinate ~P trapped 3-Phospho Glyceric acid 1 Pyruvate Succinate 1 1 Oxalosuccinate 3 Succinyl CoA Fumarate 3 2 P/O Ratio P/O ratio is defined as the number of inorganic phosphate taken to phosphorylate ADP per atom of oxygen consumed. Mechanism of Oxidative Phosphorylation Three hypothesis for the mechanism of oxidative phosphorylation has been postulated to account for the transfer of energy from the oxidation reductions of reactions involving electron transport chain (respiratory chain) to the synthesis of ATP. 1. Chemical coupling hypothesis 2. Chemiosmotic hypothesis 3. Conformational coupling hypothesis. Chemical Coupling Hypothesis This is the oldest hypothesis and it postulates that the energy yielding electron transfer process is coupled with energy requiring oxidative phosphorylation through the formation of high energy intermediate compound which is generated by the electron transport system and then subsequently utilized in the ATP formation from the ADP. In effect, it proposes the existance of specific carrier proteins called C1, C2 and C3 at each of the three ATP producing sites along with an intermediate I carried by them. At the site of release of energy sufficient to form ATP, intermediate I is combined with the carrier to form a high energy carrier and intermediate
    • 148 BIOCHEMISTRY FOR STUDENTS complex. This complex then is again linked to combine with another intermediate X to form I~X again an high energy intermediate. The I component is then finally replaced by inorganic phosphates and ADP is phosphorylated to form ATP using the energy contained in I2~X complex, which is used up. A strong objection to this effect is that no such intermediate has been found even after intensive research. This hypothesis takes the help of hypothetical carriers and hypothetical intermediates I and their effects are explained as: Effect of inhibitors: Inhibitors arrest respiration by blocking the respiratory chain at energy site I, II and III. Inhibitors of site I = Rotenone, amobarbital, piericidin Inhibitors of site II = Antimycin, BAL Inhibitors of site III = H2S, CO, CN¯ Uncouplers: Uncouplers are substances which allow electrons to continue but prevent phosphorylation of ADP to ATP. They are dinitrophenol (DNP). Uncouplers causes the hydrolysis of one of the high energy intermediates (car ~ l) resulting in the release of carrier I and energy as heat. Chemiosmotic Hypothesis This hypothesis (Peter Mitchell) assumes two points. i. The outer mitochondrial membrane is impermeable to hydrogen ions and hydroxide ions. ii. The process goes on within matrix.
    • BIOLOGICAL OXIDATION 149 During electron transport, protons are released to the outside of the mitochondria. This results in the establishment of a proton gradient across the membrane, with a high concentration of protons (H+) outside the mitochondria and low concentration of protons inside the mitochondria creating an electrochemical potential difference. This electrochemical potential difference is used to derive a vectorial membrane located ATP synthetase, or the reversal of a membrane located ATP synthetase which in the presence of inorganic phosphate and ADP forms ATP. Conformational Coupling Hypothesis According to this hypothesis (PD Bayer) the release of energy during the electron transport induces some conformational changes in the carrier protein or the coupling factor. These changes are due to the energy dependent shift in the number of location of weak bonds such as hydrogen bonds and hydrophilic interactions which normally maintain the three dimensional conformation of the proteins. Then this energy conserved in this energised conformational state is used to derive the phosphorylation of ADP by inorganic phosphorous into ATP. Simultaneously the carrier protein or the factor returns back to the original low energy conformation. This theory gets some support from the fact that inner mitochondrial membrane undergoes very rapid physical changes as the electron pass along the respiratory chain. Also this membrane shows some ultrastructural changes that accompany the addition of ADP to the respiratory mitochondria. This theory is in a way similar to the chemical coupling theory except the fact that it postulates the non-covalent bonds as the energy intermediates rather than the postulation of true high energy intermediate in the chemical theory. Shuttle System NADH is produced in the cytosol but cannot penetrate the mitochondria, i.e. the extra mitochondria NADH cannot penetrate the mitochondrial inner membrane but electron derived from it can enter electron transport chain by an indirect route called shuttles.
    • 150 BIOCHEMISTRY FOR STUDENTS Two important shuttle systems are: 1. α-glycerophosphate shuttle 2. Malate-aspartate shuttle. α-glycerophosphate shuttle: This shuttle transfers reducing equivalents from cytosol to the mitochondrial electron transport chain by the following route. Malate-aspartate shuttle: This shuttle transfer NADH from the cytosol to mitochondria by the following route.
    • METABOLISM OF CARBOHYDRATES 151 CHAPTER 8 Metabolism of Carbohydrates The major function of carbohydrate in metabolism is as a fuel to be oxidized and provide energy for other metabolic processes. In this role, carbohydrate is utilized by cells mainly in the form of glucose. It has the advantage of being cheap, easily digested and rapidly metabolized. Carbohydrate metabolism is basically the metabolism of glucose and substance related to glucose in their metabolic processes. Glucose serves as a ready source of chemical energy for humans. The sugar of blood is glucose. The digestion of carbohydrates such as starch, sucrose and lactose produces glucose, fructose and galactose which passes into blood circulation. Conversion of fructose and galactose into glucose takes place in the liver. Carbohydrates supply more than 50 percent of the energy requirement of the body. Except for ascorbic acid (vitamin C), carbohydrates are not essential to the diet, through gluconeogenesis, the body can synthesize necessary carbohydrates from certain amino acids. GLYCOLYSIS The breakdown of glucose to pyruvic acid is called glycolysis. Under aerobic condition, pyruvic acid enters mitochondria and is completely oxidized to CO2 and H2O. Whereas, under anaerobic conditions, pyruvate is converted to lactic acid. The sequence of reactions from glucose to pyruvic acid is also called Embden-Meyerhof pathway. Glucose is converted to pyruvate in 10 steps by glycolysis. Glycolysis is an extramitochondrial pathway and is carried by a group of eleven enzymes.
    • 152 BIOCHEMISTRY FOR STUDENTS Mutases are enzymes which catalyze the transposition of functional groups.
    • METABOLISM OF CARBOHYDRATES 153 Glucokinase is an inducible enzyme and has high km value for glucose whereas hexokinase is a constitutive enzyme and has low km value for glucose. Pyruvic acid has both a ketone or keto group and an acid group and hence it is a keto acid. Salient Features of Embden-Meyerhof Pathway 1. The rate limiting step in glycolysis is phosphofructokinase (PFK). PFK is stimulated by fructose-6-phosphate, AMP and ADP but is inhibited by ATP and citrate. Since one of the main object of glycolysis is to produce ATP and since the presence of excess AMP, ADP or fructose-6-phosphate means that the cell is deficient in ATP. These molecules are activator of the enzyme (PFK), stimulating it to degrade more glucose and hence more production of ATP. Consequently an excess of ATP means that the cell is catabolizing more glucose than necessary; excess ATP inhibits PFK. 2. All the reactions of glycolysis are reversible except hexokinase, phosphofructokinase and pyruvate kinase catalyzed reactions because of energy barriers. 3. Enzyme enolase is inhibited by fluoride. Since erythrocytes do not have mitochondrial enzymes to oxidize glucose aerobically, they depend on glycolysis only for their energy requirement. That is why sodium fluoride (NaF) is used in the collection of blood sugar sample because it prevents glycolysis by inhibiting the enzyme enolase. Otherwise a low result will be obtained due to glycolysis.
    • 154 BIOCHEMISTRY FOR STUDENTS 4. It is the major pathway by which glucose is metabolized in erythrocytes. 5. Glycolysis gives rise to certain intermediate compounds which are important for other biochemical processes. i. Glyceraldehyde-3 PO4: For triglycerides and phospholipid biosynthesis. ii. Acetyl CoA: Fatty acid and cholesterol biosynthesis. iii. Pyruvate: Alanine biosynthesis by transamination. Glycolysis has three principal features: 1. It is the degradative pathway whereby D-glucose is oxidized to pyruvate, which is further metabolized by either of the two routes. i. When the supply of oxygen is inadequate for complete oxidation, the pyruvate is reduced to lactate. ii. When the supply of oxygen is adequate (aerobic conditions) the pyruvate is oxidatively decarboxylated to acetyl CoA, which enters the citric acid cycle, where it is oxidized to carbon dioxide and water. 2. Glycolysis gives rise to certain intermediates which are common to other pathway such as pentose phosphate pathway. These intermediate compounds also provide sources of starting materials for the biosynthesis of substances such as triglycerides from glyceraldehyde-3-phosphate, L-alanine from pyruvate and glycogen from glucose-1phosphate. 3. Glycolysis is accompanied by the formation of ATP. Pasteur Effect Pasteur effect is the inhibition of glycolysis by oxygen. The rate limiting step in glycolysis, the phosphofructokinase, is inhibited by citrate and ATP. Crabttee Effect Crabttee effect is the inhibition of cellular respiration by high concentrations of glucose. This is due to the completion of glycolytic processes for inorganic phosphate.
    • METABOLISM OF CARBOHYDRATES 155 CITRIC ACID CYCLE The complete oxidation of acetyl moiety is effected by means of a cyclic metabolic mechanism called citric acid, also called tricarboxylic acid (TCA) cycle and Kreb’s cycle. This cycle takes place in mitochondria. The citric acid cycle operates only under aerobic conditions because it requires a supply of NAD+ and FAD which are regenerated when NADH and FADH2 transfer their electrons to O2 through the electron transport chain. TCA cycle requires the presence of oxygen, i.e. aerobic metabolism of carbohydrates and is catabolized by enzymes found in the mitochondrial fraction of the cell. Before pyruvate gains entry into the TCA cycle, it is oxidatively decarboxylated to acetyl CoA. Conversion of Pyruvate to Acetyl CoA Pyruvate is oxidatively decarboxylated to acetyl CoA by a multienzyme complex called pyruvate dehydrogenase complex. This complex enzyme system comprises of three different enzymes: i. Pyruvate dehydrogenase (29 molecule) ii. Dihydrolipoate transacetylase (1 molecule) iii. Dihydrolipoate dehydrogenase (8 molecule). which catalyze the five step reactions involved in conversion of pyruvate to acetyl CoA. The six cofactors required are (i) Mg++ ions (ii) Thiamine pyrophosphate (TPP) (iii) Lipoic acid (iv) Coenzyme A (CoASH) (v) FAD (vi) NAD+ (see page 156 for reaction). Salient Features of Citric Acid Cycle 1. Citric acid is the common pathway for the metabolism of carbohydrates, fats, and proteins; since it provides the complete oxidation of acetyl CoA to carbon dioxide and water. 2. Citrate synthetase catalyze a direct bond between the methyl carbon of acetyl CoA and carbonyl carbon of oxaloacetate. It is an irreversible reaction.
    • 156 BIOCHEMISTRY FOR STUDENTS 3. It defines the step by which citric acid, isocitric acid, α-ketoglutaric acid, succinic acid are synthesized and degraded. The stepwise mechanism of the reactions is explained below.
    • METABOLISM OF CARBOHYDRATES 157 4. Many amino acids enter the cycle at several levels either at acetyl CoA, α-ketoglutarate, oxaloacetate, succinyl CoA and fumarate. 5. The rate limiting step in the TCA cycle is the conversion of isocitrate to α-ketoglutarate. The enzyme is the citrate synthetase. The availability of acetyl CoA and oxaloacetate in plenty stimulates this enzyme while succinyl CoA by competing with acetyl CoA inhibits this enzyme. Similarly α-ketoglutaric acid, an intermediate in citric acid cycle is oxidatively decarboxylated to succinyl CoA. The enzyme involved is α-ketoglutarate dehydrogenase complex like the pyruvate dehydrogenase complex. This is an irreversible reaction forming succinyl CoA. Aresenite inhibits this reaction causing the accumulation of the α-ketoglutaric acid. Cofactors required are the same as in the conversion of pyruvate to acetyl CoA.
    • 158 BIOCHEMISTRY FOR STUDENTS Pyruvate can be channelled into TCA cycle as acetyl CoA or as oxaloacetate. This point is a switch point which controls the main function of the cycle. If pyruvate is channelled to acetyl CoA then the cycle will generate mainly energy. If pyruvate is channelled into oxaloacetate, then its main function will be to produce carbon skeletons for amino acid or fat synthesis, i.e. high levels of acetyl CoA inhibit the activity of pyruvate dehydrogenase, decreasing further synthesis of acetye CoA and the same time enhance the activity of pyruvate carboxylase, stimulating the synthesis of oxaloacetate. The reaction is summed as:Succinyl CoA ENERGETICS For each molecule of glucose, 2 pyruvates are formed. These are converted to 2 acetyl CoAs, each of which is brokendown to 3 NADH, 1FADH2 and 1 GTP. Hence, for 1 glucose molecule, 6 NADH, 2 FADH2 and 2 GTP are produced in the TCA cycle. Reactions Where ATP is Consumed Glucose to glucose-6-phosphate 1 Fructose-6-phosphate to fructose-1, 6-diphosphate 1 Reactions Where ATP is Generated Glyceraldehyde-3-PO4 to 1,3 diphosphoglycerate 2 × 3 = 6 1,3 diphosphoglycerate to 3-diphosphoglycerate 2 × 1 = 2 (Substrate level phosphorylation)
    • METABOLISM OF CARBOHYDRATES 159 Phosphoenolpyruvate to pyruvate (Substrate level phosphorylation). 2 × 1 = 2 Under Anaerobic Condition The ATP yield is 2 (Two molecules of ATP are generated in the conversion of glucose to pyruvate because NADH obtained in the glyceraldehyde-3-phosphate dehydrogenase reaction is not oxidized in mitochondria by the respiratory chain). Under Aerobic Condition Pyruvate to acetyl CoA Isocitrate to oxalosuccinate α-ketoglutarate to succinyl CoA Succinyl CoA to succinate (The substrate level phosphorylation) Succinate to fumarate Malate to oxaloacetate 2 × 3 = 6 2 × 3 = 6 2 × 3 = 6 2 × 1 = 2 2 × 2 = 4 2 × 3 = 6 ——————— Total = 30 ——————— Total number of ATP molecules formed under aerobic conditions is 38, i.e. 30 from citric acid cycle and 8 from glycolysis. Two important features of Krebs cycle are: i. Two carbon atoms enter the cycle as acetyl CoA and two carbons leave as carbon dioxide, so no net gain of carbon atom takes place. ii. The carbon atoms that leave as CO2 are not the same ones as those taken up as acetyl CoA. The tricarboxylic acid cycle or Krebs cycle serves five major functions: 1. It produces most of the carbon dioxide made in human tissues. 2. It is the source of much of the reduced coenzymes that drive the respiratory chain to produce ATP. 3. It converts excess energy and intermediate to the synthesis of fatty acids. 4. It provides some of the precursors used in the synthesis of proteins and nucleic acid. 5. Its components control directly (product precursor) or indirectly (allosteric) other enzyme system.
    • 160 BIOCHEMISTRY FOR STUDENTS This cycle is described as biochemical traffic circle, material coming to it from carbohydrate source might leave it to form fat whereas material coming to it from amino acid might leave it to form carbohydrate. The only road closed is that leading from fat to carbohydrate. Amphibolic Role of Citric Acid or Krebs Cycle Citric acid cycle is primarily a catabolic process for the final oxidation of the carbohydrates, fats and proteins into CO2 and H2O. But this cycle at the same time takes part in the various anabolic processes such as gluconeogenesis, fatty acid synthesis and amino acid synthesis by providing substrates which are the normal intermediate products of this cycle. Thus, this cycle has the dual or amphibolic role of both catalyzing the substances for energy and also taking part in synthesis. For example, the oxaloacetate and α-ketoglutarate are utilized for amino acids. Similarly, the malate and oxaloacetate are also utilized as glucose precursor in a reaction catalyzed by malate dehydrogenase first and subsequently by phosphoenol pyruvate carboxylase. Citrate is also utilized for providing acetyl CoA for fatty acid synthesis by extra mitochondrial pathway. Further succinyl CoA is utilized in the heme synthesis. Thus, all these examples establish amphibolic role of citric cycle. Hexose Monophosphate Shunt Pathway This pathway is also known as Warburg-Dickens-Lipmann pathway, pentose phosphate pathway, phosphogluconate pathway or direct oxidative pathway or reductive pathway. Though glycolysis is the principal pathway for the conversion of glucose into pyruvate in most tissues but there exists an alternative pathway. Since glucose utilization can proceed when certain reactions in the glycolytic pathway are blocked by the addition of inhibitors. Tissues where this pathway is more prominent are liver, adipose tissue, lactating mammary gland, leukocytes, testes, and adrenal cortex, etc. The enzymes of this pathway are found in the extramitochondrial cytoplasm. Importance of HMP Shunt Pathway 1. This pathway generates NADPH, which is required in the reductive synthesis of fatty acids, triglycerides and steroids.
    • METABOLISM OF CARBOHYDRATES 161 2. Pentose sugars (Ribose-5-PO4) are formed which are required in the synthesis of nucleotides and nucleic acids. 3. This pathway is important in plants which synthesize glucose from CO2 by photosynthesis.
    • 162 BIOCHEMISTRY FOR STUDENTS Energetics Energy yield of hexose monophosphate shunt pathway. Although the greatest importance of HMP shunt pathway is to provide NADPH, for each carbon of glucose oxidized
    • METABOLISM OF CARBOHYDRATES 163 to CO2. Two molecules of NADPH are reduced or 12 molecules of NADPH per mole of glucose oxidized is produced. 12 moles of NADPH are equivalent to 36 moles of ATP. Combined aerobic and anaerobic glycolysis of one molecule of glucose gives 38 ATP, whereas HMP shunt pathway yields 36 ATP. So these two pathways of glucose oxidation are almost equivalent in energy yield. GLYCOGENESIS The formation of glycogen from glucose is called glycogenesis. Under the combined act of glycogen synthetase and branching enzyme, glucose units are added to the non-reducing ends of the pre-existing glycogen by α-(1,4) and α-(1,6) linkages to form glycogen. Glucose is phosphorylated to glucose-6-PO4, by hexokinase reaction, which is then converted to glucose-6-PO4, a reaction catalyzed by the enzyme phosphoglucomutase. Glucose-1-PO4 reacts with uridine triphosphate (UTP) to form uridine diphos-
    • 164 BIOCHEMISTRY FOR STUDENTS phate glucose (UDPG). The reaction is catalyzed by the enzyme UDPG pyrophosphorylase. Now in the presence of the enzyme Glycogen synthetase, C-1 of glucose of UDPG forms a glycosidic linkage α-(1,4) with the C-4 of the preexisting glycogen molecule. The addition of glucose from UDPG to the existing glycogen molecule takes place from the non-reducing end of the glycogen molecule, thus, permitting the origin of new glycogen molecules. When the chain has been lengthened by 6 to 11 glucose molecules, a second enzyme called branching enzyme, transfers 6 glucose molecule in α-(1,4) linkages and attaches to the nearby chain in α-(1,6) linkages, thus creating a branched point in the molecule. Branching is important because it increases the solubility of glycogen and provides a large number of non-reducing sugar terminals which are the sites of activity for glycogen phosphatase, the enzyme that breaks glycogen. Glycogen Synthetase a. Glycogen synthetase-D. It is the inactive form of the enzyme. b. Glycogen synthetase. It is the active form of the enzyme. Glycogen synthetase-D, is the dephosphorylated form. It is glucose-6-phosphate dependent, i.e. it is stimulated by glucose-6-phosphate.
    • METABOLISM OF CARBOHYDRATES 165 While glycogen synthetase-I is the dephosphorylated form. It is independent of glucose-6-phosphate. Glycogen Storage Diseases These are a group of inborn error of metabolic diseases in which there is an accumulation of abnormally large amount of glycogen in the tissue due to the deficiency or absence of enzymes involved in glycogen metabolism. Various type of glycogen storage diseases are given below: The classification of these diseases are based on the name of the patient first diagnosed of that disease. Type I. II. III. Name of disease Von Geirke’s disease Pompe’s disease Cori’s disease IV. Andersen’s disease V. VI. McArdle’s disease Her’s disease Enzyme deficient Glucose-6-phosphatase α-(1,4) glucosidase Amylo-1, 6-glucosidase, i.e. debranching enzyme. 1,4 → 1,6 transglucosylase, i.e. branching enzyme Muscle glycogen phosphorylase Liver phosphorylase Glycogenolysis Breakdown of glycogen to glucose is called glycogenolysis. The breakdown of glycogen takes place in liver and muscle. In liver, the end product of glycogen breakdown is glucose whereas in muscle the end product is lactic acid.
    • 166 BIOCHEMISTRY FOR STUDENTS Under the joint action of phosphorylase [breaks only α-(1,4) linkages] and debranching enzymes [breaks only α-(1,6) linkages] glycogen is broken down to glucose. The breakdown of glycogen is initiated by the enzyme Phosphorylase, which cleaves α-(1,4) glycosidic linkages starting from non-reducing end of the glycogen molecule to give glucose 1-PO4 and this process continues until four glucose residues remain on either side of the α-(1,6) branched point. Now another enzyme Glucan transferase, transfer three glucose units from one side to another, leaving a single glucose residue at the branched point followed by debranching enzyme to break α-(1,6)-linkage. The breakdown of glycogen takes place in liver and muscle. The action of liver phosphorylase and muscle phosphorylase are explained as below. Liver Phosphorylase It exists in two forms: a. Phosphorylase: It is the active form of phosphorylase. b. Dephosphophosphorylase: It is the inactive form of phosphorylase. Activation of the inactive form involves phosphorylation of the hydroxyl group of a serine residue by a specific kinase in the presence of ATP. Inactivation of the active form is catalyzed by a specific phosphatase. The action of kinase is stimulated by c-AMP which itself is formed from ATP in the presence of adenyl cyclase. Glucagon and adrenaline stimulate glycogenolysis by increasing the activity of adenyl cyclase. Muscle Phosphorylase It exists in the following forms: a. Phosphorylase a. It is the active form of phosphorylase. It is active only in the absence of 5-AMP. It is a tetramer containing 4 molecules of pyridoxal phosphate.
    • METABOLISM OF CARBOHYDRATES 167 b. Phosphorylase b. It is the inactive form of phosphorylase. It is active only in the presence of 5-AMP. It is a dimer containing only 2 molecules of pyridoxal phosphate. Phosphorylase a contains four molecules of pyridoxal phosphate. Whereas phosphorylase b contains 2 molecules of pyridoxal phosphate. Phosphorylase in muscle is activated by epinephrine, which activates adenyl cyclase to form c-AMP, which stimulate phosphorylase kinase, key enzymes of glycolysis and gluconeogenesis in liver.
    • 168 BIOCHEMISTRY FOR STUDENTS Cori Cycle The cyclic process by which lactic acid is converted to glucose in liver and eventually reappears as muscle glycogen is known as Cori cycle. The Cori cycle is the body’s way of recycling lactic acid Liver Muscle During vigorous muscle activity, muscle glycogen is converted to lactic acid. The lactic acid diffuses from the muscle into the blood stream and transferred to the liver. In liver, lactic acid is converted to glucose by gluconeogenesis. Glucose formed in this way returns to the muscle via circulation. This cycle continues and is called Cori cycle. GLUCONEOGENESIS Gluconeogenesis is the process by which glucose or glycogen is formed from noncarbohydrate substances. The noncarbohydrate substances include glycogenic amino acids, intermediates of TCA cycle, glycerol, pyruvate, lactate, etc. gluconeogenesis is an important source for supplying glucose to various tissues when glucose is otherwise not available. Especially during
    • METABOLISM OF CARBOHYDRATES 169 fasting/starvation. Continuous supply of glucose is required for the functioning of brain, RBC, etc. even when food is not taken. The conversion of amino acids, lactate and glycerol into glucose takes place mainly in liver and kidney. Thus, liver and kidney are the major site of gluconeogenesis. Glucose-6-phosphatase does not exist in brain, adipose tissues or muscle. Therefore, these tissues are not gluconeogenic. Gluconeogenesis takes place when the energy requirements of the cell are at a minimal level and an energy source ATP is available. The production of glucose from noncarbohydrate precursors occurs by following pathways. Gluconeogenesis is regulated by four key enzymes: 1. Pyruvate carboxylase. This enzymes is stimulated by acetyl CoA and inhibited by ADP. 2. Phosphoenol pyruvate carboxy kinase. 3. Fructose-1,6-diphosphate-1 phosphatase (FDPase). This enzyme is inhibited by AMP and ADP. 4. Glucose-6-phosphatase (G6 Pase). This enzyme is stimulated by inorganic phosphate (Pi) and glucose. The conversion of pyruvate to glucose is shown on the next page 171. Insulin represses the synthesis of these four enzymes whereas glucocorticoid hormones induces their de novo synthesis. GALACTOSE METABOLISM Galactose is derived mainly from lactose of the diet. Galactose is important for the formation of glycolipids and glycoprotein and for the formation of lactose during lactation.
    • 170 BIOCHEMISTRY FOR STUDENTS Important points of galactose metabolism: 1. Conversion of galactose into glucose is the main pathway. The galactose derived from the milk sugar is readily converted into glucose in the liver. 2. Conversion of UDP-galactose into UDP-glucose is a freely reversible reaction catalyzed by UDP galactose-4-epimerase. Hence, the glucose can be readily converted into galactose in the states of galactose lack, which is the way of treating alatosemia. This does not interfere with the growth. So it is not essential in the diet. 3. Galactosemia results from the deficiency of galactoseI- phosphate-uridyl transferase deficiency than galactokinase which is normal in the RBC of galactosemic patients. 4. Galactose is needed as it is a constituent of glycolipids (cerebrosides), chondromucoids and mucoprotein. 5. Galactose is also required for the lactose synthesis in the mammary gland by the enzyme lactose synthetase. Galactosemia Inability to metabolize galactose is called galactosemia. Galactosemia is an inherited disease, generally encountered in infants, characterized by inability to metabolize galactose or lactose. This results in the accumulation of galactose in the blood and ultimately excreted in the urine. Galactosemia is due to the deficiency of the enzyme galactose-1-phosphate uridyl transferase. Galactosemia gives rise to loss in weight, mental retardation and development of cataract due to the deposition of galactitol, a reduced product of galactose, in the lens. A galactose free diet avoids these difficulties and galactose that is necessary for the synthesis of cell membranes, cerebrosides, glycolipids
    • METABOLISM OF CARBOHYDRATES 171
    • 172 BIOCHEMISTRY FOR STUDENTS and mucoproteins can be formed from glucose-1 phosphate. FRUCTOSE METABOLISM Fructose is an important source of dietary carbohydrate, accounting for approximately 20% of the total carbo-hydrate intake. Fructose is present in significant amounts in seminal fluid. It is synthesized in the prostate gland by the following
    • METABOLISM OF CARBOHYDRATES 173 reaction. In fructosuria, fructose is found in the urine, due to lack of enzyme fructokinase. LACTOSE SYNTHESIS Blood galactose is readily converted into glucose in liver. Here glucose is first converted into galactose by the pathway as above and then glucose and galactose combine to form lactose by the enzyme lactose synthetase.
    • 174 BIOCHEMISTRY FOR STUDENTS Failure to absorb dietary lactose is common in adults and is due to lactose deficiency and the irriability to hydrolyze lactose. Individuals with lactose deficiency can generally tolerate yoghurt (curd), a milk product. Yoghurt contains lactase that catalyzes the degration of lactose to glucose and galactose. URONIC ACID PATHWAY Biosynthesis of D-glucuronic acid takes place from glucose1-phosphate. UDP-glucuronic acid is required in detoxification reactions forming glucuronides (e.g. bilirubin diglucuronide) and in the synthesis of proteoglycans. Also this pathway through L-uronic acid gives rise to the synthesis of L-Ascorbic acid (Vitamin C) in the animals and other plants. These reactions occur in animals and higher plants. In man, guinea pigs and other primates, the enzyme which converts L- glunolactone to 2-keto-L-gluconate is absent, thus making ascorbic acid a vitamin for them. UDP-glucuronic acid is the active glucuronic acid. It participates into the incorporation of glucuronic acid into chondroitin sulphate and other polysaccharides. Glucuronic acid conjugates with bilirubin, steroids and certain drugs for detoxification. The compound glucose 6-PO4 is at a pivotal junction to undergo various metabolic fats such as pyruvate/lactate (glycolysis), in HMP shuntpathway, in glycogen synthesis in liver and muscle, give rise to glucuronate, ascorbic acid (uronic acid pathway).
    • METABOLISM OF CARBOHYDRATES 175 Glucose is converted to glucose-6-PO4 by two possible enzymes depending upon the tissue. One is glucokinase (found in liver) which is highly specific for the glucose and other is hexokinase (muscle and fat cells), which catalysis the phosphorylation of most hexoses, including glucose. Pentosuria Pentosuria is characterized by the increased excretion of one or more pentoses. The pentoses normally present in urine are L-xylulose, D-ribose and D-ribulose. Essential pentosuria: It is characterized by increased excretion of L-xylulose. This is due to the deficiency of enzyme L-xylulose dehydrogenase. Other types of pentosuria include alimentary pentosuria resulting in excretion of L-arabinose and xylose due to intake of large quantities of fruits and ribosuria (due to increase in excretion of D-ribose). The patients with deficiency of glucose-6-phosphate dehydrogenase when given antimalarials like primaquine that precipitates hemolysis because G-6-PD is responsbile for the maintenance of reduced glutathione level and antimalarials produces excess of free radicals as free radicals damages the RBC’s cell membrane by oxidative stress mechanism. REGULATION OF BLOOD GLUCOSE The concentration of glucose in the blood is the net resultant of two processes. 1. Rate of glucose entrance into the bloodstream 2. Rate of glucose removal from the bloodstream. Ways by which sugar is added to the blood 1. By absorption from the intestine 2. Breakdown of liver glycogen 3. By gluconeogenesis. The sources of gluconeogenesis are: amino acids, propionate, lactate, glycerol, etc. Ways by which sugar is removed from the blood 1. Conversion to liver glycogen 2. Conversion to muscle glycogen 3. In the synthesis of fats (i.e. triglycerides)
    • 176 BIOCHEMISTRY FOR STUDENTS 4. In the synthesis of glycoproteins such as nucleic acids (nucleoproteins), lactose, etc. 5. Loss in the urine. A balance of the above two processes will keep the blood sugar level within normal limits. These two processes are influenced by a number of factors under physiological conditions. The blood glucose level is most efficiently regulated by a mechanism in which liver, extrahepatic tissues and several hor-mones play an important part. Role of Liver Liver, being the centre of all metabolic activities is mainly responsible for the regulation of blood glucose level. In liver, exists the developed mechanism for uptake of glucose from the blood, conversion of glucose to glycogen for storage (glycogenesis), release of glucose from glycogen (glycogenolysis) and de novo synthesis of glucose from non-carbohydrate precursors (gluconeogenesis). Glycogenesis in liver can occur from blood glucose or any substance capable of giving rise to pyruvate. Due to the presence of glucose-6-phosphatase, liver glycogen can contribute directly to blood sugar (gluconeogenesis).
    • METABOLISM OF CARBOHYDRATES 177 Role of Extra-hepatic Tissues a. Role of muscle: Muscle glycogen does not contribute directly to the blood sugar due to the absence of the enzyme, glucose-6-phosphatase. Glycogenolysis in muscle provides glucose to blood only through the formation of lactic acid which by Cori cycle is converted to glucose in the liver. b. Role of kidney: Kidney also exerts a regulatory effect by reabsorbing glucose by the reabsorptive system of the renal tubules. When the blood glucose level rises above the renal threshold, the excess glucose appears in the urine. Role of Hormones Several hormones play an important role in the homeostatic mechanism of blood glucose level. Out of these insulin is the only hypoglycemic hormone whereas others are hyperglycemic hormones. 1. Insulin: Insulin plays an important role in the regulation of blood glucose concentration. It is secreted into the blood in response to hyperglycemia. Insulin increases the transport of glucose across the cell membranes. Insulin reduces the blood sugar level by increasing the glucose utilization by glycolysis, decreases hepatic glycogenolysis and increases glycogenesis. Hormones which keep the blood sugar level high are: 1. Epinephrine 2. Glucagon 3. Glucocorticoids 4. Thyroxine 5. Growth hormones. Mechanism by which these hormones increase the blood sugar level are: 1. By increasing the absorption of glucose from the intestines 2. By decreasing the oxidation of glucose at the tissue level 3. By preventing the synthesis of glycogen 4. By stimulating glycogenolysis 5. By potentiating gluconeogenesis. Blood sugar level is kept normal by insulin, by opposing the action of these hormones.
    • 178 BIOCHEMISTRY FOR STUDENTS 2. Glucagon: Glucagon is also called Hyperglycemicglycogenolytic hormone. Glucagon is secreted by the α-cells of the islets of Langerhans. Glucagon secretion is stimulated by hypoglycemia. It causes glycogenolysis by activating liver phosphorylase. Glucagon thus counter balances the action of insulin which is secreted into the blood when the blood glucose level is high. Glucagon acts primarily on liver and does not affect glycogen breakdown in muscles. Glucagon enhances gluconeogenesis from amino acids and lactate. 3. Epinephrine: Epinephrine stimulates glycogen breakdown in liver and muscle. The stimulation of glycogenolysis is due to its ability to activate phosphorylase. Epinephrine also inhibits muscle glycogen synthesis in liver and thus directs the production of increased blood glucose. 4. Adrenal cortex hormones: Adrenal cortex secretes glucocorticoids, which lead to gluconeogenesis which is the result of increased protein breakdown and stimulation of transaminase. It also inhibits glucose utilization in extra-hepatic tissues. 5. Anterior pituitary hormones: Growth hormones and ACTH elevate the blood glucose level. Growth hormones decrease glucose uptake by the tissues, whereas ACTH stimulates the secretion of hormones of the adrenal cortex. 6. Thyroid hormone: Thyroxine has a diabetogenic action. It increases blood glucose concentration by increased absorption of glucose from the intestines. Glycosuria The excretion of detectable amounts of reducing sugar in urine is called Glycosuria. If glucose is excreted, then the condition is called glucosuria. Glucose is filtered by the glomeruli but is completely reabsorbed by the renal tubules. The reabsorption is effected by phosphorylation in the tubular cells. The maximum rate of reabsorption of glucose by the tubules (TmG—Tubular maximum of glucose) is 350 mg/minute. When
    • METABOLISM OF CARBOHYDRATES 179 the blood levels of glucose are elevated, the filtrate contain more glucose that can be reabsorbed, the excess passes into the urine and gives rise to glucosuria. Renal Glucosuria The blood glucose level is normal, but as a defect in the reabsorption system in the tubules, kidney threshold is lowered and glucose appears in the urine. Renal glucosuria is an example of benign glucosuria. Diabetes Mellitus Diabetes mellitus is a metabolic disorder due to the deficiency of insulin, resulting in high blood glucose level and the excretion of glucose in the urine. The most important features of diabetes mellitus are: 1. The hyperglycemia and glucosuria persist during fasting. 2. Liver glycogen falls to a low level. 3. Excretion of large quantities of ketone bodies due to increased fatty acid metabolism giving rise to diabetic coma. Diabetes or diabetes mellitus is a condition where in the body does not produce enough insulin or does not properly respond to the insulin that is produced, there by keeping glucose level in the blood high. Diabetes affects nervous digestive circulatory, endocrine, urinary system but all body system are in some way affected. There is no diabetic sure but it can only be cautted. Classification 1. Type 1 diabetes: Also called childhood onset diabetes, juvenile diabetes and insulin dependent diabetes mellitus (IDDM). Type 1 diabetes is a chronic (life long) disease that occurs when the pancreas does not produce enough insulin to properly control blood glucose levels. Type 1 diabetes can occur at any age but it is most after diagnosed in children, adolescents or young adults, In this form of diabetes, the body cannot make insulin the immune system mistakenly
    • 180 BIOCHEMISTRY FOR STUDENTS attacks the cells in the pancreas that make and release insulin. As these cells die, blood glucose levels rise. People with type 1 diabetes need insulin shots. 2. Type 2 diabetes: In characterized by the inability of the body to make insulin or properly use insulin as a result, cells can not take up or utilize glucose resulting in high blood glucose level. It is a slow onset process and person having diabetes for years without knowing. Typically with type 2 diabetes, the body still make insulin, but the cells cannot use it. This is called insulin resistance. 3. Gestational diabetes: It occurs during pregnancy, labor and delivery, women who got gestational diabetes are more likely to develop type 2 diabetes. Prediabetes That condition when a person have impaired glucose tolerance where blood glucose levels are higher than normal but not high enough to be classified as diabetes. Latest autoimmune diabetes of adults (LADA) is a condition in which Type 1 diabetes develops in adults. Adults with LADA are frequently initially misdiagnosed as having type 2 diabetes based on age rather than etiology. Maturity onset diabetes of young (MODY): Condition because of defects in β cell function. According to the latest WHO guidelines two fasting glucose blood measurement about 126 mg/dl is considered diagnostic for diabetes mellitus. People with testing blood glucose level from 100-125 mg/dl is considered to have impaired fasting glucose. HbA1c given an idea about average blood glucose control over the last 120 days. Glycosylated hemoglobin (HbA1c) test is recommended for: a. checking blood glucose control in pre-diabetes. b. Monitoring blood glucose control in diabetes mellitus. The normal value of HbA1c is 4-6% correlation between HbA1c blood glucose level.
    • METABOLISM OF CARBOHYDRATES 181 Correlation between HbA1c blood glucose level HbA1c Average blood glucose level over past three months 6% 7% 8% 10% 120 150 180 240 mg% mg% mg% mg% Higher the value of HbA1c poorer the blood glucose control >6.5 = Diabetes HbA 1c <6.0 = Not diabetes in between 6-6.5 = May be pre-diabetes or risk of diabetes The extent of glycosylation of hemoglobin can be conviently monitored and used to assess the control of hyperglycemia. Analysis of hemoglobin A (the adult form of hemoglobin) reveals the extreme of minor components called hemoglobin A1. Hemoglobin A1 forms by nonenzymatic modification of hemoglobin A by glucose. Glycosylation has only minor effect on normal hemoglobin function. Glycosylation occurs continuously within the red cell, and the extent of glycosylation reflects the average glucose concentration to which the cell is exposed during its 120 days life-span. Measurement of glycosylated hemoglobin content provides a clinically useful means to assess the degree of hyperglycemia that existed over the previous several weeks. HbA1c in normal individuals (without diabetes mellitus) makes up about 6% of total hemoglobin. Individuals with controlled (blood glucose levels <10 mM, or 180 mg/dl) have levels of hemoglobin A1 of about 9% of total hemoglobin. These with less well controlled diabetes have greater than 9%, hemoglobin A1. Glycosylated hemoglobin is formed in erythrocytes by the reaction of glucose with hemoglobin. The glycosylation of hemoglobin is nonenzymatic reactions. The reaction is virtually irreversible. Its removal from the blood proceeds very slowly. The prime importance of the glycosylated hemoglobin estimation in diabetes diagnosis lies in the fact that HbA1c
    • 182 BIOCHEMISTRY FOR STUDENTS and blood glucose values of diabetics often present a different picture. Glycosylated hemoglobin fraction is not affected by the metabolic state at a given moment whereas the blood glucose level can change very rapidly. HbA1c thus makes it possible to identify hyperglycemic states which would otherwise go unrecognized. The main characteristic of the glycosylated hemoglobin fraction HBA1 is that it constitutes a kind of long-term retrospective indicator of blood glucose concentrations. This is due to the fact that the stable HbA1 is not catabolized throughout the erythrocyte lifespan (100-200 days). HbA1C gives an indication of the averaged blood glucose levels during the proceeding weeks rather than the metabolic state of the patient at the time of testing. HbA1C test estimates the portion of hemoglobin which is glycated. Increased blood glucose levels increase the glycation of hemoglobin. Glycation of hemoglobin is nonenzymatic, irreversible addition of glucose to hemoglobin. In normal individual 5-6% of the hemoglobin molecule is glycated. However, in uncontrolled diabetic patient the level can go up to more than 20% which indicates that the patient has uncontrolled hyperglycemia. Since, the formation of glycated Hb is essentially reversible and the blood level of HbA1c depend on both the lifespan of red blood cell (average 120 days) and the blood glucose concentration. Since the rate of formation of HbA1c is directly proportional to the concentration of glucose in the blood, the HbA1c concentration represents the integrated values for glucose for the proceeding 6-8 weeks. HbA1c% <6 6-7 <7 7-8 >8 Degree of glucose control Nondiabetic level Near normal glycemia Good Good control Action suggested
    • METABOLISM OF CARBOHYDRATES 183 Glycosylated hemoglobin (HbA1c) test gives an overview of diabetic control over proceeding 2-4 months. HbA1C circulates in the blood for 2-4 months before being naturally broken down by the body so the level of HbA1c at any stage can show how high the blood glucose has been over the proceedings few months. HbA1c test shows the average amount of glucose in the blood over the last three months.
    • 184 BIOCHEMISTRY FOR STUDENTS CHAPTER 9 Metabolism of Lipids PLASMA LIPOPROTEINS Lipids are water insoluble and are transported in the body in an aqueous medium in combination with various specific proteins. This results in lipid: protein complex called lipoproteins. These lipoproteins consists of triglycerides or cholesterol esters and the central core surrounded by a coat of unesterified cholesterol, phospholipid and protein. Plasma lipoproteins occur in four major forms which are: 1. High or heavy density lipoproteins (HDL) 2. Low density lipoproteins (LDL) 3. Very low density lipoproteins (VLDL) 4. Chylomicrons. Plasma lipoproteins are in dynamic state. They are continuously being synthesized and degraded with rapid exchange of both lipid and protein among themselves. Two enzymes Lecithin: cholesterol acyl transferase (LCAT) and lipoprotein lipase (also called triglyceride lipase) play a significant role in the catabolism of lipid fraction of lipoproteins. The components that are necessary for the synthesis of lipoproteins are triglycerides, cholesterol, cholesterol esters, phospholipids and apoproteins. 1. High density lipoproteins: Also called α-lipoproteins. This fraction is rich in phospholipids. 2. Low density lipoproteins: Also called β-lipoproteins. This fraction is rich in cholesterol. 3. Very low density lipoproteins: Also called α2 or pre-β-lipoproteins. This fraction is rich in triglycerides. 4. Chylomicrons: It consists of central core of triglycerides, phospholipids, and cholesterol combined with small amount of proteins.
    • METABOLISM OF LIPIDS 185 The density of lipoprotein increases with the protein content. The protein parts of lipoproteins are called apolipoproteins. Each lipoprotein differ in terms of size, density, the relative proportions of triglycerides and cholesterol esters in the core and in the nature of apoproteins on the surface. Chylomicrons Density <0.95 Protein % 1–2 TG (%) 80–95 PL (%) 3–6 Cholesterol (%) 1–3 Cholesteryl ester (%) 2–4 VLDL LDL HDL 0.95–1.006 1.091–1.063 1.063–1.21 10 25 45–55 55–65 10 3 15–20 22 30 10 8 33 5 37 15 Absorption of Fats Dietary fat is digested by the action of pancreatic lipase, present in the intestines. The lipase hydrolyses the triglycerides to 40 percent free fatty acids and glycerol, 50-57% mono-and diglycerides, 3-10% is absorbed unchanged as triglycerides. The 2-monoglycerides produced as intermediates are converted to 1-monoglycerides by an enzyme isomerase which is then digested by lipase to glycerol and free fatty acids. Of the four products of triglycerides hydrolysis, i.e. free fatty acids, glycerol, monoglycerides and diglyceride, free fatty acids and glycerol are easily absorbed as they are water soluble and are then carried away by the blood. Higher fatty acids, mono and diglycerides are absorbed with the help of bile salts in the form of water soluble molecular aggregates called mixed micelles. Inside the intestinal epithelial cell, 1-monotriglycerides are hydrolyzed by intracellular lipases to give free fatty acids and glycerol, whereas 1-monoglycerides are used for triglyceride synthesis. Higher fatty acids are largely utilized for the triglycerides synthesis inside the intestinal epithelial cells and are carried as chylomicrons which are hydrophobic water soluble triglycerides
    • 186 BIOCHEMISTRY FOR STUDENTS
    • METABOLISM OF LIPIDS 187
    • 188 BIOCHEMISTRY FOR STUDENTS covered with a layer of hydrophilic phospholipids, free and esterified cholesterol and some proteins. Fatty acids with odd numbers of carbon atoms and branched-chain fatty acids make up only a small portion of our total fatty acid intake. A high fat, low carbohydrate diet results in metabolic acidosis, whereas high protein, low carbohydrate diet results in protein imbalance with a high urinary nitrogen output, increasing carbohydrate in the diet prevents this lose of nitrogen. Oxidation of Fatty Acids The action of hormonally controlled lipase results in the hydrolysis of neutral fats to glycerol and free fatty acids. Glycerol enters the glycolytic pathway, via formation of glycerol-3phosphate by the action of ATP and glycerokinase. The fatty acids tightly bound with albumin is carried via the blood to the various tissues for oxidation. Fatty acids oxidation takes place in mitochondria. β-oxidation Fatty acids are oxidised mainly by β-oxidation. In β-oxidation, the oxidation takes place at the β-carbon atom from the carboxyl end and the β-carbon atom is oxidized to carboxyl group resulting in the formation of acetyl CoA and a fatty acid shorter by two carbon atoms. The first step in β-oxidation pathway is the activation of fatty acid to form acyl CoA by combination with coenzyme A. The enzyme acyl CoA synthetase also known as thiokinase. Acyl CoA synthesis occurs in the outer mitochondrial membrane. The acyl CoA so formed cannot penetrate the inner mitochondrial membrane to the site of fatty acid β-oxidation enzyme system. In order to cross this barrier the acyl group
    • METABOLISM OF LIPIDS 189 is transferred to carnitine. The reaction is catalyzed by carnitine acyl transferase. Acyl carnitine crosses the inner mitochondrial membrane. Now another enzyme located on the inner surface of the inner mitochondrial membrane catalyzes the reverse reaction and acyl group is transferred to intramitochondrial CoASH (Coenzyme A). Outside Inside Acyl carnitine + CoASH Acyl CoA + carnitine Once the acyl CoA is inside the mitochondria, it is metabolized by the steps given below.
    • 190 BIOCHEMISTRY FOR STUDENTS Energy Yield of Palmitic Acid Metabolized Palmitic acid is C16 acid. Palmitic acid will undergo seven steps of β-oxidation to give 8 molecules of acetyl CoA. In each sequence of β-oxidation, 5 ATP molecules are generated, (2 from FADH2 and 3 from NADH + H+, entering the respiratory chain). Since 7 steps of β-oxidation takes place, a total of 7 × 5 = 35 ATPs will be formed. Each acetyl CoA molecule on complete oxidation by citric acid cycle will give rise to 12 ATPs. So a total of 12 × 8 = 96 ATPs will be formed by the complete oxidation of 8 molecules of acetyl CoA by citric acid cycle. In the activation step, i.e. formation of palmitate oxidation is (35 + 96 – 2) = 129. β-oxidation of odd chain fatty acids yield many molecules of acetyl CoA and a terminal three carbon residue as propionyl CoA. Propionyl CoA enter the TCA cycle by the following reactions: In the formation of palmityl CoA from palmitate, 2 molecules of ATP are consumed. The net ATP yield per molecule of palmitate oxidation is (35 + 96 – 2) = 129.
    • METABOLISM OF LIPIDS 191 In addition to β-oxidation, the other pathways for fatty acid oxidation are: α -oxidation α-oxidation is a relatively minor pathway for the production of energy. Dietary fatty acids which are methylated are metabolized by this pathway. α-oxidation is important in the catabolism of branched chain and odd chain fatty acids. Phytanic acid, a metabolic product of phytol (occurs as a constituent of chlorophyll) is metabolized by oxidation. Phytanic acid, a significant constituent of milk lipids and animal fat, is metabolized by initial α-hydroxylation followed by dehydrogenation, and decarboxylation. This pathway is operative in brain and plant tissues. Refsam’s Disease Due to the deficiency of enzyme phytate α-hydroxylase, phytanic acid is not metabolized and accumulate in blood and tissues giving rise to neurological problems. This is an inborn error of metabolism. (Omega)-oxidation ω( By this pathway, ω terminus carbon is oxidized to carboxyl group to form dicarboxylic acids. Further metabolism takes place by β-oxidation. Fatty Acid Synthesis Fatty acids are synthesized by three main processes. Extra-mitochondrial De Novo Fatty Acid Synthesis This is the de novo pathway for fatty acid synthesis starting from acetyl CoA. This pathway is active in liver, kidney, brain, mammary glands, adipose tissues, etc. The enzyme is fatty acid synthetase complex, which is a multienzyme complex system containing acyl carrier protein and six enzymes to which the growing fatty acid chain is attached during de novo synthesis. This multienzyme complex molecule is a dimer consisting of two identical subunits or
    • 192 BIOCHEMISTRY FOR STUDENTS monomer. The monomer is not active only the dimer is active. The enzyme complex contains two SH group, i.e. central and peripheral. Malonyl CoA is formed from acetyl CoA by carbon dioxide fixation reaction also called carboxylation reaction. The enzyme is acetyl CoA carboxylase and it requires biotin as cofactor.
    • METABOLISM OF LIPIDS 193
    • 194 BIOCHEMISTRY FOR STUDENTS The source of carbon atoms of fatty acid in acetyl CoA. Palmitic acid is the normal end product of fatty acid synthetase complex which requires one molecule of acetyl CoA and seven molecules of malonyl CoA. Regulation of De Novo Fatty Acid Synthesis The rate limiting step in fatty acid synthesis is the carboxylation of acetyl CoA to malonyl CoA and is catalyzed by the enzyme acetyl CoA carboxylase. The enzyme consists of two components. One contains two proteins, a biotin carboxyl carrier protein (BCCP) and biotin carboxylase. The second component is transcarboxylase that catalyses the transfer of carbon dioxide from BCCP to acetyl CoA. The enzyme acetyl CoA carboxylase is activated by conversion from a monomer to a polymer. Citrate activates enzyme by causing aggregation while long chain acyl CoA inactivates it by causing it to disaggregate. Mitochondrial Synthesis of Fatty Acids This is a chain elongation pathway where the addition of acetyl CoA to the existing long chain fatty acids takes place. Palmitic acid which is a normal end product of de novo synthesis is the precursor of long chain saturated and unsaturated fatty acids. Elongation of palmitic acid to stearic acid is more abundant.
    • METABOLISM OF LIPIDS 195 Microsomal Pathway of Fatty Acid Synthesis This pathway also provides a means of elongation of both saturated and unsaturated fatty acids utilizing malonyl CoA instead of acetyl CoA. Main difference between synthesis and degradation of fatty acids: Synthesis 1. 2. 3. 4. 5. Degradation Occurs in Cytosol Acylcarrier = Acylcarrier protein Cofactors NADP Synthesized in 2C units 2C units added to α-C Occurs in mitochondria Acylcarrier = CoA Cofactor NAD, FAD Degraded in 2C units 2C units taken from the α-C end Triglyceride Synthesis Biosynthesis of triglycerides takes place in liver, adipose tissue, lactating mammary gland, intestinal mucosa, muscles and kidney. The enzymes are present in endoplasmic reticulum. In the tissues liver, kidney, lactating mammary glands, glycerol is activated by glycerokinase whereas in adipose tissues and muscles glycerokinase is absent. The formation of α-glycerophosphate comes from dihydroxy acetone phosphate, an intermediate in glycolysis, by reduction catalyzed by α-glycerophosphate-dehydrogenase. Activation of Glycerol
    • 196 BIOCHEMISTRY FOR STUDENTS Activation of Fatty Acid
    • METABOLISM OF LIPIDS 197 Cholesterol Biosynthesis Normal adult synthesizes about 1 to 1.5 gm of cholesterol per day. Whereas diet provides only 0.3 gm of cholesterol per day. In normal human adults cholesterol is found in its largest amounts in the liver (about 0.3%), skin (0.3%), brain and nervous tissues (2%), intestines (0.2%) and certain endocrine glands, with the adrenal glands containing some 10%. As much as 50% of the myelin sheath there surrounds nerves its cholesterol. The relatively high content of cholesterol in skin may be related to vitamin D formation and that in the adrenals and certain other endocrine glands to steroid hormone biosynthesis. Liver is the main site of cholesterol biosynthesis but intestines (intestinal mucosa) is also an important site of synthesis in man. The tissues where cholesterol synthesis also takes place are skin, adrenal cortex, testes, aorta, etc. The cholesterol biosynthesis takes place in the extramitochondrial compartment of the cell. The source of all the carbon atoms in cholesterol is acetyl CoA. Acetyl CoA is the fundamental or building block unit of cholesterol biosynthesis. M is derived from methyl carbon of acetyl group. C is derived from the carbonyl carbon of acetyl group.
    • 198 BIOCHEMISTRY FOR STUDENTS Synthesis of Cholesterol Takes Place in Various Stages 1. Formation of mevalonate from acetyl CoA via HMG CoA. 2. Three successive phosphorylation followed by decarboxylation to give an isoprene unit which is mainly isopentenyl pyrophosphate. 3. Condensation of six isoprene units to give C30 terpene, i.e. Squalene. 4. Cyclization of squalene to lanosterol. 5. Conversion of lanosterol to cholesterol. Cholesterol is a product of animal origin. Cholesterol occurs in egg yolk (the richest source of cholesterol), meat, liver, brain, etc. The normal serum cholesterol level is 150-250 mg percent, about 2/3rd of this is present in the esterified form. The esterification of cholesterol takes place in liver. Cholesterol is transported in the blood as lipoproteins. The highest proportion of cholesterol is found in the low density lipoprotein fraction, i.e. β-lipoprotein fraction (LDL). Cholesterol level in the blood is increased in diabetes mellitus, nephrotic syndrome, obstructive jaundice, hypoparathyroidism, myxodema and xanthomatosis. Low levels of cholesterol are found in hyperthyroidism, pernicious anemia, hemolytic jaundice, malabsorption syndrome, severe wasting, in acute infections, etc. Cholesterol cannot be catabolized to straight chain molecule or to acetyl CoA. Therefore, cholesterol cannot be used as an energy source by the cells.
    • METABOLISM OF LIPIDS 199
    • 200 BIOCHEMISTRY FOR STUDENTS
    • METABOLISM OF LIPIDS 201
    • 202 BIOCHEMISTRY FOR STUDENTS The combined risk factor of coronary heart disease (CHD) can be determined following the estimations of serum cholesterol and HDL-cholesterol. The ratio of serum cholesterol to HDL-cholesterol has predictive value in determining the risk of CHD more accurately. For normal males the ratio of 5:1 and for normal females the ratio of 4.5:1 are considered as average risk. Lower ratios significantly reduce the risk, whereas ratios of 9.5:1 and 7:1 for males and females respectively, are believed to double the risk of CHD. An inverse relationship has been observed between the risk of CHD and the concentration of HDL-cholesterol in the test serum. HDL-cholesterol represents approximately 20-25% of the total cholesterol in serum. It is also called good cholesterol, friendly cholesterol and healthy cholesterol. HDL-cholesterol may work as a scavenger of cholesterol from the tissues ridding the body of excess cholesterol. HDLcholesterol takes cholesterol away from tissues (extrahepatic) back to liver for excretion. Low HDL-cholesterol may be predictive of coronary heart diseases risk whereas high HDLcholesterol confers protection. People with high HDL levels are resistant to the development of atherosclerosis. HDL removes cholesterol from peripheral tissues and the cardiovascular system. HDL takes cholesterol away from tissues (extrahepatic) back to liver for excretion. People with high LDL levels on the other hand are prone to development of atherosclerosis. LDL is sometimes called bad cholesterol (enemy cholesterol) because it may serve as a source for the cholesterol that accumulates in atherosclerotic plagues. The various factors that influence the turnover of body cholesterol can be illustrated as follows. Cholesterol serves as a precursor of several important classes of compounds such as bile acids (liver cells), vitamin
    • METABOLISM OF LIPIDS 203 D and neutral sterols (by enteric bacteria) (coprostanol and cholestanal) steroid hormones, etc. (Progesterone, glucocorticoids, mineralocorticoids, androgens, estrogens). Conversion of cholesterol to steroid hormones is essential for life. The main steroid hormones include cortisol (a glucocorticoid produced by adrenal cortex), aldosterone (a mineralocorticoid produced by adrenal cortex), estrogen (a sex hormone produced by the ovary), testosterone (a sex hormone produced by the testes) and progesterone (a progestational hormone produced by the ovary). All steroid hormones are synthesized from pregnenolone, a C21 compound. Regulation of Cholesterol Biosynthesis The amount of cholesterol synthesized is, in part, directly related to cholesterol content in the diet. If the diet contains large amounts of cholesterol, than the organism will synthesize little if, however, the diet contains only a small amount of cholesterol, the organism can produce considerable quantities. Cholesterol biosynthesis is regulated by negative feedback mechanism. The rate limiting enzyme in cholesterol biosynthesis is HMG CoA-reductase, which catalyze the conversion of HMG CoA to mevalonic acid. Dietary cholesterol inhibits the biosynthesis of cholesterol in liver by depressing the synthesis of HMG CoA reductase in liver. Fasting inhibits cholesterol biosynthesis by diverting HMG-CoA to ketone bodies formation whereas high fat diet accelerate the cholesterol production. Atherosclerosis High levels of cholesterol are associated with atherosclerosis which is characterized by the deposition of cholesterol ester and other lipids in connective tissues of arterial walls. Factors which play a leading part in atherosclerosis are high blood pressure, obesity, smoking, lack of exercise, etc. Diet rich in saturated fatty acid, increase the plasma cholesterol concentration. Replacement of saturated fat with a fat that is rich in polyunsaturated fatty acids such as linoleic acid, decreases the plasma cholesterol concentration.
    • 204 BIOCHEMISTRY FOR STUDENTS Cornoil, peanut oil and cotton seed oil lower blood cholesterol while butter fat and coconut oil raises it. Polyunsaturated fatty acid exerts their effects by a. Stimulating cholesterol excretion into the intestines b. Stimulating the oxidation of cholesterol to bile acids c. Increasing the metabolic rate of cholesterol esters. Bile Acids Bile acids are cholic, deoxycholic and lithocholic acids. They are present in the bile in conjugation with glycine and taurine as glycocholic and taurocholic acids. Bile acids are the derivatives of cholanic acid. Deoxycholanic acid is 3,12 dihydroxy cholanic acid. Lithocholanic acid is 3-hydroxy cholanic acid. Salts of bile acids lower the surface tension and are good emulsifying agents and hence play an important role in the absorption of fats from the intestine. Bile salts are polar derivatives of cholesterol and are detergents as they contain polar and nonpolar negious. they are synthesized in the liver and stored in the gallbladder. They solubilies dietary lipids so that they can be broken down and absorbed. Bile salts are divided into two classes: primary and secondary. Primary bile salts are synthesized by humans. Secondary bile salts results from the action of intestinal bacteria on the primary bile salts. The physical and physiological propertis of the bile salts are similar. Liver synthesizes 500 mg of bile salts daily. About 94% of the intestinal bile salts is reabsorbed and 6 percent is lost in feces.
    • METABOLISM OF LIPIDS 205 Ketone Bodies The ketone bodies are: 1. Acetoacetic acid 2. β-hydroxybutyric acid 3. Acetone. The principle ketone body is acetoacetic acid which gives rise to β-hydroxybutyric acid by reduction and acetone by decarboxylation. Ketone bodies are the intermediary breakdown products of fatty acid metabolism. Under normal conditions fatty acids are oxidized to carbon dioxide and these intermediary products do not appear to any great extent in blood or urine. Ketosis Significant accumulation of ketone bodies in the blood (ketonemia) and their excretion in urine (ketonuria) give rise to a condition known as ketosis. The total blood concentration of ketone bodies in normal well fed individuals with a daily urinary excretion of less than 1 mg. Higher than normal urinary or blood concentrations are called ketonuria and ketonemia respectively. The overall condition is called ketosis. Under certain metabolic conditions such as starvation, high fat diet, severe diabetes, more fat is metabolized for energy purposes giving rise to increased formation of ketone bodies.
    • 206 BIOCHEMISTRY FOR STUDENTS Liver is the net producer of ketone bodies. In liver, ketone bodies are formed by two ways. a. HMG-CoA pathway b. Acetoacetyl CoA pathway. HMG-CoA Pathway Condensation of acetoacetyl CoA with another molecule of acetyl CoA form β-hydoxy methyl glutaryl CoA (HMG-CoA). In the presence of enzyme HMG-CoA lyase. HMG-CoA is split to acetoacetic acid and acetyl CoA. Acetoacetyl CoA Pathway Condensation of two molecules of acetyl CoA give rise to acetoacetyl CoA. In the presence of enzyme acetoacetyl CoA deacylase, acetoacetic acid is formed. Though ketone bodies are synthesized in liver but cannot be utilized by liver because the enzyme required for the activation of ketone bodies is absent in the liver. Ketone bodies pass from the liver to the blood and are oxidized by peripheral tissues. Cardiac muscle, skeletal muscle and brain prefer ketone
    • METABOLISM OF LIPIDS 207 bodies for energy utilization. Before the ketone bodies are utilized by these tissues they must be activated. Acetoacetic acid is activated by two ways. a. Kinase activation b. Transferase reaction. Kinase Activation Transferase Reaction (See Below) Danger of Ketosis Acetoacetic acid and β-hydroxybutyric acid are fairly strong acids and are buffered when present in blood or tissues. Their excretion in the urine results in the loss of buffer cations. Since
    • 208 BIOCHEMISTRY FOR STUDENTS ketone bodies are negatively charged, their excretion from the body is accompanied by excretion of positively charged ions usually sodium as sodium salts, which depletes the alkali reserve of the body leading to fall in plasma bicarbonate level, giving rise to fall in pH. This leads to ketoacidosis, which may be cause of danger in uncontrolled diabetes mellitus. Fatty Livers Significant accumulation of triglycerides in the liver leads to a condition known as fatty liver. Normally, the liver contains 5% of the lipids. Normal liver is rich in glycogen and not in fat. But under physiological and pathological conditions the lipid content rises to 25–30%. The increased fat in liver may result from: a. Factors associated with increased free fatty acid (FFA) levels—Such as (i) Starvation, (ii) Diabetes mellitus (severe, uncontrolled), (iii) Ketosis, (iv) Pregnancy (toxicemia, etc.). Here the increased FFA mobilization leads to increased triglyceride synthesis and accumulation. b. Due to the deficiency of lipotropic factors such as choline, methionine, Vitamin E, essential fatty acids, vitamin B6 or pantothenic acid. c. Other intoxicating agents such as carbon tetrachloride, chloroform, phosphorus, arsenic, lead, alcohol. Fatty liver is associated with uncontrolled diabetics chronic alcohol intake obesity and protein malnutrition. Biochemical Basis of Fatty Liver Fatty liver falls into two groups. 1. In one group there is some primary factor causing an increase in free fatty acid either due to increased mobilization from adipose tissue or increased hydrolysis of lipoproteins or chylomicrons by lipoprotein lipase. This increased free fatty acid level leads to increased synthesis of triglycerides. The production of lipoproteins from triglycerides specifically the chylomicrons and VLDL, does not keep pace with the triglyceride synthesis allowing their accumulation to result in fatty liver. This is the mechanism
    • METABOLISM OF LIPIDS 209 in starvation, uncontrolled diabetes, ketosis and toxicemia of pregnancy. 2. In the other group the defect lies somewhere in the production of plasma lipoprotein; such a block could be at any one or more of the following sites: i. Apoprotein synthesis. ii. Lipoprotein synthesis from lipid and apoproteins. iii. Synthesis of lipids-specifically the phospholipids. iv. Secretory mechanism of the lipoproteins. Substances which prevent or relieve such abnormal accumulation of lipids in the liver are called lipotropic factors. They are choline, betaine, methionine, ethomolanine, inositol. Role of Liver in Lipid Metabolism Though liver is not the sole organ of lipid metabolism yet it has the complete enzyme systems to carry out the following major activities. 1. Synthesis and degradation of fatty acid (β-oxidation). 2. Synthesis of triglycerides. 3. Synthesis of cholesterol and its derivatives such as bile acid. 4. Phospholipid synthesis. 5. Synthesis of plasma lipoproteins such as VLDL and HDL. 6. Ketone bodies formation.
    • 210 BIOCHEMISTRY FOR STUDENTS CHAPTER 10 Metabolism of Proteins Twenty amino acids are present in dietary proteins. These amino acids are present in L-configuration. L-form of amino acid is the physiological active form of the amino acid. The transport of L-amino acids is energy dependent and require ATP, Na+, K+, Mn++ and vitamin B6. While D-form of amino acid is physiological inactive and is transported by diffusion. DIGESTION AND ABSORPTION Two important features of digestion are: 1. It breakdown, the nondiffusible bigger molecules into diffusible smaller molecules (amino acids). 2. During digestion, the biological specificity of a protein is destroyed, i.e. they are no longer antigenic, thus averting allergic reactions to food. Digestion of Protein by Various Enzymes Proteins are hydrolyzed to their constituent amino acids by the action of variety of enzymes. 1. Pepsin: It converts proteins to proteoses and peptones. 2. Trypsin: It cleaves peptide bonds involving carboxyl groups of arginine and lysine. 3. Chymotrypsin: It cleaves peptide bonds involving carboxyl groups of phenylalanine, tyrosine and tryptophan. 4. Carboxypeptidases: It cleaves proteins and peptides from the carboxyl end. 5. Aminopeptidases: It cleaves proteins and peptides from the amino end. 6. Dipeptidases: It cleaves dipeptides.
    • METABOLISM OF PROTEINS 211 Absorption L-form is absorbed at much faster rate than the D-form. All amino acids are absorbed by active process. Active process requires ATP, pyridoxal phosphate, Mn++, Na+ and K+. Sources of amino acids in the body pool are: 1. Dietary proteins. 2. Intracellular synthesis. 3. Tissue protein breakdown. Dietary proteins serve three broad functions: 1. Their consituent amino acids are used for the synthesis of the body proteins. 2. The carbon skeletons of the amino acids are oxidized to yield energy. 3. Their carbon and nitrogen atoms may be used to synthesize other nitrogen containing cellular constituents as well as many non-nitrogen containing metabolites.
    • 212 BIOCHEMISTRY FOR STUDENTS How the Amino Acids are Metabolized within the Cell? Metabolism Anabolic Phase It is a synthetic phase 1. Protein Biosynthesis Tissue proteins, blood proteins, enzymes, hormones. 2. Synthesis of nonprotein nitrogen substances takes place such as creatine, purines, pyrimidines, glutathione, choline, etc. Catabolic Phase It is a breakdown phase 1. Transamination. 2. Oxidative deamination. 3. Decarboxylation. 4. Utilization of nitrogen residue. i. Glutamine synthesis ii. Urea cycle. Transamination It involves the transfer of an amino group from an amino acid to the keto group of the keto acid forming a new amino acid and a keto acid. The reaction is reversible and is catalyzed by transaminase enzymes. These enzymes are also known as aminotransferases. The general reaction is represented as: The transaminases require pyridoxal phosphate as the coenzyme. The pyridoxal phosphate acts as a carrier of amino group from amino acid to keto acid. The reaction involves the formation of Schiff’s base. The mechanism is represented as:
    • METABOLISM OF PROTEINS 213 Transaminases are present in practically all the tissues. The most abundant of these are the glutamate-oxaloacetate transaminase (GOT) or aspartate transaminase (AST) and glutamate-pyruvate transaminase (GPT) or alanine transaminase (ALT). GPT is predominent in liver whereas, GOT is predominent in heart. Determination of the concentration of GOT and GPT in serum is used to assess the degree of cardiac and liver damages. Examples Alanine, aspartic acid and glutamic acid participate most in transamination. Lysine and threonine do not participate in transamination. Transamination, by converting amino acids to keto acids (which are prominent in TCA cycle), provide an important link between the protein and carbohydrate or fat metabolism. By this way amino acids are sources of energy to the body by keto acids which undergo complete oxidation. Also transamination reactions appear to play two major roles in amino acid metabolism. i. To serve as a means for the interconversion of number of amino acids to increase the amount of one that may be in short supply. ii. To channel the amino groups of amino acids, ultimately to glutamate and aspartate. Decarboxylation Amino acids are decarboxylated to give amines. The enzyme is decarboxylase. It requires pyridoxal phosphate as cofactor.
    • 214 BIOCHEMISTRY FOR STUDENTS Decarboxylation of amino acids give rise to some of the biologically active amines (biogenic amines such as histamine, serotonin and α-amino butyrate). Histidine Histamine Tyrosine Tyramine Glutamic acid γ-aminobutyric acid (GABA) Tryptophan Tryptamine 5-hydroxy tryptophan 5-hydroxy tryptamine (serotonin) Arginine Agotomin (in bacteria only). Oxidative Deamination Oxidative deamination involves the removal of α-amino group of amino acids to their corresponding keto acids. The enzyme is amino acid oxidase. In the amino acid oxidase reaction, the amino acid is first dehydrogenated by the flavoprotein of oxidase forming an amino acid. This spontaneously adds water to decompose the corresponding α-keto acid with the loss of α-amino nitrogen as ammonia. For L-form of amino acids, the enzyme is L-amino acid oxi- dase. It is FMN dependent. For D-form of amino acids, the enzyme is D-amino acid oxidase. It is FAD dependent. UREA CYCLE (KREBS-HENSELEIT CYCLE) The deamination of amino acids produces ammonia which is toxic. By Krebs cycle it is converted to urea, a nontoxic compound, which is transported via the blood to the kidneys and
    • METABOLISM OF PROTEINS 215 excreted in the urine. Urea formation takes place mainly in the liver. Two molecules of ammonia and one molecule of CO2 are converted to urea for each turn of the cycle. Urea is synthesized in the liver. One nitrogen of urea is derived from ammonium ion, and the second is derived from aspartate. The carbonyl group is derived from carbon dioxide (as bicarbonate). Overall Reaction: NH3 + CO2 + 3ATP + 3H2O → Urea + 2ADP + AMP + 2Pi + PPi Various stages of urea cycle are: 1. Synthesis of carbamoyl phosphate 2. Synthesis of citrulline 3. Synthesis of arginine This is divided into two parts: a. Synthesis of argininosuccinic acid b. Cleavage of argininosuccinic acid. 4. Synthesis of urea. Synthesis of Carbamoyl Phosphate The first step in urea synthesis, is the formation of carbamoyl phosphate. The enzyme is carbamoyl phosphate synthetase. The enzyme requires biotin and N-acetyl glutamic acid (AGA) for activity (i.e. cofactors). AGA is the modifier of the enzyme.
    • 216 BIOCHEMISTRY FOR STUDENTS It acts on the noncatalytic site and keeps the enzyme in active configuration.
    • METABOLISM OF PROTEINS 217 Synthesis of Citrulline Carbamoyl phosphate and ornithine combines to form citrulline. The reaction is catalyzed by ornithine transcarbamylase. Synthesis of Arginine a. Synthesis of argininosuccinic acid b. Cleavage of argininosuccinic acid. Argininosuccinic acid is formed from citrulline and aspartic acid. The reaction is catalyzed by enzyme argininosuccinate synthetase. Argininosuccinic acid so formed is cleavaged by enzyme argininosuccinase to arginine and fumaric acid.
    • 218 BIOCHEMISTRY FOR STUDENTS Synthesis of Urea Enzyme arginase splits arginine to urea and ornithine. Ornithine so formed again enters the cycle at the second step and hence, the cycle continues. In urea cycle, everytime a citrulline passes out of the mitochondria ornithine passes in. This is what happens and the protein in the mitochondrial membrane that allows this to happen is called an ornithine citrulline exchanger. Urea cycle is a route for biosynthesis of arginine, a semiessential amino acid. Link between urea cycle and TCA cycle is shown below: The urea cycle eliminates excess ammonia. Ammonia is derived mainly dietary amino acids that are not used promptly for protein synthesis. A human consuming 100 g of protein daily excretes about 16.5 g of nitrogen per day. Urea is the chief nitrogen metabolite of humans, accounts for 80-90%, of excreted nitrogen. Urate and ammonium ions are other end products.
    • METABOLISM OF PROTEINS 219 Hyperammonia Hyperammonia or hyperammonemic syndrome is because of increased level of ammonia in the blood. The urea is formed from ammonia by urea cycle. So any deficiency or defect of urea cycle enzymes give elevated levels of ammonia. Hyperammonemia gives rise to mental retardation. Metabolism of Individual Amino Acids According to metabolic fates of their skeletons amino acids are classified in following way: Glucogenic Amino Acids Those amino acids which give rise to the intermediates of carbohydrate metabolism. The product may be pyruvic acid, fumaric acid, oxaloacetic acid, α-ketoglutaric acid, etc. The glucogenic amino acids are glycine, alanine, asparatic acid, glutamic acid, arginine, cysteine, histidine, proline, serine, methionine, valine. All the nonessential amino acids are glucogenic in character. The amount of glucose derived from protein can be calculated by estimating nitrogen and glucose excreted in the urine. The glucose to nitrogen ratio in urine was found to be 3.65 in diabetic animals which were fed proteins only. Therefore the amount of glucose derived from 100 gm of protein is 3.65 × 16 = 58.4 gm. The factor 16 is because of the average nitrogen content of proteins is 16%. This suggests that 58% of protein is glycogenic. The glycogenic character of proteins is taken into account while calculating diet schedule of diabetic patients are made. Ketogenic Amino Acids Those amino acids which give rise to the intermediates of fat metabolism. The product may be acetyl CoA and acetoacetyl CoA, i.e. ketone bodies.
    • 220 BIOCHEMISTRY FOR STUDENTS
    • METABOLISM OF PROTEINS 221 The ketogenic amino acids are leucine, isoleucine, phenylalanine and tyrosine lysine. Phenylalanine and tyrosine tryptophase are both glucogenic and ketogenic amino acids. These amino acids yield acetoacetic acid and fumaric acid which can be converted to acetic acid and pyruvic acid respectively. GLYCINE It is a nonessential amino acid and is synthesized by the living cells. Glycine contains no asymmetric carbon atom and hence does not exist in D or L-form. Glycine is glycogenic amino acid. Metabolism 1. Glyoxalate pathway: It is the main pathway by which glycine is metabolized:
    • 222 BIOCHEMISTRY FOR STUDENTS Hyperoxaluria The metabolic defect in this disease is due to the disorder of glyoxalate metabolism where glyoxalic acid is not oxidized to formic acid, but is converted to oxalate, giving rise to increased excretion of oxalate in the urine. 2. Serine pathway: Glycine picks up one carbon moiety and is converted to serine. This signifies the glycogenic character of glycine. 3. Hemoglobin synthesis: Glycine participates in the synthesis of heme part of hemoglobin. Glycine + Succinyl CoA → α-amino-β-ketoadipic acid 4. Glycine-choline cycle: See page 223. 5. Purine synthesis: The entire molecule of glycine is incorporated in the synthesis of purines. The source of C-4, C-5 and N-7 in purine skeleton is glycine. 6. Synthesis of glutathione: Glutathione is tripeptide, i.e. it contains three amino acids which are glutamic acid, cysteine and glycine. γ-glutamic acid—Cysteine—Glycine
    • METABOLISM OF PROTEINS 223
    • 224 BIOCHEMISTRY FOR STUDENTS 7. Creatine synthesis: Glycine participate in the synthesis of creatine. The other two amino acids are arginine and methionine. 8. Conjugation reaction: For the formation of bile acids, glycine is important. Glycine + Cholic acid → Glycocholic acid The following diagram signifies that though glycine is nonessential but it is metabolically most active amino acid. In detoxification reactions of body. Glycine + Benzoic acid → Hippuric acid. Compounds which are toxic detoxified by these reactions.
    • METABOLISM OF PROTEINS 225
    • 226 BIOCHEMISTRY FOR STUDENTS Glycinuria This disease is due to the decreased reabsorption of glycine because of defect in renal tubular transport of glycine. Glycinuria is characterized by high excretion of glycine in the urine and tendency to form oxalate renal stones. Sulfur Containing Amino Acids The sulfur containing amino acids are methionine, cystine and cysteine. Methionine is an essential amino acid. The deficiency of it causes negative nitrogen balance and loss in weight in man, whereas, cystine and cysteine are nonessential or dispensible amino acids. Cysteine is synthesized from methionine and cysteine from cystine. The presence of cystine in the diet decreases methionine requirement in that it relieves the demand for the formation of cystine. The structure of methionine, cysteine and cystine are: METHIONINE Methionine is the principal methyl donor in the body. The transfer of methyl group of methionine to appropriate donors is called transmethylation.
    • METABOLISM OF PROTEINS 227 The active form of methionine that functions in methylation reaction is S-adenosyl methionine also called active methionine. It is a high energy compound. Methionine + ATP S-adenosyl methionine. The various transmethylation reactions carried by methionine are: a. Guanidoacetic acid to creatine b. Norepinephrine to epinephrine c. Dimethylethanolamine to choline d. Nicotinic acid to N-methylnicotinic acid e. Phosphatidyl ethanolamine to phosphatidyl choline. CYSTEINE AND CYSTINE Cysteine and cystine are related by oxidation-reduction reactions. Mercaptoethanolamine goes in the coenzyme A synthesis whereas cysteic acid on decarboxylation gives taurine, which conjugates with bile acids to produce taurocholic acids. Metabolic Role of Cysteine 1. It is a major constituent of the proteins of hair and hooves and the keratin of skin. 2. It forms a part of many proteins in which it takes part in the formation of disulphide bond such as in insulin. 3. It takes part in the synthesis of coenzyme A. 4. It is a part of glutathione. 5. It is a precursor of taurine that conjugates with cholic acid.
    • 228 BIOCHEMISTRY FOR STUDENTS Metabolism of Cystine and Cysteine Cystinuria Excessive excreting of cystine in urine takes place. Also there is defect in renal reabsorption mechanism for lysine, arginine and ornithine. This is an inherited disease. Cystinosis Deposition of cystine crystals in many tissues and organs takes place. Aminoaciduria is also present.
    • METABOLISM OF PROTEINS 229 Active Sulfate Coenzyme adenosine-3'-phosphate-5'-phosphosulfate or 3'phosphoadenosine-5-phosphosulfate (PAPS) is called active sulfate. The structure of active sulfate is given below. Active sulfate sulfating agent Active sulfate is involved in the sulphonation of phenols and of hexosamine derivatives as in the formation of chondroitin sulfate and in other sulfanaling transfer reactions, i.e. sulfolipids or sulfatides. They are formed from cerebrosides by reaction with active sulfates. Phenol, steroids also react with PAPS giving respective sulf ate derivatives which are eliminated in the urine. This detoxification mechanism takes place in liver. PHENYLALANINE AND TYROSINE Both phenylalanine and tyrosine are aromatic amino acids. Tyrosine is an hydroxylated phenylalanine. The structures of phenylalanine and tyrosine are:
    • 230 BIOCHEMISTRY FOR STUDENTS Phenylalanine is an essential amino acids whereas tyrosine is nonessential amino acid. Phenylalanine can be converted to tyrosine but tyrosine cannot give rise to phenylalanine. Hence, the requirement of tyrosine can be met by the adequate amount of phenylalanine in the diet. Phenylalanine and tyrosine are ketogenic amino acids. Metabolism of Phenylalanine and Tyrosine Phenylalanine and tyrosine are involved in the synthesis of a number of important compounds which include thyroxine, melanin, epinephrine and norepinephrine. Conversion of phenylalanine to tyrosine takes place in the liver. The enzyme is phenylalanine hydroxylase. The enzyme requires molecular oxygen. Fe++, NADPH and biopterin as cofactor. The reaction is explained below: Major pathway by which phenylalanine and tyrosine are metabolized are given as follows:
    • METABOLISM OF PROTEINS 231
    • 232 BIOCHEMISTRY FOR STUDENTS
    • METABOLISM OF PROTEINS 233
    • 234 BIOCHEMISTRY FOR STUDENTS Phenylalanine and tyrosine metabolism give rise to the synthesis of the important hormones, thyroxine and triiodotyrosine. Synthesis of melanine Formation of Thyroxine (T4) and Triiodotyrosine (T3) The complete metabolism of phenylalanine and tyrosine is summed as below: Inborn errors of metabolism associated with phenylalanine metabolism.
    • METABOLISM OF PROTEINS 235 Inborn Error of Metabolism There are a number of metabolic abnormalities which are congenital, present throughout life and hereditary. Such abnormalities are represented as in born error of metabolism. In some of these conditions failure of a metabolic step leads to the excretion of intermediate products which cannot be further metabolize along the metabolic path because of specific enzyme deficiency but which normally readily metabolizes. Inborn errors Phenylketonuria Tyrosinosis Enzyme deficit Phenylanine hydroxylase, p-hydroxy phenylpyruvatehydroxylase. Alkaptonuria Homogentisic acid oxidase Albinism Tyrosinase. Various blocks in the metabolism of phenylalanine giving rise to different inborn errors of metabolism are shown below: Phenylketonuria Phenylketonuria is an inborn error of metabolism associated with phenylalanine metabolism. The enzyme deficit is phenylalanine hydroxylase. This enzyme catalyze the conversion of
    • 236 BIOCHEMISTRY FOR STUDENTS phenylalanine to tyrosine. Due to the deficiency of the enzyme phenylalanine hydroxylase, the main pathway for the metabolism of phenylalanine via tyrosine is blocked and the minor alternate pathway takes place. The various metabolites that accumulate in the blood and excreted in the urine by the minor pathway are explained below. Phenylketonuria results in severe mental deficiency and the children suffering from this disease are mentally retarded because, the metabolites of phenylketonuria, i.e. phenylpyruvic acid, phenyllactic acid and phenylacetic acid, inhibit the formation of serotonin, a brain potent metabolite. Tyrosinosis Tyrosinosis is an inborn error of metabolism associated with phenylalanine metabolism. The enzyme deficit is p-hydroxy phenylpyruvic acid hydroxylase. Due to the deficiency of this enzyme, p-hydroxy phenylpyruvic acid is not converted to homogentisic acid, resulting in the accumulation of p-hydroxy phenylpyruvic acid in blood and the excretion of p-hydroxy phenylpyruvic acid and its reduction product, p-hydroxyphenyllactic acid in the urine.
    • METABOLISM OF PROTEINS 237 Alkaptonuria Alkaptonuria is an inborn error of metabolism associated with phenylalanine metabolism. The enzyme deficit is homogentisic acid oxidase. The deficiency of homogentisic acid oxidase causes a block in the metabolic pathway at homogentisic acid, resulting in its accumulation in the blood and excretion in the urine. Alkaptonuria is characterized by the excretion of urine which upon standing gradually becomes darker in color and finally turns black. Accumulation of homogentisic acid in body fluid and its deposition in cartilages and other connective tissues give rise to a condition called ochronosis. Albinism Albinism is an inborn error of metabolism associated with phenylalanine metabolism. The enzyme deficit is tyrosinase. Due to the deficiency of enzyme tyrosinase, the conversion of tyrosine to melanin formation is blocked. TRYPTOPHAN Tryptophan is an essential amino acid. It is the only amino acid containing indole ring. Tryptophan has the metabolic distinction of giving rise to nicotinic acid, a number of vitamin B-complex group, during its course of metabolism. It is ketogenic in nature. Metabolism of Tryptophan Tryptophan is metabolized by the following way. The metabolism takes place in liver. Synthesis of niacin The major pathway by which tryptophan is metabolized to niacin is given page 238. This pathway gives rise to the synthesis of niacin. 60 mg of tryptophan gives rise to 1 mg of nicotinic acid in the human body. In diet, tryptophan is not in sufficient amount to meet the requirement of this vitamin.
    • 238 BIOCHEMISTRY FOR STUDENTS Synthesis of serotonin This is another important pathway by which tryptophan is metabolized. Serotonin is also called 5-hydroxytryptamine (5HT), Enteramine or Thrombocytin.
    • METABOLISM OF PROTEINS 239 Carcinoid Syndrome: Increased excretion of 5-hydroxy indole acetic acid (5-HIAA) due to increased production of serotonin give rise to carcinoid syndrome. Normally one percent of tryptophan is metabolized by this pathway but in the carcinoid patients 60 percent of the tryptophan is metabolized by this pathway. Normal excretion of 5-HIAA is 2-10 mg per day but in carcinoid syndrome patients it may go as high as 501000 mg per day. The patient of these syndrome exhibit chronic diarrhea. Synthesis of Indole and skatole This is the minor pathway by which tryptophan is metabolized. The synthesis of indole and skatole takes place in the large intestines due to certain bacteria. The foul smell of the feces is due to these. Synthesis of melatonin Hartnup Disease This is an inborn error associated with tryptophan metabolism. The enzyme deficient is tryptophan pyrrolase. This disease is characterized by three symptoms: a. Pellagra like dermatitis b. Intermittent cerebellar ataxia c. Mental retardation. Urine of the patient contains large amount of tryptophan, indole acetic acid and its glutamine conjugate. Feces also contain large amounts of tryptophan. In Hartnup disease there is a deficiency of nicotinic acid because tryptophan is not available for the synthesis of nicotinic acid.
    • 240 BIOCHEMISTRY FOR STUDENTS LEUCINE, ISOLEUCINE AND VALINE These are the branched amino acids. Metabolism of Branched Chain Amino Acids Branched chain amino acids are metabolized as given below: Maple Syrup Urine Disease This is an inborn error of metabolism, in infants, resulting in the block in the metabolism of leucine, isoleucine and valine. Due to the deficiency of enzyme oxidative decarboxylase (i.e. step 2), oxidative decarboxylation of α-keto acids does not take place and hence branched chain keto acid derivatives of leucine, isoleucine and valine accumulate in blood and appear in urine. The odour of urine of such infants resembles that of maple syrup.
    • NUCLEIC ACID—CHEMISTRY AND METABOLISM 241 CHAPTER 11 Nucleic Acid—Chemistry and Metabolism Nucleoprotein belongs to the category of conjugated proteins, the nucleic acid part is the prosthetic group and the protein part consists of protamines and histones, which are basic in nature. The successive degradation of the nucleoproteins is shown below: Bases present in nucleic acids are purines and pyrimidines
    • 242 BIOCHEMISTRY FOR STUDENTS Pyrimidine Bases Three main pyrimidine bases are: 1. Uracil = It is 2,4-dioxy pyrimidine. 2. Thymine = It is 2,4-dioxy-5 methyl pyrimidine. 3. Cytosine = It is 2-oxy-4-aminopyrimidine. The oxypurines and oxyprimidines exist in enol and keto form. Enol form is called lactim form and keto form is called lactum form. Lactum form is predominent of the two. Purine Bases Two bases are: 1. Adenine = It is 6-amino purine 2. Guanine = It is 2-amino-6-oxypurine. Their structures are: Pentose Sugars The pentose sugars present are D-ribose and D-2-deoxyribose. Both sugars occur as furanose form in nucleotides.
    • NUCLEIC ACID—CHEMISTRY AND METABOLISM 243 Nucleoside Base-sugar unit is called nucleoside. Nucleoside = Base – Sugar Purine bases are attached at N-9 position to sugar moiety whereas pyrimidine bases are attached at N-1 position to sugar moiety. The nature of linkage is α-glycosidic linkage. Base Sugar Nucleoside Adenine Guanine Uracil Cytosine Thymine D-ribose D-ribose D-ribose D-ribose D-deoxyribose Adenosine Guanosine Uridine Cytidine Thymidine Nucleotides Nucleotides are phosphorylated nucleosides. They are represented by base-sugar-phosphate unit. Nucleotides = Base-sugar-phosphoric acid. Base Sugar Phosphate Nucleotide Adenine D-ribose Phosphoric acid Guanine D-ribose Phosphoric acid Adenylic acid. Adenosine monophosphate (AMP) Guanylic acid. Guanosine monophosphate (GMP) Contd...
    • 244 BIOCHEMISTRY FOR STUDENTS Contd... Base Sugar Phosphate Nucleotide Hypoxanthine D-ribose Phosphoric acid Uracil D-ribose Phosphoric acid Cytosine D-ribose Phosphoric acid Thymine 2-deoxyD-ribose Phosphoric acid Inosinic acid. Inosine monophosphate (IMP) Uridylic acid. Uridine monophosphate (UMP) Cytidylic acid. Cytidine monoposhphate (CMP) Thymidylic acid. Thymidine monophosphate (TMP) NUCLEIC ACIDS Nucleic acid are polynucleotide and the nucleotides are linked by means of 3',5'-phosphodiester bonds. Nucleic acids are of two types: 1. Deoxyribose nucleic acid (DNA) 2. Ribose nucleic acid (RNA). Hydrolytic products of DNA and RNA Deoxyribose Nucleic Acid (DNA) DNA is a polymer of 2-deoxyadenylic acid, 2-deoxyguanylic acid, 2-deoxycytidylic acid and thymidylic acid. It is represented as Base-deoxyribose-phosphate.
    • NUCLEIC ACID—CHEMISTRY AND METABOLISM 245
    • 246 BIOCHEMISTRY FOR STUDENTS Structure of DNA Watson and Crick proposed the helix structure of DNA, in which the two polynucleotide chains are wound about a common axis in the form of a double helix. These two polynucleotide chains are antiparallel, i.e. they run in opposite directions. The backbone of DNA, which is a constant throughout the molecule, consists of deoxyriboses linked by 3', 5' phosphodiester bridges. Polynucleotide chains are so oriented that an adenine is always located in the space opposite a thymine and a guanine is opposite a cytosine. This positioning of base is
    • NUCLEIC ACID—CHEMISTRY AND METABOLISM 247 called base pairing or base complementarity. There exists three hydrogen bonds between guanine and cytosine. The hydrogen bonding involves the keto and amino groups of purine and pyrimidine bases. DNA double helix is stabilized by hydrogen bonding. In DNA, purine and pyrimidine bases carry genetic information whereas sugar and phosphate groups perform a structural role. Ribose Nucleic Acids (RNA) RNA is a polymer of ribonucleotides. RNA is made up of a ribosephosphate backbone to which the various bases are attached. RNA, like DNA, exhibits polarity. The 5’ hydroxyl group points towards the 5’ end and 3’-hydroxyl group points to the 3’ end of the molecule. The sequence of bases along the sugar-phosphate backbone determines the primary structure (information content) of RNA, and this is the factor that distinguish one RNA from another. RNA is present in three forms. All the three forms are present as single strand, each has characteristic molecular weight and sedimentation coefficient. 1. Transfer or soluble RNA 2. Messenger RNA 3. Ribosomal RNA. Transfer or Soluble RNA It comprises 10-20% of the total RNA of the cell. 1. It is present in the soluble fraction of the cell. 2. It is involved in the transfer of amino acids. Each amino acid has a specific t-RNA. 3. It is a small molecule containing 75 to 90 nucleotides. 4. It has a heterogenous base composition. 5. It has a clover leaf structure and possess a specific triplet nucleotide known as anticodon, which is complimentary to the 3 bases on m-RNA called codon.
    • 248 BIOCHEMISTRY FOR STUDENTS The structure of transfer RNA is Messenger RNA Messenger RNA is synthesized in the nucleus during transcription in which sequence of bases in one strand of DNA is transcribed in the form of a single strand of m-RNA. The sequence
    • NUCLEIC ACID—CHEMISTRY AND METABOLISM 249 of bases in, m-RNA strand is complimentary to that of DNA. After transcription, m-RNA passes into cytoplasm and then on to ribosomes where it serves as a template for the sequence of amino acids during biosynthesis of proteins. Ribosomal RNA It comprises 50-80% of the total cellular RNA. Ribosomes carry ribosomal RNA. It has a homogenous base composition. Ribosomal RNA is required to bind m-RNA and specific enzymes utilized for peptide bond synthesis. Various fractions of ribosomes are 30s and 50s. Some Biologically Important Nucleotides 1. Adenosine diphosphate (ADP), adenosine triphosphate (ATP). They are sources of high energy phosphate bonds and take part in oxidative phosphorylation. 2. Inosine diphosphate (IDP) and inosine triphosphate (ITP) participates in phosphorylation reactions. 3. Guanosine triphosphate (GTP) and Guanosine diphosphate (GDP) participates in citric acid cycle. 4. Uridine diphosphate glucose (UDPG) participate in gluconeogenesis. 5. NAD+ and coenzyme A are of biomedical importance and are synthesized from amphobolic intermediate. 6. Cyclic AMP and cyclic GMP serves the secondary messenger function. 7. CDP-Acyl glycerol in lipid biosynthesis acts as high energy intermediates. Purines and Pyrimidines Metabolism Precursors of Purine Ring N-1 is derived form amino nitrogen of aspartate. N-3 and N-9 derived from amide nitrogen of glutamine. N-2 and C-8 are derived from format.
    • 250 BIOCHEMISTRY FOR STUDENTS C-6 is derived from respiratory carbon dioxide. C-4, C-5 and N-7 are derived from glycine. Precursors of Pyrimidine Ring N-3 is derived from amide nitrogen of glutamine. C-2 is derived from respiratory carbon dioxide. N-1, C-4, C-5 and C-6 are derived from aspartate. Biosynthesis of the Purine Ribonucleotides The starting material for the de novo synthesis of purine ribonucleotide is an activated form of D-ribose-5-phosphate on which purine ring is built up step-by-step. D-ribose-5-phosphate, derived from pentose phosphate pathway is converted to 1-pyrophosphate ribose-5-phosphate (PPRP) by the transfer of pyrophosphate group from ATP to C-1 of ribose.
    • NUCLEIC ACID—CHEMISTRY AND METABOLISM 251 The first ribonucleotide formed by this pathway is inosinic acid which is a precursor of adenylic acid and guanylic acid.
    • 252 BIOCHEMISTRY FOR STUDENTS Biosynthesis of Pyrimidine Nucleotides This biosynthetic pathway involves the formation of pyrimidine ring first from its chain precursors followed by the attachment of D-ribose-5-phosphate moiety. The first pyrimidine ribonucleotide formed is uridylic acid (UMP) which is a precursor of cytidine nucleotides and thymidine deoxynucleotides. Orotic Aciduria It is an inherited metabolic disorder of pyrimidine biosynthesis, characterized by accumulation of orotic acid in blood and its excretion in urine due to deficiency of enzyme orotidylic acid phosphorylase and orotidylic acid decarboxylase. Salvage Pathway De novo synthesis of purines is the main pathway by which purines bases are synthesized in the body. But there exists another pathway in the body called salvage pathway by which purine nucleotides are also formed. Salvage pathway involves the synthesis of purine nucleotides from free purine bases and purine nucleotides which
    • NUCLEIC ACID—CHEMISTRY AND METABOLISM 253
    • 254 BIOCHEMISTRY FOR STUDENTS are salvaged from dietary sources and tissue breakdown. This pathway is mainly operative in leukocytes, brain, etc. which are not capable of de novo synthesis of purine nucleotides. Since 90 percent of the free purines formed by man are salvaged and recycled by this pathway and hence this pathway is important in purine economy in the vertebrates. Salvage reactions promoted by the action of two enzymes which convert free purines into corresponding purine nucleotides for reuse are Adenine phosphoribosyl transferase and Hypoxanthine-guanine phosphoribosyl transferase (HGPR Tase). The reactions performed by these enzymes are as follows: Lesch-Nyhan Syndrome Deficiency of enzyme hypoxanthine-guanine phosphyribosyl transferase (HGPR Tase) give rise to Lesch-Nyhan syndrome, a genetic disorder. This enzyme catalyze the transfer of ribose phosphate group of 5 PRPP to either guanine, xanthine or hypoxanthine. When this enzyme is deficient guanine, xanthine and hypoxanthine are not salvaged and hence degraded to uric acid. Catabolism of Purines This first step in the catabolism of purines is their hydrolytic deamination to hypoxanthine, i.e. adenine is converted to
    • NUCLEIC ACID—CHEMISTRY AND METABOLISM 255 hypoxanthine and guanine to xanthine. In the second step both xanthine and hypoxanthine are oxidized to uric acid. In man, the end product of purine catabolism is uric acid. Whereas in lower primates, the enzyme uricase, hydrolyze uric acid to allantoin. Catabolism of Pyrimidines The catabolism of pyrimidines takes place mainly in the liver and the breakdown pathway is represented as: The breakdown of pyrimidines gives rise to the formation of β-alanine and β-aminoisobutyric acid. β-alanine is metabolized to acetate, carbon dioxide and ammonia, whereas β-aminoisobutyric acid is metabolized to propionic acid, which in turn gives rise to succinic acid. β-alanine is an important constituent of pantothenic acid and therefore, of coenzyme A. Also β-alanine is a component of carnosine and anserine which are synthesized by the muscle and brain.
    • 256 BIOCHEMISTRY FOR STUDENTS Regulation of Purine Synthesis Purine biosynthesis is regulated by feedback inhibition at the following sites: a. Feedback inhibition of 5-phosphoribose pyrophosphate synthetase by AMP, GMP and IMP. b. Feedback inhibition of amidotransferase by the final products of the pathway ATP, ADP, AMP, GTP, GDP, GMP, IMP, IDP and IMP. c. Inosinic acid (IMP) is at a branched point in the synthesis of AMP and GMP. AMP inhibits the conversion of IMP to adenylosuccinate by inhibiting the enzyme adenylosuccinate dehydrogenase. Similarly, GMP inhibits the conversion of IMP to xanthylic acid by inhibiting the IMP dehydrogenase. d. ATP is a substrate in the synthesis of GMP whereas GTP is a substrate in the synthesis of AMP. This reciprocal substrate relationship regulates the synthesis of AMP and GMP, i.e. GMP synthesis is accelerated when AMP levels are high.
    • NUCLEIC ACID—CHEMISTRY AND METABOLISM 257 Regulation by Pyrimidine Biosynthesis The committed step in pyrimidine biosynthesis is the formation of N-carbamoyl aspartate from aspartate and carbamoyl phosphate. The enzyme is aspartate transcarbamylase. This enzyme is feedback inhibited by CTP, the final product in the pathway. The second control site is carbamoyl phosphate synthesis which is feedback inhibited by UMP. Gout Gout is a metabolic disease, the salient biochemical feature of which is hyperuricemia. As a result of hyperuricemia, large amounts of uric acid (as sodium salt) are deposited in the joints and tissues (tophi). Gout is of two types: 1. Metabolic gout 2. Renal gout. Metabolic Gout i. Primary metabolic gout ii. Secondary metabolic gout. Primary metabolic gout: This is due to inherited metabolic defect in purine metabolism leading to increased rate of conversion of glycine to uric acid. Secondary metabolic gout: This is due to increased catabolism of nucleic acids, i.e. polycythemia, leukemia, etc.
    • 258 BIOCHEMISTRY FOR STUDENTS Renal Gout i. Primary renal gout ii. Secondary renal gout. Primary renal gout: In this type of gout, the defect lies in the kidney where there is faulty enzymatic transport of urates by the renal tubules. The rate of excretion of uric acid is lowered. Secondary renal gout: This is due to glomerulonephritis or some other destructive process leading to kidney failure.
    • VITAMINS 259 CHAPTER 12 Vitamins In addition to oxygen, water, proteins, fats, carbohydrates and inorganic salts, a number of organic compounds are also necessary for the life, growth and health of animals including man. These compounds are known as the accessory dietary factors or vitamins and are only necessary in very small amount. Vitamin cannot be produced by the body and hence, must be supplied. Vitamins are defined as organic compounds, occurring in natural food either as such or as utilizable precursors which are required in minute amounts for normal growth, maintenance and reproduction. They differ from other organic foodstuffs in that they do not enter into the tissue structure and do not undergo degradation for the purpose of providing energy. The absence of these results in deficiency disease. Most of the vitamins are supplied by the diet. Very few vitamins which are synthesized in the intestine belongs to the vitamin B group. Vitamins which are synthesized in the intestinal flora are: vitamin K, thiamine, riboflavin, pyridoxine, folic acid, niacin and biotin. But the entire requirement of these vitamins are not met by the endogenous synthesis. Vitamin deficiencies are must often the result of consuming monotonous diet, i.e. diet-based on limited number of food sources. The requirements for vitamins are usually greatest during the neonatal period. The vitamins have been classified into: 1. Fat soluble vitamins: They are soluble in fat solvents. Vitamins in this group are vitamins A, D, E and K.
    • 260 BIOCHEMISTRY FOR STUDENTS 2. Water soluble vitamins: They are water soluble and includes vitamin C and vitamins of B-complex. Most of the vitamins form the integral part of coenzymes. Fat soluble vitamins are stored in our fat deposits (liver and adipose tissue) and water soluble vitamins are constantly flushed from our bodies. Therefore, we can do without lipid soluble vitamins for a resonable amount of time but we must keep replacement the water soluble vitamins. Vitamins acts as coenzyme, antioxidants, (free radical quenching agents) as signalling agents in the cells, as regulator of gene expression and as redox. Vitamin Like Compounds Those compounds which are highlighted because of their known role as coenzymes in prokaryotes eukaryotes or roles as a probiotic (growth promoting substance) in higher animals are defined as vitamin like. Vitamin like substances are taurine, queuosine, coenzyme Q, pteridines (other then folic acid), such as biopterin and the molybdenum containing pteridine cofactor, pyrroloquinoline quinone (PQQ). Vitamins as Coenzymes Vitamins Active form Functions performed Thiamine Thiamine pyrophosphate Aldehyde group transfer Hydrogen group transfer Hydrogen group transfer Acyl group transfer Hydrogen transfer Riboflavin Flavin mononucleotide (FMN) Flavin adenine dinucleotide (FAD) Pantothenic acid Coenzyme A Nicotinamide Nicotinamide adenine dinucleotide (NAD) Nicotinamide adenine dinucleotide phosphate (NADP) Pyridoxine Pyridoxal phosphate Hydrogen transfer Amino group transfer Contd...
    • VITAMINS 261 Contd... Biotin Biocytin Folic acid Tetrahydrofolic acid, i.e. Folacin Cyanocobalamin Cobamides Lipoic acid Lipoyl lysine Carboxyl group transfer, i.e. CO2 fixing Methyl, methylene, formyl or formimino group transfer Alkyl group transfer Acyl group transfer FAT SOLUBLE VITAMINS VITAMIN A Retinol, growth promoting vitamin, anti-infective vitamin, antixerophthalmia. Structure Vitamin A occurs in two forms, vitamin A1 and vitamin A2. The structure of vitamin A1 is: Vitamin A2 contains an additional double bond between C-3 and C-4. Vitamin A2 is 40 percent active to vitamin A1. Certain carotenes called provitamins A are converted into vitamin A in the body. β-carotenes give rise to two molecules of vitamin A whereas α- and γ-carotenes give rise to one molecule each of vitamin A. Functions 1. The most important function of vitamin A is in the visual cycle (page 262). Retina contains conjugated protein rhodopsin. Rhodopsin consists of protein ‘opsin’ and vitamin A1 aldehyde. Rhodopsin is the major light receptor of rod cells. Under
    • 262 BIOCHEMISTRY FOR STUDENTS 2. 3. 4. 5. 6. the influence of light rhodopsin is converted to transretinal and opsin. Transretinal is inactive in the resynthesis of rhodopsin. Transretinal is converted to transretinal by reductase which is also inactive in rhodopsin synthesis, is passed into blood stream. During resynthesis of rhodopsin, which occurs in dim light and in the dark, active cis-retinal enters the retina from the blood and is oxidized to cis-retinal by retinal action of retinal reductase. Now cis-retinal couples with opsin to form rhodopsin. The visual process involves the removal of active retinal isomer from the blood by the retina which returns the inactive isomer to the circulation. In the maintenance of proper health of epithelium tissues. For the stability and integrity of cellular and subcellular membranes. Necessary for the synthesis of mucopolysaccharides as it helps in the incorporation of sulfur in chondroitin sulfate. It is also involved in the nucleic acid metabolism. It is also involved in the election transport chain and in oxidative phosphorylation. Sources Provitamin sources: Food rich in carotenes such as carrot, papayas, tomatoes, etc. Readymade or preformed sources: Fish liver oils such as shark, cod, halibut fish, liver oils, egg yolk, butter, milk products. Visual cycle
    • VITAMINS 263 Daily Requirement 5000 IU Deficiency Disease Deficiency of vitamin A gives rise to night blindness. Hypervitaminosis A Excessive intake of vitamin A gives rise to hypervitaminosis A. The symptom of this toxicity include anorexia, irritability, headache, peeling of skin, vomiting.
    • 264 BIOCHEMISTRY FOR STUDENTS VITAMIN D The term vitamin D does not refer to a single dietary factor but to a number of chemically related compounds, all of which have the property of preventing or curing rickets. The two most active substances in this respect are vitamin D2 and vitamin D3. Structure Vitamin D2 is also known as ergocalciferol and vitamin D3 as cholecalciferol. Irradiation of 7-dehydrocholesterol with ultraviolet radiations produces cholecalciferol whereas irradiation of ergosterol produces ergocalciferol. Vitamin D2 differs from vitamin D3 with respect to the double bond additionally present in the side chain at position 20 and 21. The biologically active form of vitamin D are 25-hydroxy cholecalciferol and 1,25-cholecalciferol. Liver converts cholecalciferol to 25-hydroxy cholecalciferol (25 HCC) whereas kidney converts 25-HCC to 1,25 dihydroxy cholecalciferol (1,25-DHCC). Another important active form formed by the kidney is 24,25-dihydroxy cholecalciferol (24,25-DHCC) but very little is known of biological function of this form. 1,25-dihydroxy cholecalciferol perform the following functions: i. It promotes calcium absorption from the intestine ii. It promotes mobilization of calcium from bones. Functions 1. The basic action of vitamin D is to increase the absorption of calcium and phosphorus from the intestines.
    • VITAMINS 265 2. Vitamin D promotes resorption of bone and mobilization of calcium from bones. 3. Vitamin D increases excretion of phosphate by the kidney. Sources Fish liver oils (i.e. cod liver oil, shark liver oil, halibut liver oil), egg yolk, milk. Daily requirement 400 IU infants and children 100 IU adults 400 IU pregnancy and lactation Deficiency Disease Deficiency of vitamin D gives rise to rickets in children and osteomalacia in adults. In rickets, there is a fall in intestinal absorption of calcium and phosphate, increased excretion of urinary phosphate and loss of calcium from the bones, leading to softness and deformities of bones. In rickets and osteomalacia, there is an increase in serum alkaline phosphatase activity. Hypervitaminosis D Doses above 1500 units per day for long period rise to vitamin D toxicity. Excessive intake of vitamin D causes loss of appetite, nausea, irritability, excessive mobilization of calcium from bones into blood. VITAMIN E (Antisterility vitamin, fertility factor) Vitamin E refers to a group of compounds having vitamin E activity and are known as tocopherols. Four unsaturated alcohols, i.e. α, β, γ and δ tocopherols occur in nature. These tocopherols differ slightly in structure in their side chain, α-tocopherol is most potent of them.
    • 266 BIOCHEMISTRY FOR STUDENTS Structure The structure of α-tocopherol is: They are thermostable and sensitive to the effects of oxidizing agents and ultraviolet rays. Functions 1. Tocopherols act as powerful antioxidants: a. They prevent the autoxidation of vitamin A and carotenes. b. They prevent the formation of fatty acid peroxidases in tissues due to the autoxidation of unsaturated fatty acids with oxygen. c. They protect the lipids of biological membranes against oxygen by acting as antioxidants (i.e. prevent the peroxidation of polyunsaturated fatty acids that occur in membranes throughout the body). 2. Vitamin E prevents rancidity. Sources Wheat germ oil, corn oil, peanut oil, soyabean oil, sunflower oil, egg yolk, leaves of spinach, alfalfa, sweet potatoes. Daily Requirement 15 IU Vitamin E intake is related to the intake of polyunsaturated fatty acids, i.e. 0.4 mg per gm of polyunsaturated fatty acids. Deficiency Disease Deficiency of vitamin E gives rise to sterility in rats. VITAMIN K (Coagulation factor) Two naturally occurring vitamin K are vitamin K1 and vitamin K2. Both are naphthaquinone derivatives.
    • VITAMINS 267 Structure Vitamin K1 is phylloquinone and is chemically known as 2-methyl-3-phytyl-1, 4-naphthaquinone. Vitamin K2 is farnesoquinone and is chemically known as 2-methyl-3 difarnesyl-1, 4-naphthaquinone. Vitamin K is thermostable can withstand reduction and is rapidly oxidized both in acidic and alkaline medium. It is completely destroyed by ultraviolet radiations. Certain compounds having vitamin K activity are called vitamines. Vitamines are synthetic compounds possessing vitamin K activity (Example: Menadione). Menadione is more potent than vitamin K1 on weight basis. Functions 1. In the synthesis of prothrombin. 2. It is involved in oxidative processes taking place in photosynthesis of plant kingdom. 3. It is involved in electron transport chain and is involved in oxidative phosphorylation. Vitamin K cycle
    • 268 BIOCHEMISTRY FOR STUDENTS Sources Vitamin K1, is present in alfalfa, spinach, cabbage, cauliflower, egg yolk, liver. Vitamin K2 is present in putrifying fish. It is also synthesized by intestinal flora. Daily Requirement Sufficient amounts of vitamin K are synthesized by intestinal bacteria so there is no dietary requirement under physiological condition. Deficiency Disease Vitamin K deficiency gives rise to hypoprothrombinemia which leads to prolongation of prothrombin time. WATER SOLUBLE VITAMINS Water soluble vitamins includes vitamin C and members of vitamin B complex. Vitamin C (Ascorbic acid, anti-scorbutic vitamin) Structure
    • VITAMINS 269 Vitamin C, which is hydrophilic, acts as an antioxidant in solution. Vitamin C is a powerful reducing agent and is oxidized to dehydroascorbic acid. Both forms are biologically active. It is stable in acidic solution at low temperatures but undergoes destruction in alkaline solution when in contact with air. Vitamin C is not synthesized by man and its entire requirement is met by diet. The most rich sources of vitamin C in the body are adrenal cortex, corpus luteum, pituitary, pancreas, liver, etc. Functions 1. Participation in the hydroxylation of proline and lysine present in collagen, an intracellular cementing substance. 2. Participates in the synthesis of steroid hormones both in adrenal cortex and corpus luteum. 3. Participates as cofactor in the following reaction: a. In phenylalanine metabolism. p-hydroxy phenylpyruvic acid → homogentisic acid b. Dopamine → Norepinephrine c. Folic acid → Folinic acid. 4. Vitamin C is necessary for the synthesis of carnitine in the liver. 5. Necessary for the absorption of iron by reducing ferric form to ferrous form. 6. In tissue respiration, i.e. oxidation-reduction phenomenon. 7. In bile acid formation, vitamin C is required at the 7-αhydroxylase step. 8. Ascorbic acid may act as water soluble antioxidant and inhibit the formation of nitrosamine. Sources Citrus fruits such as lemon, orange, pineapple, etc. Indian goosebury, green pepper, cauliflower, tomatoes, spinach, potato. Daily requirement: 60-80 mg. Deficiency Disease Deficiency of vitamin C gives rise to scurvy. The early manifestation in man are swelling of joints, hemorrhage in skin,
    • 270 BIOCHEMISTRY FOR STUDENTS muscle, gastrointestinal tract, inflammation of gums, ulceration, etc. In scurvy, vitamin C level in blood falls down. Vitamin B Complex The members of this group are: 1. Thiamine (B1). 2. Riboflavin (B2). 3. Pantothenic acid (B3). 4. Choline (B4). 5. Niacin (B5). 6. Pyridoxine (B6). 7. Biotin (B7). 8. Folic acid (B9). 9. Cyanocobalamin (B12). 10. Para amino benzoic acid. 11. Inositol. 12. Lipoic acid. The vitamins of this family have been grouped together because of the following fulfilment: 1. Usually present in yeast. 2. Present in the outer covering of seeds and cereals. 3. Synthesized by the microorganisms in the intestines. 4. They are water soluble. 5. Required in minute amounts and their deficiencies give rise to ordinary manifestations. 6. They usually serve as a coenzyme of various enzyme systems. Foods that are poor sources of one of the B vitamins level to be the poor sources of several B vitamins. THIAMINE (Vitamin B1, anti-neuritic vitamin, anti-beriberi factor, aneurin) Structure
    • VITAMINS 271 Thiamine consists of a substituted pyrimidine ring joined by a methylene bridge to substituted thiazole ring. Thiamine is water soluble. It is thermolabile, destroyed in alkaline medium but thermostable in acidic medium. Thiamine occurs in the cells largely as its active coenzyme form, i.e. thiamine pyrophosphate, also called cocarboxylase. Functions Thiamine pyrophosphate (TPP) is used mainly as a coenzyme in the carbohydrate metabolism. 1. In oxidative decarboxylation of α-ketoacids. Pyruvic acid and α-ketoglutaric acid which are intermediates in the carbohydrate metabolism are oxidatively decarboxylated to acetyl CoA and succinyl CoA respectively. Pyruvic acid α-ketoglutaric acid Acetyl CoA Succinyl CoA 2. In transketolation reaction. An intermediate step in hexose monophosphate shunt pathway where transfer of glycolaldehyde group from D-xylulose5-phosphate and glyceraldehyde. D-xylulose-5-PO4 + D-ribose-5-PO4 D-sedoheptulose-PO4 + D-glyceraldehyde-PO4 3. In yeast TPP acts as coenzyme for nonoxidative decarboxylation of α-keto-acids, i.e. pyruvate to CO2 and acetal dehyde. Sources Yeast, outer coating of seeds, cereals, legumes, wheat, pork, eggs. Vitamin B1 is also synthesized during germination. Daily Requirement: 1-1.4 mg. Thiamine requirement rises with a rise in caloric intake of food, i.e. 0.5 mg of vitamin B1 for every 1000 calories in the form of carbohydrates.
    • 272 BIOCHEMISTRY FOR STUDENTS Deficiency Disease Deficiency of vitamin B2 gives rise to beriberi in man, and polyneuritis in birds and animals. Rise in blood pyruvic acid is the best biochemical test for vitamin B1 deficiency. RIBOFLAVIN (Vitamin B2, lactoflavin) Structure Riboflavin is a derivate of isoalloxazine, i.e. dimethyl-isoalloxazine attached to ribotyl group. Riboflavin is thermolabile, destroyed in alkaline medium, active in acidic medium. Its aqueous solution gives a yellowgreen fluorescence. Functions 1. Riboflavin is present as such in retina where it plays a part in light adaptation. 2. Riboflavin exists as component of two coenzymes called flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) which acts as coenzymes or prosthetic group of many flavoprotein enzymes. Examples of flavoprotein containing FMN and FAD as prosthetic groups are:
    • VITAMINS 273 Flavin Mononucleotide (FMN) a. Warburg yellow enzyme b. L-amino acid oxidase c. Cytochrome C reductase. Flavin Adenine Dinucleotide (FAD) a. b. c. d. e. D-amino acid oxidase Xanthine oxidase Glycine oxidase Thiophorase oxidase Acyl CoA dehydrogenase. Sources Yeast, milk, egg, meat, fish, liver, kidney, green leafy vegetables. Daily requirement: 0.4-1.4 mg Deficiency Disease Riboflavin deficiency does not give rise to any clear cut disease but the important deficiency symptoms are choliosis, fissures in lips, mouths, inflammation of the tongue, dermatitis. Riboflavin is excreted mostly in fecal matter. In urine, a pigment known as urochrome is excreted which is very much related to riboflavin. NIACIN (Pellagra preventive factor) Structure
    • 274 BIOCHEMISTRY FOR STUDENTS Niacin is a pyridine derivative, thermostable and is not rapidly oxidized. Functions Niacinamide is a component of nicotinamide adenine dinucleotide (NAD+) and niacinamide adenine dinucleotide phosphate (NADH4) and acts as coenzyme for many anaerobic dehydrogenases by accepting hydride ions during oxidation of their substrates. Enzymes requiring NAD+ or NADH coenzymes are: a. Glyceraldehyde-3-PO4 dehydrogenase b. Lactate dehydrogenase c. Ketose reductase d. Malic dehydrogenase. Enzymes requiring NAD+ or NADPH as coenzymes are: a. Isocitrate dehydrogenase b. Glucose-6-PO4 dehydrogenase c. Aldolase reductase. Sources Yeast, meat, liver, kidney, eggs, fish, legumes. Nicotinic acid is also synthesized during tryptophan metabolism, 60 mg of tryptophan gives rise to 1 mg of niacin. Deficiency Disease Deficiency of niacinamide gives rise to pellagra in man and black tongue in dogs. Pellagra affects the skin, central nervous system and the gastrointestinal tract. The three important symptoms of pellagra are diarrhea, dermatitis and dermentia. Diets such as corn maize give rise to niacin deficiency and ultimately to pellagra because corn or maize are distinctly deficient in tryptophan. The important excretory products of nicotinic acid in human urine are nicotinic acid as such, nicotinamide, N-methylniacinamide, nicotinuric acid and 6-pyridone of nicotinic acid.
    • VITAMINS 275 PANTOTHENIC ACID Structure Pantothenic acid is a peptide like compound formed from pantoic acid and β-alanine. It is thermostable and resistant to oxidation. Functions Pantothenic acid is a part of coenzyme, which serves as carrier of acyl group in enzymatic reactions. 1. Fatty acid oxidation 2. Fatty acid synthesis 3. Pyruvic acid oxidation 4. Biological acetylations 5. Cholesterol biosynthesis 6. In acyl carrier proteins. Sources Yeast, liver, kidney, egg yolk, molasses. Daily Requirements Synthesized by the intestinal flora in sufficient amount to meet the requirement. PYRIDOXINE (Antiachrodynia factor) Structure Vitamin B6 refers to a group of pyridoxine, pyridoxal and pyridoxamine compounds having similar biological activity. Pyridoxine is most abundant in plants, and pyridoxal and pyridoxamine are most abundant in animal tissues.
    • 276 BIOCHEMISTRY FOR STUDENTS Pyridoxal and pyridoxamine are thermolabile and photolabile. Pyridoxine can be converted to pyridoxal and pyridoxamine but pyridoxal and pyridoxamine cannot be converted back to pyridoxine. All are biologically active but only the phosphate esters of pyridoxal and pyridoxamine can function as coenzymes. Functions Pyridoxine containing enzymes is important in amino acid and protein metabolism. 1. Pyridoxine acts as coenzymes for decarboxylation reactions: a. Histidine → Histamine b. Tyrosine → Tyramine c. 5-hydroxy tryptophan → 5-hydroxy tryptamine (5HT) d. Glutamic acid → γ-amino butyric acid (GABA) e. α-amino-β-keto adipic acid → β-amino levulinic acid 2. As coenzyme in transamination reaction. 3. As coenzymes in dehydrases reactions. Serine → Pyruvic acid Threonine →α-ketobutyric acid 4. As coenzymes in trans-sulfurases reaction. Homocysteine → Serine 5. As coenzymes for desulfuration reactions. Cysteine → Pyruvic acid Homocysteine → α-ketobutyric acid 6. In tryptophan metabolism, the conversion of kynurenine to anthranilic acid requires pyridoxal phosphate as coenzyme.
    • VITAMINS 277 In vitamin B6 deficiency, it is converted to xanthurenic acid. So measurement of xanthurenic acid is an indication of vitamin B6 deficiency. 7. Also helps in the transport of amino acids to the cell membrane and is also necessary for the absorption of amino acids from the intestinal mucosa. 8. Also involved in the conversion of linolenic acid to arachidonic acid, hence, it is essential for essential fatty acid synthesis. Sources Yeast, liver, egg yolk, rice polishings. Synthesized by the microflora of the intestines. Daily requirement: 2-2.5 mg. BIOTIN Structure Biotin contains fused imidazole and thiophene. Biotin is present in food both in free and in combined form with proteins. The combined form is liberated by proteolytic enzymes. Biotin is also synthesized by intestinal flora.
    • 278 BIOCHEMISTRY FOR STUDENTS Functions Biotin is required in carbon dioxide fixation reactions. Reactions where biotin is involved are: 1. Acetyl CoA — Malonyl CoA (Fatty acid synthesis) 2. Pyruvic acid — Oxaloacetic acid (Glucongenesis) 3. Propionyl CoA — D-methyl malonyl CoA 4. CO2 + NH3 — Carbamyl phosphate (Urea cycle) 5. In purine ring synthesis, i.e. the C-6 position in purine ring skeleton. Structure of coenzyme A Sources Yeast, egg yolk, milk, molasses, chocolate, tomato, peanuts. Daily requirement: 200 μg. Deficiency Disease Raw egg induces biotin deficiency because it contains a protein ‘avidin’ which tightly binds biotin and prevents its absorption from the intestine.
    • VITAMINS 279 FOLIC ACID Structure Folic acid consists of three components: i. Pteridine nucleus ii. Para-aminobenzoic acid. iii. Glutamic acid. Folic acid occurs in nature in three types: a. Pteroyl monoglutamate which contains only one molecule of glutamic acid. b. Pteroyl triglutamate which contains three molecules of glutamic acid. c. Pteroyl heptaglutamate which contains seven molecules of glutamic acid. Before folic acid can function as coenzyme in various metabolic reactions it must be reduced to tetrahydrofolic acid. Folic acid→Dihydrofolic acid→Tetrahydrofolic acid (TH4). The various activated forms of folic acid are: N5-formyl TH4, N10-formyl TH4 N5-10-methenyl TH4, N5-10-methylene TH4. 5 N -TH4 contains formyl group at 5-position. It is also called folinic acid. TH4 is biologically inactive except that it has only one role, i.e. in the formylation of glutamic acid in histidine metabolism whereas N10-TH4 and N510-TH4 are the biologically most active forms of tetrahydrofolic acid. Folic acid coenzymes are collectively known as folacin. Functions Folic acid coenzymes are involved in the transfer and incorporation of single or one carbon moiety.
    • 280 BIOCHEMISTRY FOR STUDENTS One carbon moieties are: i. Formyl group and formate group, i.e.—CHO and —HCOOH ii. Hydroxymethyl group, i.e. —CH2OH iii. Methyl group, i.e. —CH3 iv. Formimino group, i.e. —CH=NH They are inter convertible: —CH 2OH —CHO —-COOH Sources of one carbon moieties are: i. α-carbon of glycine gives rise to —CHO group ii. Histidine gives rise to —CH=NH group iii. Choline donates —CH3 group via betaine (choline as such cannot donate methyl group) iv. Biotin gives rise to —CH3 group v. β-Carbon of serine gives rise to —CHO group. Utilization of one carbon moiety: 1. Conversion of ethanolamine to choline. 2. Conversion of glycine to serine. 3. Conversion of norepinephrine to epinephrine. 4. Conversion of guanidoacetic acid to creatine. 5. Conversion of uracil to thymine. 6. Conversion of ribonucleotides to deoxyribonucleotides. 7. In the formation of N-formylmethionine transfer RNA. 8. In purine synthesis, i.e. C-3 and C-8 positions come through one carbon moiety. FIGLU excretion test (Formiminoglutamic acid excretion test). This is a diagnostic test for finding folic acid deficiency. In the histidine metabolism, the conversion of N-formiminoglutamic acid to glutamic acid is a folic acid dependent step. When folic acid deficient patients are given an increased load of histidine, there is an increased excretion of formiminoglutamic acid in urine due to the nonconversion of above step due to folic acid deficiency. So increased histidine load test is used to find the folic acid deficiency.
    • VITAMINS 281 Sources Yeast, liver, kidney, green vegetables. Daily requirement: 0.4-0.8 mg. Deficiency Disease The main deficiency symptoms are anemia, i.e. the reduced ability to produce red blood cells. CYANOCOBALAMIN (Vitamin B12, antipernicious factor, castle’s extrinsic factor) Structure Cyanocobalamin is made up of two components. The larger component is corrin ring system, containing four pyrrole rings. One of the pair of pyrrole rings is joined directly. Cobalt is coordinated to the four nitrogen of the pyrrole rings, the second component is a ribonucleotide, 5,6 dimethyl benzimidazole joined to corrin by a nitrogen atom of nucleotide and cobalt atom and by an ester linkage between the 3 phosphate group of the ribonucleotide and a side chain of the corrin ring. Replacement of cyanide group by hydroxy group, nitro group, and methyl group forms hydroxy cobalamin, nitro cobalamin and methyl cobalamin respectively. All of them possess vitamin B12 activity. But hydroxycobalamin also called vitamin B12 is more potent because it is readily absorbed and its concentration in blood rises very early. Vitamin B12 acts in the form of three coenzymes, called cobamides. The three types of cobamides are: 1. Cobamide I (where CN¯ is replaced by dimethyl benzimidazole group). 2. Cobamide II (where CN¯ is replaced by benzimidazole group). 3. Cobamide III (where CN¯ is replaced by adenyl group).
    • 282 BIOCHEMISTRY FOR STUDENTS Function Cobamide coenzymes are involved in: 1. Conversion of L-methylmalonyl CoA to succinyl CoA. 2. Conversion of glutamic acid to β-methyl aspartic acid. 3. Conversion of ribonucleotides to dexyribonucleotides. Sources Liver, kidney, meat, fish, egg yolk. Also synthesized by intestinal bacteria. Daily Requirement: 3-4 μg. Deficiency Disease Deficiency of vitamin B12 gives rise to pernicious anemia. Pernicious anemia is not simply the result of vitamin B12 deficiency in the diet but is caused by a lack of specific glycoproteins in the gastric juice called the intrinsic factor. This protein binds vitamin B12 and is transported.
    • VITAMINS 283 Also, deficiency of vitamin B12 gives rise to increased excretion of methyl malonic acid in urine. ANTIVITAMINS Antivitamins are substances which possess structural similarity to certain vitamins but behave antagonistically to these vitamins when introduced into the body, thereby preventing the normal function of these vitamins. Examples of vitamins with their antivitamins are Thiamine: Pyrithiamine, Riboflavin: Isoflavin, Pyridoxine: Isonicotinic acid hydrazide (INH), Folic acid: Aminopterin, vitamin K: Dicoumarol.
    • 284 BIOCHEMISTRY FOR STUDENTS CHAPTER 13 Acid-base Balance ACID-BASE BALANCE The acid-base balance of the body is basically the metabolism, of hydrogen ions. When these hydrogen ions are produced in the body, there are many ways by which they are handled and excreted by the body so as to maintain the pH of the body fluids constant. Electrolyte Composition of Plasma Cations mEq/L Na+ K+ Ca++ Mg++ 142 5 5 3 Total 155 Anions Cl¯ HCO¯ 3 HPO¯ 4 SO¯ ¯ 4 Organic acids Proteinate Total mEq/L 103 27 2 11 6 16 165 Three mechanisms for the maintenance of acid-base balance are: 1. Buffer systems of the body fluids. 2. Lungs (Respiration). 3. Kidneys (Renal mechanism). Buffer Systems of the Body Fluids The chief acids produced in the body are H2CO3, HHb, HHbO2, proteins and various organic acids such as lactic acid,
    • ACID-BASE BALANCE 285 pyruvic acid, citric acid, are taken by the chemical buffer systems of the body. The buffer systems of the body fluids represent the first mechanism involved in the regulation of pH. The buffer systems of the various body fluid compartments are: The important buffers in the plasma are bicarbonate: carbonic acid buffer. In the RBC, the hemoglobin buffer system predominates whereas proteins and phosphate buffer represent the buffers of intracellular fluids. When protons are added to the body fluids, they are taken up by the buffer bases (anions) to form buffer acids, and when there is a deficit of protons they are given to the body fluids by the buffer acids. Bicarbonate Carbonic Acid Buffer It is the most important buffer system of nonvolatile acids entering the extracellular fluids because of two reasons. 1. It is present in high concentration than the other buffer systems. 2. The production of H2CO3, is effectively buffered and is disposed by the lungs as CO2. Such acids, e.g. HCl, H2SO4, lactic acid, etc. react with NaHCO3 as follows:
    • 286 BIOCHEMISTRY FOR STUDENTS The pH of the buffer system is given by ⎧ B+ HCO − ⎫ 3 pH = pk + log ⎨ ⎬ ⎩ H 2 CO 3 ⎭ The pH of the body fluids depends on the ratio of BHCO3 and H2CO3. As long as the ratio remains 20:1, the pH is normal. Phosphate Buffer Phosphate buffer plays a minor part in blood but is more important in the kidney in regulating pH. It is important in raising plasma pH through excretion of H2PO4 by the kidney. ¯ HPO − 80 4 = − H 2 PO 4 20 At pH 7.4, plasma has four parts dihydrogen phosphate to one part of monohydrogen phosphate. At pH 5.8, the ratio is ten parts of monohydrogen phosphate to one part of dihydrogen phosphate. This variation in pH is made use of in the removal of hydrogen ions by the kidney. Protein Buffer At the pH of the blood, the plasma proteins are anions but act as weak acids. H¯ Protein ⇔ H+ Protein In plasma, protein buffer plays a much smaller part than bicarbonate buffer but in the cells proteins form the most important buffering system. Lungs The level of H2CO3 in the plasma is under the control of lungs. The respiratory mechanism, compensates for disturbance of acid-base balance by regulating H2CO3. Whenever, there is acidosis (i.e. hydrogen ions in blood increase), pH is decreased, with a lowered HCO¯ : H2CO3 ratio. The lung ventilation is 3 increased, alveolar pCO2 is decreased and H2CO3 is increased
    • ACID-BASE BALANCE 287 which increases the HCO¯: H2CO3 ratio and the pH returns 3 to normal. In alkalosis, when the pH is increased, the ratio HCO¯: 3 H2CO3 is high. Lung ventilation is decreased, the pCO2 of the alveolar is increased and H2CO3 in the blood and other fluids is increased which lowers the HCO¯ : H2CO3 ratio and 3 thus pH returns towards normal. Kidney Kidney plays the important role in regulating both the electrolyte concentration and the acid-base balance of the body fluids. The main mechanism by which the kidney maintains the pH of the body fluids is by regulating secretion of H+ ions which is linked up with the conservation of base in the form of sodium bicarbonate, the formation of acid phosphates and generation of NH4+ ions by the kidney tubules. Various acids such as lactic acids, ketone bodies, sulfuric acid, phosphoric acid are taken by bicarbonate for neutralization as soon as they are formed. This shows that the bicarbonate is the alkaline reserve of the body. These acids buffered with Na+ are first removed by glomerular filtration. The cation is then reabsorbed by the renal tubules in exchange of H+ which are secreted. Mechanism of H+ excretion. The H+ secreted by the tubular cells are handled in three principal ways. 1. Bicarbonate reabsorption 2. Ammonium ion production 3. Acidification of the urine. Bicarbonate Reabsorption Mobilization of H+ for tubular secretion is accomponished by the ionization of carbonic acid. In the proximal tubule, the
    • 288 BIOCHEMISTRY FOR STUDENTS exchange of H+ against sodium bicarbonate takes place. The formation of carbonic acid is catalyzed by the enzyme carbonic anhydrase. Ammonium Ion Production Another mechanism of the conservation of cation is the production of NH3. NH3 formation takes place in the distal tubules. NH3 formed enters the tubular filtrate, combines with H+ to form NH4+ ions. The NH4+ then replace Na+ ions. The Na+ ions is reabsorbed by the H+-Na+ exchange and re-enters the plasma as NaHCO3. The NH4+ ions are excreted in urine as NH4Cl.
    • ACID-BASE BALANCE 289 Acidification of the Urine After all the bicarbonate has been absorbed, the H+ ion secretion then proceeds against NaHPO4. The exchange of Na+ ion for secreted H+ ion changes Na2HPO4 to NaH2PO4. Na+ and HCO¯ return to plasma and H+ is excreted in urine 3 to maintain normal acid-base balance and electrolyte concentration. As long as the ratio of bicarbonate to carbonic acid is 20:1 in blood, pH of blood is normal. Any variation in the ratio, will disturb the acid-balance of the blood and leads to acidosis or alkalosis. Disturbances in acid-base balance can be classified broadly under two headings: 1. Acidosis 2. Alkalosis. Acidosis (a) Respiratory acidosis (b) Metabolic acidosis Alkalosis (a) Respiratory alkalosis (b) Metabolic alkalosis. Respiratory Acidosis Respiratory acidosis is due to increase in H2CO3 in the blood, resulting in lowering of pH of the blood. This is compensated
    • 290 BIOCHEMISTRY FOR STUDENTS by increase reabsorption of HCO¯ in the renal tubules. Such 3 condition occurs in pneumonia, asthma, etc. Metabolic Acidosis This is due to the decrease in HCO3 in the blood with no change in H2CO3. This is compensated by elimination of more CO2 (hyperventilation). Such condition occurs in uncontrolled diabetes with ketosis. Anion Gap Anion gap is defined as the difference between the plasma sodium and potassium concentrations and the sum of the chloride and bicarbonate. The anion gap is made up of the difference between the sum of unmeasured cations such as ionized calcium and magnesium and of unmeasured anions such as phosphate, urate, organic acids and plasma proteins. Since the sum of plasma cation and anion concentrations must be equal to maintain electrochemical neutrality. The normal value of anion gap is 10-15 mEq/L (average 12 mEq/L). Anion gap may change because of an alteration in any of the quantities on the right hand side of the equation. A rise in anion gap is usually due to a rise in the unmeasured anions in diabetic ketoacidosis or renal failure. Small increase may occur in alkalosis due to unmeasured anionic equivalence of plasma proteins. A reduced anion gap is caused by a low plasma albumin concentrations. Anion Gap The anion gap is a mathematical approximation of the difference between the anions and cations routinely measured in serum. Routine electrolyte measurement include Na+, K+, Cl¯ and HCO3 and unmeasured cations include Ca+2, Mg+2 and ¯ ¯ ¯ unmeasured anions, i.e. PO4 3, SO4 2, protein–1, organic acids.
    • ACID-BASE BALANCE 291 If the Cl¯ and HCO3 conc are summed and substracted ¯ from total of Na+ and K+ conc the difference comes about 10-15 mmol/L (average 23 mmol/L). a. If anion gap exceeds 17 mmol/L, i.e. increased concentration of unmeasured anions • Diabetes mellitus • Alcoholism • Starvation • Salicylate • Uremia. b. If anion gap less than 10 mmol/L, i.e. increase in unmeasured cations or a decreases in unmeasured anions • Lithium intoxication • Multiple myeloma • Hypoalbuminemia. Respiratory Alkalosis This is due to decrease in H2CO3 in the blood. This is compensated by decreased reabsorption of HCO¯ by the renal tubules. 3 Such condition occurs in hepatic coma. Metabolic Alkalosis This is due to increase in HCO¯ in the blood giving rise to 3 increase in pH of the blood. This is compensated by retention of CO2 in the blood. The increased pH of blood leads to retany. It can occur in Cushing’s syndrome. Urine Normal Respiratory acidosis Respiratory alkalosis Metabolic acidosis Metabolic alkalosis 6-65 ↓ ↑ ↓ ↑ pH Plasma [HCO¯] [H2CO3] H2CO3 3 mEq/L mEq/L ———— [HCO¯] 3 2.5 ↑ ↓ ↓ ↑ 51.25 ↑ ↓ ↓ ↑ 1 <1 >1 <1 >1 : : : : : 20 20 20 20 20
    • 292 BIOCHEMISTRY FOR STUDENTS CHAPTER 14 Water and Mineral Metabolism BIOLOGICAL IMPORTANCE OF WATER 1. Water is an essential constituent of cell structures and provides the media in which the chemical reactions of the body takes place and substance are transported. 2. It has a high specific heat for which, it can absorb or gives off heat without any appreciable change in temperature. 3. It has a very high latent heat. Thus, it provides a mechanism for the regulation of heat loss by sensible or insensible perspiration on the skin surface. 4. The fluidity of blood is because of water. Water comprises 70% of the lean body mass of the adult. Lean body mass = weight of the body—fat content of the body. Water is present in the body both inside and outside the cells. Strictly speaking there are two water compartments in the body. i. Intracellular water: Water present inside the cell. ii. Extracellular water: Water present outside the cell. Extracellular fluid is further divided into: 1. Plasma: It comprises 7.5% of the body weight. 2. Interstitial fluid: It comprises 20 percent of the body weight. 3. Dense connective tissue, i.e. water content in the bones and cartilages. It comprises 15% of the body weight. 4. Transcellular fluid (intracellular fluid): It comprises 2.5% of the body weight.
    • WATER AND MINERAL METABOLISM 293 The volume of fluids in various compartments of the body can be found out by various methods such as isotopic dilution, injection of dyes, heavy water, antipyrine, etc. Total distribution of water in the human body. percent Intracellular water Extracellular water i. Plasma ii. Interstitial iii. Bones and cartilages iv. Transcellular water 55 45 7.5 20 15 2.5 ml/kg of body weight 335 270 45 120 90 15 The quantity of water in the body depends upon the body weight. The daily water requirement is about 1 ml/Kcal, i.e. a requirement of 2000 Kcal necessitates a water intake of 2000 ml. Infants have proportionately more water loss and should be allowed about 150 ml water for each 100 Kcal. Thirst is a good guide for adequate fluid intake. The source of water in the body are as follows (Figures in bracket indicate the average water intake). 1. Water by drinking (1200 ml). 2. Water present in food (1000 ml). 3. Metabolic water, i.e. water formed in the body by the oxidation of food stuffs, i.e. oxidation of carbohydrates, fats and proteins (Amino acids). The amount of water produced in the body from metabolism is about 200 to 450 ml daily. 100 gm of carbohydrates, fats and proteins yield 85 ml, 107 ml and 41 ml of water each. Normal diet provides 300 ml of metabolic water daily. Water is lost from the body by the following routes. Figures in bracket indicate the average quantity of the water loss. 1. Water lost through skin both as sensible and insensible perspiration (600 ml). Insensible perspiration is so called because one is not aware of it; it evaporates as it is formed.
    • 294 BIOCHEMISTRY FOR STUDENTS On the other hand, with vigorous activity especially in hot weather, we lose much additional water through visible perspiration. Sensible perspiration is called active sweating. It depends upon: i. Habits ii. Type of activity. If metabolic rate is high, then water loss will be high. Greater the respiration rate, the higher will be metabolic rate, i.e. more loss. Water loss depends upon: 1. Metabolic rate 2. Climate conditions 3. Water lost through lungs in expired water (400 ml). The loss of water through lungs depends upon: i. Rate of respiration ii. Temperature and humidity of atmosphere iii. Water lost through kidney in urine. iv. Water lost through intestines in feces (100 ml). Water loss is proportional to the function of metabolic rate. Kidney is the most important guardian of the water content of the body. The water loss through skin, lungs and feces are not controlable but there is an automatic feedback mechanism by the kidney. Certain volume of urine has to be lost by the kidney and it is called minimum urine volume or obligatory excretion. Kidney controls the excretion of waste products and to dissolve them minimum urine volume of 500 ml is needed. The 500 ml constitutes the 2 percent of body weight which has to be lost even when the body does not take any water. Water content depends upon body weight and water loss depends upon metabolic rate and both are not correlated. Minimum water excreted by the kidney depends upon: i. Concentrating power of the kidney ii. Quantity of water products. Healthier the kidney, the greater will be concentrating power of the kidney.
    • WATER AND MINERAL METABOLISM 295 Concentrating power of the kidney decreases in certain diseased conditions such as: i. Obstruction to the flow of kidney ii. Chronic nephritis iii. Any dehydration iv. Shock. Dehydration Dehydration results: 1. When the water intake is less than the body needs. This occurs in no food of fluid intake. 2. When the fluid loss from the body is abnormally high, e.g. excessive perspiration in hot weather, severe diarrhea, vomiting, fever, with increased loss from the skin, severe burns with accompanying water losses and in uncontrolled diabetes with frequent urination. Dehydration is corrected by electrolytes and water. Edema Edema is the accumulations of water in the body. It occurs when the body is unable to excrete sodium in sufficient amounts. This is not unusual in diseases of the heart when the circulation is impaired or when the kidneys are unable to excrete waste normally. Edema also occurs following prolonged protein deficiency because tissues are no longer able to maintain normal water balance. MINERALS Minerals are inorganic substances. Minerals are present in all body tissues and fluids. Unlike carbohydrates, fats and proteins, mineral elements do not furnish energy. Unlike vitamins, the minerals are not destroyed in food preparation. However, they are soluble in water so that some loss will occur if cooking liquids are discarded. In contrast to the organic substances, which can be considered as energy sources, the inorganic substances do not supply any energy. Their presence is necessary for the maintenance of certain physiochemical conditions which are essential for life.
    • 296 BIOCHEMISTRY FOR STUDENTS Principal minerals required by the body are sodium, potassium, calcium, magnesium, phosphorus, sulfur and chlorine. These comprises 70% of the total mineral of the body contents. In addition, copper, zinc, cobalt, manganese, moly- bdenum, iodine, fluorine. Basic functions performed by the minerals are: i. As structural components of body tissues. ii. In the maintenance of acid-base balance. iii. In the regulation of body fluids. iv. In transport of gases. v. In muscle contractions. Iron Total iron content in the body is 3.5 gm. 70% of this iron is present in hemoglobin. Biologically important compounds of iron are hemoglobin, myoglobin, cytochromes, catalases, peroxidase. In all these compounds iron is present as heme form or porphyrin form. In addition to these iron is present in nonheme form called nonheme iron. Nonheme iron is present as ferritin (a stored form of iron) and transferrin (a transport form of iron). Functions 1. As hemoglobin, in the transport of oxygen. 2. In cellular respiration, where it functions as essential component of enzymes involved in biological oxidation such as cytochromes c, c1, a1, etc. Absorption of Iron The maximum absorption of iron is not more than 10 percent of the iron content of the diet. In the food, iron is present in ferric form either as ferric hydroxides or in combination with ferric organic compounds. Acidity of gastric juice results in the liberation of ferric form. Ferric form is reduced to ferrous form by the reducing substances such as glutathione, vitamin C and cysteine present in the food absorption.
    • WATER AND MINERAL METABOLISM 297 The regulation of iron absorption is governed by mucosal block theory. According to this theory, ferrous ions on entering the mucosal epithelial cell are oxidized to ferric ions which combines with a protein called apoferritin to form ferritin (also known as siderophilin). Apoferritin is a glycoprotein containing sialic acid, galactose, mannose as the carbohydrate moieties. Each molecule of apoferritin combines with 2 atoms of ferric, iron to form ferritin. This ferritin is a stored form of iron. The amount of apoferritin present in the mucosal cells is the controlling factor. Factors which affect iron absorption are: 1. Low phosphate diet increases iron absorption whereas high phosphate diet decreases iron absorption by forming insoluble iron phosphates. 2. Iron in ferrous form is more soluble and is readily absorbed than the ferric form. 3. Phytic acid and oxalates decreases iron absorption by forming iron phytate and iron oxalate. No absorption of iron takes place under following conditions: 1. Any condition of partial or total gastrectomy 2. Dissertion of small intestine 3. Achlorohydria 4. Profuse diarrhea 5. Malabsorption syndrome.
    • 298 BIOCHEMISTRY FOR STUDENTS Iron is transported in the plasma as Fe+++ form in combination with β-globulin called transferrin also known as sidoferrin. The iron in this form is called protein bind iron (PBI). The entire iron in the plasma is in the protein bound iron. The protein bound iron in: Adults : 120-140 μg per 100 ml of blood Females : 90-120 μg per 100 ml of blood. The plasma iron content is the net resultant of the following: i. Rate of RBC destruction ii. Rate of iron absorption from intestines iii. Rate of apoferritin synthesis iv. Rate of erythropoiesis v. Extent of blood losses. Iron is stored in the body as ferritin. Ferritin can bind up to 4000 iron atoms per molecule. If iron is taken in abnormally large amounts, the excess is deposited in liver as hemosiderin. Excessive accumulation of iron in liver, lungs, pancreas, heart are other tissues results in hemosiderosis, when this is accompanied by bronze pigmentation of the skin, the condition is called hemochromatosis. Sources Meat, heart, kidney, spleen, egg yolk, fish, dates, nuts, legumes, molasses, spinach, cooking of food in iron vessels. Daily requirement: 10-15 mg. Calcium Calcium is present in the body in the largest amount of all the minerals present in the body. Calcium comprises 2% of the body weight. RBC is devoid of calcium. The normal serum level is 9-11 mg percent. Calcium is present in three forms: 1. Ionized form: This form is phy siologically active form.
    • WATER AND MINERAL METABOLISM 299 2. Protein bound fraction: This form is physiologically inert. 3. In combination with citrates: Protein bound fraction is nondiffusible whereas other two fractions are diffusible. Absorption of Calcium 1. Calcium salts are more soluble in acidic media than the alkaline media. Greater the acidity, the more will be the absorption. 2. Certain foodstuffs contain phytic acid (present in cereals) and oxalates (present in spinach) which inhibit calcium absorption by forming insoluble calcium salts. 3. When fat absorption is not proper, the free fatty acids present react with calcium to form soaps (calcium salts of fatty acid) will hinders absorption. 4. On a high protein diet the calcium absorption will be more. 5. Vitamin D is necessary in the diet to promote the absorption of calcium. 6. The optimum calcium phosphorus ratio in the diet should be 1:1. Functions 1. Calcium along with phosphorus is essential for bones and teeth formation. 2. In blood coagulation: Calcium activates the conversion of prothrombin to thrombin. 3. In milk clotting. 4. In enzyme activation: Calcium activates large number of enzymes such as adenosine triphosphatase (ATPase), succinic dehydrogenase, lipase, etc. 5. In muscle contraction. 6. In normal transmission of nerve impulses. 7. In neuromuscular excitability.
    • 300 BIOCHEMISTRY FOR STUDENTS Regulation of Blood Calcium Level 1. Indirect factors: Those factors which have an effect on calcium absorption. Under this comes dietary factors which have been discussed in the absorption of calcium. 2. Direct factors: Those which have direct effect on blood calcium. These are: a. Hormones i. Parathyroid hormone regulates the concentration of ionized serum calcium. ii. Calcitonin lowers calcium level by inhibiting bone absorption and thus decreases the loss of calcium from bones. b. Serum proteins Decrease in serum proteins will result in decrease in total calcium level as most of the calcium bound to protein will be less. c. A reciprocal relationship exists between calcium and phosphorus in the blood. Increase in serum phosphorus causes decrease in serum calcium and vice versa. Sources Dairy products such as milk, cheese are the best sources. It is also present in lentils, nuts. Daily Requirement Adults 800 mg In females during 2nd and 3rd semester of pregnancy and lactation 1200 mg Infancy 350-540 mg Children from 1 to 8 years 0.8-1.2 gm. Rickets Deficiency of vitamin D gives rise to rickets in children. The main symptom of rickets is, insufficient calcification by calcium phosphate of the bones in growing children. The bones, therefore, remain soft and deformed by the body weight.
    • WATER AND MINERAL METABOLISM 301 Osteoporosis Osteoporosis is a disease of demineralization or decalcification of the bones. It is a condition when calcium is withdrawn from the bones. The bone becomes week and porous and hence breaks. It is more prevalent in older women than in men. Sodium Sodium is the principle cation of the extracellular fluid. Functions 1. In the regulation of acid-base balance. 2. In the maintenance of osmotic pressure of the body fluids. 3. In the preservation of normal irritability of muscles and permeability of the cells. The normal serum sodium level is 133-146 mEq/L. Increased level of sodium in the serum is called hypernatremia. Hypernatremia occurs in: i. Cushing disease ii. Administration of ACTH iii. Administration of sex hormones iv. Diabetes insipidous v. After active sweating. Low levels of sodium in serum is called hyponatremia. Hyponatremia occurs in: i. Acute Addison’s disease ii. Vomiting, diarrhea iii. Severe burns iv. Intestinal obstruction v. Nephrosis vi. Any situation where there is active sweating and we take plain water.
    • 302 BIOCHEMISTRY FOR STUDENTS Potassium Potassium is the principal cation of the intracellular fluid. Functions 1. 2. 3. 4. Intracellular cation in acid-base balance In muscle contraction, particularly in cardiac muscle Conduction of nerve impulse Cell membrane function. The normal concentration of potassium in the serum is 3.5-5.5 mEq/L. Increased level of potassium in serum is called hyperkalemia. Hyperkalemia occurs in: i. Addison’s disease ii. Advanced chronic renal failure iii. Dehydration iv. Shock. Low levels of potassium in serum give to hypokalemia. Hypokalemia occurs in: i. Diarrhea ii. Metabolic alkalosis iii. Familial periodic paralysis Potassium is required during glycogenesis. This potassium is withdrawn from the extracellular fluid giving rise to hypokalemia. Phosphorus Functions 1. Phosphorus along with calcium is essential for bones and teeth. 2. Buffering action, i.e. phosphate buffers. 3. In the formation of high energy compounds, i.e. ATP. 4. In the synthesis of RNA and DNA. 5. In the synthesis of phospholipids. 6. In the synthesis of phosphoproteins. Absorption The absorption of phosphorus is related to that of calcium. Normally 1/3rd of the ingested phosphorus is passed in the feces and 2/3rds in the urine. A high calcium diet, diminishes
    • WATER AND MINERAL METABOLISM 303 the phosphorus absorption by forming insoluble calcium phosphates. Phosphorus is present in the blood as: i. Inorganic phosphorus ii. Organic phosphorus iii. Lipid phosphorus. The normal serum inorganic phosphorus level is 2.5-4 mg%. It is higher in children, the value being 4-6 mg%. Increase in serum phosphorus is found in: i. Chronic nephritis ii. Hypoparathyroidism Decrease in serum phosphorus is found in: i. Rickets. ii. Hyperparathyroidism iii. De Toni-Fanconi syndrome. Sulfur Sulfur is present in three amino acids. Methionine, cystine and cysteine and thus it is present in all proteins in the body. Connective tissue, skin, hair and nails are especially rich in sulfur. Also thiamine and biotin (member of vitamin B complex) and coenzyme A contain sulfur in these molecules. Diet which is adequate in protein meets the daily requirement of sulfur. Copper Total copper content in the human body is 100-150 mg. It is present in almost all the tissues of the body. Liver is the richest source of copper. Functions 1. Copper is an important constituent of certain enzymes such as, cytochromes, cytochrome oxidase, catalase, peroxidase, ascorbic acid oxidase, uricase, tyrosinase, cytosolic superoxide dimutase, etc. 2. Necessary for growth and bone formation.
    • 304 BIOCHEMISTRY FOR STUDENTS 3. Necessary for formation of myelin sheaths in the nervous systems. 4. Helps in the incorporation of iron in hemoglobin. 5. Helps in the absorption of iron from GI tract. 6. Helps in the transfer of iron from tissues to the plasma. Copper is present in the plasma as ceruloplasmin. The concentration of ceruloplasmin in plasma is 23-40 mg %. The copper containing protein in RBC is erythrocuperin, in liver it is hepatocuperin and in brain it is cerebrocuperin. Like iron, copper is conserved and reutilized by the body. Ceruloplasmin It is a blue colored copper containing metalloprotein with α2¯ globulin. It contains 8 atoms of copper bound per molecule. The reduced form is colorless. It is glycoprotein containing 8-10 units of sialic acid residues per molecule. About 90-95 % of the total copper in the plasma is present in the ceruloplasmin molecule and remainder is bound to albumin. Ceruloplasmin has oxidase activity and thereby facilitates the incorporation of ferric iron into transferrin. Vitamin C is utilized as hydrogen donor. Increased levels are seen in acute infections. Chronic conditions such as rheumatoid arthritis, cirrhosis and in postoperative stages. Malnutrition also has increased levels. Wilson disease shows decrease serum levels in both copper and ceruloplasmin. Sources Molasses, nuts, legumes, shell fish. Daily Requirement: 2-5 mg. Wilson Disease or Hepatolenticular Degeneration In Wilson’s disease, large amount of copper is deposited in liver, brain, etc. total copper content in the plasma and
    • WATER AND MINERAL METABOLISM 305 ceruloplasmin bound copper content decrease. There is an increased excretion of copper in the urine. Some time copper is also deposited in renal tubules giving rise to renal tubular degeneration. The salient features of which are glycosuria and amino aciduria. Zinc Zinc is an important constituent of pancreas. Functions 1. Zinc is a constituent of certain enzymes such as carbonic anhydrase, carboxypeptidase, alkaline phosphatase, lactate dehydrogenase, alcohol dehydrogenase, superoxide dimutase, retinene reductase, DNA and RNA polymerase. 2. Necessary for taste buds. 3. Necessary for fertility of mice. 4. Necessary for tissue repair and wound healing. 5. Necessary for protein synthesis and digestion. 6. Necessary for optimum insulin action as zinc is the integral constituent of zinc. Fluoride Functions 1. It gives strength to enamel tissues. 2. It prevents the bacterial action to the teeth. 3. Necessary for the health of teeth. Fluoride ions inhibits all those enzymes which needs Mg++ also, i.e. inhibition of glycolysis reactions. On enolase, it has the maximum inhibition activity. Addition of fluoride salts in water is known as fluoridation. Fluorosis Excessive intake of fluorine gives rise to fluorosis. Deficiencies of fluorine seem to increase the incidence of dental caries whereas excessive concentrations of fluorine in the drinking water causes corrosion of the enamel of the teeth, a process known as mottling.
    • 306 BIOCHEMISTRY FOR STUDENTS CHAPTER 15 Xenobiotics A xenobiotic is a compound that is foreign to the body. The principle classes of xenobiotics includes drugs, chemical carcinogens and other pollutants and insecticides. Metabolism of Xenobiotics 1. Phase I: The major reaction involved is hydroxylation catalyzed by cytochrome P-450. In addition to hydroxylation, deamination, dehalogenation, desulphuration are included in this phase. Cytochrome P-450 is hemoprotein. Highest concentration of which is present in water. 2. Phase II: The hydroxylated compounds produced in phase I are converted into various polar metabolites by conjugation with glucuronic acid, sulfate, acitate, glutathione or methylation. The overall purpose of two phases of metabolism of xenobiotics is to increase their water solubility and thus facilitates their excretion from the body. Liver is the main site for detoxification process though kidneys also participate to some extent. Detoxified products are excreted in urine or feces.
    • XENOBIOTICS 307 Detoxification mechanism is classified under four main types: a. Oxidation b. Hydrolysis c. Reduction d. Conjugation. Oxidation A large number of foreign compounds are destroyed in the body by oxidation. a. Primary alcohols are oxidized through aldehyde to acids Secondary alcohols are oxidized to ketones. Chloral, used as hypnotic, is oxidized to trichloroacetic acid. Primary aromatic amines undergo oxidation to corresponding acids. Sulfur of organic sulfur compounds is oxidized to sulfates. Hydrolysis Hydrolysis of esters, amide glucosides, etc. brings about significant changes in the alteration of foreign molecule in the body.
    • 308 BIOCHEMISTRY FOR STUDENTS Reduction Reduction is less common than oxidation in the body. Picric acid Picramic acid Conversion of—S—S—linkages to —SH groups. Conversion of azocompounds to amines Reduction of double bonds. Conjugation Conjugation process usually includes oxidation, reduction and hydrolysis of foreign substances although, some compounds are conjugated without previous alteration. a. Bilirubin is conjugated and excreted as the glucuronoids. Bilirubin + Glucuronic acid Bilirubin mono and diglucuronides b. Benzoic acid is conjugated with glycine and excreted as hippuric acid.
    • XENOBIOTICS 309 c. Phenylacetic acid is conjugated with glutamine to form phenylacetyl glutamine. l l d. Phenolic compounds are conjugated with sulfates.
    • 310 BIOCHEMISTRY FOR STUDENTS CHAPTER Nutrition 16 Food is the prime necessity of life. The purpose of food is to provide fuel which when broken down by oxidation gives energy required for performing vital activities. A balanced diet must provide for the maintenance of the body as well as energy requirements and where necessary, for growth and reproduction. Essential elements lost from the body excretion must be replaced. Essential nutrients are those that cannot be synthesized in adequate amounts (if at all) and are required in the diet. All the calories in the food comes only from the carbohydrates, fats, proteins and not from the vitamins, minerals and water though they are also essential components of food. The unit of energy is kilocalorie, which is 1000 times the small calorie. One calorie is defined as the amount of heat required to raise the temperature of 1 gm of water by 1°C. The calorie value of the foodstuffs can be determined by the Bomb calorimeter. Foodstuff Carbohydrates Fats Proteins Heat of combustion (Kcal/gm) In Bomb In the calorimeter organism 3.8-4.2 9.0-9.6 5.0-5.3 3.8-4.2 9.0-9.6 4.0-4.5 Corrected value 4 9 4 Caloric Value of Food The food we eat is rarely of pure carbohydrate, pure fat or pure protein, but a mixture of these.
    • NUTRITION 311 The caloric value of a mixed food depends on its composition and digestibility. The appropriate caloric value of a food can be calculated by simple formula. Caloric value = 4 (Carbohydrates + Proteins) + 9 Fats (in Kcal/gm). Respiratory Quotient (RQ) Respiratory quotient is defined as the ratio of the volume of carbon dioxide produced by the oxidation to the volume of oxygen consumed for the oxidation. RQ = Volume of carbon dioxide produced Volume of oxygen consumed RQ depends upon the type of foodstuffs being metabolized. For carbohydrates: RQ is 1 For fats: RQ is low, because fats are deficient in oxygen as compared to carbohydrates, therefore to oxidize fat, more amount of oxygen is required, e.g. the oxidation of tristearin. 2C57H110O6 + 163 O2 → 114 CO2 + 110 H2O RQ = 114 = 0.7 163 For proteins: RQ of proteins has been found to be 0.80. For proteins, it is not possible to write equation because the exact structure is not known in many cases. On a mixed diet, RQ is found to be 0.85. Very low values of RQ are found in diabetes, when large amount of glucose and ketone bodies are excreted in the urine. High values of RQ are found when carbohydrates are converted into fats and deposited in the adipose tissue. Hence, it is clear that RQ gives some indication of the type of food being metabolized.
    • 312 BIOCHEMISTRY FOR STUDENTS The respiratory quotients for various mixtures of fats and carbohydrates are given as follows: RQ Percent fats Percent carbohydrates 1 0 100 0.89 20 80 0.83 20 60 0.77 60 40 0.74 80 20 0.71 100 0 Basal Metabolic Rate (BMR) By basal metabolism, we mean the amount of energy required just to maintain the body processes when a person is at complete rest. This lowest amount of energy is called basal metabolic rate. BMR is measured in terms of heat production. The higher the rate of metabolism, the more is the heat production. To measure the BMR, the following conditions are necessary: 1. He should be in the postabsorptive state 2. He should be physically relaxed 3. He should be awake 4. He should be in an environment having the temperature 20-25°C 5. His body temperature should be normal. The following factors which influence BMR are: 1. Surface area: Larger the surface area, the higher would be BMR 2. Age: BMR decreases with age 3. Sex: Woman has lower BMR than men 4. State of nutrition: BMR is lowered in starvation and undernutrition 5. Hormonal action: BMR increases in hyperthyroidism, decreases in hypothyroidism Specific Dynamic Action (SDA) Specific dynamic action of foodstuff is defined as the extra amount of heat produced over and above the caloric value of the foodstuff when burnt inside the body.
    • NUTRITION 313 When protein equivalent to 100oC is ingested, on metabolization, its production is 130oC. This extra 30oC is due to SDA of protein which is derived at the expense of tissue metabolizing the foodstuff. Thus, the SDA of proteins is 30 percent. Similarly, the SDA of carbohydrates is 5 percent and that of fat is 13 percent. On the mixed diet, the SDA is reduced to about 10-12 percent. While calculating the energy requirement for daily activities 10 percent of the total calories is added to provide energy for the expenditure of SDA. Four basic food values contribute to nutrient essential for a complete and balanced diet. 1. Milk group: Provides high quality protein, calcium, phosphorus, riboflavin and vitamin D. 2. Meat group: Supplies protein of high biologic value, niacin, thiamine, vitamin B12, heme iron and minerals. 3. Vegetable fruit group: Supplies ascorbic acid, carotene, other water soluble vitamins, minerals and fiber (roughage). No vegetable protein has a high biologic value but when properly mixet it is possible for one protein to complement another. 4. Cereal group: High in carbohydrates for energy needs, also include vitamins, fiber and iron if the cereals are not refined. Proteins of this group do not have a high biologic value as animal protein. Biological Value of Proteins The biological value of a protein is a measure of the degree to which its nitrogen can be used for growth or maintenance of total body function. The biologic value of a dietary protein is a measure of the extent to which it satisfies the amino acid requirement for growth or the maintenance of total body function. In general, animal proteins have a high biologic value, a major exception is gelatin which lacks the essential amino acid, tryptophan and therefore has no biologic value. Vegetable
    • 314 BIOCHEMISTRY FOR STUDENTS proteins have a low biologic value because each one has a low level of one or more essential amino acid. The biological value is expressed as: = N intake - N loss in feces, sweat, urine × 100 Nintake - N loss in feces = N retained × 100 N absorbed Biological Value of Protein It is the ratio between the amount of N retained and N absorbed during specific internal. BV = Retained N × 100 Absorbed N Net Protein Utilization (NPU) NPU = Retained N × 100 Intake N NPU is a better index to assess nutritional quality and availability of a protein. Net Dietary Protein Value (NDPV) NDPV = Intake of N × 6.25 × NPU. This will assess both quality and quality of protein in the diet. The biological value of a protein signifies: 1. The presence and amounts of various essential amino acids 2. The digestibility of protein 3. Availability of digested products. In general, animal proteins have a high biological value because of its high essential amino acid contents whereas vegetable proteins have a low biological value because of low contents of one or more essential amino acids.
    • NUTRITION 315 Caloric Requirement The daily caloric requirement of the body is the sum total of basal energy demands and energy required for the additional work of the day. During growth, pregnancy, convalescence, caloric demand of the body increases and extracalories must be provided. Carbohydrates Carbohydrates are the main energy source in human nutrition. Carbohydrates supply about 55-70% of the total require- ment of the human body. The widespread use of carbohydrate rich food is due to the fact that they are relatively inexpensive. They are far cheaper than fats or proteins of the same caloric value. Carbohydrates are not strictly essential since all carbohydrates can be synthesized from dietary amino acids. The most important carbohydrate in the food of man is starch. Starch is the main constituent of all cereals. Another important carbohydrate of our food is sucrose. Glucose, fructose and other monosaccharides are present in many foods. Fats Fats are the most concentrated source of energy because of their high caloric values. Fats provide about 20-25% of the total caloric requirement of the body. Out of this, at least one percent of the fat should comprise of essential fatty acids. The purpose of essential fatty acids is: 1. Essential fatty acids lower the serum cholesterol level. 2. Essential fatty acids support good growth. In general, animal fats are poorer in essential fatty acids. It is recommended that less than 10% of total calories be obtained from saturated fatty acids and less than 10 percent be from polyunsaturated fatty acids. Proteins The main purpose of proteins is to provide tissue repair and synthesis. For energy purpose, its effect is secondary. The quality of protein depends upon its amino acids make up.
    • 316 BIOCHEMISTRY FOR STUDENTS Depending on the essential amino acids content they are classified as: 1. Complete proteins: It contains all the essential amino acids in adequate amounts and support good growth in the young experimental animals. 2. Partially complete proteins: Proteins partially lacking in one or more essential amino acids. They cannot support growth in young experimental animals but can maintain nitrogen balance. 3. Incomplete proteins: They completely lack in essential amino acids. They do not support growth and nitrogen balance. All the proteins of animal origin are complete proteins. On the average the nitrogen content of dietary protein is 100 × nitrogen content (g) = protein (g). 16 In other words 6.25 × N (g) = protein (g). 16% by weight, thus Dietary Fiber Dietary fiber is defined as those components of food that cannot be broken down by human digestive enzymes. Vegetables, wheat and most grain fibres are the best sources of water insoluble cellulose, hemicellulose and legnin. Fruits, oats, and legumes are the best source of water soluble fibers pectins, gums, etc. Another form of carbohydrate in the diet is as dietary fiber. This form of carbohydrate is not digested. They are of two types; insoluble and soluble. Soluble fiber foms a gel-like solution when combined with water. Soluble fiber slows down the passage of food through digestive tract. Oatbran, dried beans vegetable and pulp of fruits are rich sources of soluble fiber. Some type of soluble fiber appears to decrease cholesterol absorption. Insoluble fiber facilitates the movement of food though the digestive tract but it tends to bind with certain minerals during digestion and decrease their absorption. The bran that
    • NUTRITION 317 covers wheat, rice, coss and other plants whole grain is rich in insoluble fiber. Overall view of catabolic pathways of diet. Almost all dietary carbohydrates is of plant origin. Dietary carbohydrate can be divided into available (absorbable) and unavailable (fiber) varities. Some types of fibers have a high capacity for water absorption and increases the bulk of the stool. Protein-Calorie Malnutrition (PCM) Two forms of protein-calorie malnutrition are kwashiorkor and marasmus. They are seen in infants and young children in Africa and Latin America due to severe poverty or as a result of parental ignorance regarding infant feeding or child neglect. Kwashiorkor It is a disease caused by malnutrition specifically by prolonged insufficient intake of necessary proteins in infants. The infants obtain enough calories but the high carbohydrate food does not supply enough protein. The infant fail to grow. The chief characteristic of kwashiorkor are lack of appropriate cellular developments, edema, diarrhea, poor growth, low plasma protein levels, muscle wasting, edema, diarrhea and increased
    • 318 BIOCHEMISTRY FOR STUDENTS susceptibility to infection. Marasmus This occurs in infants who are weaned very early and who are fed diets which are low in calories as well as protein. Since severe malnutrition has occurred very easily in life, brain cells develop less giving rise to mental retardation. Protein calorie malnutrition can be prevented with protein rich foods. Marasmus is defined as inadequate intake of both protein and energy. Kwashiorkor is defined as inadequate intake of protein in the presence of adequate energy intake. Marasmic Kwashiorkor This occurs when the clinical features of both marasmus and kwashiorkor are present. Edema is present and body weight is less than 60 percent of standard weight. Skin and hair changes and fatty liver, characteristics of kwashiorkor are found. Vitamins Vitamins are organic compounds, essential for health and necessary for the maintenance of proper activity of the body. Vitamins are present in the naturally occurring foods and are required in very small amounts. A complete diet provides all the necessary vitamin requirements of the body. Deficiency of vitamins gives rise to various deficiency diseases. Minerals Inorganic substances do not supply energy but their presence is necessary for the maintenance of certain physiochemical conditions which are essential for life. Minerals cannot be synthesized and required minerals are therefore dietary essentials.
    • NUTRITION 319 Water as an Essential Nutrient Humans can live without oxygen for only a matter of minutes. Water is the next most essential requirement for life. Death from dehydration following within several days without fluid intake. Death can occur when there is excessive water loss because of diarrhea, etc. The massive diarrhea of cholera can kill in 8 hours. Water balance exists when fluid intake is equivalent to output. Metabolic water is produced when protons, electrons and oxygen react during metabolism. FOOD VALUES Ingredients calories Rice Atta Dal (Avg) Vegetables (Avg) Vegetables (Green) Potatoes Orange Banana Mutton Fish Liver (Goat) Egg Milk (Buffalo) Milk (Cow’s) Milk (DMS) Milk (Skimmed fresh) Cheese (Processed) Bread Cornflakes Soyabeans Cream Butter milk Sugar Honey Sago Protein gm (1) Qty (2) CHO gm gm gm (3) 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 ml ml ml ml Fat Calories (4) (5) (6) 6.5 12.1 24.1 2.0 2.0 1.6 0.6 1.2 18.5 17.0 20.0 13.3 4.3 3.2 3.2 2.5 24.1 7.8 6.8 43.2 — 0.8 — 0.3 0.2 0.4 1.7 1.2 0.2 0.4 0.1 0.1 0.4 1.33 1.3 0.3 13.3 8.8 4.1 3.5 4.1 25.1 0.7 3.8 195 36.0 1.1 — — 0.2 97.0 69.4 60.0 6.0 3.3 22.6 11.4 27.2 — 1.8 — — 5.1 4.4 4.7 4.6 6.3 51.9 88.2 22.9 — 0.5 100.0 79.5 87.1 345 341 359 34 25 97 48 116 194 87 107 173 117 67 63 48 22 248 367 432 324 15 400 320 351 In order to make the diet schedule of a person, first the total caloric requirement is estimated from the table given on next page. Out of this, one gm per kg body weight should
    • 320 BIOCHEMISTRY FOR STUDENTS be provided in the form of proteins and fats each approximately and rest all are given as carbohydrates. Caloric Requirement Activity Normal man Normal woman Growing child 1 year 2 years 3 years Calories Protein (g) Sedentary Moderate Hard work Sedentary Moderate Hard work Pregnancy 2400 2800 3900 1900 2200 3000 +300 1200 4-6 years 7-9 years 10-12 years 1600 1800 2100 Calcium (g) Iron (mg) 55 .4-.5 20 45 .4-.5 03 +10 1.0 40 .4-.5 16 to 80 17 18 20 22 23 41 The detailed menu can be prepared roughly from the table given below. Normal Balanced Diet for Adult Man Foodstuffs Cereal Pulse Leafy vegetables Other vegetables Roots and tubers Fruit Milk Fat and oil Meat/fish Egg Sugar/jaggary Peanuts/fat Sedentary work Veg Nonveg (g) (g) 400 70 100 75 75 30 200 35 — — 30 400 55 100 75 100 30 100 40 30 30 30 Moderate work Veg Nonveg (g) (g) 475 80 125 75 75 30 200 40 — — 40 475 65 125 75 100 30 100 40 30 30 40 Hard work Veg Nonveg (g) (g) 650 80 125 100 100 30 200 50 — — 55 50/30 650 65 125 100 100 30 100 50 30 30 55 50/30
    • NUTRITION 321 Normal Balanced Diet for Adult Woman Foodstuffs Sedentary work Veg Nonveg (g) (g) Moderate work Veg Nonveg (g) (g) Cereal Pulse Leafy vegetables Other vegetables Roots and tubers Fruit Milk Fats and oil Meat/fish Egg Sugar/jaggary Peanuts/fat 300 60 125 75 50 30 200 30 — — 30 — 250 70 125 75 75 30 200 35 — 30 30 — 200 45 125 75 50 30 100 35 30 30 30 — 353 55 125 75 75 30 100 45 30 — 30 — Hard work Veg Nonveg (g) (g) 475 70 125 100 100 30 200 40 — 30 40 40/25 475 55 125 100 100 30 100 45 30 — 40 40/25 For Growing Child Foodstuffs 1 – 3 yrs Veg Nonveg (g) (g) Cereal 150 Pulse 50 Green leafy veg 50 Roots and tubers 30 Fruit 50 Milk 300 Meat/fish/egg — Sugar and jaggary 30 150 40 50 30 50 200 30 30 4-6 yrs 7-9 yrs 10-12 yrs Veg Non- Veg Non- Veg Nonveg veg veg (g) (g) (g) (g) (g) (g) 200 60 75 50 50 250 — 40 200 50 75 50 50 200 30 40 250 250 70 60 75 85 50 50 50 50 250 200 — 30 50 50 420 70 100 75 50 250 — 50 420 60 100 75 50 200 30 50 Balanced Diet for Pregnant Lady Foodstuffs Cereal Pulse Roots and tubers Sedentary work Veg Nonveg (g) (g) 350 60 50 340 45 50 Moderate work Veg Nonveg (g) (g) Hard work Veg Nonveg (g) (g) 400 70 75 525 70 100 400 55 75 525 55 100 Contd...
    • 322 BIOCHEMISTRY FOR STUDENTS Contd... Foodstuffs Sedentary work Veg Nonveg (g) (g) Moderate work Veg Nonveg (g) (g) Green leafy veg Other veg Fruit Milk Fats/oil Sugar/jaggary Meat/fish Egg Peanuts/fat 150 75 30 325 30 40 — — — 150 75 30 325 35 40 — 30 — 150 75 30 225 35 40 30 30 — 150 75 30 225 50 50 30 — — Hard work Veg Nonveg (g) (g) 150 100 30 225 40 50 — 30 40/25 150 100 30 225 45 50 30 40 40/55 1500 CALORIES DIABETIC DIET CHART Total Food for Day Foodstuffs (1) Wheat flour (unsifted) Bread Milk (DMS) Green vegetables Fruit Dal/lean meat, fish, chicken Cottage cheese/egg Salted biscuits Ghee/oil Butter MEAL PLAN Breakfast Milk Butter Veg gm Nonveg gm (2) (3) 125 50 400 500 125 50 125 50 400 500 125 150 35 15 10 5 1 15 10 5 1 cup 1 tea spoon Household measures (4) 5 small chapattis 2 slices 2 small glasses 1 small 2 katori cooked dal 1½ serving of mutton 2 tea spoons 1 tea spoon Protein-65 gm (approx) Fiber-11.5 gm Bread : 2 slices Cheese/egg : 1 Contd...
    • NUTRITION 323 Contd... Foodstuffs Veg gm (2) (1) Mid morning Fruit Lunch Chapatti Nonveg gm (3) 1 3 (small) Dal 1 katori Tea Tea without sugar 1-2 Biscuits Dinner Chapattis: Cooked veg Salad Curd Dal Bedtime Milk Household measures (4) Tea: 1 cup (without sugar) Cooked vegetable and salad Oil/ghee : 1 tea spoon 2 1 katori 1 katori 1 katori 1 cup 2000 Calories Diabetic Diet (Vegetarian/Nonvegetarian) Carbohydrates = 280 gm, Fats = 64 gm, Proteins = 74 gm Foodstuffs Atta Vegetables Milk Curd Cheese Egg Dal Meat/fish/chicken Bread Ghee Butter Fruit Quantity in gm 200 450 400 ml 200 25 1 No. 30 50/120/100 100 15 15 1 No. Contd...
    • 324 BIOCHEMISTRY FOR STUDENTS Contd... Foodstuffs Quantity in gm Condiments Salt Tea leaves 10 15 7 Breakfast Milk Bread Butter Cheese Egg 150 50 (2 slices) 8 25 1 No. Mid morning Tea of coffee milk 50 Lunch Atta Vegetable Dal Meat/fish/chicken Curd Ghee 100 250 30 55/120/100 100 5 Evening Tea of coffee milk Bread Butter 50 50 (2 slices) 7 Dinner Atta Vegetable Curd Fruit Ghee Milk 100 200 100 1 No. 5 150
    • NUTRITION 325 1800 Calories Low Cholesterol Diet (Vegetarian/Nonvegetarian) Carbohydrate = 312 gm, Fat = 30.6 gm, Proteins = 70 gm Foodstuff Quantity in gm Skimmed milk Curd (Skimmed) Bread Atta or rice Dal (for veg) (for nonveg) Fruit Vegetables Oil Sugar Salt Tea leaves Fish/chicken (for nonveg) 600 ml 200 100 150 50 25 2 No. 500 25 25 10 7 100/80 Breakfast Skimmed milk Fruit 250 ml + sugar 1 No. Mid morning Tea or coffee milk 50 ml + sugar Lunch Atta or rice Fish/chicken Vegetables Curd Fruit Oil 70 100/80 250 100 1 Evening Tea or coffee milk Bread (50 ml + sugar) 50 gm Dinner Atta or rice Vegetables Curd Oil 75 250 100 100 Bedtime Skimmed milk 250 ml
    • 326 BIOCHEMISTRY FOR STUDENTS CHAPTER 17 Organ Function Tests LIVER FUNCTION TESTS The various function tests that are in existence depending upon the array of activities by the liver are enumerated below: Metabolic Function A. Carbohydrate metabolism: a. Galactose tolerance tests (oral and intravenous). b. Fructose tolerance tests. B. Lipid metabolism: a. Serum cholesterol: Free and esterified form of cholesterol estimations. b. Estimation of fecal fats. C. Protein metabolism: a. Total proteins, A/G ratio, prothrombin time. b. Flocculation tests: Thymol turbidity test, zinc sulfate test, colloidal gold test, cephalin cholesterol flocculation test, formal gap test. Detoxification and Protective Functions A. Conversion of ammonia to urea: Estimation of blood urea and blood ammonia. B. Formation of bilirubin diglucuronide: Estimation of serum bilirubin (direct and indirect), icteric index, urinary estimation of bilirubin and urobilinogen. C. Hippuric acid test. Excretory Functions Bromsulphalein (BSP) test.
    • ORGAN FUNCTION TESTS 327 Storage Functions A. B. C. D. Glycogen estimation. Lipid estimation. Estimation of vitamin A, D, and B12. Estimation of serum iron and serum iron binding capacity. Hematologic Function Estimation of prothrombin time and factors VII, IX and X. Cellular Structural Studies Liver biopsy: The importance of liver function tests are: 1. To assess the severity of liver damage. 2. To differentiate different types of jaundice. 3. To find out the presence of latent liver diseases. There are hosts of test to evaluate the functions of liver but those that are commonly employed have got significance for assessing the conditions of patients. The first group of tests regarding the secretory, excretory, and enzymatic functions are: Serum bilirubin estimation, bilirubin and urobilinogen in urine, BSP excretion test, serum alkaline phosphatase estimation and SGPT. The second group meant for assessing the protein synthetic functions are: Total proteins estimation, A/G ratio and prothrombin time. The final and the third group that are meant for lipid metabolic functions are: Estimation of serum cholesterol and determination of free and esterified cholesterol ratio. Estimation of Serum Bilirubin Bilirubin is estimated by van den Bergh’s reaction involving Diazo reagent. van den Bergh’s reaction consists of two parts, the direct and indirect reactions. A. Direct reaction a. Immediate direct reaction: An immediate development of violent color in 10/30 seconds.
    • 328 BIOCHEMISTRY FOR STUDENTS b. Delayed direct reaction: In which color appears from five to thirty minutes and then develops slowly to a maximum. No direct reaction may be observed. B. Indirect reaction, bilirubin bound to albumin is estimated. Interpretations Normal serum bilirubin level is 0.2-0.6 mg %. Immediate direct reaction—in obstructive jaundice. Indirect/Delayed direct reaction—in hepatic jaundice. Direct reaction—in hepatic jaundice. The three types of jaundice has been discussed in Chapter 5. Determination of serum bilirubin gives a measure of the intensity of the jaundice. Higher values are found in obstructive jaundice than in hemolytic jaundice. Estimation of Urine Bilirubin Urine bilirubin is qualitatively estimated by Fouchet reagent and interpretations. Estimation of Urine Urobilinogen Urine urobilinogen is qualitatively estimated by Ehrlich reagent and interpretations. Bromsulphalein Excretion Test (BSP Test) A measured amount of dye is injected intravenously. The liver rapidly removes the dye and excretes in the bile. If the liver function is impaired, the excretion is delayed and larger proportion of dye remains in the serum. It is very sensitive test and is most useful in liver cell damage without jaundice, in cirrhosis and chronic hepatitis. In healthy adults not more than 5% of the dye should remain in blood, but the bulk of dye is removed in twenty five minutes. In hepatic diseases, cirrhosis, 40–50% of dye retention takes place. Also abnormal retention of dye in hepatocellular or obstructive jaundice takes place.
    • ORGAN FUNCTION TESTS 329 Estimation of Serum Alkaline Phosphatase Normal level is 3-12 King-Armstrong units or 3-4 Bodansky units. Increased level of alkaline phosphatase is found in postnecrotic disease, cirrhosis, carcinoma of liver, obstructive jaundice, hepatocellular jaundice, bone disease and may go up to 200 KA units. Serum alkaline phosphatase activity is high in obstructive jaundice but remains unchanged in hemolytic jaundice. So estimation of this activity may be of help to identify the type of jaundice. Estimation of Serum Glutamic Pyruvic Transaminase (SGPT): Alanine Transaminase (ALT) Normal SGPT level is 9-39 IU. Increase of SGPT activity is a more specific indicator of cell damage than that of SGOT. Increased levels of SGPT are common in hepatocellular damage and obstructive jaundice. Determination of Serum Proteins and A/G Ratio Serum proteins estimation yields most useful information in chronic liver disease. The liver is the site of albumin, fibrinogen and some of α- and β-globulins synthesis. The normal serum protein level is 6.0-8.0 g% Albumin level is 3.5-5.5 g% Globulin level is 2.0-3.5 g% The normal A/G ratio is 1.2:1. In advanced liver disease, the albumin is decreased and globulin is increased so that albumin-globulin ratio is reversed. Serum proteins are decreased in malnutrition, liver damage. Low serum albumin is found in severe liver damage due to the impaired ability of the liver to form albumin. Prothrombin Time Prothrombin time is the time required for clotting to take place in citrated plasma to which optimum amounts of thromboplastin and calcium has been added.
    • 330 BIOCHEMISTRY FOR STUDENTS Prothrombin is formed by the liver cells, vitamin K being required. When bile salts are not present in the intestine, the absorption of vitamin K from the intestine is impaired. Prothrombin time is used in liver disease and jaundice. In jaundice and liver disease, the prothrombin time is prolonged. The normal prothrombin time is 16-18 seconds. Estimation of Total and Esterified Cholesterol Liver synthesizes, esterifies and excrete cholesterol into bile. So cholesterol level is affected in liver disease. Normal cholesterol level is 150-250 mg%. Esterified form is 60-70% of the total. Cholesterol level is decreased in hepatitis, cirrhosis, hyperthyroidism, malabsorption syndrome in severe wasting in acute infections, pernicious anemia, etc. Cholesterol level is increased in obstructive jaundice, intrahepatic obstruction, myxedema, lipid storage disease, atherosclerosis, nephrotic syndrome, diabetes mellitus, xanthomatosis. Liver Biopsy Histophathological studies of liver biopsy reveals various pathological states of liver cells. RENAL FUNCTION TESTS Kidney plays an important role in the maintenance of acid base-balance and volume of water in the body. It serves an important function of excretion of products of metabolism and other harmful substances. Renal function tests are done to assess the functional capacity of kidney. The aims of renal function tests in clinical biochemistry are to detect impairment of renal function as early as possible. The kidney regulates the chemical composition of body fluids by selective filtration of blood through the glomerular basement membrane.
    • ORGAN FUNCTION TESTS 331 The movement of molecules through the membrane is dependent upon their size, plasma concentration and electrical change. In healthy kidneys, most proteins are too large to cross the basement membrane. Damage to glomerular basement membrane in the kidney can alter its permeability. This enables large protein molecules such as albumin to pass through the membrane into the urine resulting in proteinuria. The size of protein molecule detected in the urine may give an indication of underlying kidney dysfunction causing proteinuria. Following tests are generally done to assess the renal function: 1. Concentration test (specific gravity test) 2. Dilution test 3. Phenolsulfonphthalein (PSP) test 4. Urea clearance test 5. Inulin clearance test 6. Creatinine clearance test 7. Renal blood flow. Microalbumin Detection of sustained elevations of albumin (>20 μg/min) in the urine indicates kidney damage and if left untreated can result in irreversible damage. Microalbuminuria (20-200 μg/ min) is an early marker of renal damage and enables preventive measures to be taken. Measurement of urinary albumin levels is an important diagnostic test in conditions associated with increased risk of renal failures, e.g. diabetes, essential hypertension, nondiabetic renal disease and pregnancy. Concentration Test This is designed to test the concentrating power of the kidneys. The capacity of the kidney to concentrate urine is one of the most sensitive tests for early loss of function. It is also the simplest test to perform since it does not require any laboratory facilities.
    • 332 BIOCHEMISTRY FOR STUDENTS When the kidney loses its capacity to do osmotic work, the urinary solids must be excreted in more dilute solution, amount of solids to be excreted, greater volume is required to accommodate the same. The specific gravity of urine, which in the normal individual fluctuates widely in response to fluid intake becomes confined within normal limits until it is fixed at approximately 1010. These changes are usually expressed as polyuria. The day night ratio of urine volume, normally 3-4 to 1 tends towards 1:1 ratio. The advantage of this test is that it is useful for the detection of renal defect where the blood urea is normal. Dilution Test In addition to the loss in the power of the kidneys to produce concentrated urine, there is also an impairment in its ability to excrete dilute urines. This later fact has been used in dilution test. In this test no water is taken after midnight, the bladder is emptied at 7 AM and the patient is given 120 ml of water to drink in 30 minutes. Urine samples are collected hourly for next four hours, i.e. at 8, 9, 10 and 11 AM. The volume and specific gravity of each specimens are measured. In the normal individual, approximately 1200 ml of urine will be excreted during this time and the specific gravity of at least one specimen should fall to 1003 or below. With impaired renal function such a low specific gravity is not reached and small volumes may be less than 100 ml and the specific gravity of urine may not fall below 1010. Phenolsulfonphthalein (PSP) Test PSP test indicates a general loss of nephron function. This test consists of intramuscular injection of solution of PSP, a dye that is eliminated only by the kidneys, the amount of this dye in urine can be estimated colorimetrically. The time of its first disappearance in the urine and the quantity eliminated within a definite period are taken as a measure of the functional capacity of the kidneys. The test is harmless, simple and for general purposes the most satisfactory of the functional tests.
    • ORGAN FUNCTION TESTS 333 In the normal individuals 25% or more of the dye will be excreted during the first 15 minutes after intravenous injection and not less than 70% will be excreted during the two-hour period. The rate at which the dye appears in the urine depends both on the renal blood flow and the action of tubular epithelium in removing the dye from the blood. Percentage of impairment of PSP excretion is as follows: a. Slight: 52-40% excretion of injected dye b. Moderate: 39-25% excretion of injected dye c. Marked: 20-11% excretion of injected dye. Urea Clearance Test Urea clearance is defined as the number of ml of blood which contains the urea excreted in a minute by the kidneys. Urea clearance = where mg urea excreted per minute UV = mg urea per ml of blood B U = mg urea per ml of urine B = mg urea per ml of blood V = ml urine excreted per minute. The clearance is a ratio of the urinary excretion to the average blood level determinated simultaneously. Such a ratio is correlated more directly with the progress of kidney disease and shows a deviation from normal in early renal damage. Normal urea clearance on average is 75 ml per minute when rate of excretion of urine is 2 ml or more per minute. This is maximum urea clearance. Maximum urea clearance = = observed urea clearance × 100 average normal standard urea clearance UV × 100 = 100 UV B 75 B 75 If the quantity of urine excreted is less than 2 ml per minute then the clearance is found to be 54 ml per minute and this clearance is called standard urea clearance.
    • 334 BIOCHEMISTRY FOR STUDENTS Standard urea clearance = observed urea clearance × 100 average normal standard urea clearance UV B × 100 = 100 U V = 54 B 54 V Grading renal function on the basis of urea clearance value is as follows: Urea clearance Renal function Over 70 Normal 70-40 Mild deficit 40-20 Moderate Below 20 Severe deficit Below 5 Coma Inulin Clearance Test This test is done to find the glomerulus filtration rate. Inulin is filtrated by the glomerulus but it is neither secreted nor absorbed by tubules. Inulin is given subcutaneously or by intravenous infusion. The amount of inulin excreted in each minute (U in V) is equal to the amount filtered by the glomeruli. The concentration of inulin in the glomerular filtrate is equal to that in the plasma. So the clearance value of inulin is same as glomerular filtration rate. Normal rate is 110 to 150 ml per minute. Glomerular filtration rate (ml per minute) = Inulin clearance = UV ⎞ ⎛ C = ⎜C = ⎟ B ⎠ ⎝ mg inulin per 100 ml urine × ml urine passed per minute mg inulin per 100 ml plasma Creatinine Clearance Test Creatinine clearance test is also done to find out the glomerular filtration rate but the situation is complicated in human beings
    • ORGAN FUNCTION TESTS 335 because a portion of creatinine is secreted by tubules. This portion rises unpredictably when failure of filtration occurs in renal disease. Normal value for creatinine clearance is 95-105 ml per minute. Renal Blood Flow Renal blood flow can be determined by using para-amino hippuric acid, which at low blood concentration is removed almost completely by tubular excretion in a single circulation through kidney. Effective renal blood flow as calculated from this type of clearance procedure is about 1000 to 1150 ml per minute or expressed as plasma flow about 600 to 700 ml per minute. PANCREATIC FUNCTION TEST The important constituents of pancreatic juice are: 1. Enzymes a. Carbohydrates splitting enzymes such as α- and β-amylases. b. Proteolytic enzymes consist of trypsinogen, chymotrypsinogen and peptidase. They are converted to trypsin, chymotrypsin and carboxypeptidase respectively. Other proteolytic enzymes include deoxyribonuclease (DNAase) and ribonuclease (RNAase). c. Fat splitting lipase acts on neutral fats and phospholipids liberating fatty acids and glycerol. 2. Bicarbonate. 3. Water. Bicarbonate and water are the major components of pancreatic juice. Daily volume varies from 1500-3000 ml. The bicarbonate and the fluid is dependent on the hormone secretion. This hormone is secreted mainly as a result of stimulation by HCl. Enzyme secretion in the pancreatic juice is not under the control of secretion but of another hormone pancreozymin.
    • 336 BIOCHEMISTRY FOR STUDENTS The test most commonly employed in pancreatic dysfunction are: 1. Determination of enzymes in serum and urine. The enzyme studies are amylase and lipase. 2. Examination of stool. Determination of Serum Amylase Serum amylase is estimated by two methods. Sacchrometric Method Serum is incubated with a starch solution and the amount of reducing substances present is determined before and after the incubation. The difference gives a measure of amylolytic activity of the serum. Somogyi’s Iodine Test The time required for complete digestion of a certain amount of starch is determined by periodic testing with iodine. Normal serum amylase value is 80-200 Somogyi units per 100 ml. It is increased in acute pancreatitis, as high as 1000 Somogyi units per 1,000 ml and even more. Low serum amylase has been found in abscess of liver, acute hepatocellular damage, cirrhosis of liver, cholecystitis. Amylase is usually absent in newborn. Urinary Amylase Variation in urinary amylase reflects alteration in serum amylase so long as kidneys are functioning normal. In renal disease, serum amylase may be increased and urine amylase is low. Normal value is 1-3 ml/minutes. It is elevated in acute pancreatitis, obstruction of pancreatic duct and in cases of pancreatic carcinoma. Determination of Serum Lipase Serum lipase hydrolyzes the esters of long chain fatty acids containing 8–18 carbon atoms.
    • ORGAN FUNCTION TESTS 337 Serum lipase parallels change in amylase but rises later and lasts longer. The increase is more pronounced. Serum lipase value is elevated in all conditions in which amylase is elevated. Serum lipase value is more informative than amylase in pancreatic cancer. Urinary Lipase Lipase is excreted by the kidneys and can be demonstrated in the urine. It is elevated in: 1. Hemorrhagic pancreatitis. 2. Some cases of renal impairment. Stool Examination i. Fat in stool ii. Nitrogen in stool. Fat in Stool Ingested fat is normally split by pancreatic lipase into fatty acids and glycerol and the products of hydrolysis are absorbed by intestinal tract. Therefore, in stool, the neutral fat, free fatty acids and soaps are relatively 6 gm/24 hr. Increase in neutral fat (steatorrhea) to 11% represents deficiency of fat splitting enzyme. Nitrogen (Protein in Stool) a. Clastro colic fistula b. Obstructive jaundice Nitrogen (protein in stool) Total fecal nitrogen is 0.25-2 g/day. Increased nitrogen content is found in pancreatic insufficiency. In addition to these, various other tests such as provocative test, secretion test. Pancreozymin test, vitamin A tolerance test and fat absorption test are also helpful in pancreatic dysfunction.
    • 338 BIOCHEMISTRY FOR STUDENTS GASTROINTESTINAL (GIT) FUNCTION TEST D-xylose Excretion Test Function test for: GIT Condition and Limitations Gives true results only when kidneys are normal. Method The patient is given 5 gm of D-xylose orally, urine samples are collected for the next 5 hours and a blood sample is collected after 2 hours. Xylose is absorbed in the intestine, reaches liver and excreted by kidneys. The blood level reaches around 35 mg% after excretes arount 1.5 gm of xylose in urine with in five hours. Result Lower values of blood and urine indicate malabsorption due to mucosal damage.
    • IMMUNOLOGY 339 CHAPTER 18 Immunology INTRODUCTION The main function of immune system is to prevent or limit infections by microorganisms such as bacteria, viruses, fungi and parasites. Protection is provided primarily by cellmediated and antibody mediated (humoral) arms of immune system. Two other major components of immune system are complement and phagocytosis. Cell mediated arm consists of T lymphocytes (e.g. helper T cell and cytotoxic T cell) and humoral arm consits of B lymphocytes. B lymphocytes when activated convert into plasma cells which in turn produce antibodies. The main functions of antibodies are to: 1. Opsonize bacteria 2. Neutralize toxins and virus cell mediated immunity: (i) inhibits organisms sich as fungi, parasites and intercellular bacteria, it also kills virus infected cells and tumor cells, (ii) regulates antibody response. Natural and Acquired Immunity Natural immunue is resistance not acquired through contact with an antigen. It is nonspecific this immunity does not improve after exposure to organism in contrast to acquired immunity, also natural immune response have no memory in contrast to long-term memory of acquired immunity. Active and Passive Immunity Active immunity is resistance induced after contact with foreign antigens, e.g. microorganism. In this, the host actively
    • 340 BIOCHEMISTRY FOR STUDENTS produces immune response consisting of antibodies and activatedd helper and cytotoxic lymphocytes. Main advantage of acute immunity is that response is long-term but major disadvantage is its slow onset. Passive immunity is resistance-based on antibodies performed in another host. Performed antibodies against certain viruses (e.g. rabies and hepatitis A and B) can be injected to limit viral multiplication other forms of passive immunity are IgG passed from mother to fetus during pregnancy and IgA passed from mother to newborn during breastfeeding. Antigens Antigens are molecules that react with antibodies whereas immunogens are molecules that induces an immune response. In most cases, antigens are immunogens, but there are certain exceptions, e.g. haptens. Haptens are molecules that are not immunogenic but can reach with specific antibody. Haptens are usually small molecules and are not protein in nature, e.g. penicillins, catechol.
    • IMMUNOLOGY 341 as A. B. C. D. Features of molecules that determine immunogenicity are follows: Molecular weight Complexity of chemical structure Foreigness Genetic constitution of host. Origin of Immune Cells During postnatal life, stem cells reside in bone marrow and differentiate into erythroid, myeloid and lymphoid series. The latter give rise to 2 populations in ratio of 3:1 as T and Blymphocytes. Thymic Selection Negative Selection CD-4+, CD-8+ cells, bearing antigen receptors for self-proteins are killed by programmed cell death called apoptosis. This refer to clonal deletion.
    • 342 BIOCHEMISTRY FOR STUDENTS Positive Selection CD-4+, CD-8+ cells, bearing antigen receptors that do not react with self MHC proteins are also killed. These two processes produce cells that are selected for their ability to react both with foreign antigens via antigen receptors and with self MHC proteins. T Cells Within the thymus, T cells differentiates under the influence of thymic hormones (thymosine) to express surface glycoproteins, e.g. CD-3, CD-4, CD-8 (CD-cluster of differentiation). CD-3 is present on all T cells. But CD-4 and CD-8 are present on different populations of T cells. Activation of T Cells Activation of helper T cells requires that they recognize a complex on the surface of antigen presenting cells (APC) like macrophages. B cells dendritic cells, Langerhans cells within the cytoplasm of APC’s let say, macrophage, foreign protein is cleaved into small peptic. These small peptides are then associated wtih MHC II proteins. This complex of small peptides and MHC II molecules are transported to cell surface of macrophage, where this complex is presented to receptors on CD-4+ helper T cells. Similar events occur within a virus infected cell, cleaved viral peptide associates with a class I MHC molecule and complex is transported to the surface and viral antigen is presented to the receptor on CD-8+ cytotoxic cell. Further steps in activation include the following: 1. Interaction of antigen with TCR (that is present on T cells) specific for that antigen. 2. IL-1 produces by macrophages is also necessary for activation. 3. For full activation of helper T cells an additional “costimulatory signal” is required, i.e. B protein present on surface of APC must interact with CD-28 protein on helper T cell.
    • IMMUNOLOGY 343 Generally speaking class I MHC proteins present endogenously synthesized antigens, e.g. viral proteins whereas class II MHC proteins present the antigens of extracellular microorganisms that have been phagocytowed, e.g. bacterial proteins. There are two subpopulations of helper T cells. These are named as Th1 and Th2. These two types of cells are orginated from Th-O cells also known as native helper T cell. This gives use to different types under influence of different interleukins. FUNCTIONS OF T CELLS Effector Functions of T Cells 1. Delayed hypersensitivity against intracellular bacteria like mycobacterium tuberculosis. It is shown by TH cells. 2. Cytotoxicity: By it body destroys virus infected cells, tumor cells and allograft rejection. T cells take part in this by releasing performs. 3. Antibody dependent cellular cytotoxicity (ADCC). Antibody bound to surface of infected cell is recognized by IgG receptors on the surface of macrophages and NK cells.
    • 344 BIOCHEMISTRY FOR STUDENTS
    • IMMUNOLOGY 345 Regulatory Function of T cells 1. Antibody production: Helper T cells secrete IL-4 (B cell growth factor) and IL-5 (B cell differentiation factor) which are responsible for production of antibodies. 2. Cell mediated immunity: Helper T cells also secrete IL-2 (T cell growth factor) which acts on the same cell at IL-2 receptor, thus increasing the cell mediated immunity. B Cells B cells constitute about 30% of circulating pool of small lymphocytes and their life-span is short. B cells precursors after originating from fetal cover migrate to bone marrow and they do not require thymus for maturation. There are two stages of development of B cells: 1. Antigen independent 2. Antigen dependent. Antigen independent phase include: Stem cells → pre B cells → B cells. Antigen depedent phase includes: Activates B cells → plasma cells. Activation of B Cells 1. When antigen binds to surface IgM present on B cell surface, endocytosis of that takes place this antigen is processed and appear on the surface again in conjugation with class II MHC proteins. This complex is recognized by helper T cells and this produces IL-4, IL-5 which are B cell growth factor and differentiation factor respectively. 2. Costimulatory signal is necessary CD-28 on T cell must interact with B-7 on B cell. This signal stimulate T cell to produce IL-2. 3. CD-40 L on T cell must interact with CD-40 on B cell. This interaction is required for class switching from IgM to IgG. Macrophages These are derived from bone marrow and exist as both free and fixed.
    • 346 BIOCHEMISTRY FOR STUDENTS a. Free macrophages: Wandering macrophages, e.g. monocytes. b. Fixed macrophages, e.g. Kupffer cells (liver) Langerhans cells (skin) Neurological cells (brain) Dust cells (lung). Macrophages migrate to the site of inflammation under the influence of Csa, Anaphylatoxin. There are three main functions of macrophages: 1. Phagocytosis 2. Antigen presentation 3. Cytokine production, e.g. IL-1, TNF. Natural Killer Cells 1. These are called so because they are active without prior exposure to virus. 2. These are nonspecific. 3. These kill without antibodies but presence of antibodies enhance its efficiency. 4. They kill virus infected cells and tumor cells by secreting performs and these are similar to cytotoxic T cells. 5. IL-12 and gamma interferon are potent activator of NK cells. 6. NK cells are 5-10% of peripheral lymphocytes. 7. NK cells have no memory. No T cell receptor (TCR), no requirement of MHC proteins and no passage through thymus for maturation. Differences between T cells and B cells Characters 1. 2. 3. 4. 5. 6. 7. 8. IgM on surface CD-3 on surface Immunoglobulin synthesis Regular of antibody synethsis IL-2, 4, 5, gamma interferon synthesis Effector of cell mediated immunity Maturation in thymus Maturation in bursa equivalent T cells B cells ✗ ✓ ✗ ✓ ✓ ✓ ✓ ✗ ✓ ✗ ✓ ✗ ✗ ✗ ✗ ✓
    • IMMUNOLOGY 347 Brief Description about Cytokines 1. Interleukin 1 Source: Macrophages Function: • activator of Th cells • endogenous pyrogen 2. Interleukin 2 Source: Th1 subset of helper T cell Function: T cell growth factor (TCGF) 3. Interleukin 4 Source: Th2 subset of helper T cells Function: B cell growth factor (BCGF) 4. Interleukin 5 Source: Th2 subset of helper T cells Function: B cell differentiation factor (BCDF) 5. Gamma interferon Source: Th1 subset of helper T cells Function: Stimulate phagocytosis and killing by macrophages and NK cells 6. Tumor necrosis factor (TNF) Source: Macrophages Function: Causes necrosis of tumors 7. Transforming growth factor β (TGF-β) Source: T cells, B cells, macrophages Function: Anticytokine actions. Antibodies Antibodies are globulin proteins (immunoglobulins) that react specifically with the antigen that stimulated their production. Antibodies are gamma globulins. Structure Immunoglobulins are glycoproteins made up of light (L) and heavy (H) polypeptide chains. Molecular weight of H and L chains are respectively 50,000 and 25,000.
    • 348 BIOCHEMISTRY FOR STUDENTS Shape: Y shaped Consists of: Four polypeptide chains • Two heavy chains • Two light chains Linked by: Disulphide bond. Specialties 1. An individual antibody molecule always consists of identical H and identical L chains. 2. L and H chains are subdivided into variable and constant regions: L chain: H chain: Constant Variable Constant Variable region region region region — — — — CL VL CH VH 3. L chains are of two types kappa (κ) and lambda (λ). 4. H chains are of five types gamma (γ), alpha (α), mu (μ), delta (δ) and epsilon (ε). 5. Variable regions are responsible for the binding of antigens whereas constant regions are responsible for: a. Complement activation b. Various biologic functions. 6. IgG and IgA have three Ch domains and IgM and IgE have four. Effect of Proteolytic Enzymes 1. Papain: It breaks the immunoglobulin molecules in hinge region, above disulphide interchain bond. Thus producing two identical Fab fragments and one Fc fragments. 2. Pepsin: It breaks the immunoglobulin molecules at hinge region below the interchain disulphide bond, thus, producing one Fab fragment and many Fc fragment.
    • IMMUNOLOGY 349 Isotype, Allotype, Idiotype Isotype Isotypes are defined by antigenic differences in the their constant regions like IgG, IgA, IgD, IgM, IgE are different isotype, also IgG1, G2, G3, G4 and IgA1, A2 are different isotypes. Allotype Allotypes are additional antigenic features immunoglobulins that vary among individuals. e.g. γ H chain contains an allotype called Gm k L chain contains an allotype called Inv. Idiotypes Idiotypes due to variation in variable region of H and L chain individual CDR which differ with each other are known as idiotypes.
    • α γ + – – + – – 5. H chain 6. Placental passage 7. Allergic response 8. Found in secretions 9. Opsonization 10. Antigen receptor on B cell 11. J chain +/– – – + – – Mono or dimer – Monomer Lowest 3. Molecular weight _ 15% IgA 4. Structure Highest Highest (75%) IgG 2. Serum conc 1. Percentage of total Property + + + – – – μ Pentamer Highest _ 9% IgM Properties of human immunoglobulins – + – – – – δ Monomer – _ 0.2% IgD – – – – + – ε Monomer – Lowest Lowest (0.004%) IgE 350 BIOCHEMISTRY FOR STUDENTS
    • IMMUNOLOGY 351 About idiotypes, immunological network are made by Jerne, which consists of idiotype, anti-idiotype. Antibody Diversity This is due to: 1. Multigene organization 2. Combinatorial joining 3. Junctional flexibility 4. Somatic hypermutation. Antibody Class Switch (IL-4) Mature B cells 1st express both IgM and IgD following stimulation. Other isotypes are also produced so, it is clear that there is switch from IgM and IgD to other classes. • Eight types of genes are present in definite sequence in C chain from 5’-3’, • Each of these C genes, have a switch site towards 5’ end which are 1-2 k base pair length but not present at 5’ end of Cδ So, this leads to expression of IgM and IgD together rest are switched from this. Hybridoma and Monoclonal Antibodies Hybridoma is a hybrid cell capable of producing monoclonal antibodies. When one clone of antibody producing cells secrete a particular type of antibody against a particular antigenic determinant. It is called as monoclonal antibodies. • Kohler and Milstein in 1975, first produced monoclonal antibodies from hybridoma cells. They showed that cultured splenic cells from mouse immunized with specific antigen can be fused with that of cultured mouse myeloma
    • 352 BIOCHEMISTRY FOR STUDENTS cells in 5:1 ratio. The hybrid cells so produced will remain immortal in culture like myeloma cell and produce monoclonal antibodies like immunized splenic cells. Uses 1. Diagnostic purpose: • Bacterial viral diseases • Blood grouping. 2. Therapeutics: • Anticancer therapy • Immunosuppression in organ transplantation. MHC Molecules • • • • • Also known as HLA complex Discovered by Dausset Present in all nucleated cells Coded by genes present on chromosome 6 3 groups Class I 2000 kb ABC Class II 1000 kb DP, DQ, DR Class III 1000 kb • Class I codes protein for antigen recognition and binding by T cells • Class II codes protein for antigen recognition and binding by Th cells • Class III: Do not participate in major histocompatibility but some other products like tumor necrosis factor, heat shock protein, complement component, etc. Class I Molecule • Long glycoprotein polypeptide chain. 3 regions in chain α1 α2 α3 and associated with other molecule β2 microglobulin, i.e. non MHC molecule Ag binding site is present between α1 and α2. • This codes protein for antigen recognition and binding by cytotoxic T cells • This only expresses endogenous antigen like that of viral antigens.
    • IMMUNOLOGY 353 Class II Molecule • It is heterodimer consisting of α1 α2 β1 β2 Ag binding site is present between α1 and β1 • This codes protein for antigen recognition and binding by Helper T cells • This only expresses exogeneous antigen like that of bacterial antigens. *Class I includes 3 multiple gene loci A, B, C while Class II includes DP, DQ, DR. MHC Polymorphism • Need: Large variety of antigens to be present so wide range of antigens binding sites on these molecules • This is due to following reasons: • Multiple gene loci • Multiple alleles for a locus • Codominant expression. Complement System • The complement system consists of approximately 20 proteins that are present in normal human serum. The complement refers to ability to those proteins to complement, i.e. augment, the effecsts of other components of immune system. So, this is important component of our innate host defences. • There are three main effects of component of complement: 1. Lysis of bacteria, tumors 2. Generation of mediators 3. Opsonization. • Complement system is heat labile while antibodies are heat stable. • This consists of C1 + Ca C1 is formed by C1q C1r C1s.
    • 354 BIOCHEMISTRY FOR STUDENTS Activation of Complement There are two pathways of activation of complement: 1. Classical pathway 2. Alternate pathway. Classical Pathway This is due to Ag-Ab complex CH2 domain of Fc portion of Ab remains hidden when it is exposed due to binding of Ag. This activate complement Ag-Ab complex ↓ activates C1q → C1r → C1s ↓ ↓ Due to active esterase activing of C1S ↓ Activation of C4 → C4a + C4b then of C2 → C2a + C2b Now this C4b, 2b acts as C3 convertase ↓ Which activate C3 → C3a + C3b ↓ Chemotactic Opsonization and anaphylactic C3b attach to C3 convertase, to form C5 convertase ↓ which activate C5 → C5a + C5b Chemotactic ↓ and anaphylactic C5b joins C5 convertase then sequential attachment of C6 → C9 ↓ Membrane attach complex → lysis
    • IMMUNOLOGY 355 Alternate Pathway • This may include properdin, cobra-venom, IgA, IgE, bacterial endotoxin, yeast cell wall, etc. • Properdin: protein in serum ↓ combines with zymosan ↓ Mg2+ properdin-zymosan complex ↓ This activates C3 directly rest sequences are repeated to form membrane attack complex (MAC) with the help of factor B and factor D. Biologic Effects 1. 2. 3. 4. 5. Opsonization — C3b Chemotaxis — C5a Anaphylatoxin — C3a C4a C5a Cytolysis Enhancement of antibody production.
    • 356 BIOCHEMISTRY FOR STUDENTS CHAPTER 19 Cancer Cancer cells are characterized by three properties: 1. Diminished control of growth, i.e. loss of contact inhibition. 2. Invasion of local tissues. 3. Spread metastasis to other body parts cancer specially refers to malignant tumor for benign tumor properties (2) and (3) are absent. Certain genes controlling growth and interactions with other normal cells are apparently abnormal in structure of regulation in cancer, e.g. isolation of BRCA-1 gene which increases susceptibility to breast and ovarian cancer. Agents Causing Cancer Cancer is second most common cause of death in USA after cardiovascular diseae. Agents causing cancer fall into three main categories: 1. Radiant energy 2. Chemical compounds 3. Some virus. Radiant Energy UV rays, X-rays, γ-rays are mutagenic and carcinogenic. UV rays cause pyridine dimers to form. Also X-rays and γ-rays causes free radicals to form in tissues. These free radicals damage the DNA and macromolecules. Chemical Compounds Approximately 80% of human cancers are caused by environmental factor, principally chemicals. These include polycyclic
    • CANCER 357 aromatic hydrocarbons, aromatic amines, nitrosam-ines, arsenic, cadmium, chromium, D-actinomycin, aflatoxin B1. Viruses Viral oncogenesis is also important aspect to study. These viruses include adenovirus. Herpes virus, retrovirus, EpsteinBarr virus (EB-virus). Changes during Malignant Transformation 1. 2. 3. 4. 5. 6. Alteration of morphology Loss of contact inhibition of growth Loss of anchorage dependence Loss of contact inhibition of movement Increased rate of glycolysis Diminished requirement for growth factors. Oncogenes of Rous Sarcoma Virus
    • 358 BIOCHEMISTRY FOR STUDENTS Various Mechanisms of Activation of Proto-oncogenes to Oncogenes 1. Promotor insertion: Certain retroviruses lack oncogenes but may cause cancer over a longer period of time than those containing oncogenes. Promotor is inserted upstream to myc gene. The integrated ds-cDNA is called provirus. 2. Enhancer insertion: In this case, provirus is inserted down stream from the myc gene or upstream from it but oriented in reverse direction. 3. Chromosomal translocation: a. Philadelphia chromosome: Involves translocation in chromosome 9 and 22 and causes chronic myelogenous leukemia. b. Burkitt lymphoma: Involves translocation in chromosome 8 and 14. This is fast growing cancer of human B-lymphocytes. 4. Gene amplification: Administration of anticancer drug methotrexate, inhibitor of enzyme dihydrofolate reductase. Resistance to this drug implies the amplification of gene for dihydrofolate reductase. 5. Point mutation: Point mutation is also able to convert protooncogene to oncogene. Tumor Suppressor Genes RB1 • • • • It is involved in genesis of retinoblastoma Gene is located on chromosome 13 and 14 pRB is nuclear phosphoprotein Unphosphorylated species of pRB binds certain viral proteins, forming complexes that inactive them. p53 • Acts as “guardian of genome” or “gurdian of tissue” • p53 is nuclear phosphoprotein • p53 binds various viral proteins forming inactive complexes.
    • CANCER 359 Mechanism of Action of Oncogenes Cancer Chemotherapy 1. 2. 3. 4. 5. Compound Vincristine and vinblastine (from chrysanthemum) Cisplatin (metallo-organic compound of platinum) Alkylating agents Antimetabolites Antitumor antibiotics Treatment use Kaposi’s sarcoma Carcinoma of lung Myeloma Leukemia Hodgkin’s disease
    • 360 BIOCHEMISTRY FOR STUDENTS CHAPTER Hormones 20 Hormones are defined as the chemical substances formed in one part of the body, carried in the blood stream to the other organs or tissues where they exert the action. The main function of hormones is to catalyze and control various metabolic reactions. They resemble enzymes in two ways: 1. They are catalyst and needed in very small amount. 2. They are not used in the reaction and hence, resemble enzymes. They differ from enzymes in several aspects. 1. Enzymes are utilized at the site where they are produced, while in case of hormones the site of origin is far from the site of action (target organ). 2. Hormones have to be discharged into blood stream whereas enzymes show their action in the cell. 3. Hormones are not only proteins but also have diverse structure. Hormones can be proteins, small peptides, single amino acids or steroids whereas enzymes are only protein in nature. Hormone Action General features of hormone classes: Group I Types Solubility Receptor Transport proteins Group II Steroids, iodothyronines calcitriol Lipophilic Intracellular Yes Polypeptides proteins glycoproteins Hydrophilic Plasma membrane No
    • HORMONES 361 Group I (Steroid) Hormones Action These hormones diffuse through plasma membrane of all cells and encounter specific receptors in target cells. This hormone receptor complex undergo activation process. Now this binds to hormone response element (HRE) of DNA and activate the inactivated specific genes. To the 3’ end of HRE there is promotor element (PE), which is to the 5’ end of structural gene. In this way, information is transferred and there is formation of specific proteins to elicit metabolic response. Group II (Peptide) Hormones These have membrane receptors and information is carried in cell by secondary messengers. For example, we are considering adenyl cyclase system here the interaction of hormone with its receptor results in activation/inactivation of adenyl cyclase. This is coupled to a protein which is having intrinsic GTPase activity. By this activation of adenylyl cyclase. c-AMP formation takes place which activate protein kinase, which in turn phosphorylate proteins to phosphoproteins, which elicit physiological effects. Cyclic AMP (3',5' Cyclic Adenylic Acid) Cyclic AMP is formed from ATP by the enzyme adenyl cyclase. Hormones which activates adenyl cyclase are: 1. Epinephrine, (more in muscle than liver) 2. Norepinephrine 3. Glucagon, (brings about a greater increase of c-AMP in liver than in muscle) 4. Thyroid. Thus, by activating the enzyme adenyl cyclase, they increase the level of c-AMP. c-AMP is destroyed by the enzyme phosphodiesterase to 5'-AMP.
    • 362 BIOCHEMISTRY FOR STUDENTS Hormone which activates phosphodiesterase is insulin. Thus the level of c-AMP is the result of these two enzymes. Functions 1. c-AMP stimulates glycogenolysis and inhibits glycogenesis. 2. c-AMP acts like a second messenger. 3. To stimulate specific kinase enzymes. Lipolysis is controlled by the amount of c-AMP present in the tissues. The process that destroy or preserve c-AMP has an effect on lipolysis. The enzyme phosphodiesterase is inhibited by caffeine, xanthine theophylline, etc. giving rise to accumulation of c-AMP in tissues. This leads to increased lipolysis giving rise to more FFA in the plasma. The enzyme adenyl cyclase is inhibited by insulin, nicotinic acid and prostaglandins. Hormones are classified structurally into three groups: 1. Amino acid derivatives: Those hormones derived from amino acid thyrosine such as epinephrine, norepinephrine and thyroid hormones. 2. Peptide: Protein hormones: Those hormones containing large proteins or medium size peptides such as insulin, glucagon, parathormone, calcitonin, pituitary hormones. 3. Steroid hormones: They are all derived from cholesterol and contain steroid nucleus, i.e. cyclopentanoperhydrophenenthrene nucleus. Progestrogen, estrogen, androgen, mineralocorticoid and glucocorticoid.
    • HORMONES 363
    • 364 BIOCHEMISTRY FOR STUDENTS Glucocorticoids (cortisol), mineralocorticoids (aldosterone), and progestrogens (progesterone) all contain 21C carbon atoms and are referred as C-21 steroids. The androgens (testosterone) are C-19 steroids while estrogens (estradiot) are C-18 steroids. Androgens and estrogens contain a potential keto group at C-17 position and hence are called 17-ketosteroid. Steroids, hormones are divided into three types: i. Glucocorticoids: They primarily affect metabolism of carbohydrates, fats and proteins but are most important in adaptation to stressful situations, examples: cortisol, cortisone and corticosterone. ii. Mineralocorticoids: Those hormones which are important for the regulation of salt balance, i.e. reabsorption of Na+ and excretion of K+ and water distribution in tissues. Examples: Aldosterone, II-deoxycortisol and II-deoxycorticosterone (DOC). iii. The adrenal androgens: Those which are important in the development of sexual hair and libido in females. INSULIN Insulin is an anabolic protein hormone. It is isolated from pancreas. Its crystalization requires traces of zinc. The major metabolic actions of insulin are centered in liver, muscle and adipose tissue. Liver Glucose is freely permeable to liver cells. Insulin induces some of the enzymes of gluconeogenesis. Insulin stimulates glycolysis by stimulating the synthesis of following enzymes: 1. Glucokinase 2. Phosphofructokinase 3. Pyruvate kinase. Insulin depresses gluconeogenesis by depressing the synthesis of the following enzymes: 1. Pyruvate carboxylase 2. Phosphoenol pyruvate carboxykinase
    • HORMONES 365 3. Fructose-1, 6-diphosphatase 4. Glucose-6-phosphatase. Muscle and Adipose Tissue Insulin stimulates the metabolism giving rise to: 1. Increased glycogen deposition 2. Increased glycolysis 3. HMP shunt pathway is stimulated 4. Increased fatty acid synthesis. In adipose tissues, insulin increases, fatty acid synthesis from Acetyl CoA (glycolysis ↑) and NADPH (HMP Shunt pathway ↑) and triglyceride synthesis from glycerophosphate. In adipose tissues, insulin inhibits the release of free fatty acids. Since the liberation of fatty acids from adipose tissues is stimulated by c-AMP, insulin depresses c-AMP level and hence, inhibits fatty acid release giving rise to enhanced lipogenesis and triglyceride synthesis. Insulin Receptors Insulin receptors has been studied in great detail using biochemical and recombinant DNA techniques. These are glycoprotein in nature. It is heterodimer consisting of two subunits called as α and β. This is represented as α2β2. The two subunits are linked by disulphide bonds. α-subunit is extracellular and it binds insulin. β-subunit is transmembrane protein and serves the purpose of signal transduction cytoplasmic portion of β-subunit has tyrosine kinase activity and an autophosphorylation site. • The human insulin receptor gene is located on chromosome 19 • Insulin receptor density is 20,000 per cell • Both subunits are extensively glycosylated • Effect of binding of insulin to insulin receptor: 1. There is conformational change in receptor 2. The receptors make cross links 3. Receptors are internalized 4. One or more signals are generated (See on page 366).
    • 366 BIOCHEMISTRY FOR STUDENTS Diabetes Insulin deficiency results in diabetes mellitus. As a result of insulin lack, glucose transport is impaired and hence, hyperglycemia occurs. 1. Key enzymes of glycolysis are depressed, whereas the enzymes of gluconeogenesis are activated which contributes to hyperglycemia. 2. Uptake of amino acids is depressed, the level of amino acid in blood increases, glycogenolysis takes place which add glucose to the blood. 3. Protein synthesis is decreased. Since, protein synthesis requires ATP consumption, ATP production is decreased because of glycolysis. 4. Fatty acid synthesis and triglyceride synthesis is depressed because of decrease in acetyl CoA, ATP, NADPH and glycerophosphate. Increased lipolysis of stored lipids give rise to free fatty acids which interfere with several steps of carbohydrate phosphorylation in muscle.
    • HORMONES 367 5. Glycogen synthetase is depressed because of depression of glycogen synthetase by the activation of phosphorylase. Fatty acids in high concentrations inhibit fatty acid synthesis by feedback inhibition of acetyl CoA carboxylase-step. Increased level of acetyl CoA from fatty acids activate pyruvate, and hence, stimulate gluconeogenesis. Fatty acids also stimulate gluconeogenesis by entering TCA cycle, which produces citrates. Citrates inhibit glycolysis at phosphofructokinase step. Fatty acids inhibit the citrate synthetase and pyruvate dehydrogenase and hence, inhibit TCA cycle. The level of ketone bodies and cholesterol increases. GLUCAGON Glucagon also called hyperglycemic, glycogenolytic hormone. Glucagon is secreted by the α-cells of the islets of Langerhans. 1. Glucagon increases blood glucose by accelerating glycogenolysis in the liver. This action is mediated through c-AMP. Glucagon increases c-AMP level, which in turn activates phosphorylase kinase which results in glycogenolysis. 2. Glucagon stimulates gluconeogenesis in the liver via c-AMP which stimulates the enzyme pyruvate carboxykinase. 3. Glucagon inhibits synthesis of fatty acids and cholesterol in the liver. It activates lipase in the liver, which results in increased free acid liberation from liver triglycerides. 4. Glucagon stimulates the release of glycerol and free fatty acid from adipose tissue. Glucagon does not stimulate glycogen breakdown in the muscles. TRIIODOTHYRONINE (T3) AND THYROXINE (T4) Thyroid gland contains iodized glycoprotein thyroglobulin’, hydrolysis of which gives triiodothyronine and tetraiodothyronine (thyroxine). They are also abbreviated as T3 and T4. T3 is 5-10 times biologically active than T4.
    • 368 BIOCHEMISTRY FOR STUDENTS In plasma, thyroxine (T4) is transported as thyroxine binding globulin (TBG) and thyroxine binding prealbumin (TBPA) whereas triiodothyronine (T3) is poorly binded to plasma. The structures of these hormones are: Metabolic Effects 1. Calorigenic effect: Increases rate of energy exchange and oxygen consumption of all tissues. BMR increases. 2. Protein metabolism: Protein metabolism which leads to positive nitrogen balance. 3. Carbohydrate metabolism: a. Increases the rate of intestinal absorption of glucose b. Hyperglycemia may also be associated with increased degradation of insulin c. Thyroxine enhances gluconeogenesis d. Glycolysis, Krebs cycle and HMP pathway are enhanced. 4. Lipid metabolism: Stimulates fat breakdown. Release of unesterified FFA from adiposes tissues with consequent increase in their concentration in blood. CALCITONIN Calcitonin secretion into the blood is regulated by the high level of serum calcium.
    • HORMONES 369 Calcitonin lowers calcium level. The main effect of calcitonin is to decrease the loss of Ca++ from the bones and hence, it opposes the action of parathyroid. Parathyroid Parathyroid hormonal secretion maintains the concentration of ionized calcium in the plasma. Secretion of parathyroid is regulated by the concentration of ionized serum calcium and it varies inversely with the concentration of serum Ca++. Administration of parathyroid results in: 1. Increased serum calcium concentration. This results from: (a) Increased absorption of Ca++ from the intestine (b) Increased rate of mobilization of Ca++ from the bones (c) Increased renal reabsorption of calcium. 2. Increased phosphorus excretion in the urine. The metabolism of Ca and P are interrelated. As the level of one rises the excretion of other is increased. PARATHORMONE Parathormone regulates Ca and P metabolism. The control is exerted by negative feedback mechanism whereby hypocalcemia stimulates and hypercalcemia inhibits the release of the hormone. Metabolic action: 1. Increases serum Ca 2. Decreases serum Pi 3. Increases urinary PO4 4. Increases citrate content of blood plasma, kidney and bone. Ionized calcium: 1. Increased absorption of Ca++ from intestines (in presence of adequate amount of vitamin D) 2. Increased renal tubular absorption of Ca++ from bones 3. Increased renal tubular absorption of Ca++ Effect on Skeleton In the presence excess parathormone, reabsorption of bone occurs and Ca++ increases in blood.
    • 370 BIOCHEMISTRY FOR STUDENTS THYROID GLAND Antithyroid Drugs Reduction of hypersecretion of thyroid hormones in hyperthyroidism can be achieved by drugs which acts in different ways on hormone synthesis and release. 1. Drugs which inhibit trapping of iodide by thyroid By metabolic poisons, e.g. cyanide dinitrate By monovalent anions, e.g. chlorate, perchlorate, pertechnitate, thiocyanate, periodate, nitrate. 2. Drugs inhibiting oxidation and organic binding of iodine and formation of T3 and T4, e.g. thiouracil, carbamizole, propyl thiouracil. 3. Iodine/iodide: Acts mainly by reducing release of thyroid hormones. 4. β-adrenergic blocking drugs like propanolol, atenolol, reduced symptoms of hyperthyroidism. 5. Radioactive I131: It is given to destroy overactive thyroid tissue. 6. Inhibiting oxidation, e.g. PABA, sulphonamides.
    • PROTEIN BIOSYNTHESIS 371 CHAPTER 21 Protein Biosynthesis It is now established that DNA is the macromolecule that ultimately controls every aspect of cellular function primarily through protein synthesis. DNA → RNA → Protein The flow of biological information is clearly from one class of nucleic acid to another from DNA to RNA and from their to protein. The process of protein biosynthesis is also called translation because the language consisting of four base pairing letters of the nucleic acid is converted into that comprising the twenty letters of amino acids in the proteins. It is a very complex process which requires more than 100 macromolecules. Transfer RNA molecules, activating enzymes, soluble factors and m-RNA are required, in addition to ribosomes. Proteins are synthesized in the amino to carboxyl direction by the sequential addition of amino acids to the carboxyl end of the growing peptide chain. Site of protein synthesis is ribosomes. Protein biosynthesis takes place in four major steps. Each step requires specific enzymes and cofactors. 1. Activation 2. Initiation 3. Elongation In the cytosol requiring t-RNAs, amino acids, ATP and Mg++ ions. In ribosomes m-RNAs, initiation factors. IF1, IF2 and IF3 as well as GTP and Mg++ ions. Two elongation factors EF-T and EF-G are required. GTP provides the energy.
    • 372 BIOCHEMISTRY FOR STUDENTS 4. Termination Requiring some releasing factors for releasing the synthesized proteins from ribosomes in the cytoplasm. ACTIVATION STEP The formation of a peptide bond between the amino group of one amino acid and the carboxylic group of the other amino acid is not favorable thermodynamically as such. This barrier is overcome by the activation of the carboxylic group of the amino acid molecule. The activated intermediates in protein synthesis are amino acid esters in which the carboxylic group of an amino acid is linked to either the 2' or 3'-hydroxyl group of the ribose moiety at the 3' end of t-RNA. This amino group can migrate rapidly between 2' and 3'-hydroxyl group. This is called acyl t-RNA. This activation, besides facilitating the peptide bond formation, is also important because only the t-RNAs can recognize the codon message carried by the m-RNA. The amino acid themselves are not able to do such decoding. The activation is catalyzed by a class of enzyme called aminoacyl-t-RNA synthetase, each of which is highly specific for one amino acid and its corresponding t-RNA.
    • PROTEIN BIOSYNTHESIS 373 In this reaction, pyrophosphate cleavage of ATP takes place to yield AMP and pyrophosphate. The transfer of an amino acid to t-RNA takes place in two steps. In the first step, ATP reacts with amino acid to form amino adenylic acid in which the 5'-phosphate group is linked in an acid anhydride bond with the carboxyl group of the amino acid. This high energy anhydride bond activates the carboxyl group. In the next step, the amino acyl group is transferred to the t-RNA to give aminoacyl-t-RNA and adenylic acid (AMP). The new ester linkage between the t-RNA and the amino acid formed at the expense of ATP is a high energy bond. The pyrophosphate so formed may undergo subsequent hydrolysis to orthophosphate, thus, utilizing ultimately two high energy phosphate bonds. This overall activation reaction is essentially reversible. The specificity of the aminoacyl t-RNA synthetase indicates that this enzyme must possess two different very specific sites for binding amino acid and its corresponding t-RNA. There is also a third site for binding ATP. These enzymes are so specific that there is 1 in 10,000 chance of an error under intracellular condition, however these enzymes can still be fooled by certain nonbiological amino acid analogs such as p-fluorophenylanine and ethionine which are incorporated in place of phenylalanine and methionine.
    • 374 BIOCHEMISTRY FOR STUDENTS INITIATION OF POLYPEPTIDE CHAIN (IN RIBOSOMES) In E. coli and other prokaryotes, the polypeptide synthesis begins with the methionine. This enters after its free amino group has been formylated (i.e. blocked) and activated as formylmethionine t-RNA. This is enzyme catalyzed reaction in which N10 formyl tetrahydrofolate acts as a formyl group donar. The free methionine does not take part in the initiation. The reaction takes place is: Methionine + t-RNA Methylene t-RNA N10 formyltetrahydrofolate Formyl-met-t-RNA + + Met-t-RNA Tetrahydrofolate The t-RNA carrying methionine occurs in two forms. Only one form designated as met-t-RNA, is capable of accepting N-formyl group. The other one cannot accept formylmethionine-t-RNA but only methionyl-t-RNA. The enzyme catalyzing this reaction—transformylase is also specific and does not formylate methionine residue attached to the other species of t-RNA i.e. t-RNAmet. The blocking of free amino group has significance, i.e. does not allow the amino acid to be inserted into the chain during the process of elongation. So, it can only be used to start the protein synthesis. However, it has been found that the initiating residue in the cytoplasmic protein synthesis of eukaryotic cells is unacetylated-methionine residue bound to one specific t-RNA-met, the initiator t-RNA. As studied and described for the bacteria E. coli the initiation process takes place in the following steps (as mentioned on page 375). The ribosomes separates into 50s and 30s subunits. The 30s subunits reacts with the initiation factor-3 (IF-3) to form a complex, the complex then binds with m-RNA to which is then added a molecule of initiation factor-1 (IF-1). The f met-t-RNA and GTP binds to initiation factor-2 (IF-2) to form a complex of f-met-t-RNA-GTR-IF-2. This complex
    • PROTEIN BIOSYNTHESIS 375
    • 376 BIOCHEMISTRY FOR STUDENTS then binds with the complex formed of 30s subunit IF-3, m-RNA and IF-1 to form what is called the initiation complex. To this is then combined the 50s subunit to form a complex 70S functional ribosome. In this process, GTP is hydrolyzed to GDP + Pi and the three initiation factors IF-1, IF-2 and IF-3 are dissociated from the ribosome. The binding of the specific m-RNA molecule on small 30s subunit is also very accurate. It is believed that each m-RNA contains one or more ribosomal binding site. Each site contains a specific nucleotide sequence which helps in the correct positioning of the m-RNA molecule on the 30s subunit. Each m-RNA has at least one ribosomal site for the production of one polypeptide chain. A m-RNA coding for 3 polypeptide chain will, therefore, contain 3 sites. This process of initiation ensures that the initiating aminoacyl m-RNA is correctly placed on the P site and positioned at the initiation codon AUG so that the ribosome starts translating the correct point on m-RNA. The P and A sites are the two carriers on the 70s ribosome into which t-RNA molecules can be inserted. The sites (P-peptidyl site and A-aminoacyl site) are bounded partially by the 50s and 30s subunits and a specific m-RNA codon. It is this m-RNA codon in relation to these sites which determines the correct binding of the specific aminoacyl t-RNA molecule. The sites themselves, however, can allow the attachment of any aminoacyl t-RNA. It has been established that translation of codons on m-RNA begins in 5'-3' direction. The initiation factors are proteins which can be extracted with strong salt solution. They have a molecular weight of about 9,000, 55,00 and 21,000 respectively. As seen, these factors keep on undergoing attachment and release reaction on the 30s subunits. ELONGATION It begins when: — The initiating t-RNA is bound on the P site, with its anticodon pairing with the triplet codon on the m-RNA. — A site is free. This process takes place in steps:
    • PROTEIN BIOSYNTHESIS 377 Binding of the Incoming Aminoacyl-t-RNA on the ASite It occurs on the A-site of the functional 70s ribosomal complex. As studied in prokaryotes, the incoming aminoacyl-t-RNA binds to a specific protein present in the cytoplasm called as elongation factor T (EF-T). This consists of two subunits, EFTu and EF-T3. The combination of EF-T and GTP in the next step results in the formation of EF-Ts free. This now combines with t-RNA carrying the new amino acid to form a ternary complex EF-Tu-GTP-aminoacyl-t-RNA. This complex then binds to the ribosome in such a way that aminoacyl-t-RNA is positioned correctly on A-site with its anticodon bound to codon on the m-RNA in the site A. The energy required for the positioning of the new aminoacylt-RNA is provided by the GTP which undergoes. hydrolysis
    • 378 BIOCHEMISTRY FOR STUDENTS and then leaves the ribosomes as EF-Tu-GTP complex after the aminoacyl-t-RNA has been properly placed on the Asite. It is stated that the carrier protein EF-T does not bind with the initiating f-met-t-RNA. The process is blocked by tetracylines. Peptide Bond Formation With the f-met-t-RNA on the P-site and new aminoacyl-t-RNA on the A-site, peptide bond is formed by the nucleophilic attack of the amino group of the incoming amino acid on the carboxylic group of the f-met-t-RNA present in the P-site. This reaction is catalyzed by the enzyme peptidyl transferase resulting into formation of dipeptidyl-t-RNA on the A-site, i.e. the amino acid on the initiating t-RNA is transferred on the A-site of t-RNA leaving an empty t-RNA on the P-site. The energy provided is given by the high energy ester bond between f-methionine and t-RNA. TERMINATION After the complete synthesis of the polypeptide chain, the termination of the chain is signalled by one of the three special
    • PROTEIN BIOSYNTHESIS 379 termination codons on m-RNA. After the attachment or the incorporation of last amino acid into the polypeptide chain, the chain is still attached to t-RNA by its carboxyl terminal on the A-site. The release of chain from here is mediated by the releasing factors symbolized as R1, R2 and R3. They bind to the ribosome to cause a shift of the polypeptidyl-t-RNA from A-site to P-site; the ester bond between the polypeptide chain and the last t-RNA is then hydrolyzed apparently by the action of peptidyl transferase enzyme. Once, the polypeptide chain is released the last t-RNA and m-RNA also leave the ribosome which then dissociates into 50S subunits the new polypeptide chain is again started. The exact details of this step are not yet clearly known. The chain probably leaves the ribosome as a folded molecule with tertiary structure because the ribosome is found to contain several enzymes, in addition to protein synthesizing agents. Post-translational Processing of Polypeptide Chain After the polypeptide chain has been synthesized completely it undergoes certain changes to yield its biologically active form. Most of the proteins do not have a formyl group at the amino terminal. So, it is thought that formyl group is removed by the action of the enzyme deformylase. Some proteins do not have methionine as the amino terminal residue. It is believed to be removed afterwards by the enzyme methionine aminopeptidase. Still further some proteins are acylated at their N-terminal residue after they are synthesized. The disulphide bonds are similarly formed in reactions catalyzed in microsomes to allow them the tertiary structural modifications. Energy Requirements of Protein Synthesis 2 ATP bonds are required in the activation of amino acid. i. GTP is hydrolyzed in the binding of aminoacyl-t-RNA to A-site. ii. GTP is hydrolyzed in the translocation ribosomes. Total of 4 high energy bond = 4 × 7.3 = 29.2 Kcal, for each peptide bond synthesized. And each bond gives about 50 Kcal on hydrolysis. Hence, it is highly energy consuming process
    • 380 BIOCHEMISTRY FOR STUDENTS to generate peptide bond. Infact, it is the most expensive process in cells which is biosynthetic. Inhibitors of Protein Synthesis Many inhibitors used in human beings for treating infections act by inhibiting the protein synthesis in the prokaryotes. Examples are: tetracycline, chloramphenicol, puromycin, streptomycin, neomycin, etc. Inhibitor Chloramphenicol Streptomycin Tetracycline Puromycin Fusidic acid Site of action Block peptidyl transfer in 70s ribosome. Binds to 30s subunit to affect initiation. Inhibits binding of incoming aminoacyl-t-RNA to A-site. Reacts with peptidyl-t-RNA to give puromycin-peptidylt-RNA. Inhibits translocation. CODON Genetic experiments have shown that a group of three bases in fact, codes for one amino acid. This group of bases is called codon. General Characteristic of Genetic Code 1. Each code word consists of three nucleotide bases arranged in a definite sequence, i.e. it is a triplet. 2. There are in all 64 code words out of which 61 code for various amino acids. These are called nonsense codons. The other three codons do not code for any particular amino acid which are signals of chain termination. They are UAG, UAA and UGA. 3. There are more than one codon for many amino acids such as 4 codons each for glycine and alanine and 6 codons each for arginine, leucine and serine. This property is called as degeneracy of genetic code. Only tryptophan and methionine have only one codon for them.
    • PROTEIN BIOSYNTHESIS 381 4. Out of the triplet codon, the first two bases are very specific as far as the sequence is concerned. But third one is not that specific. For example—GCU, GCA and GCG, all are specific codons for the alanine. Each codon has two same bases at first and second position but the third one is different. Thus the third base tends to be loose and wobbles about. Third base wobbles. Further when the two amino acids have two codons each of which have first two bases common, the third becomes the determining one and in such case the third position is filled by purine base in one and a pyrimidine base in the other. For example—for histidine and glutamic acid, there are two codons for each. Histidine CAU, CAC Glutamic acid CAA, CAG. In those, the third base is purine base in both the codons for glutamic acid and third base in both the codons are a pyrimidine base for histidine. The nucleotide sequence is from 5' to 3' end and as the third base lies on the 3' end. 5. In the codon messages, no signals are required to indicate the end of one codon and the beginning of the other codon, i.e. they are identical in all species of life right from virus to man. This has been tested in more than one way. The codon specific for serine, i.e. is identical in virus, bacteria, lower animal and even in man. This refers to the universality of codon, i.e. many amino acids are designated by more than one triplet. Only tryptophan and methionine are coded by one triplet only. REGULATION OF GENE EXPRESSION Gene, carrying the genetic information, expresses itself by leading to the formation of a specific protein through the process of transcription and translation. Hence, the regulation of gene expression in effect means the regulation of protein
    • 382 BIOCHEMISTRY FOR STUDENTS synthesis. This regulation can therefore, takes place at the following two levels. 1. Transcription control, i.e. the formation of m-RNA from the gene is the point of regulation. 2. Translation control, i.e. the synthesis of the proteins from m-RNA is the point of regulation. However, in most bacteria and prokaryotes transcriptional control is the main regulatory method. Transcription Control The basic process of protein synthesis is regulated by induction and or repression. Depending upon the metabolic state of the organism, certain enzymes, i.e. the proteins are either induced or repressed. Jacob and Monard proposed the concept of operon to explain the phenomenon of induction and repression as the means of transcription control of protein synthesis. Initially, this hypothesis named as operon model was postulated with particular reference to lactose metabolism regulation in E. coli by the genetic pathway. This hypothesis postulates the existance of an operon as the group of functionally related structural genes lying contigious to each other in the chromosomes which can be turned off and on coordinately by the same regulatory gene. Since they explained the protein synthesis with particular reference to lactose. They proposed the lactose operon (Lac operon) as the model for regulatory mechanism. This explained the induction of three protein brought about by the lactose and the repression of these proteins by the presence of glucose in the following way. There are present structural genes which are responsible for transcription m-RNA for three proteins, i.e. β-galactosidase, permease and transacetylase. These three functional genes are under the regulation of an inhibitory locus—now called as regulatory gene. This is then the operator locus on the chromosome of DNA adjacent to the structural genes. The regulatory gene normally excerts an inhibitory influence on the structural genes preventing them from transcripting the
    • PROTEIN BIOSYNTHESIS 383 various m-RNAs by means of a protein molecule called repressor. This represser then binds with the operator to inhibit the transcription under normal circumstances. The binding of the repressor, which is reversible to the operator interferes with the bindings of RNA polymerase on to the promotor, another locus present in the vicinity of operator. There appears to be some overlappings in the limits of the promotor and operator. To initiate the protein synthesis and when the repressor molecule is not bound to the operator, the transcription is carried out uninterrupted by the structural gene. Thus according to this operon model, the repressors control the rate of transcription of DNA into RNA. DNA Repair The maintainance of integrity of the information in DNA molecules is of great importance. The mechanisms responsible for this monitoring mechanism in E. coli includes 3’ to 5’ exonuclease activity. Repair is of four types: 1. Mismatch repair 2. Base excision repair 3. Nucleotide excision repair 4. Double strand break repair. 1. Mismatch repair Problem: Mismatching refers to copying errors. Solution: 1. Methyl directed strand cutting 2. Exonuclease digestion. 2. Base excision repair Problem: Chemical/radiation damage to a single base Solution: Base removal by N-glycosylase, abasic sugar removal replacement. 3. Nucleotide excision repair Problem: Chemical/radiation damage to DNA segment Solution: Removal of an approximate 30-nucleotide base pairs and replacement. 4. Double strand break repair Problem: Chemotherapy, oxidative free radicals, ionizing radiation Solution: Synapses, unwinding alignment, ligation.
    • 384 BIOCHEMISTRY FOR STUDENTS Applied Pyrimidine dimers can be formed in the skin cells of human exposed to unfiltered ultraviolet sunlight in the rare genetic disease xeroderma pigmentosum, the cells cannot repair the damaged DNA resulting in extensive accumulation of mutations that leads to skin cancers. The most common form of this disease is caused by the absence of UV-specific endonuclease. DNA replication The biosynthesis of a duplicate copy of DNA prior to cell division (DNA → DNA). DNA repair The removal and synthesis of short segments of DNA damaged by chemical or physical agents or of DNA synthesized with errors during replication. DNA recombination The exchange of gene segments between different DNA molecules. DNA transposition A nonclassical type of genetic recombination involving the movement of a gene from one location to another on the same chromosome or to a different chromosome.
    • INSTRUMENTATION 385 CHAPTER Instrumentation 22 COLORIMETRY Colorimetry depends upon the measurement of the amount of color, i.e. intensity of color, produced during a chemical reaction in which the substance being estimated takes part quantitatively. The intensity of color produced is proportional to the concentration of the reacting substances and it is possible to measure the concentration of the substance by determining the depth of the color. Laws governing the absorption of light are governed by Lambert’s and Beer’s which are as follows: Lambert’s Law Proportion of light absorbed by the substance is independent of the intensity of the incident light. Beer’s Law Absorption depends only on the number of absorbing molecules through which the light passes. Mathematical derivation is given as: log I = KCL I0 where C K L Optical density = KCL I0 I = = = = = Concentration Constant Thickness Incident light Emergent light
    • 386 BIOCHEMISTRY FOR STUDENTS Optical Density It is defined as the logarithmic ratio of the incident light to that of emergent light. I OD = log 0 I Transmission It is defined as the ratio of the intensity of transmitted light to that of incident light. I T = 0 I Relationship between optical density and transmission: I OD = log 0 I I = log T 100 = log %T = 2 – log T When transmission is 100%, the optical density is 0. Colorimeter comes under the visible range. Actually what we are measuring in the colorimeter is the maximum absorption of the light. We select a particular filter for a particular color, so that the maximum absorption of the light should be their. ELECTROPHORESIS Electrophoresis is defined as the migration of charged particles in the solution under the influence of electric field. The rate of migration is directly proportional to the number of charges present on the component. Proteins are colloidal particles and charged either positive or negative which depends on the pH of the solution. In acidic medium, it acts as cation and in alkaline medium as anions. If uncharged particles are charged, then they can be separated. If a potential difference is applied
    • INSTRUMENTATION 387 across them, current will flow and cations move towards cathode and anion towards anode. Migration depends upon: i. pH of buffer ii. Net charge of amphoteric particle iii. Temperature iv. Voltage and current. ISOTOPES AND THEIR APPLICATION Isotopes are atomic species having similar atomic numbers but different atomic weights due to the difference in the nucleus of atom. As the atomic number is same, isotopes have the same chemical properties but different physical properties. Isotopes are of two types: 1. Stable or nonradioactive isotopes 2. Unstable or radioactive isotopes Stable or nonradioactive isotopes: They do not emit any radioactive radiations. Unstable or radioactive isotopes: They emit radioactive radiations, i.e. α, β or γ-rays. Radioactivity It is the phenomenon where radioactive substances emit α, β or γ-rays on disintegration. α-rays: They consists of doubly charged helium atoms. They have least penetration power of all the three particles with greatest ionization power, because they have heavy particles they cannot penetrate much. β-rays: They are fast moving electrons having ionization power less than α-particles and less than γ-rays. γ-rays: They are electromagnetic waves with maximum penetration power but less ionization power of all the three particles. Measurement of Radioactivity 1. Geiger-Müller counter: The radiations entering the gas mixture produce ions. When α, β or γ-rays collide with gas atoms or molecules the cation and anion move to cathode and anode
    • 388 BIOCHEMISTRY FOR STUDENTS respectively and produce electric impulse which is proportional to the activity of the radioactive substance. 2. Proportional counter: It is same as Geiger-Müller counter except the gas is not a mixture but a single monoatomic gas. It can differentiate between α, β and γ-rays. However, it is less sensitive. 3. Scintillation counter: In this method, no gas or mixture of gas is used. The radiations are allowed to full over fluorescent substance, e.g. crystals or on a liquid organic solvent (liquid scintillation counter). The photons emitted are allowed to pass through a photo multiple tube that converts this light into electricity and amplified using 10 to 15 diodes whose strength is proportional to the radioactivity of the substance. Units of Radioactivity Radiation absorption dose: Since the radioactive rays can produce ions inside the tissues also, long exposure to these rays can be dangerous. 1 rd = 100 ergs of energy/gm of tissue. 1 roentgen = 1 rd (practically). Application of Isotopes a. In biochemistry, isotopes are used in working out metabolic pathways, i.e. cholesterol synthesis from acetic acid, purine synthesis from glycine. Also some commonly used stable isotopes are used in the determination of turnover of different metabolic activities in the body. b. Determination of total plasma volume and total blood volume in the body. c. Determination of average life of RBC. d. In iodine metabolism. ELECTROMETRIC DETERMINATION OF pH The pH of solutions can be determined more accurately by potential measurements of certain electrodes than by the use of indicators. They give rapid and accurate results. Common
    • INSTRUMENTATION 389 electrical methods for pH determination depend upon the use of hydrogen or glass electrode. Hydrogen Electrode It consists of a small platinum strip coated with platinum block and absorbs hydrogen gas. A platinum wire welded to the electrode makes contact with the outer circuit, the Pt strip is surrounding by glass tube with inlets and outlets for H2 which is admitted at 1 atmosphere. 2H H2 (On Pt surface) (Remain on Pt) 2e¯ + 2H+ (Pass into solution from electrode) Since H2 is admitted at constant pressure, the solution tension of hydrogen atoms has a constant value. If electrode 1 is maintained constant by immersion in 1 N H+ ions, then potential will vary depending on the H+ ions concentration around electrode 2. The electrode with H2 at 1 atmosphere in a 1 N H+ solution is called hydrogen electrode and is arbitrarily assigned a potential, Eno of Zero under all conditions and is used as a standard reference for other electrodes. EMF = En + Eno If potential difference between the normal H2 electrode and the electrode in the unknown solution is known for a given temperature, pH of the solution can be calculated from the formula: EMF pH = , where T is absolute temperature. 0.00019837T Calomel Electrode It consists of metallic mercury in contact with Hg2Cl2 in KCl solution. Potential varies with the saturation of KCl solution but for a given temperature and KCl concentration, the potential of the calomel electrode against the normal hydrogen electrode is constant, i.e. at 25°C, the potential of saturated
    • 390 BIOCHEMISTRY FOR STUDENTS calomel electrode is 0.2458 Volts. Now, if a hydrogen electrode is placed in the solution of unknown pH and connected with a saturated calomel electrode, the potential difference registered will be more than that would have been obtained against a normal hydrogen electrode by 0.2458 Volts. The pH of unknown solution can, therefore, be calculated as: pH = EMF − Encalomel 0.00019837T Glass Electrode This method of determining pH is rapidly replacing the hydrogen electrode procedure. It is not affected by oxidizing or reducing agent. It is based on the principle that when a glass membrane separates two different solutions differing in pH, a potential difference is found to exist between the two surfaces of the glass. It consists of a thin walled glass bulb made out of a special type of low melting point glass. It is filled with normal HCl solution in contact with Ag/AgCl electrode. The platinum wire dipping in the electrolyte passes out of the glass tube and the bulb is placed in the solution, whose pH is to be measured. The potential is measured against a standard calomel electrodes. pH Meter A glass electrode is made up of a bulb containing solution of known pH into which is dipping an Ag/AgCl electrode. The bulb is fragile as it is made up of a thin layer of glass. The glass electrode and calomel electrode both are dipped in a solution of unknown pH. The electrodes are connected by potentiometer. ESTIMATION OF NITROGEN CONTENT BY MICROKJELDAHL METHOD Any nitrogen containing substance on digestion with concentrated H2SO4 is converted into ammonium sulfate. From the ammonium sulfate so formed, the ammonia is distilled off, by treating with strong alkali. The evolved NH3 is trapped
    • INSTRUMENTATION 391 in a suitable indicator, which on titration with standard H2SO4 gives the amount of ammonia trapped and thus by back calculation, the nitrogen content is found out. Reaction The whole procedure for nitrogen estimation is divided into following three parts: a. Digestion b. Distillation c. Estimation. Digestion A known weight or the volume of the organic compound is taken in a small micro-kjeldahl flask, followed by 2 ml of concentrated H2SO4. To this, a pinch of CuSO4 (acts as catalyst) and K2SO4 (raises boiling point and prevents bumping) are added. The mixture now looks black. The flask is placed on a microburner and is heated slowly over a small flame. In the initial stage, a low flame is used and later on a strong flame is used till the solution is completely digested and acquires a slight blue tinge. The time of digestion depends upon the nature of nitrogen in the unknown substance (i.e. complexity of the nitrogen). Under similar conditions, run a blank which contains all the reagents except test material.
    • 392 BIOCHEMISTRY FOR STUDENTS Distillation The contents of the digested material are transferred quantitatively into a distillation jacket. The digested flask is washed with distilled water and the contents poured into the distillation jacket followed by 10 ml of 40 percent NaOH. A steady stream of steam is bubbled into the distillation jacket. The ammonia evolved is trapped in boric acid: Tashiro’s indicator. In acidic pH, the indicator is violet in color. In alkaline solution, it is green in color. Estimation The trapped ammonia is estimated by titrating against N/70 H2SO 4. The color of the indicator becomes green after the ammonia has been absorbed into it. This is titrated against N/70 H2SO4 until the solution becomes violet again. Reaction Indicator Calculation Proteins contain 16% nitrogen, i.e. 16 mg of nitrogen is present in 100 mg of protein. 1 mg of nitrogen is present in 6.25 mg of protein = 1 liter of 1N-NH3 1 liter of 1N H2SO4 1 liter of 1N H2SO4 = 14 gm of nitrogen = 14 mg of nitrogen 1 ml of 1N H2SO4 1 ml of N/70 H2SO4 = 0.2 mg of nitrogen. If x be the difference between the titre value for test solution and blank.
    • INSTRUMENTATION 393 If 0.2 ml of serum is digested initially than 0.2 ml of serum produces 0.2 × x mg of nitrogen. 1 ml of serum produces 0.2 × x mg of nitrogen. 0.2 100 ml of serum produces 0.2 × x mg of nitrogen. 2 The results can be converted into proteins by multiplying by factor 6.25. Chromatography is defined as the analytical technique for separating compounds on the basis of differences in affinity for a stationary and a mobile phase. The difference in affinity involves the process of either adsorption or partition. In adsorption chromatography, the binding of a compound to the surface of the solid phase takes place whereas in partition chromatography, relative solubility of a compound in two phases results in the partition of the compound in two phases. Thus, all types of chromatography known so far have been grouped in either of the two mentioned form.
    • 394 BIOCHEMISTRY FOR STUDENTS As all the experimental techniques have got their own way of representations, chromatographic method has also entirely different notation by which results are represented: They are known as Rf. Rf expresses the relative rate of movement of solutes and solvents. Rf is defined as the ratio of the distance travelled by the compound at its point of maximum concentration to the distance travelled by the solvent. Both the distances are measured from the point of application of the sample: Rf = Distance travelled by the solute Distance travelled by the solvent Rf value has no unit. Rf is always less than one. Rf value of different compounds are entirely different. Just as melting point, boiling point and other physical constants are different for different compounds, so is the case with Rf values. The Rf values vary with the solvent used, i.e. two solvents will give two values. Thus, Rf value is always quoted with reference to the solvents used. In paper chromatography, the analysis of an unknown substance is mainly done by the flow of solvents on specially designed filter paper. One of the two solvents is imiscible or partially miscible in the other solvent. The solvent rises up by the capillary action and by adsorption on the paper, the separation is effected by differential migration of the mixture of substances. This occurs due to difference in partition coefficients. When the solvent moves over the spot, two type of forces are involved. They are: 1. Propelling force: This assists in the propagation of the substances in the direction of the flow of the solvent. 2. Retarding force: This tries to drag the substances behind towards their point of application. The Rf value, the distance through which the substances move on the paper under the influence of the solvent is due to the net resultant of these two types of forces.
    • INSTRUMENTATION 395 In biological mixture separation: 1. The mixture is available in very small quantity. 2. Mixture is usually made up of proteins, cannot be subjected to high temperature, high or low pH because they get denatured. 3. Substances having melting points or boiling points very close to each other. Their solubilities are also very closely related. All these difficulties can be overcome by using the chromatographic techniques.
    • INDEX 397 Index A Absorption 18 calcium 299 fats 185 iron 296 Acetoacetyl CoA pathway 206 Acetyl number 62 Acid phosphates 143 base balance 284 Acidification of urine 289 Acrolein formation 60 Action of acids 27 amylases starch 47 dilute alkali 34 Activation of B cells 345 fatty acid 196 glycerol 195 T cells 342 Active and passive immunity 339 sulfate 229 sulfate sulfating agent 229 Adenosine diphosphate 249 triphosphate 249 Adenylic acid 373 Adrenal androgens 364 cortex hormones 178 Aerobic dehydrogenases 141 Agents causing cancer 356 α-glycerophosphate shuttle 150 Alanine transaminase 213, 329 Albinism 237 Aldaric or saccharic acid 35 Aldonic acid 34 Aldoses 20 Alkaline phosphatase 137 Alkaptonuria 237 Amino acid derivatives 362 molecule 372 sugars 26 Ammonium ion production 288 Amphibolic role citric acid or Krebs cycle 160 Amylopectin 46 Amylose 46 Anaerobic dehydrogenases 122, 141 Andersen’s disease 165 Anion gap 290 Anterior pituitary hormones 178 Antiachrodynia factor 275 Antibody class switch 351 diversity 351 Antigens 100, 340 Antioxidant system 71 Antipernicious factor 281 Antisterility vitamin 265 Antithyroid drugs 370 Antivitamins 283 α-oxidation 191 Application of isotopes 388 Arachidonic acid 57 α-rays 387 Aspartate transaminase 213 Atherosclerosis 203 B B cells 345 Balanced diet pregnant lady 321 Banana 319 Barford’s test 38 Basal metabolic rate 312 Base excision repair 383 Beer’s law 385
    • 398 BIOCHEMISTRY FOR STUDENTS Benedict’s qualitative reagent 30 reagent 34 Bicarbonate 335 carbonic acid buffer 285 reabsorption 287 Bile acids 204 Binding of incoming aminoacylt-RNA 377 Biochemical basis of fatty liver 208 changes in jaundice 117 Biological importance of water 292 oxidation 140 value of proteins 313, 314 Biophysics 1 Biosynthesis of purine ribonucleotides 250 pyrimidine nucleotides 252 Biotin 277 Biuret reaction 95 Blood buffers 9 group substances 52 Bohr effect 112 β-oxidation 188 β-rays 387 Bread 322 Breakdown of hemoglobin 113 Brief description cytokines 347 Bromsulphalein excretion test 328 Brownian motion 17 Buffer 4 systems of body fluids 284 Burkitt lymphoma 358 Butter 322 C Calcitonin 368 Calcium 298 Calomel electrode 389 Caloric requirement 315, 320 value of food 310 Calories diabetic diet 323 chart 322 Calories low cholesterol diet 325 Calorigenic effect 368 Cancer 356 cells 356 chemotherapy 359 Carbohydrate 19, 315 metabolism 326, 368 Carbon monoxide poisoning 105 Carcinoid syndrome 239 Carcinoma of lung 359 Cardiolipin 65 Castle’s extrinsic factor 281 Catabolism of purines 254 pyrimidines 255 Catalases 143 Catalytic site oractive sites of enzymes 134 Cell mediated immunity 345 Cellular structural studies 327 Cellulose 47 Cephalins 64 Cereal group 313 Cerebrosides or glycolipids 66 Ceruloplasmin 304 Characteristics of coenzymes 121 Characterization of fats 61 Chemical compounds 356 coupling hypothesis 147 Chemiosmotic hypothesis 148 Chemistry of amino acids 74 and proteins 74 carbohydrates 19 lipids 53 Cholesterol biosynthesis 197 Chondroitin sulfates 51 Chylomicrons 184 Citric acid cycle 155 Classification and functions of lipids 54 Classification of amino acids 75 carbohydrates 19 enzymes 122 proteins 85 Coagulation factor 266 Codon 380
    • INDEX 399 Coenzyme ubiquinone 145 Coenzymes 121 Colloids 16 Colorimetry 385 Competitive inhibition 131 Complete proteins 316 Compound lipids 62 Concentration test 331 Conformational coupling hypothesis 149 Conjugated proteins 86 Conjugation reaction 224 Constitutive enzymes 136 Conversion of pyruvate to acetyl CoA 155 Copper 303 Cori cycle 168 Cori’s disease 165 Cottage cheese/egg 322 Crabttee effect 154 Creatine phosphokinase 137 synthesis 224 Creatinine clearance test 334 Crystalloids 16 Curd 323 Cushing’s syndrome 291 Cyanocobalamin 281 Cyclic amp 361 Cysteine ® pyruvic acid 276 and cystine 227 Cystinosis 228 Cystinuria 228 Cytochromes 145 Dense connective tissue 292 Deoxyribose nucleic acid 244 Derived lipid 68 proteins 86 Determination of serum amylase 336 lipase 336 Detoxification and protective functions 326 Dextrins 48 Diabetes 366 mellitus 179 Diagnostic value of plasma enzymes 137 Dialysis 16 Dietary fiber 316 Differences between T cells and B cells 346 Digestion and absorption 210 of protein various enzymes 210 Dihydrofolic acid 279 Dilution test 332 Distillation 392 Disulphide bonding 94 DNA ® RNA ® protein 371 recombination 384 repair 383 replication 384 transposition 384 Double strand break repair 383 Dust cells 346 D-xylose excretion test 338 E D Daily requirement 300 Dal 323 Dal/lean meat 322 Danger of ketosis 207 De Toni-Fanconi syndrome 303 Decarboxylation 213 Deficiency disease 265 Degree of glucose control 182 Dehydration 295 results 295 Dehydrogenases 141 Denaturation 96 E. coli 382 Edema 295 Effect of enzyme concentration 128 negative ions 96 pH 128 positive ions 96 proteolytic enzymes 348 salt concentration 95 skeleton 369 substrate concentration 124 temperature 129 Effector functions of T cells 343
    • 400 BIOCHEMISTRY FOR STUDENTS Egg 319, 322 Eicosanoids 73 fatty acid derivatives 59 Electrolyte composition of plasma 284 Electrometric determination of pH 388 Electrophoresis 88, 386 pattern of normal serum 97 Embden-Meyerhof pathway 153 Energetics 158 Energy requirements of protein synthesis 379 yield of palmitic acid metabolized 190 Enhancer insertion 358 Enolphosphates 143 Enzyme activity 129 induction 135 inhibitions 130 specificity 122 Enzymes 120, 335 Epinephrine 178 Essential amino acids 80 fatty acids 57 pentosuria 175 Esterified cholesterol 330 Estimation of Serum alkaline phosphatase 329 bilirubin 327 glutamic pyruvic transaminase 329 urine bilirubin 328 urobilinogen 328 Excretory functions 326 Extra-mitochondrial de novo fatty acid synthesis 191 F Factors influencing rate of enzymatic reaction 124 Fat in stool 337 soluble vitamins 259, 261 Fats 315 energy source 56 oil 322 Fatty acid 55 synthesis 191 livers 208 Ferrous protoporphyrin 105 Fertility factor 265 Fibrous proteins 85 Figlu excretion test 280 Fish 319 chicken 322 Flavin adenine dinucleotide 273 mononucleotide 273 Flavonucleotides 141 Fluoride 305 Fluorosis 305 Folic acid 279 Food values 319 Formal titration 82 Formiminoglutamic acid excretion test 280 Free radicals 72 Fructose metabolism 172 Fruit 322, 323 Functions of amino acids 79 carbohydrates 19 hemoglobin 104 iron 296 plasma proteins 98 proteins in body 97 T cells 343 G Galactose metabolism 169 Galactosemia 170 Gamma interferon 347 Gangliosides 67 Gastrointestinal function test 338 Geiger-Müller counter 387 Gene amplification 358 General characteristic of genetic code 380 Gestational diabetes 180 Ghee/oil 322 Gibbs donnan equilibrium 15
    • INDEX 401 Glass electrode 390 Globin 109 Globular proteins 85 Glucagon 178,361, 367 Glucocorticoids 364 Glucogenic amino acids 219 Gluconeogenesis 168, 176 Glucose oxidase 35 Glutamate oxaloacetate transaminase 213 pyruvate transaminase 213 Glutamic acid 279 Glycine 221 choline cycle 222 Glycinuria 226 Glycogen 48 storage diseases 165 synthetase 164 Glycogenesis 163 Glycogenolysis 165 Glycolysis 151 Glycosides formation 40 Glycosuria 178 Glyoxalate pathway 221 Gout 257 γ-rays 387 Green vegetables 322 Growing child 321 Guanidinophosphates 143 Guanosinediphosphate 249 Guardian of genome 358 tissue 358 H Hartnup disease 239 H-chains 99 Hematologic function 327 Heme 105 Hemochromatosis 298 Hemoglobin 102, 103 cooperativity 110 gun hill 112 synthesis 222 variants 111 Hemolytic or pre-hepatic jaundice 116 Henderson-Hasselbalch equation 5, 6 Heparin 50 Hepatic porphyria 115 Hepatocellular or hepatic jaundice 116 Her’s disease 165 Heteropolysaccharides 49 Hexose monophosphate shunt pathway 160 High density lipoproteins 184 energy compounds 143 or heavy density lipoproteins 184 HMG-CoA pathway 206 Hodgkin’s disease 359 Homogentisic acid oxidase 237 Homopolysaccharides 45 Hormone action 360 classes 360 response element 361 Hormones 360 Hyaluronic acid 49 Hybridoma and monoclonal antibodies 351 Hydrogen bonding 94 electrode 389 ion concentration 1 Hydrogenation 60 Hydrolysis 307 Hydroperoxidases or peroxidases 142 Hydrophobic interactions 94 Hyperammonia 219 Hyperoxaluria 222 Hyperparathyroidism 303 Hypertonic solutions 12 Hypervitaminosis A 263 D 265 Hypotonic solutions 12 Hypoxanthine-guanine phosphoribosyl transferase 254 I Immunoglobulin 99 Immunology 339
    • 402 BIOCHEMISTRY FOR STUDENTS Importance of HMP shunt pathway 160 Inborn error of metabolism 235 Incomplete proteins 316 Inducible enzymes 136 Inherited erythropoietic porphyria 114 Inhibitors of protein synthesis 380 Initiation of polypeptide chain 374 Inosine diphosphate 249 triphosphate 249 Instrumentation 385 Insulin 364 receptors 365 Intensity of color 385 Interstitial fluid 292 Intracellular fluid 292 Inulin clearance test 334 Invert sugar 44 Iodine number 61 Ionized calcium 369 Iron 296 Isocitrate dehydrogenase 137 Isoelectric point of amino acids 83 Isoenzymes 136 Isotonic solutions 12 Isotopes and application 387 Isotype, allotype, idiotype 349 J Jaundice 116 K Kaposi’s sarcoma 359 Ketogenic amino acids 219 Ketone bodies 205 Ketoses 21 Ketosis 205 Kidney 284, 287 Kinase activation 207 Krebs-Henseleit cycle 214 Kupffercells 346 Kwashiorkor 317 L Lactate dehydrogenase 136 Lactose 42 synthesis 173 Lambert’s law 385 Langerhans cells 346 Latest autoimmune diabetes of adults 180 Lecithins 63 Lesch-Nyhansyndrome 254 Leucine, isoleucine and valine 240 Line-weaver burk equation 126 Linoleic acid 57 Lipid metabolism 326, 368 Liver 364 biopsy 327, 330 function tests 326 goat 319 phosphorylase 166 Low density lipoproteins 184 Lungs 286 respiration 284 M Macrophages 345 Malate-aspartate shuttle 150 Malignant transformation 357 Maltose 41 Maple syrup urine disease 240 Marasmickwashiorkor 318 Marasmus 318 Maturation in bursa equivalent 346 thymus 346 Maximum urea clearance 333 Mcardle’s disease 165 Meal plan 322 Meat group 313 fish 322 Mechanism of action oncogenes 359 H+ excretion 287 oxidative phosphorylation 147 Messenger RNA 248
    • INDEX 403 Metabolic acidosis 290, 291 action 369 alkalosis 291 effects 368 function 326 gout 257 role of cysteine 227 Metabolism of branched chain amino acids 240 carbohydrates 151 cystine and cysteine 228 individual amino acids 219 lipids 184 phenylalanine and tyrosine 230 proteins 210 tryptophan 237 xenobiotics 306 Methemoglobin 110 Methionine 226 Methionione 144 Method of determining km 128 MHC molecules 352 polymorphism 353 Microalbumin 331 Microsomal pathway of fatty acid synthesis 195 Mid morning 323 Milk 322 buffalo 319 cow’s 319 DMS 322 group 313 Milliequivalent 14 Mineralocorticoids 364 Minerals 295, 318 Mismatch repair 383 Mitochondrial synthesis of fatty acids 194 Mixed function oxidases 142 Monosaccharides 19 Mountain sickness 104 Mucoproteins and glycoproteins 52 Muscle adipose tissue 365 fat cells 175 phosphorylase 166 Mutarotation 28 Mutton 319 Myoglobin 112 N Natural and acquired immunity 339 killer cells 346 Net dietary protein value 314 protein utilization 314 Neurological cells 346 Niacin 273 Ninhydrin reaction 82 Nitrogen 337 balance 80 Noncompetitive inhibition 133 Nonessential amino acids 80 Nonrepetitive secondary structure 94 Normal balanced diet for adult man 320 woman 321 Nucleic acid 244 chemistry and metabolism 241 Nucleoside 243 Nucleotide excision repair 383 Nucleotides 243 Nutrition 310 O Obstructive or post-hepatic jaundice 119 Oligosaccharides 40 Opsonize bacteria 339 Optical density 386 Orange 319 Organ function tests 326 Organic pyrophosphates 144 Origin of immune cells 341 Orotic aciduria 252 Osazone formation 30 Osmosis and osmotic pressure 12 Osmotic pressure 12, 98 Osteoporosis 301 Oxidases 140 Oxidation 307 of fatty acids 188 Oxidative deamination 214 phosphorylation 146 Oxidoreductases 122 Oxygenases 142
    • 404 BIOCHEMISTRY FOR STUDENTS Oxygenation curves for hemoglobin and myoglobin 111 P Pancreatic function test 335 Pantothenic acid 275 Para-aminobenzoic acid 279 Parathormone 369 Parathyroid 369 Partially complete proteins 316 Pasteur effect 154 Peanuts/fat 322 Pellagra preventive factor 273 Pentose sugars 242 Pentosuria 175 Peptide 361 bond formation 378 pH meter 390 of buffer 387 Phenolsulfonphthalein test 332 Phenylalanine and tyrosine 229 hydroxylase 235 Phenylketonuria 235 Phenylthiohydantoin derivative 90 Philadelphia chromosome 358 Phosphate buffer 286 Phosphatidic acid 63 Phosphatidyl inositol 64 Phospholipids 63 Phosphorus 302 Physiological jaundice or neonatal jaundice 119 Plasma 292 lipoproteins 184 proteins 98 Plasmalogens 65 Polenske number 62 Polysaccharides 45 Pompe’s disease 165 Porphins 102 Porphyria 114 Porphyrins 103 Post-translational processing of polypeptide chain 379 Potassium 302 Potatoes 319 Precipitation reactions 95 Precursors of purine ring 249 pyrimidine ring 250 Prediabetes 180 Primary antioxidants 71 metabolic gout 257 renal gout 258 Process of lipid peroxidation 72 Promotor element 361 insertion 358 Propelling force 394 Properties of colloidal solutions 16 fats 60 Proportional counter 388 Prostaglandins 58 Protein biosynthesis 212, 371 buffer 286 in stool 337 metabolism 326, 368 calorie malnutrition 317 Proteins 85, 315 Prothrombin time 329 Pteridine nucleus 279 Purine bases 242 synthesis 222 Purines and pyrimidines metabolism 249 Pyridine nucleotides 141 Pyridoxine 275 Pyrimidine bases 242 dimers 384 Pyrroloquinoline quinone 260 R Radiant energy 356 Radiation absorption dose 388 Rancidity 60 Reactions monosaccharides 26 proteins 94 with nitrous acid 82 Refsam’s disease 191
    • INDEX 405 Regulation Blood calcium level 300 glucose 175 by pyrimidine biosynthesis 257 cholesterol biosynthesis 203 de novo fatty acid synthesis 194 gene expression 381 purine synthesis 256 Regulatory function of T cells 345 Reichert meissel number 62 Renal blood flow 335 function tests 330 glucosuria 179 gout 258 mechanism 284 Repressor 383 Respiratory acidosis 289, 291 alkalosis 291 chain 144 phosphorylation 146 quotient 311 Retarding force 394 Riboflavin 272 Ribose nucleic acids 247 Ribosomal RNA 249 Rickets 300,303 Rochelle salt 30 Role of extra-hepatic tissues 177 hormones 177 kidney 177 liver 176 liver lipid metabolism 209 muscle 177 surface tension 17 S Sacchrometric method 336 Salad 323 Salted biscuits 322 Salvage pathway 252 Sanger’s method 89, 90 Saponification 60 Schiff’s base 212 Scintillation counter 388 Secondary antioxidants 71 metabolic gout 257 renal gout 258 Semiessential amino acids 80 Serine pathway 222 Serum creatine phosphokinase 138 glutamate oxaloacetate transaminase 138 pyruvate transaminase 138 lactate dehydrogenase 138 Shuttle system 149 Sialicacids 51 Sickle cell hemoglobin 112 Simple lipids 54 proteins 85 Sodium 301 Somogyi’s iodine test 336 Specific dynamic action 312 Sphingomyelins 66 Starch 45 Stereochemistry 21 Stereospecificity 123 Steroids 69, 361 Stool examination 337 Storage functions 327 Structure of coenzyme 278 DNA 246 hemoglobin 106 proteins 88 Substrate level phosphorylation 146 specificity 123 Sugar/jaggary 322 Sulfatides (sulpholipids) 68 Sulfur 303 containing amino acids 226 Supersecondary structures 94 Surface tension 17 Synthesis of arginine 217 carbamoyl phosphate 215 citrulline 217 glutathione 222 heme 105 indole and skatole 239
    • 406 BIOCHEMISTRY FOR STUDENTS melanine 234 melatonin 239 niacin 237 nonprotein 212 serotonin 238 urea 218 T T cells 342 Tea 323 Terpenes 69 Tertiary antioxidants 71 Tetrahydrofolic acid 279 Thiamine 270 pyrophosphate 271 Thymic selection 341 Thyroid 361 gland 369 hormone 178 Timnodonic acid 57 Total food for day 322 Transamination 212 Transcellular fluid 292 Transcription control 382 Transfer or soluble RNA 247 Transferase reaction 207 Triglyceride synthesis 195 Tryptophan 237 Tumor suppressor genes 358 Turnover number 130 Tyndall effect 17 Tyrosinosis 236 U Uncompetitive inhibition 134 Under aerobic condition 159 Units of radioactivity 388 Urea clearance test 333 cycle 214 Uridine diphosphate glucose 249 Urinary amylase 336 lipase 337 Uronic acid 34 pathway 174 V van Der Waal’s forces 94 Van’thoff’s law 12 Varicyate porphyria or mixed 115 Vegetable fruit group 313 Vegetables 319 Vegetarian/nonvegetarian 323 Very low density lipoproteins 184 Viscosity 18 Vitamin A 261 B complex 270 B12 281 B2, lactoflavin 272 C 268 D 264 E 265 K 266 like compounds 260 Vitamins 259, 318 coenzymes 260 Voltage and current 387 vonGeirke’s disease 165 W Water 335 and mineral metabolism 292 essential nutrient 319 soluble vitamins 260, 268 Wheat flour 322 Wilson disease or hepatolenticular degeneration 304 X Xenobiotics 306 Z Zinc 305