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An Easy Guide for

Practical Biochemistry

An Easy Guide for

Practical Biochemistry

Divya Shanthi D’Sa

MBBS, DFH

Lecturer Department of Biochemistry Shimoga Institute of Medical Sciences Shimoga, Karnataka, India

Sowbhagya Lakshmi

MSc, PhD

Professor of Biochemistry Shimoga Institute of Medical Sciences Shimoga, Karnataka, India

®

JAYPEE BROTHERS MEDICAL PUBLISHERS (P) LTD St Louis (USA) • Panama City (Panama) • New Delhi • Ahmedabad • Bengaluru Chennai • Hyderabad • Kochi • Kolkata • Lucknow • Mumbai • Nagpur

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2/B, Akruti Society, Jodhpur Gam Road Satellite Ahmedabad 380 015, Phones: +91-79-26926233, Rel: +91-79-32988717 Fax: +91-79-26927094, e-mail: [email protected] 202 Batavia Chambers, 8 Kumara Krupa Road, Kumara Park East Bengaluru 560 001, Phones: +91-80-22285971, +91-80-22382956, Rel: +91-80-32714073 Fax: +91-80-22281761, e-mail: [email protected] 282 IIIrd Floor, Khaleel Shirazi Estate, Fountain Plaza, Pantheon Road Chennai 600 008, Phones: +91-44-28193265, +91-44-28194897, Rel: +91-44-32972089 Fax: +91-44-28193231, e-mail: [email protected] 4-2-1067/1-3, 1st Floor, Balaji Building, Ramkote Cross Road Hyderabad 500 095, Phones: +91-40-66610020, +91-40-24758498, Rel:+91-40-32940929 Fax:+91-40-24758499, e-mail: [email protected] No. 41/3098, B & B1, Kuruvi Building, St. Vincent Road Kochi 682 018, Kerala, Phones: +91-484-4036109, +91-484-2395739, +91-484-2395740 e-mail: [email protected] 1-A Indian Mirror Street, Wellington Square Kolkata 700 013, Phones: +91-33-22651926, +91-33-22276404, +91-33-22276415 Fax: +91-33-22656075, e-mail: [email protected] Lekhraj Market III, B-2, Sector-4, Faizabad Road, Indira Nagar Lucknow 226 016, Phones: +91-522-3040553, +91-522-3040554 e-mail: [email protected] 106 Amit Industrial Estate, 61 Dr SS Rao Road, Near MGM Hospital, Parel Mumbai 400 012, Phones: +91-22-24124863, +91-22-24104532, Rel: +91-22-32926896 Fax: +91-22-24160828, e-mail: [email protected] “KAMALPUSHPA” 38, Reshimbag, Opp. Mohota Science College, Umred Road Nagpur 440 009 (MS), Phone: Rel: +91-712-3245220, Fax: +91-712-2704275 e-mail: [email protected]

North America Office 1745, Pheasant Run Drive, Maryland Heights (Missouri), MO 63043, USA Ph: 001-636-6279734 e-mail: [email protected], [email protected] Central America Office Jaypee-Highlights Medical Publishers Inc., City of Knowledge, Bld. 237, Clayton, Panama City, Panama Ph: 507-317-0160 An Easy Guide for Practical Biochemistry © 2010, 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 authors and the publisher. This book has been published in good faith that the material provided by authors is original. Every effort is made to ensure accuracy of material, but the publisher, printer and authors will not be held responsible for any inadvertent error(s). In case of any dispute, all legal matters are to be settled under Delhi jurisdiction only. First Edition: 2010 ISBN 978-81-8448-793-0 Typeset at JPBMP typesetting unit Printed at Ajanta Offset & Packagins Ltd., New Delhi

Dedicated to

Students

Preface Biochemistry, a fascinating subject that deals with every function and every reaction of the body. Clinical biochemistry plays a tremendous impact on the diagnosis and treatment of patients. Medical students should be aware of the practicals, diagnostic parameters and their estimations. They should acquire sound knowledge about the diagnostic reports and its implications which aids in diagnosis and prognosis of the disease. Biochemistry is the most fast growing subject, extensively applicable to understand the disease at molecular level. Estimations of various biochemical parameters definitely give an insight to understand the normal metabolism and its aberrations leading to diseases, which forms the foundation for medicine. Biochemistry should be encouraged in relation to health and disease which will make the subject more interesting and fascinating to the students. We are hopeful that this practical biochemistry book will help the medical students to envisage about the various facts encountered in the reactions in the body. Dear students let us admit that ‘Biochemistry’ is rarely a medical graduates’ favorite given the fact that it is non‘Clinical’, extensive, volatile and there is little in it to arouse any amount of interest in the medical graduates. In our teaching biochemistry to undergraduate medical students we have realized practicals is where they have shown some amount of enthusiasm towards biochemistry.

viii An Easy Guide for Practical Biochemistry Our main aim is to make this book simple and attractive to the undergraduate medical students as far as possible which is apparent from the title of the book. Towards achieving this goal, each practical session has been reorganized such that it becomes easy to understand. Wherever possible, the subject is presented in tabular format such that it becomes very concise; and all test results are given in color such that the simple task of reading the book itself realizes one doing the practicals. Fundamental concepts and principles behind each experiment are explained in a simple way. Our only genuine concern is to help you to understand the subject in an easy and organized way such that this little knowledge comes as a big help not only in your exams but also in your future medical career. We will be glad to accept constructive criticism and fruitful suggestion to make this book a better one.

Divya Santi D’Sa Sowbhagya Lakshmi

Acknowledgments The authors are indebted to student community who constantly motivate and make us stay updated with the latest knowledge. It is our proud privilege to express our gratitude to our colleagues Dr Govindaswamy, Dr I C Vinay, Dr Jyothi R S and Mr Anil Kumar Suryavamshi involved in reviewing the manuscript of this book. I take this opportunity to thank computer operators Mrs Veena Jayaram and Mr Sunder for their help in preparing the manuscript. We are also very grateful to our lab technicians Mr Vasant, Mr Shankar, Mr Girish and Miss Rajeshwari for their valuable assistance. The authors are indebted to Jaypee Brothers Medical Publishers (P) Ltd., New Delhi, for bringing our effort to a proper shape in a way that it could become a good pocket companion to the students. We are very grateful to Shri Jitendar P Vij, Chairman and Managing Director and publishing team of M/s Jaypee Brothers Medical Publishers (P) Ltd., for their sincere efforts and cooperation.

Contents SECTION 1: LABORATORY RULES AND REGULATIONS 1. Laboratory Hazards and First Aid ............................. 3 2. Laboratory Safety Rules ............................................ 10 3. Specimen Collection and Processing ...................... 16 4. Glasswares Used in Biochemistry Laboratory ...... 20 SECTION 2: QUALITATIVE TESTS 5. Qualitative Analysis of Carbohydrates .................. 29 6. Qualitative Analysis of Proteins .............................. 48 7. Nonprotein Nitrogenous Substances ...................... 74 8. Qualitative Analysis of Normal Urine .................... 82 9. Analysis of Abnormal Constituents in Urine ........ 99 10. Hemoglobin and its Derivatives ............................ 117 11. Spot Tests .................................................................. 123 SECTION 3: QUANTITATIVE TESTS 12. Principles of Colorimetry ........................................ 129 13. Estimation of Blood Sugar ...................................... 138 14. Estimation of Blood Urea ........................................ 144 15. Estimation of Urine Creatinine .............................. 148 16. Estimation of Serum Inorganic Phosphate .......... 154 17. Estimation of Serum Total Proteins ...................... 159

xii An Easy Guide for Practical Biochemistry SECTION 4: DEMONSTRATION PRACTICALS 18. Chromatography ...................................................... 169 19. Electrophoresis ......................................................... 174 20. Glucose Tolerance Test .......................................... 181 21. Estimation of Serum AST and ALT ...................... 188 22. Estimation of Serum Cholesterol ........................... 192 23. Estimation of Plasma Ascorbic Acid ..................... 195 24. Flame Photometer .................................................... 197 25. CSF Analysis ............................................................. 200 26. Estimation of Albumin in Urine ............................ 204 Appendices .................................................................. 207 Index ........................................................................... 269

Chapter

1

Introduction to Biophysics 

Introduction to Biophysics (Measurement and Accuracy)

     

Meaning of Biophysics Importance of Biophysics in Nursing Concept of Unit Fundamental and Derived Units Systems of Units Units of Length, Weight, Mass and Time

INTRODUCTION Modern biophysics combines state-of-the-art physical measurements with computational models to understand the detailed physical mechanisms underlying the behavior of complex biological systems. Biophysics is a growing enterprise worldwide, driven primarily by the widespread realization of the major contribution that can be made to biological science by a combination of truly state-of-the-art physical measurements with modern molecular biology. The field occupies a unique and central position at the intersection of the biological, chemical, physical, and computational sciences.

1

 Biophysics in Nursing Biophysics is intrinsically interdisciplinary. Biophysics takes a quantitative, physical, non-phenomenological approach to biology that is firmly rooted in the principles of condensed-phase physics and physical chemistry. Biophysicists are driven primarily by their curiosity about how biological systems work at the molecular level. While they routinely employ the methods of molecular biology, their primary focus is on development of novel structural and dynamical tools that enable uniquely incisive studies of systems ranging in complexity from single proteins in vitro to the complex interactions of biopolymers in live cells. Biophysicists as a group most often develop the novel, sophisticated experimental methods that reveal molecular level details with unprecedented clarity. The state of the art in X-ray crystallography, solution phase and solid-state NMR, atomic force microscopy, single-molecule methods, EPR, and fluorescence microscopy continues to evolve in ways that better elucidate biological structure and function. In parallel, biophysicists are developing powerful new computational tools based on firmly established physical principles that are sufficiently accurate to greatly enhance insights from experiment. Just as the tools of molecular biology gradually become useful to biophysicists, overtime the new tools developed by biophysicists gradually find widespread use among all biological scientists.

MEANING OF BIOPHYSICS The term Biophysics was first used in 1892 by Karl Pearson in his book The Grammar of Sciences. “Biophysics is defined as the science where there is application of the laws of physics to life process.” “Biophysics is the application of physical principles and methods to the study of the structure of living organisms and the mechanisms of the life process.” “It is the science of living physics; the forms of physics applies the knowledge of physics to explain biological questions, such as the transmission of nervous impulses or muscle control.” “Biophysics is branch of science that deals with study of physical or biophysical principles and their application to health sciences.”

2

Introduction to Biophysics  IMPORTANCE OF BIOPHYSICS IN NURSING Study of biophysics immensely benefits the nurses, because it helps them to acquire: 1. Practical, functional knowledge of physical principles that underline nursing procedures and the operation of machinery that nurses use. 2. Technical knowledge from the science of physics that applies specifically to nursing performance and understand certain biomedical phenomenon like how does a suction apparatus operates? What is the most efficient way to move a heavy object or a patient? How does air get in and out of the lungs?, etc. In addition study of biophysics helps a nurse understand following contents of nursing:

Measurement • •

Accuracy in preparation of medications Assessment of patients by measurement of vital signs.

Motion • •

Inertia in accidents Physiological reaction to high velocity centrifuges.

Gravity • • •

Circulation of blood • Postural drainage Postoperative position • ESR estimation Dependent position for edema patient

Center of Gravity • •

Body mechanics Crutch walking

• Lifting and turning patients

Specific Gravity •

Underwater exercises

• Examination of the body fluids

3

 Biophysics in Nursing Force • Torques in traction • Muscle action • Vector addition and analysis in traction Pressure • Suction • Internal and external respiration • Positive pressure • Oxygen therapy ventilation • Administration of irrigation and parenteral fluid Heat • Thermometry • •

Steam inhalation Thermography

Light and Sound • Actions of lenses • Microscopy • Refraction • Audiometery Electricity • Patient monitors, ECG, EEG, EMG • Electrosurgical procedures • Use of transistors in apparatus

• Application of heat and cold application • Basal metabolism • Autoclave and sterilization

• • • •

Use of mirrors in apparatus Ophthalmoscope Visual fields Human audibility

• Diathermy • Electric shock therapy • Cardiac pacemakers

Work and Energy • Circulation of blood • Pulse formation • Work done by heart and skeletal muscles Molecular Physics • Artificial kidney • Colloidal dispersions • Surface tension of antiseptics • Viscosity of blood

4

Introduction to Biophysics  Atomic Physics • High energy radiation • X-ray therapy • Radioisotopes • Tracer studies of metabolism • Precautions in use of radioactive material • Half-life in radiotherapy CONCEPT OF UNIT Nursing care demands several measurement tasks like measuring vital signs, patient’s height, weight, body mass index, 24 hours fluid balance, and so many others. In this situation, a nurse takes a measurement of physical quantity and compare measured value of physical quantity with a standard to determine its relationship with that standard. The standards of measurement is called a unit. “The unit of any measurement is defined as a conventional quantity used as the reference or standard of measurement to which measurements with that unit can be compared.” FUNDAMENTAL AND DERIVED UNITS The unit of measurement is fixed by definition and is independent of such physical conditions as temperature, humidity, etc. The numerical value of a physical quantity, therefore, refers to the number of standard unit of measurement. For example, when we say that a patient’s temperature is 38°C, it means that the patient’s temperature is 38 times the unit of measurement, called degree Celsius (°C). Thus measurement of any quantity has two characteristics—a numerical value and a unit. For example, you measure the birth weight of a baby as 3.5 kg. Then 3.5 is the numerical value and Kg is the unit. Although the number of physical quantities that we measure is very large, we do not need a very large number of standards to compare every measurement. It is so because all the physical quantities are not independent quantities in so far as their measurement is concerned. For example, velocity of a body is measured in units of length (meter) and time (seconds). A few independent standards have been chosen to fix the units of certain physical quantities. The measurement of most of the other physical quantities can be expressed in terms of

5

 Biophysics in Nursing these independent standards. These independent standards are length, mass and time. Such units fixed by independent standards are called fundamental units. For example, – One meter: the unit of length, – One kilogram: the unit of mass and – One second: the units of time are fundamental units. “Fundamental units are those units, which can neither be derived from one another, nor can they be further resolved into any other units.” Units of measurement of many physical quantities such as density, speed, volume, pressure and force can be derived from these fundamental or basic units using physical equations. These units are called derived units. For example, the unit of volume is cubic meter which is derived from the unit of length. Speed is defined as distance covered per unit time and its unit is m/s. The unit of speed is derived from units of length and time. “Units of various physical quantities, which can be expressed in terms of the fundamental units of mass, length and time, are called derived units.”

SYSTEMS OF UNITS There are several systems of units that have been used for measuring physical quantities. The commonly used systems are the CGS (Centimeter gram second), the FPS (Foot pound, second), the MKS (Meter kilogram, second) and the SI (System internationale). They differ from each other because different standards of measurement are used for fundamental quantities. Table 1.1 contains the standards of measurement for fundamental quantities in these systems. The two systems of measurement most frequently used in nursing practice are the MKS (also called metric) and the FPS (also called English). You may note from Table 1.1 that the units for these physical quantities are the same in the metric and SI systems.

6

Introduction to Biophysics 



Table 1.1: Systems of units with their standards of measurement

Physical quantity

CGS system

FPS system

MKS system

Length Mass Time Temperature Electric current Light intensity Amount of substance

Centimeter (cm) Foot (f) Meter (m) Gram (gm) Pound (d) Kilogram (kg) Second (s) Second (s) Second (s) — Fahrenheit (F) Celsius (°C) — — — — — — — — —

SI system Meter (m) Kilogram (kg) Second (s) Kelvin (K) Ampere (A) Candela (Cd) Mole (mol)

FUNDAMENTAL UNITS IN VARIOUS SYSTEMS Unit of Length Length can be defined as the distance between two points in space. The unit of length in English system is the foot. The unit of length in the Metric system is the meter. In health care system one can observe use of both the system like patient’s hight recorded in feet, whereas small size papule on skin is measured in millimeters. Similarly, in microscopic work, a very small unit– micron is used. The micron is 1/1,000 mm. The various multiples of units of length are listed in Table 1.2 for both Metric and English systems.



Table 1.2: Multiples of units of length in English and Metric systems English system

Metric system

12 inches = 1 foot 3 feet = 1 yard 5 ½ yard = 1 rod 1,760 Yard = 1 mile 5,280 feet = 1 mile

10 10 10 10 10 10 10

millimeters (mm) = 1 centimeter (cm) centimeter (cm) = 1 decimeter decimeter = 1 meter (m) meters = 1 decameter decameters = 1 hectometer hectometers = 1 kilometer (km) kilometers = 1 myriameter

Note: 1 feet = 12 inches = 30 cm (1 inch = 2.5 cm)

7

 Biophysics in Nursing Unit of Mass and Weight The mass of a body refers to the quantity of matter contained in it. The unit of mass in Metric system and SI system is the kilogram (kg). A physical balance ordinarily measures the mass of a body. Table 1.3 shows unit of mass in the English system and Metric system. Some of these units are used in measuring food items for special diets, amount of drugs, weights of patients, etc. Although commonly we use the terms mass and weight in the same sense, the two terms have different meanings in physics. In physics, concept of mass and weight are different. Mass of a body is the quantity of matter contained in it. On the other hand, weight is defined as the gravitational force with which a body is pull towards the center of the earth. Mathematically, we write W=m×g where, ‘W’ denotes the weight of the body, ‘m’ is its mass and ‘g’ is the acceleration due to gravity. In the SI system, the unit of weight is Newton. Since, the value of the acceleration due to gravity varies with the distance of an object



Table 1.3: Multiples of units of mass in the Metric and English systems English system Troy units 24 grains = 1 pennyweight 20 pennyweight = 1 ounce 12 ounces = 1 pound Avoirdupois units 27.34 grains = 1 dram 1 6 drams = 1 ounce 16 0unces = 1 pound 25 pounds = 1 quarter 4 quarters = 1 hundredweight 20 hundredweight = 1 short ton 2,240 pounds = 1 long ton Apothecaries unit 20 grains = 1 scruple 3 scruple = 1 dram 8 drams = 1 ounce 12 ounces = 1 pound

8

Metric system 10 milligrams = 1 centigram 10 centigram = 1 decigram 10 decigrams = 1 gram 10 grams = 1 decagram 10 decagrams = 1 hectogram 10 hectograms = 1 kilogram 1,000 kilograms = 1 metric ton

Introduction to Biophysics  from the center of the earth, the weight of the object changes with its position on the earth. For example, an object at sea level weighs more than it does on a high mountain because the value of the acceleration due to gravity of the earth on the object is greater at sea level. The mass, however, remains the same everywhere. The mass of an object is measured by a physical balance whereas its weight is measured by a spring balance. Mass is a scalar quantity while weight is a vector quantity because it is directed towards the center of the earth. Mass of a person is the same on the earth as well as on the moon, but weight of the person is different at these two places because their pulls on the person are different. A person weighs six times more on earth than on the moon. Whereas mass and weight have the same numerical value, it is important in solving problems to indicate the unit specifically, as one of force (weight) or as one of mass. one gram (gm) is a unit of mass; one gram weight is unit of weight (Tables 1.4 and 1.5).

Units of Time The unit of time is the second and is based on the natural clock. The natural clock is governed by the time taken by the earth to complete



Table 1.4: Conversion of weight and measurements Weight

1 ounce = 8 drams 12 ounces = 1 pound 1000 microgram (Mcg) = 1 milligram (mg) 1000 milligram (mg) = 1 gram (gm) 1000 grams (g) = 1 kilogram (kg) 1 kilogram (kg) = 2.2 pounds 1 grain = 60 milligram (mg) 1 dram = 4 grams (g) 1 ounce = 30 grams 1 pound = 375 grams 1 milligram = 1/60 grains (gr)

Fluid volume 60 minimus = 1 fluid dram 8 fluid drams = 1 fluid ounce 20 fluid ounce = 1 pint 2 pints = 1 quart (1000ml) 8 pints = 1 gallon 1 milliliters (ml) = 15 – 16 minims (15–16 drops) 1 liter = 35 fluid ounce 1 fluid ounce = 30 ml 1 fluid dram = 4 ml 1 gallon = 4.5 liter 1 minimus = 0.04 ml = 1 drop 1 pint = 500 ml Household measurements 1 teaspoonful = 4 or 5 ml = 1 fluid dram = 60 drops

9

 Biophysics in Nursing



Table 1.5: Prefixes and symbols used with SI units and Metric units Prefix Tera Giga Mega Kilo Hecto Deca Meter Deci Centi Milli Micro Nano Pico Femto Atto

Symbol T G M K H Da m D C Mm u n p f A

Power 1012 109 106 103 102 10 1 10–1 10–2 10–3 10–6 10–9 10–12 10–15 10–18

Value (in meter) 1,000,000,000,000 1,000,000,000 1,000,000 1,000 100 10 1 .1 .01 .001 .000001 .000000001 .000000000001 .000000000000001 .000000000000000001

one revolution around the moon. According to this clock, one second is defined as (1/86400) part of a mean solar day; a solar day is the period between noons of two consecutive days and a mean solar day is the average solar day over a year, which is 24 hours. Since one hour contains 60 minutes and one minute contains 60 seconds, a mean solar day of 24 hours would have 24 × 60 × 60 = 86400. Thus, one second is 1/86400th part of a mean solar day. Let us consider some of the measurements of time you make in the course of your work. You will see that the second’s hand of your watch is sufficiently accurate for recording a patient’s pulse rate (number of pulse beats per minute). However, for studying the heart beat of a patient by electrocardiography, greater accuracy in the measurement of time is required. In this case the beating of the heart must be accurately measured in tenths or hundredths of a second. In nursing practice, you may come across situations when a measurement taken in Metric unit must be changed to the corresponding English unit and vice versa. For this reason, approximate equivalents commonly used in the hospital are given in Table 1.6.

10

Introduction to Biophysics 



Table 1.6: Conversion between Metric and English systems

Weight

Length

Volume

1 gram = 15 grains 2.54 centimeter = 1 inch 1 cubic centimeter = 15 minims 4 grams = 1 dram 1 meter = 39.37 inches 4 cubic centimeter = 1 fluid dram 30 grams = 1 ounce 30 cubic centimeter = 1 fluid 454 grams = 1 pound ounce 1 kilogram = 2.2 pounds

QUESTIONS Q 1. Discuss the meaning and importance of biophysics in nursing. Q 2. Discuss the concept of units and fundamental and derived units. Q 3. Describe the different system of the units. Q 4. List of the basic units of length, weight, mass and time. BIBLIOGRAPHY 1. Cantor CR, Schimmel PR. Biophysical Chemistry, WH Freeman, San Francisco, 1980;1-3. 2. Cantor CR, Schimmel PR, Freeman WH. San Francisco, Biophysical Chemistry 1980;1-3. 3. Dogonadze RR, Urushadze ZD. Semi-classical method of calculation of rates of chemical reactions proceeding in polar liquids. J Electroanal Chem 1971;32:235-45. 4. Eugenie V. Mielczarek, Elias Greenbaum, Robert S Knox. Biological Physics. New York. American Institute of Physic, 1993. 5. Flitter HH, Rowe HR. An Introduction to Physics in Nursing. St. Lous: The CV Mosby Company, 1995. 6. Glaser R, Biophysics, Springer, 2001. 7. Glaser R. Biophysics: An Introduction. Springer Verlag, Heidelberg, 2004. 8. Glaser, Roland. Biophysics: An Introduction (Corrected ed.). Springer, 2004;11-23. 9. Gomber KL, Gogia KL. Fundamental Physics. Ambala: Paedeep Publishers, 2004. 10. Goyal RP, Tripathi SP. Oncise Physics, New Delhi: Selina Publishers, August 2007. 11. Hobbie RK, Roth BJ. Intermediate Physics for Medicine and Biology (4th Edition). Springer, 2006. 12. Hobbie RK, Roth BJ. International Physics for Medicine and Biology (4th ed.). Springer, 2006. 13. Lal S. Principles of Physics. Ambala: Paedeep Publishers, 2004.

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 Biophysics in Nursing 14. Meyer B Jackson. Molecular and cellular biophysics. New York: Cambridge Publication, 2006. 15. Nicolas Rashevsky, Mathematical biophysics. . Rev. ed., University of Chicago Press, 1948. 16. Perutz MF, Electrostatic Effects in Proteins, Science, 1978;201:1187-91. 17. Perutz MF. “The haemoglobin molecule”. Proceedings of the Royal Society of London. Series, 1969; B 173 (31): 113-40. 18. Perutz MF. Proteins and Nucleic Acids, Elsevier, Amsterdam, 1962. 19. Perutz MF. Proteins and Nucleic Acids: Structure and Function. Amsterdam: Elsevier, 1962. 20. Philip C, Nelson. Biological Physics (Updated Edition). WH Freeman, 2007. 21. Phillips R, Kondev J, Theriot J. Physical Biology of the Cell. Garland Science, Oxford, 2008. 22. Rashevsky, N., Mathematical Biophysics: Physico- Mathematical Foundations of Biology - Vol.2, New York: Dover Publications, Third Revised Edition, 1960. 23. Rodney MJ, Cotterill. Biophysics: An Introduction. Wiley, 2002. 24. Ruch TC, Fulton JF. Medical physiology and biophysics. Saunders 1974:1232. 25. Sneppen K, Zocchi G. Physics in Molecular Biology. Cambridge University Press, 2005. 26. Sneppen K, Zocchi G. Physics in Molecular Biology (1 ed.). Cambridge University Press 2005;10-17. 27. Volkenshtein MV, Dogonadze RR, Madumarov AK, Urushadze ZD, Kharkats Yu.I. Theory of Enzyme Catalysis. Molekuliarnaya Biologia (Moscow)1972; 6: 431-9 (In Russian, English summary).

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Section 1

Laboratory Rules and Regulations

1

Laboratory Hazards and First Aid

Biochemical parameters aids in diagnosis and prognosis of diseases. The medical students should have the knowledge of the various tests, diagnostic investigations done in biochemistry laboratory. They should also be aware of all potential hazards and the safety measures.

HAZARDS The student is surrounded by many dangers (Fig. 1.1) such as: • Broken glassware. • Corrosive reagents. • Mechanical hazards. • Poisonous fumes that could be inhaled. • Inflammable chemicals. • Gas leakages. • Electrical hazards.

SAFETY MEASURES • Lab coat has to be worn in order to protect oneself from corrosive splashes. • One has to be careful while handling gases. • Blood, urine, CSF and other biological fluids should be handled with great care as they are potential sources of infections like HIV and Hepatitis.

4

An Easy Guide for Practical Biochemistry

Fig. 1.1: Signs of some laboratory hazards

• Chemical work involving irritating chemicals and dangerous infectious materials should always be conducted under hoods with good exhaust and adequate ventilation. • Safety cabinets and hoods should be used while handling corrosive reagents. • Electrical heater and other electrical appliances should be checked and insulated frequently. Damage should be rectified immediately. • Bunsen burner should never be used around inflammable material like ether, and acetone.

Laboratory Hazards and First Aid

5

• General health should be maintained at all costs as an effective means for keeping natural immunity and resistance. • Eating and drinking in the laboratory must be avoided.

CARE WHILE PIPETTING • Exercise great care and take precautions while mouth pipetting. The mouth of the pipette should be plugged with cotton or piece of rubber while filling. • One should not be engaged in conversation or other disturbances. • Automatic dispensers and automatic pipettes must be used for pipetting acids, alkalis, corrosive solutions and poisonous solutions. • The hands should be kept free of cuts and abrasion. • Hands should be washed with soap water followed by washing with disinfectant material. • Pipettes and other instruments employed should be placed immediately in disinfectant solution.

PRECAUTIONS REGARDING FIRE SAFETY • Open flames should not be left unattended. • Any leakage of gas should be properly attended and reported. • Smoking should be strictly prohibited. • Burning match sticks should not be thrown in waste baskets. IN CASE OF ACCIDENTAL FIRE • Sand or blanket should be used to put off the small fire. • For longer blazes, “Fire Extinguishers” have to be used.

6

An Easy Guide for Practical Biochemistry

• Water should NOT be used on electrical fire. • Water should NOT be used on a fire caused by organic solvents such as ether, alcohol, petrol, etc. • While trying to escape from fire, in case if it cannot be extinguished quickly, it is safe to stay close to the floor and crawl by covering mouth with damp cloth.

ACCIDENTAL SWALLOW OF CORROSIVE SOLUTIONS 1. If the corrosive solution swallowed is an acid: • Spit the corrosive solution. • Promptly rinse the mouth. • Antidotes such as 8% magnesium hydroxide (milk of magnesia) or egg white mixed in water can be used orally to neutralize the acid. • Seek medical help immediately. 2. If the corrosive solution swallowed is an alkali: • Promptly rinse the mouth. • Antidotes such as lemon juice or 5% acetic acid can be taken orally to neutralize the alkali. • Seek medical help immediately. INHALATION OF CORROSIVE GASES Take the student to fresh air and seek medical help immediately. BURNS From strong acids: • First wash with LOTS of water and then wash with 5% sodium carbonate or 5% ammonium hydroxide. • Seek medical help immediately.

Laboratory Hazards and First Aid

7

From strong alkalies: • Wash immediately with LOTS of water and later with 5% boric acid or dilute acetic acid solution. • Seek medical help immediately.

LABORATORY FIRST AID Laboratory first aid refers to the immediate help given to an injured person. The first aid kit should contain: • Cotton wool and gauze • Roller bandage • Scissors • Acetic acid • Milk of magnesia • Spirit • Adhesive tapes • Disinfectant solution • 5% sodium carbonate.

PRECAUTIONS TO BE TAKEN WHILE HANDLING CHEMICALS • All chemicals should be considered as potentially dangerous and should be handled carefully. One should be aware that accidental injuries can occur either from direct contact through skin, by inhaling vapors, powder or swallowing by mistake while pipetting. • Clear labeling of all the bottles containing chemicals and reagents should be done and their potential hazards should be noted on the label. • Reagent bottles should be held with both hands and should not be carried by holding their necks. • The reagent bottles in use should be kept on shelf at the eyelevel of the user.

8

An Easy Guide for Practical Biochemistry

• Corrosive chemicals should be opened with great care and added slowly to water with continuous cooling and stirring as these substances can destroy the living tissue, e.g. Strong acids like sulfuric acid, hydrochloric acid and strong alkalies like potassium hydroxide, sodium hydroxide, etc. • Automatic dispensers should be used to dispense acids, alkalies and corrosive liquids. • Toxic chemicals such as cyanides, barbiturates must be kept locked in cupboards and mouth pipetting of these should be avoided at any cost. • Some organic solvents are highly toxic to certain organs, e.g. Benzene is toxic to bone marrow; carbon tetrachloride and halogenated hydrocarbons are toxic to liver, etc. Hence, their use should be minimized in assays. • Precautions must be taken while handling carcinogenic substances such as benzidine, orthotoulidine. Bottles containing such substances have to be labeled as CARCINOGENIC. Skin contact with them must be strictly avoided and rubber or plastic gloves should be used while handling these substances. • One has to be careful while handling explosive substances. Certain precautionary measures must be followed like: 1. Perchloric acid should be kept in fume cupboard. 2. Picric acid should be stored in a container of water tightly closed with cork or rubber stopper. 3. Ether should be kept in brown or dark bottles away from sunlight since on exposure to sunlight they form peroxides, that when raised to certain sufficient concentration cause violent explosion.

Laboratory Hazards and First Aid

9

4. Cylinder containing inflammable gases like hydrogen, propane, acetylene should be kept outside the laboratory when not in use.

IN CASE OF ACCIDENTS IN THE LABORATORY • One should not be panic. • Alarm should be raised as soon as possible. • The laboratory should be evacuated, to minimize further damage to property. • Gas and electricity connections have to be turned off immediately. • In case of fire attacks, fire extinguishers should be used to tackle them. • In case of large fires, the fire brigade has to be called.

WASTE DISPOSAL Chemical Waste • Neutralization of acids and alkalies should be done prior to their washing in the sink. • Organic solvents should be stored in metal drums and later it must be washed off. • Some chemicals can be cleared or disposed by INCINERATION. Radioactive Waste Expert opinion has to be taken for the disposal of radioactive waste, and their guidelines have to be strictly followed. Flushing radioactive substances down the sink can be very dangerous as they pollute the underground water table.

10 An Easy Guide for Practical Biochemistry

2

Laboratory Safety Rules

Some rules are NOT made to be broken. That is true for the rules used in a biochemistry lab.

GENERAL GUIDELINES 1. Conduct yourself in a responsible manner at all times in the laboratory. 2. Follow all written and verbal instructions carefully. If you do not understand a direction or part of a procedure, ASK YOUR TEACHER BEFORE PROCEEDING WITH THE ACTIVITY. 3. Do not touch any equipment, chemicals, or other materials in the laboratory area until you are instructed to do so.

Laboratory Safety Rules 11 4. Be prepared for your work in the laboratory. Read all procedures thoroughly before entering the laboratory. Never fool around in the laboratory. 5. Always work in a well-ventilated area. 6. Observe good housekeeping practices. Work areas should be kept clean and tidy at all times. 7. Be alert and proceed with caution at all times in the laboratory. Notify the teacher immediately, if you observe any unsafe conditions. 8. Dispose all chemical wastes properly. Never mix chemicals in sink drains. Sinks are to be used only for water. Check with your teacher for disposal of chemicals and solutions. 9. Keep hands away from face, eyes, mouth and body while using chemicals or lab equipment. Wash your hands with soap after performing all experiments. 10. Any time when chemicals, heat, or glassware are used, students must wear safety goggles. 11. Dress properly during a laboratory activity. Long hair, dangling jewelry, and loose or baggy clothing are hazardous in the laboratory. Long hair must be tied back, and dangling jewelry and baggy clothing must be secured. Shoes must completely cover the foot. No sandals allowed in the laboratory. 12. A lab coat should be worn during laboratory experiments.

ACCIDENTS AND INJURIES 13. Report any accident (spill, breakage, etc.) or injury (cut, burn, etc.) to the teacher immediately, no matter how trivial it is. Do not panic.

12 An Easy Guide for Practical Biochemistry 14. If a chemical splashed into your eye(s) or on your skin, immediately flush with running tab water for at least 20 minutes.

HANDLING CHEMICALS 15. All chemicals in the laboratory are to be considered dangerous. Avoid handling chemicals with fingers. Do not taste, or smell any chemicals. 16. Check the label on all chemical bottles twice before removing any of the contents. Take only as much chemical as you need. 17. Never return unused chemicals to their original container. 18. Never remove chemicals or other materials from the laboratory area. 19. Never pipette by mouth.

HANDLING GLASSWARE AND EQUIPMENT 20. Never handle broken glass with your bare hands. Use a brush and dustpan to clean up broken glass. Place broken glass in the designated glass disposal container. 21. Examine glassware before each use. Never use chipped, cracked or dirty glassware. 22. If you do not understand how to use an equipment, ASK THE TEACHER FOR HELP! 23. Do not immerse hot glassware in cold water. The glassware may shatter.

Laboratory Safety Rules 13

HEATING SUBSTANCES 24. Heated glassware remains very hot for a long time. They should be set aside in a designated place to cool, and picked up with caution, using tongs. 25. Never look into a container that is being heated as there are chances of it getting splashed to the face or eyes.

Fig. 2.1

14 An Easy Guide for Practical Biochemistry

AFTER EXPERIMENTS 26. Clean all the glassware you have and put them on the shelf in a proper order. 27. Wipe and clean the table. 28. Put all chemicals back to their respective places. 29. Put off the gas burner.

Fig. 2.2

PIPETTING TECHNIQUES 1. Use a pipette bulb to draw liquid above the calibration mark. 2. Remove the bulb and cover the pipette with your forefinger. 3. Dry the pipette tip with a tissue.

Laboratory Safety Rules 15 4. Rotate the pipette using the thumb and the other fingers to let in air so that the liquid drains slowly until the meniscus reaches the calibration mark. 5. To deliver the liquid, hold the pipette vertically and let the pipette tip touch the wall of the receiving container. 6. When the delivery is completed, touch the tip of the pipette to the wall of the container. Caution: Always keep the pipette tip under liquid surface when you draw up liquid. Never use the bulb to blow air inside the pipette, this will introduce dust and make the pipette dirty.

16 An Easy Guide for Practical Biochemistry

3

Specimen Collection and Processing

Biological samples like blood, saliva, CSF, pleural fluid, ascitic fluid, synovial fluids, kidney stones, gallstones and urine samples are used for analysis of various biochemical parameters which aid in diagnosis of various diseases. Blood is the most frequently used body fluid for analytical purposes. Ideally all estimations should be performed within 1-2 hr after collection. Whenever, a delay of more than 2 hours is anticipated, the serum and plasma samples have to be refrigerated at 4°C and if the delay is more than 6 hr, it is refrigerated at - 20°C. For extracting serum, allow the blood to clot at room temperature for 20- 30 minutes. Loosen the clot by a stick, and centrifuge for 10 minutes at 3000 RPM. Separate the serum and label it. They can be used later for analysis. Special care should be taken to avoid hemolysis. Hemolyzed samples alter the values of many chemical estimations because of the release of RBC contents, which can cause color change. False high results may be obtained because of hemolysis. Hemolyzed samples affect bilirubin and enzyme estimations giving erroneous results.

COLLECTION OF BLOOD Blood is collected by: • Venipuncture • Arterial puncture • Capillary puncture

Specimen Collection and Processing 17 Arterial and venous blood specimens differ in some important aspects. Venipuncture is more commonly performed for obtaining blood. Disposable needles are used to eliminate the hazards of infections. Sterilization of the puncture site is done by using 70% alcohol or ether. Whole blood, serum or plasma can be selected depending upon the methods by which the biochemical parameters are to be investigated. Usually serum/plasma is preferred.

ANTICOAGULANTS Blood starts clotting within few minutes after it is removed from the body unless an anticoagulant is used to stop the process of clotting. Blood with anticoagulant is known as ‘WHOLE BLOOD’. Serum is the fluid portion of clotted blood while plasma is the fluid portion of unclotted blood. Various anticoagulants are used depending on the parameters to be analyzed. Blood is collected and mixed with some chemicals that prevent clotting. These chemicals are called anticoagulants. Most of the anticoagulants used in the laboratory, act by binding calcium as an insoluble salt. Oxalates, citrates and EDTA chelate calcium ion. The routinely used anticoagulants are: 1. Potassium oxalate 2. Double oxalate 3. Ammonium oxalate 4. Sodium citrate 5. Heparin

18 An Easy Guide for Practical Biochemistry 6. EDTA (ethylene diamine tetra acetate) 7. Sodium fluoride (to prevent glycolysis; used for glucose estimation) 8. Acid citrate dextrose (ACD).

COLLECTION AND PRESERVATION OF URINE SPECIMENS Collection of Urine Samples • The urine samples should be collected in clean and dry containers. • For most of qualitative tests, a random urine sample is satisfactory. • Morning specimen is desirable for normal analysis. • Repeated urine samples are necessary to test for orthostatic proteinuria. • 24 hours sample is preferable for determination of calcium, uric acid, urinary protein and ketosteroids, as the concentrations vary at different times of the day. The patient is instructed to collect the urine sample from morning 8’o clock to the next day morning 8’o clock. Preservation of Urine Samples Several changes like urinary decomposition, precipitation of phosphates, crystallization of uric acid and bacterial action may alter the urinary composition, if it is kept for long periods, especially in the collection of 24 hours urine samples. Also urine may become alkaline due to precipitation of uric acid and urates.

Specimen Collection and Processing 19 Various preservatives are used depending on the analysis of parameters in urine. The common ones are concentrated hydrochloric acid (HCl) toluene and liquid petroleum. Before carrying out any estimation in urine, the urinary deposits must be mixed well. The total volume is measured which is required to calculate the amount of constituents of urine excreted/day and to calculate output per unit time in clearance tests.

20 An Easy Guide for Practical Biochemistry

4

Glasswares Used in Biochemistry Laboratory

Various types of glasswares are used in the biochemistry laboratory to measure, prepare and transfer or to keep solutions, reagents and samples. These glasswares may be volumetric (graduated) or non-volumetric (non-graduated). Volumetric glasswares include flasks, measuring cylinders, pipettes, etc. Non-volumetric glasswares include beakers, funnels, bottles and test tubes, etc.

LABORATORY GLASSWARES Beakers Beakers are wide, straight sided cylindrical vessels available in a wide range of volumes from 50 ml to several liters. They are used mainly for the preparation of the solutions and reagents. Flasks These have capacities of 25- 500 ml. Different types of flasks are available. a. Conical flasks (Erlenmeyer type): These are used for performing titration, and for boiling the solutions. Evaporation is minimum because of the conical shape. b. Flat- bottomed round flasks: These are used mainly for heating the liquids.

Glasswares Used in Biochemistry Laboratory 21 c. Round bottomed flasks: These can withstand high temperature. So they are used to evaporate samples to dryness, distillation of water, alcohol and other organic compounds. d. Volumetric flasks: They are flat bottomed, pear-shaped vessels with long narrow necks with a specific volume mark and fitted with a stopper. Graduated (Measuring) Cylinder Graduated (measuring) cylinders are narrow, straight side vessels that are used to measure specific volumes. They are available in sizes ranging from 10 ml to several liters. A high degree of accuracy is not possible because of their wider bore. Burettes Burettes are long, graduated tubes with a stop cork at one end, available in capacities of 10 to 100 ml. These devices are used to deliver known volumes of liquid into a container accurately. By measuring from one graduated line to another graduated line, one can deliver even fractional volumes (less than 1 ml) of liquid with a high degree of accuracy. They are used mainly for titrations and also to dispense corrosive reagents. Funnels Funnels are used to transfer liquids/solids into container, separation of solids from liquids. Funnels usually have short or long, thin stems. These funnels are used with filter paper to remove particles from solutions. Funnels with wide mouthed stems that allow solids to pass through easily are used for transferring solids into a container.

22 An Easy Guide for Practical Biochemistry Bottles Reagent Bottles Reagent bottles are cylindrical with a narrow neck fitted with stopper, available in various capacities of 25-2000 ml. They are made up of plain white or amber colored glass. Amber colored bottles are useful to store certain light sensitive chemicals like silver nitrate. Drop Bottles Drop bottles have a narrow neck with a slotted glass stopper, available in 50-100 ml capacities. They are used for delivery of drops of solutions such as stains and indicator solutions and are made up of white or brown glass. Wash Bottles Wash bottles are usually plastic bottles with a delivery tube at the top.

PIPETTES Pipettes are used for delivery of accurate and controlled volumetric measurements. They are of various types differing in their levels of accuracy and precision which includes complex adjustable or automatic pipettes. Manual Pipettes a. To deliver type of pipettes (TD): These pipettes must be held vertically and the tip must be placed against the side of the accepting vessel to drain liquid by gravity. Common pipettes included under TD type are, graduated and volumetric pipettes.

Glasswares Used in Biochemistry Laboratory 23 b. Graduated pipettes: These pipettes are available from 0.1-10 ml capacity, e.g. Mohr pipettes and serological pipettes. Mohr pipettes are glass tubes of uniform diameter with a tapered delivery tip, have graduations made at uniform intervals but well above the tapered delivery tip. These are mainly used for pipetting distilled water and reagents. However, 0.1 ml and 0.2 ml pipettes are used for pipetting specimen like blood, serum or plasma. The serological pipettes are either of TD or blowout pipettes. c. Volumetric pipettes: These pipettes are not graduated but designed specially to deliver a specific quantity of the specimen. They have an open- ended bulb holding the bulk of the liquid, a long glass tube at one end that has the mark to describe the extent to which the pipette is to be filled and a tapered delivery portion. These pipettes hold and deliver only the specific volumes indicated at the upper end of the pipette. These are used mainly to pipette specimen and standards, and are very accurate. Pipettes must be refilled or rinsed out with the appropriate solvent after the initial liquid has been drained from the pipettes. d. Micropipettes: Micropipette can deliver volumes ranging from 1-500 μl. It consists of capillary tubing with a line demarcating a specific volume. These are filled to the line by capillary action. e. Pasteur pipettes: This pipette has a rubber bulb attached to the top of a glass tubing. It is tapered at the tip and is especially useful in delivering urine samples.

24 An Easy Guide for Practical Biochemistry Auto Pipettes Sucking and blowing with mouth is not done in auto pipettes. A mechanical plunger does this work. These are frequently used in the laboratory to repeatedly add a specific volume of a reagent. They are mainly of push button type (Eppendorf type) and are piston operated devices to dispense liquid. These pipettes may be of fixed volume type or variable volume adjustable type. a. Fixed volume type: The volume of the fluid sucked is fixed. Different pipettes are used to pipette different fixed volumes. It can dispense fixed volumes of 10 μl, 20 μl, 50 μl, 100 μl, 200 μl, 1000 μl as required. b. Variable volume adjustable type: The volume of fluid to be dispensed can be adjusted with the adjusting screw as required. Variable volumes, e.g. 20- 200 μl, 100- 1000 μl are available. Test Tubes Test tubes are of uniform thickness which can withstand mechanical and thermal shocks. Tubes with rim are preferred when reagent in the test tube is directly heated on the flame using a test tube holder. Test tubes are available in capacities of various volumes. Outer diameter × length (mm): i. 10 × 75 mm – used for testing, identification of biochemical substances and as well as for centrifugation. ii. 15 × 125 mm – used for most of biochemistry tests. iii. 18 × 150 mm – for heating the reaction mixture directly on flame.

Glasswares Used in Biochemistry Laboratory 25 Centrifuge Tubes Centrifuge tubes are either graduated or plain. They are usually conical shaped and their size is usually 17 × 120 mm. Folin-Wu Tubes Folin-Wu tubes have markings at 12.5 ml and 25 ml; they have a bulb at the bottom with a constriction. They are used for determination of blood sugar by Folin- Wu method. Dispensers Dispensers are used to dispense large fixed volumes of reagents. They are usually used to dispense strong acids and alkalies. Desiccators Desiccators are used to keep solid or liquid materials dry. They usually have an area at the bottom where a desiccant (water absorbing material) is placed, which removes the water of hydration from compounds.

CLEANING OF GLASSWARE Glassware should be thoroughly rinsed with tap water and cleaned with some detergents. Finally, it should be rinsed with tap water followed by distilled water. The apparatus can be dried quickly by rinsing with alcohol followed by ether. The cleaned glassware except the graduated glassware is dried in a hot air oven. Dichromate-Sulfuric acid mixture (chromic acid) is used for cleaning glasswares which removes even the last traces of grease.

Section 2

Qualitative Tests

Qualitative Analysis of Carbohydrates 29

5

Qualitative Analysis of Carbohydrates

DEFINITION Carbohydrates are defined as polyhydroxy alcohols with an aldehyde or ketone as the functional group.

CLASSIFICATION Carbohydrates are classified according to the number of sugar molecules in them as monosaccharides, oligosaccharides and polysaccharides. Monosaccharides Monosaccharides are also called as simple sugars. They have only one potential sugar group. They consist of a single polyhydroxy aldehyde or ketone unit, and thus cannot be hydrolyzed into simpler form. They may be subdivided into different groups as follows: 1. Depending upon the number of carbon atoms they possess, e.g. No. of carbon

Type of sugar

Aldoses

Ketoses

1 2 3 4

Trioses Tetroses Pentoses Hexoses

Dihydroxyacetone Erythrulose Ribulose, xylulose Fructose

5

Heptoses

Glyceraldehyde Erythrose Ribose, xylose Glucose, galactose, mannose Glucoheptose

Sedoheptulose

30 An Easy Guide for Practical Biochemistry 2. Depending upon the functional groups: • Aldehyde (CHO) - Aldoses • Ketone (C=O) - Ketoses. Oligosaccharides Oligosaccharides consist of a short chain of monosaccharide units (2 to 10 units), joined together by a characteristic bond called glycosidic bond. Oligosaccharides are subdivided into different groups based on the number of monosaccharide units present. Type of No. of oligosaccharide monosaccharide

Example

Type of monosaccharide present

Disaccharide

Two

Maltose Lactose Sucrose

Glucose + Glucose Glucose + Galactose Glucose + Fructose

Trisaccharide

Three

Raffinose Glucose + Galactose + Fructose

Tetrasaccharide

Four

Stachyose

2 molecules of Galactose + Glucose + Fructose

Pentasaccharide

Five

Verbascose

3 molecules of Galactose + Glucose + Fructose

Disaccharides are classified as: • Reducing disaccharides: In reducing disaccharides one of the functional groups is free. e.g. Maltose, Lactose • Non- reducing disaccharides: Non-reducing disaccharides do not have free functional group. The potential functional groups are involved in glycosidic linkage. e.g. Sucrose, Trehalose

Qualitative Analysis of Carbohydrates 31 Polysaccharides Polysaccharides are carbohydrates having more than ten monosaccharide units. They are also called glycans or complex carbohydrates. They are classified into two types according to the type of monosaccharide units present. 1. Homopolysaccharides: Made up of repeated units of same type of monosaccharide units. e.g. Starch, glycogen, cellulose, inulin, dextrins, dextrans 2. Heteropolysaccharides: Made up of different types of monosaccharide units and their derivatives. e.g. Agar, gum, pectins, glycosaminoglycans such as hyaluronic acid, heparin sulfate, keratin sulfate, chondroitin sulfate.

FUNCTIONS OF CARBOHYDRATES 1. 2. 3. 4.

Carbohydrates are the main source of energy in the body. Storage form of energy (starch and glycogen) Excess carbohydrate is converted to fat. Glycoproteins and glycolipids are components of cell membranes and receptors. 5. They form structural basis of many organisms.

REACTIONS OF CARBOHYDRATES Chemical properties of carbohydrates are used as principles in identification of these substances. The functional group of the sugar molecule takes part in most of the chemical reactions.

32 An Easy Guide for Practical Biochemistry Reaction with Alkalies Weak Alkalies With weak alkali, the free functional group in monosaccharide gets tautomerized and forms enediol, which is a strong reducing agent. Carbohydrates such as sucrose, which do not contain free functional groups are not enolized by alkali and relatively stable in alkali solution. Strong Alkalies On boiling with strong alkali, the aldehyde polymerizes to form resin which is called caramelization. Thus, glucose loses its reducing property. Reaction with Acids Weak Acids These have no appreciable action on sugars. Strong Acids With strong acids, sugar undergoes dehydration forming furfural. The pentoses yield furfural and hexoses give hydroxy- methyl furfural. Keto- hexoses like fructose yield greater amount of furfural derivative than aldo- hexoses like glucose.

TESTS FOR CARBOHYDRATES In order to understand and remember easily, the various tests of carbohydrates are explained in the beginning as general tests. Their responses to individual carbohydrates are given later. Learning this way will also aid in the examination, when an unknown solution is given for qualitative analysis.

Qualitative Analysis of Carbohydrates 33 The various tests for carbohydrates are given below: 1. Molisch test: specific test for carbohydrates 2. Iodine test: specific test for polysaccharides 3. Benedict’s test: specific test for reducing substances 4. Barfoed’s test: specific test for monosaccharides 5. Seliwanoff’s test: specific test for ketohexoses 6. Osazone test: to differentiate the reducing sugars on the basis of crystal formation. Physical Properties 1. 2. 3. 4.

Color: Colorless except Starch which is pale white Clarity: Clear except Starch which is cloudy Odor: Odorless Reaction to litmus: Neutral

Chemical Tests Molisch Test Principle: Carbohydrates, when treated with concentrated Sulfuric acid undergo dehydration to form furfural/ hydroxymethyl furfural derivatives which on condensation with alphanaphthol form colored products (chromogens). Experiment

Observation

Take 2 ml of given Violet ring at the junction solution in a clean of the two liquids is dry test tube; add formed. 1-2 drops of Molisch reagent. Mix. Incline the test tube slightly and overlay 2 ml of conc. sulphuric acid along the sides of the test tube so as to form two layers.

Inference Given solution is a carbohydrate.

34 An Easy Guide for Practical Biochemistry Molisch reagent: A 5% solution of alpha naphthol in ethyl alcohol. Points to Remember: • This is a general test for all carbohydrates. • Molisch test is given by sugars with at least five carbons because it involves furfural derivatives which are five carbon compounds. • Rapid pouring of sulfuric acid down the test tube leads to water- acid interaction which produces heat and can cause charring of carbohydrates, resulting in the formation of black ring. Therefore, acid should be layered very slowly and carefully to minimize this interaction. • Charring occurs due to the precipitation of carbon as a result of dehydration of the carbohydrate. This occurs as a result of the action of concentrated sulfuric acid on it. • Impurities in the reagent tend to give a green ring, which is negative test. • A green ring even in absence of carbohydrates is due to excess of alpha naphthol. • In case of oligo and polysaccharides, they are first hydrolyzed to monosaccharides by acid, which undergoes dehydration to form furfural or its derivatives. • Some proteins and lipids can also give positive Molisch test. This occurs if these substances have a bound carbohydrate moiety attached to them, e.g. albumin. Iodine Test Principle: The test depends upon the property of adsorption possessed by the large polysaccharide molecules which adsorb the smaller iodine molecules on their surface to form the blue colored complex of ill-defined chemical nature. The property of adsorption decreases on heating, the complex dissociates and, therefore, the color disappears.

Qualitative Analysis of Carbohydrates 35 Iodine reagent: 0.5 ml of iodine diluted to 5 ml with distilled water. Experiment Take 2 ml of given solution in a test tube. Add 2-3 drops of Iodine solution.

Observation

Inference

Blue color is formed.

Given solution is a polysaccharide.

Points to Remember: • This is a specific test for polysaccharides. • The amylose component of starch has a helical structure. When it is treated with iodine solution, Iodine is trapped inside the coil and the complex has an intense blue color. When the amylose solution is heated the helical conformation is disrupted and loses its capacity to bind iodine. On cooling the original conformation is regained and the capacity to bind iodine is also recovered. • Sometimes the color may not reappear on cooling as small amounts of iodine added may vaporize away during heating. Benedict’s Test (Reduction under alkaline condition) Principle: In mild alkaline medium reducing sugars undergo tautomerization to form enediols which reduce cupric ions to cuprous ions. Cuprous hydroxide is formed. During the process of heating cuprous hydroxide is converted to cuprous oxide which gives different shades of colored precipitate depending upon the concentration of the sugar.

36 An Easy Guide for Practical Biochemistry Benedict’s Qualitative Reagent contains • Copper sulfate: provides cupric ions. • Sodium carbonate: makes the medium alkaline. • Sodium citrate: chelates cupric ions and releases it slowly for reduction. Thus prevents the precipitation of cupric ions as cupric hydroxide by forming cupric sodium citrate complex. It acts as a stabilizing agent. Improves the shelflife of the reagent by preventing an interaction between sodium carbonate and copper sulfate, which may otherwise get precipitated as cupric carbonate. Experiment Take 5 ml of Benedict’s reagent in a test tube. Add 8 drops of the given solution. Mix and boil for 2 min. over a small flame. Allow to cool spontaneously.

Observation

Inference

Brick-red precipitate is formed

Given solution is a reducing sugar.

Points to Remember: • This test is also a semi-quantitative test which can be reported as under: Observation

Inference

Sign

Approx. sugar

No change of blue color: - no precipitate.

Absence of reducing sugar

-

Nil

Green precipitate.

Presence of reducing sugar

+

up to 0.5 %

Yellow precipitate.

Presence of reducing sugar

++

0.5 to 1 %

Orange precipitate.

Presence of reducing sugar

+++

1 to 1.5 %

Red precipitate

Presence of reducing sugar

++++

2%

Brick red precipitate.

Presence of reducing sugar

++++

>2%

Qualitative Analysis of Carbohydrates 37 • Color of the precipitate depends on the concentration of sugar present.

Fig. 5.1

• Frequently used as screening test for Diabetes mellitus. • It gives positive result in the presence of other reducing substances like ascorbic acid, glutathione, salicylates, uric acid, glucuronides and homogentisic acid. • Benedict’s quantitative reagent contains potassium thiocyanate and potassium ferrocyanide in addition to copper sulfate, sodium carbonate and sodium citrate present in Benedict’s qualitative reagent. Benedict’s tests (In case of sucrose) Since sucrose is a non-reducing sugar, it does not give a positive Benedict’s test. Hence, the below given procedure has to be followed. Acid Hydrolysis Principle: Heating in an acidic environment leads to the hydrolysis of the glycosidic bonds present in disaccharides or polysaccharides. HCl Sucrose ——— → Glucose + Fructose

38 An Easy Guide for Practical Biochemistry Procedure: To 5 ml sucrose Add 8-10 drops of Conc. Hydrochloric acid. Boil for 3 min. cool. Divide the solution into two parts. Neutralize one part by adding 10 drops of 20% sodium carbonate. Benedict’s tests after acid hydrolysis: Experiment

Observation

Inference

Take 5 ml of Benedict’s reagent in a test tube; add 8 drops of neutralized hydrolysate solution. Boil for 2 min.

Brick-red precipitate is formed

On acid hydrolysis Sucrose is converted to reducing Monosaccharides (Glucose + Fructose)

Benedict’s test (In case of starch) Starch being a non- reducing sugar does not give positive Benedict’s test. Therefore, Benedict’s test should be conducted after acid hydrolysis. Acid hydrolysis To 5 ml of starch Add 8-10 drops of Conc. Hydrochloric acid. Boil for 5 min. in a conical flask. Cool. Neutralize the solution by adding 10 drops of 20% sodium carbonate. Points to Remember: • Acid hydrolysis of starch does not abruptly lead to the formation of glucose. • Starch on hydrolysis by acid gives the following products,

Qualitative Analysis of Carbohydrates 39 Starch → soluble starch → amylodextrins → erythrodextrins → achrodextrins → maltose → glucose. • On enzyme (amylase) hydrolysis, maltose is the major end product. • Neutralization is required to protect the sodium carbonate in the reagent to form enediol. Benedict’s test after acid hydrolysis: Experiment Take 5 ml of Benedict’s reagent in a test tube; add 8 drops of neutralized hydrolysate solution. Boil for 2 min.

Observation

Inference

Brick-red precipitate is formed

On acid hydrolysis starch gives reducing sugars.

Barfoed’s Test ( Reduction under acidic medium) Principle: In mild acidic medium reducing sugars undergo tautomerization to form enediols which reduce cupric ions to cuprous ions. Cuprous hydroxide is formed. During the process of heating cuprous hydroxide is converted to cuprous oxide which gives red precipitate. Since acidic medium is unfavorable for reduction, stronger reducing agents like monosaccharides give reduction within 30 seconds. Barfoed’s reagent: Copper acetate in glacial acetic acid.

40 An Easy Guide for Practical Biochemistry Experiment

Observation

Inference

Take 2 ml of Barfoed’s reagent in a test tube; add 1 ml of given solution. Mix and boil for 30 seconds. Allow it to cool at room temperature.

Floating red precipitate is formed.

Given solution is a monosaccharide

Points to Remember: • It is a specific reduction test for reducing monosaccharides. • Time for heating is one of the important factors in this reaction. • If the boiling period exceeds 30 seconds, disaccharides will be hydrolyzed to monosaccharides and red colored precipitate of cuprous oxide will be formed. • Helps to differentiate reducing monosaccharides from reducing disaccharides. Seliwanoff’s Test Principle: Carbohydrates are dehydrated to form furfural derivatives by hydrochloric acid present in Seliwanoff’s reagent. Furfural derivative of ketosugar condenses with resorcinol to form a chromogen (cherry red color). Seliwanoff’s reagent: 50 mg of resorcinol in 33 ml of concentrated hydrochloric acid and diluted to 100 ml with water.

Qualitative Analysis of Carbohydrates 41 Experiment

Observation

Inference

Take 3 ml of Seliwanoff’s reagent in a test tube; add 1 ml of given solution. Boil for 30 seconds and allow it to cool at room temperature.

Cherry red color is formed.

Given solution is a ketosugar

Points to Remember: • This test is specific for ketohexoses only. • Useful in differentiating aldohexoses and ketohexoses. • The test will be answered by fructose, sucrose and other fructose containing carbohydrates. • A similar color may also develop in case of glucose or maltose if the boiling is prolonged due to transformation of glucose into fructose by the catalytic action of the hydrochloric acid. • This test is very sensitive even for 0.1 % fructose. In the presence of glucose along with fructose sensitivity decreases. • It serves as an indirect test for sucrose because sucrose gets hydrolyzed by the hydrochloric acid present in the Seliwanoff’s reagent to fructose and glucose; the liberated fructose reacts with the reagent to give a positive reaction.

42 An Easy Guide for Practical Biochemistry Seliwanoff’s test (for sucrose) • In case of sucrose, the below procedure has to be followed. Experiment

Observation

Inference

Take 3 ml of Seliwanoff’s reagent in a test tube; add 1 ml of acid hydrolysate. Boil for 30 seconds.

Cherry red color is formed

Hydrolyzed sucrose contains a ketosugar.

Osazone Test Principle: Phenyl hydrazine at 100°C and at pH of 5 reacts with carbonyl group of the reducing sugars to form a soluble phenyl hydrazone which on further reactions forms insoluble osazones. Osazone mixture: To 1 part of Phenyl hydrazine Hydrochloride add 2 parts of sodium acetate and few drops of glacial acetic acid. Sodium acetate acts as a buffer to maintain the pH. Phenyl hydrazine HCl reacts with C1 and C2 of a reducing sugar first to form phenyl hydrazone and then to form osazones. To 5 ml of given solution add 3 spatula of osazone mixture. Mix well and keep in boiling water bath for 5 min. cool. Take out some crystals on a slide and observe under microscope. In case of lactose and maltose, mix well and keep in boiling water bath for 30 min. air cool and observe the crystals under microscope.

Qualitative Analysis of Carbohydrates 43 Points to Remember: • Osazones are solid yellow crystalline substances which have got characteristic structures when observed under the microscope. • All reducing sugars will form osazone with excess of phenyl hydrazine when kept at boiling temperature. • Hydrazones are highly water soluble, but osazones are insoluble. • Acetic acid and sodium acetate are useful as buffer to maintain the pH 5, appropriate for the formation of osazones. • Osazones of monosaccharides are insoluble in hot water, and will form crystals in hot condition. • Osazones of disaccharides are soluble in hot water. So they form crystals only when the test tube is cooled. • Glucose, fructose and mannose differ structurally with respect to 1st and 2nd carbon atoms. During osazone formation phenyl hydrazine reacts with the 1st and 2nd carbon atoms and hence the structural difference between these three sugars is masked. Hence glucose, fructose and mannose form the same needle shaped osazone crystals. • In the sucrose molecule, the active groups on 1st and 2nd carbon atoms of constituent glucose and fructose molecules are not free (no free aldehyde/ketone group). Hence, sucrose does not produce osazone crystals. • But it produces osazone crystals when the solution is kept in a boiling water bath for 30 minutes because of the hydrolysis of sucrose to glucose and fructose. Importance and significance • To identify the reducing sugar that is excreted in the urine especially during the period of lactation. To differentiate glucose and lactose that is excreted in the urine.

44 An Easy Guide for Practical Biochemistry Osazones

Minimum time for formation of crystals

Appearance of crystals

Glucosazone

5 minutes

Needle shaped/broom-stick shaped/hay stack or sheaves of corn appearance

Fructosazone

2 minutes

Needle shaped/broom-stick shaped/hay stack or sheaves of corn appearance

Lactosazone

30 minutes

Powder-puff shaped/cotton ball/badminton ball shaped/ pincushion with pins/hedgehog shaped or flower of touch-me-not plant shaped crystals

Maltosazone 30-40 minutes

Sunflower shaped/star shaped crystals

Qualitative Analysis of Carbohydrates 45 • For standardizing and characterization of glucose. • To differentiate lactose and maltose, which cannot be done by routine test. The carbohydrates to be studied in the lab are: 1. Glucose • Glucose is a monosaccharide, an aldohexose and a reducing sugar. • The sources of glucose are cane sugar, starch, etc. 2. Fructose • Fructose is a monosaccharide, a ketohexose, and a reducing sugar. • The sources of fructose are fruits, cane sugar, inulin, honey, etc. 3. Lactose • Lactose is a milk sugar. • Present in breast milk and is a good source of energy for the newborn. • Composed of β- D glucose and β- D galactose. • Linked by β 1-4 glycosidic linkage. • Digested by lactase. (Lactase is deficient in lactose intolerance). • Lactose may be seen in the urine of pregnant and lactating women. 4. Maltose • Maltose is composed of two molecules of α D glucose. • Linked by α 1-4 glycosidic linkage. • Sources are germinating seeds and malt. • Digested by maltase present in intestinal juice. 5. Sucrose • Sucrose is composed of α D glucose and β D fructose. • Sources of Sucrose are cane sugar, etc.

46 An Easy Guide for Practical Biochemistry • Linked by α β 1, 2 glycosidic linkage. • In sucrose the linkage is between the functional groups of glucose and fructose. Since, there is no free functional group sucrose is non-reducing. On hydrolysis the linkage is cleaved and it becomes a reducing sugar. • Digested by sucrase. 6. Starch • Starch is a plant polysaccharide. • The sources of starch are storage parts of plants like potato, seeds, cereals and tubers. • Composed of amylose and amylopectin component. • The individual glucose units in amylose are linked by α 1-4 glycosidic linkages. • Amylopectins have branching points linked by α 1-6 glycosidic linkages. • Starch is a non-reducing sugar. The test results for different carbohydrates are summarized below: Test

Molisch

Iodine

Benedict’s

Glucose

+

-

+

Barfoed’s Seliwanoff’s +

-

Fructose

+

-

+

+

+

Lactose

+

-

+

-

-

Maltose

+

-

+

-

-

Sucrose

+

-

- (+ after acid hydrolysis)

-

+ after acid hydrolysis

Starch

+

+

- (+ after acid hydrolysis)

-

-

Qualitative Analysis of Carbohydrates 47 IDENTIFICATION OF UNKNOWN CARBOHYDRATE

Flow chart 5.1: Scheme for identification of unknown carbohydrate

48 An Easy Guide for Practical Biochemistry

6

Qualitative Analysis of Proteins

DEFINITION Proteins are defined as sequence specific polymers of L α-amino acids linked by peptide bonds. They are complex organic substances made up of carbon, hydrogen, oxygen and nitrogen. Some proteins also contain sulfur and phosphorus.

CLASSIFICATION 1. Simple proteins: made up of only amino acids. Ex: Albumin, globulin and protamines 2. Conjugated proteins: made up of amino acids and a nonprotein part which is known as the prosthetic group. e.g. Glycoproteins: immunoglobulins Chromoproteins: hemoglobin Lipoproteins: occur in blood and on cell membranes- HDL, LDL, VLDL Metaloproteins: hemoglobin, cytochrome Nucleoproteins: histones Phosphoproteins: casein of milk and vitelline of egg yolk 3. Derived proteins: derived from native (naturally occurring) proteins. They are of two types: • Primary derived proteins: formed by agents such as heat, acids, alkalies, etc. which cause only slight changes

Qualitative Analysis of Proteins 49 in the protein molecule and its properties without hydrolytic cleavage of peptide bond. e.g. Proteons, metaproteins. • Secondary derived proteins: formed in the progressive hydrolytic cleavage of the peptide bonds of proteins into smaller molecules. Ex: proteoses, peptides and peptones.

FUNCTIONS OF PROTEINS 1. Maintain the structural integrity of bones, tendons, hair and teeth- collagen, elastin, keratin. 2. Acts as enzymes (catalytic function). 3. Hormones (regulatory function). 4. Antibodies- immunoglobulins (defense protein). 5. Coagulation factors. 6. Carrier proteins (e.g. albumin, thyroglobulin) 7. Contractile element in the muscle (actin, myosin) 8. Proteins act as intracellular buffer in maintaining the acid-base balance.

REACTIONS OF PROTEINS Reactions of proteins are studied as: 1. Precipitation reactions of proteins 2. Color reactions of proteins. Precipitation Reactions of Proteins Proteins are large molecules with variable sizes, shapes and charges. Solubility of a protein depends on the proportion and distribution of polar hydrophilic groups and non-polar hydrophobic groups in the molecule. Proteins form colloidal systems in aqueous medium. A colloid is a system in which the particles have diameters in

50 An Easy Guide for Practical Biochemistry the range of 1-200 millimicron. The stability of protein in solution will depend mainly on the charge and the degree of hydration (shell of water molecules around the particles). Polar groups of the protein (-NH2, COO–, OH–) tend to attract water molecules around them to form a shell of hydration. Types of Colloids 1. Suspensoids 2. Emulsoids Suspensoids: Suspensoids are stabilized by the electrical charges over the surface of the molecule. Emulsoids: Emulsoids are stabilized by: 1. Electric charge over the surface of the molecule 2. Hydration shell around the molecule Proteins can be precipitated by: 1. Removing their shell of hydration 2. Neutralization of electrical charges 3. Denaturation (disorganization of native protein, loss of biological activity) 4. Bringing them to isoelectric pH Any factor which neutralizes the charge or removes the shell of hydration will cause precipitation of proteins. Importance of Precipitation of Proteins 1. Precipitation is used to separate proteins from biological fluids like blood, plasma, CSF, etc. before estimation of important chemical constituents such as urea, sugar, creatinine, etc. because proteins interfere in their estimation.

Qualitative Analysis of Proteins 51 2. Precipitation can also be used under well defined conditions to separate a particular protein from a mixture of proteins, e.g. precipitation of albumin from serum at full saturation with ammonium sulfate. Amino Acid Amino acids are organic substances, having two functional groups–amino group and carboxyl group. Amino group is basic while carboxyl group is acidic in nature. The proteins to be studied in the lab are: 1. Albumin 2. Casein Precipitation by Salts Principle: Addition of neutral salts like ammonium sulfate leads to adsorption of hydration shell along with neutralization of surface charges leading to protein precipitation. This is known as ‘salting out’. a. Half-saturation with ammonium sulfate:

52 An Easy Guide for Practical Biochemistry Experiment Take 3 ml of protein solution in a test tube. Add equal volume of saturated ammonium sulfate solution Mix and allow to stand for 5 min. Filter it. Perform Biuret test with the filtrate by adding 3 ml of 40% sodium hydroxide and 2-3 drops of 1% copper sulfate.

Observation

Inference

a. White precipitate a. Protein in the is formed. given solution is b. No white precipitated. precipitate b. Protein in the is formed. given solution is not precipitated by half-saturation with ammonium sulfate. a. No violet color is formed. b. Violet color is formed

Note: Filterate which contains protein gives violet color with Biuret test.

b. Full saturation with ammonium sulfate: Experiment Take 3 ml of protein solution in a test tube. Add solid ammonium sulfate in small quantities at a time with mixing until the solution is saturated. Allow to stand for 5 minutes.

Observation a. White precipitate is formed. b. No white precipitate is formed.

Inference a. Protein in the given solution is precipitated. b. Protein in the given solution is not precipitated by full saturation with ammonium sulfate. Contd...

Qualitative Analysis of Proteins 53 Contd... Experiment Filter it. Perform Biuret test with the filtrate by adding 3 ml of 40% sodium hydroxide and 2-3 drops of 1% copper sulfate.

Observation

Inference

a. No Violet color is formed. b. Violet color is formed

Note: • Solid ammonium sulfate is added in small quantities at a time with mixing until the solution is saturated, i.e. there should be some undissolved salt in the bottom of the test tube. • Filtrate which is not having any trace of protein gives blue color with Biuret reagent.

Discussion: • Solubility of a protein depends on ionic concentration of the medium. Therefore, the presence of very small quantities of salts will increase the solubility of a protein by diminishing protein interaction. This is called ‘Saltingin’. • Filtrate contains high concentration of ammonium ions which interfere in Biuret test by forming a deep blue cuprammonium ions [Cu (NH3)4++] which obscure the violet color produced by proteins. This can be overcome by the use of 40% sodium hydroxide instead of 10% sodium hydroxide. • Depending on the surface area of the protein, the amount of salt required is variable. Higher the molecular weight of a protein the salt required for precipitation is lesser.

54 An Easy Guide for Practical Biochemistry • Smaller molecules like albumin have relatively a large surface area. They hold more water molecules around them. Hence, a higher concentration of salt is required for precipitation of albumin. Thus, albumin is precipitated by full saturation. • Casein and gelatin are precipitated by half saturation because they have high molecular weight. Isoelectric Precipitation Principle: The solubility of proteins is minimum at their isoelectric pH as the protein molecules become electrically neutral at this pH. Note: Perform the test with only casein. Experiment

Observation

Inference

Take 3 ml of casein solution in a test tube. Add 3 drops of bromocresol green indicator. Mix. Add 1% acetic acid drop by drop until a light green color is obtained which indicates that the pH is close to 4.6.

A curdy green precipitate will be formed.

At pH 4.6, casein is precipitated.

Points to Remember: • The pH at which the molecule carries no net charge is known as isoelectric point or isoelectric pH (pI) • At isoelectric pH 1. The net charge is zero. 2. No mobility in an electric field.

Qualitative Analysis of Proteins 55

• • • • •

•

•

3. Least soluble. 4. Buffering capacity and viscosity will be minimum. 5. Precipitation will be maximum. pH range of bromocresol green is 4- 4.6. Isoelectric pH of casein is 4.6. Isoelectric pH of human albumin is 4.7. Proteins have minimum solubility at the isoelectric point. Casein forms a flocculent precipitate at its isoelectric pH 4.6; and redissolves in highly acidic or alkaline solutions. When milk is curdled, the casein forms a white curd, because lactic acid produced by the fermentation process, lowers the pH to the isoelectric point of casein. Casein is precipitated from milk and the supernatant is called whey. Isoelectric point of albumin is 4.7. At this pH the color of the solution is dark green. If the color is not brought to light green, i.e. (pH 4.6) of casein, albumin may form a curdy dark green precipitate at pH 4.7. So while performing the isoelectric precipitation test for casein, bring the pH of the solution to 4.6 (light green) to avoid interference of albumin.

Precipitation by Organic Solvents Principle: Proteins in solution form hydrogen bonds with water. Organic solvents like acetone, ether or ethanol when added to a protein solution in water, reduce the concentration of water molecules available for keeping the proteins in solution and thus decrease the number of hydrogen bonds. The dielectric constant of the medium is also reduced

56 An Easy Guide for Practical Biochemistry causing aggregation, precipitation and denaturation of proteins. Experiment

Observation

Inference

Take 1 ml of protein solution in a test tube. Add 2 ml of ethanol. Mix.

A white precipitate is formed.

Protein is precipitated by organic solvents.

Points to Remember: • For the precipitation of protein by alcohol, protein should be in electrolyte form. This may be achieved by dissolving the protein in saline. Precipitation by Alkaloidal Reagents Principle: The negatively charged ions of the alkaloids neutralize the positive charge on the protein causing denaturation which results in precipitation. Experiment a. Take 2 ml of protein solution in a test tube. Add picric acid drop by drop.

Observation A thick yellow precipitate is formed.

Inference Protein is precipitated by alkaloidal reagent.

Contd...

Qualitative Analysis of Proteins 57 Contd... Experiment

Observation

Inference

b. Take 2 ml of protein solution in a test tube. Add trichloroacetic acid drop by drop.

A white precipitate is formed

Protein is precipitated by alkaloidal reagent.

c. Take 2 ml of protein solution in a test tube. Add sulfosalicylic acid drop by drop.

A white precipitate is formed.

Protein is precipitated by alkaloidal reagent.

Points to Remember: • Tungstic acid, phosphotungstic acid, trichloroacetic acid, picric acid, sulfosalicylic acid and tannic acid are powerful protein precipitating agents. These acids lower the pH of the medium, when proteins carry net positive charges. These protein cations are electrostatically complexed with negatively charged ions to form proteintungstate, protein-picrate, etc. to form thick flocculent precipitate. • Tanning in leather processing is based on the protein precipitating effect of tannic acid.

58 An Easy Guide for Practical Biochemistry • This test using sulfosalicylic acid is commonly employed for preliminary screening of urine for the presence of proteins. It is also used to identify proteins in CSF. • For estimation of blood constituents photometrically, proteins interfere with the analysis. This is avoided by an initial protein precipitation by alkaloidal reagents. Precipitation by Heavy Metal Ions Principle: Proteins exist as negatively charged ions (anions) in pH higher than their isoelectric pH (generally in an alkaline medium). To such a solution if salt of heavy metals are added, positively charged metal ions can complex with protein anion and metal proteinates are formed which get precipitated. Experiment

Observation

Inference

a. Take 2 ml of protein solution in a test tube. Add 10% lead acetate solution drop by drop.

White precipitate is seen.

Protein is precipitated by heavy metals like lead.

b. Take 2 ml of protein solution in a test tube. Add 5% mercuric nitrate solution drop by drop.

White precipitate is seen.

Protein is precipitated by heavy metals like mercury.

Qualitative Analysis of Proteins 59 Points to Remember: • Salts of iron, copper, zinc, lead, cadmium and mercury are toxic, because they tend to precipitate normal proteins of the gastrointestinal wall. • This test principle is used in treating heavy metal poisoning. • Based on this principle, raw egg white is used as an antidote in mercury poisoning and then an emetic is given to remove Hg++ ions which are held by albumin. • If the sample solution is significantly alkaline, its pH should be adjusted to 7- 7.5 to avoid formation of metal hydroxides, which interfere with the test. • Avoid adding excess of heavy metal ions as this may redissolve the precipitate due to absorption by the protein molecules, which will give them a positive charge. Precipitation by Heat and Acid (Heat and Acetic Acid Test) Principle: On heating the protein loses its structure and becomes denatured to form a coagulum. It is precipitated after the addition of acetic acid, which provides the suitable pH to get the maximum precipitate. Experiment

Observation

Take albumin A white coagulum is seen solution upto ¾ th at the upper heated test tube. Hold the portion, which gets test tube over a flame intensified after the in a slanting position addition of acetic acid. and boil the upper portion of the albumin solution. The lower portion serves as control. Add 1% acetic acid drop by drop.

Inference Indicates the presence of heat coagulable protein like albumin.

60 An Easy Guide for Practical Biochemistry Points to Remember: • Proteins have specific structural organizations. • When a protein is heated, its physical, chemical and biological properties are changed due to breaking up of certain bonds and the resultant change in the conformation of its molecules. This process is known as denaturation. • However, when the coagulable proteins are heated at their isoelectric pH, a series of changes occur involving dissociation of the protein subunits (disruption of quaternary structure), uncoiling of the polypeptide chains (disruption of tertiary and secondary structure) and matting together of the uncoiled polypeptide chains (coagulation). • Denaturation is sometimes reversible, but coagulation is an irreversible process. Some proteins when heated, though denatured, are still soluble. They may be precipitated by bringing to isoelectric pH. • Proteins are easily denatured when subjected to heat treatment. Denatured proteins are less soluble than the native proteins. • Albumin and globulin are easily coagulated by heat, near or at their isoelectric point. On addition of acetic acid, there is a decrease in pH; when pH approaches the isoelectric pH of albumin/globulin, coagulation occurs spontaneously since the solution is pre-heated. Acetic acid is added: • To provide suitable pH to get maximum precipitate. • To differentiate between protein and interfering substances mainly phosphates. • If the precipitate persists and deepens after the addition of acetic acid it is due to proteins. If it disappears it indicates the presence of phosphates.

Qualitative Analysis of Proteins 61

COLOR REACTIONS FOR PROTEINS Proteins are high molecular weight macromolecules made up of amino acid residues linked by peptide bonds. All together there are 20 types of amino acids found in proteins. Due to the presence of the polypeptide bonds and different amino acids residues in their molecules, they react with a variety of reagents to form colored products. These are known as color reactions of proteins. These reactions are of importance in qualitative detection and quantitative estimation of proteins and their constituent amino acids. Albumin • • • • • •

Human albumin is a simple protein. Found in milk, eggs and plasma. Soluble in water. Constitutes the major part of (60%) plasma proteins. Synthesized only by liver. Due to its low molecular weight and high concentration, albumin is responsible for 70-80% of the osmotic pressure of plasma. • Albumin contains all the essential amino acids in required amounts. • It is the best example for complete protein. So it is known as a first class protein, a protein of high biological value. Functions of Albumin 1. Maintenance of colloidal osmotic pressure in both vascular and extravascular space. 2. Albumin acts as a transport protein for free fatty acids, bilirubin, calcium and most of the drugs.

62 An Easy Guide for Practical Biochemistry Casein • Casein is the chief protein of milk. • It is a conjugated protein (phosphoprotein) with a phosphate group attached to the hydroxyl group of serine and threonine residues. • It is rich in most of the essential amino acids and is of high nutritive value. • Its isoelectric pH is 4.6. • Curdling of milk involves the concept of isoelectric precipitation. Physical Properties of Albumin 1. 2. 3. 4.

Color: Pale white Clarity: Cloudy Odor: Odorless Reaction to litmus: Neutral.

Physical Properties of Casein 1. 2. 3. 4.

Color: Pale white Clarity: Cloudy Odor: Characteristic odor Reaction to litmus: Alkaline.

Chemical Properties Biuret Test Principle: Cupric ions in alkaline medium form a violet colored complex with peptide bond nitrogen. Copper sulfate is converted to cupric hydroxide which chelates with peptide linkage in proteins to give the purple color. Biuret Reagent: Contains sodium potassium tartarate and copper sulfate.

Qualitative Analysis of Proteins 63 Experiment

Observation

Inference

Take 2 ml of protein solution in a test tube. Add 2 ml of 5% sodium hydroxide. Mix and add one or two drops of 1% copper sulfate.

Violet color is formed

Indicates the presence of peptide linkage.

Points to Remember: • It is a general test for proteins. • The reaction is so named since Biuret (NH2-CO-NH-CONH2) formed by the condensation of two molecules of urea when heated at 180° C also answers this test. • The minimum requirement for a positive test is the presence of two peptide bonds in the molecule (three amino acids). • Individual amino acids and dipeptides do not answer this test. • This test is positive for all compounds containing more than one peptide linkage (- CO-NH-) e.g. proteins and their hydrolytic products (metaproteins, proteoses, peptones, polypeptides except dipeptides and amino acids). • This test is also positive for substances which contain two carbamyl (-CONH2) groups joined directly or through a single atom of nitrogen or carbon. Hence non- proteins, e.g. oxamide and biuret give positive test. • This reaction can be used for quantitative estimation of proteins. • Insoluble protein like keratin gives negative Biuret test.

64 An Easy Guide for Practical Biochemistry Precautions: • Care must be taken that not more than 2 drops of dilute copper sulfate be added; otherwise blue color (due to excess formation of cupric hydroxide) will develop instead of violet color. • Magnesium sulfate and ammonium sulfate interfere with this test. Therefore, the test should not be carried out with solutions containing these salts. Application: • It is a common and delicate test for the identification of protein in a biological material. Ninhydrin Test Principle: Ninhydrin reacts with free α amino acids to give a bluish purple colored complex called Ruhemann’s purple. Ninhydrin is a powerful oxidizing agent and causes oxidative decarboxylation of α amino acids producing an aldehyde. The reduced Ninhydrin (hydrindantin) then reacts with ammonia and another molecule of Ninhydrin and produces bluish purple colored complex. Alpha amino acids + Ninhydrin →

Aldehyde + CO2 + NH3 + Hydrindantin

Hydrindantin + NH3 + Ninhydrin → Bluish purple colored complex

Ninhydrin reagent: 0.2% of Ninhydrin in acetone. Experiment

Observation

Inference

Take 1ml of protein solution in a test tube. Add 10 drops of Ninhydrin. Heat to boiling.

Bluish purple color is formed.

Indicates the presence of free α-amino acids.

Qualitative Analysis of Proteins 65 Points to Remember: • This test is positive for all α- amino acids. • Proline and hydroxy proline are imino acids and they have no α amino group. Hence, they give a yellow color with Ninhydrin. • Amino acids with amide group like glutamine and asparagine give brown color. • Ninhydrin test may be used as an additional test to confirm the presence of protein in a solution. • This test is positive for all amino acids containing free amino and carboxylic groups. Hence, it is positive for proteins, peptones, peptides. • If Biuret test is negative and Ninhydrin test is positive in a given solution, it indicates that free α amino acids are present in the given solution. • This test is used to detect amino acids in chromatography. Xanthoproteic Test Principle: The benzene ring system in tyrosine and tryptophan undergo nitration on treatment with strong nitric acid at elevated temperature forming a yellow precipitate. The yellow precipitate turns orange due to ionization, in alkaline medium. Experiment Take 3 ml of protein solution in a test tube. Add 1ml of conc. Nitric acid and mix. Heat the solution for one minute and cool it under tap water. Observe the color. Divide the contents into two parts.

Observation

Inference

In acid medium yellow color is formed.

Indicates the presence of aromatic amino acids. (Tyrosine and tryptophan).

66 An Easy Guide for Practical Biochemistry Experiment

Observation

To one part of the solution, add 2 ml of 40% sodium hydroxide till the solution to make it alkaline. Mix well and observe.

In alkaline medium orange color is formed.

Inference

Points to Remember: • This is a specific test for aromatic amino acids. • Yellow color is due to the formation of nitro derivatives of benzene ring containing amino acids. • This reaction is also the basis of yellow stain in skin by nitric acid. • Nitration of phenylalanine under these conditions normally does not take place and phenylalanine alone gives a weak positive result. Millon’s Test (Cole’s mercuric nitrite test) Principle: Sodium nitrite reacts with Sulfuric acid to form nitrous acid (reacting acid). The protein gets precipitated by the mercuric sulfate. The reacting groups (phenol group of tyrosine) which get exposed on boiling, reacts with nitrous acid to form mercury phenolate. This gives red color precipitate. Millon’s reagent: Contains 10% mercuric sulfate (prepared in 10% Sulfuric acid) and 1% sodium nitrite.

Qualitative Analysis of Proteins 67 Experiment

Observation

Inference

Take 1ml of protein solution in a test tube. Add 1ml of 10% mercuric sulfate. Boil gently for 30 seconds. Cool it under tap water. Add 3 drops of 1% sodium nitrite solution. Mix and observe.

Red coagulum is formed.

Indicates the presence of hydroxyphenyl group (tyrosine).

Points to Remember: • This test is specific for hydroxy phenyl group of tyrosine. • This test cannot be employed to detect tyrosine in urine because chlorides which are normally present in urine interfere with the reaction by forming unionized mercuric chloride. • This test is given by phenols or phenolic substances such as salicylic acid. • Heat coagulable proteins give red precipitate, whereas smaller molecules of proteins like peptones give red colored solution without precipitate. Aldehyde Test for Indole Nucleus (Hopkins- Cole- Adam’s Test) Principle: Mercuric sulfate causes mild oxidation of indole group of tryptophan, which condenses with an aldehyde to give the colored complex.

68 An Easy Guide for Practical Biochemistry Experiment

Observation

Inference

Take 3 ml of protein solution in a test tube Add 2 drops of 0.2% formalin and a drop of 10% mercuric sulfate. Add carefully 3 ml of conc. Sulfuric acid along the sides of the test tube.

A purple or violet color is formed at the junction of the two liquids.

Indicates the presence of Indole ring (tryptophan).

Points to Remember: • It is a specific test for indole nucleus/ring. • Sulfuric acid with mercuric sulfate is used as an oxidizing agent in this reaction. • Tryptophan is an essential amino acid and its presence indicates a good nutritive value of the protein. • Casein and egg albumin give a positive test. Sakaguchis Test for Guanidine Group Principle: In alkaline medium α- naphthol combines with the guanidine group of arginine to form a complex which is oxidized by sodium hypobromite to form a bright red colored complex. Experiment

Observation

Inference

Take 3 ml of protein solution in a test tube. Add 5-6 drops of 40% sodium hydroxide and 4 drops of Molisch reagent. Mix and add 10 drops of bromine water.

Bright red color is formed.

Indicates the presence of arginine.

Qualitative Analysis of Proteins 69 Points to Remember: • The test is specific for guanidine group of arginine. • Sodium hypochlorite can be used instead of sodium hypobromite. • This test is given by albumin, globulin and gelatin as they contain arginine. • Avoid addition of excess of α- naphthol, as it masks the color development. Sulfur Test for Cystine and Cysteine Principle: On boiling with sodium hydroxide the sulfur present in the protein is converted to inorganic sodium sulfide. This reacts with lead acetate to form a black precipitate of lead sulfide. R-SH + 2 NaOH → R-OH + Na2S + H2O Na2S + (CH3COO)2Pb → PbS + 2CH3+COONa Experiment

Observation

Inference

Take 2 ml of protein solution in a test tube. Add 2 ml of 40% sodium hydroxide. Boil for one minute. Cool it under tap water. Then add 5 drops of lead acetate.

Black or brown color precipitate is formed.

Indicates the presence of cystine or cysteine residues.

Points to Remember: • This test is specific for –SH (thiol) group of cysteine and cystine. • It is a test for sulfur- containing amino acids.

70 An Easy Guide for Practical Biochemistry • Methionine does not answer the test since the sulfur in methionine cannot be easily split with alkali since, it is in thio-ether linkage. • Albumin and keratin will answer this test, but casein, deficient in sulfur containing amino acid will not. • Avoid excess of lead acetate solutions, which will form white precipitate. Pauly’s Test for Histidine and Tyrosine Principle: Diazobenzene sulfonic acid reacts with imidazole ring of histidine to form a cherry red colored diazotized product under alkaline condition. With the hydroxyphenyl group of tyrosine an orange-red colored product is obtained. Experiment Take 1 ml of 0.5% sulfanilic acid in a test tube. Add 1 ml of 0.5% sodium nitrite solution. Mix. After standing for 1 minute, add 2 ml of protein solution. Mix. Cool under tap water and then add 1ml of 10% sodium carbonate to make the solution alkaline.

Observation

Inference

Cherry red color is formed.

Histidine is present.

Orange red color is formed.

Tyrosine is present.

Qualitative Analysis of Proteins 71 Points to Remember: • This test is specific for imidazole group of histidine. • It is also positive for phenolic hydroxyl group. • A positive Pauly’s test and negative Millon’s test indicate the presence of histidine. Molisch Test for Carbohydrate Moiety in Proteins Principle: Carbohydrates, when treated with Conc. sulfuric acid undergo dehydration to form furfural derivatives which on condensation with alpha-naphthol forms colored products. Molisch reagent: A 5% solution of alpha naphthol in ethyl alcohol. Experiment

Observation

Inference

Take 2 ml of protein solution in a clean dry test tube; add 1-2 drops of Molisch reagent. Mix. Incline the test tube slightly and add 2 ml of conc. Sulfuric acid along the sides of the test tube so as to form two layers.

Violet ring at the junction of the two liquids is formed.

Indicates the presence of bound carbohydrate.

Point to Remember: • Egg albumin has bound carbohydrate.

72 An Easy Guide for Practical Biochemistry Test for Organic Phosphorus ( Neumann’s Test) (Test with casein solution) Principle: On boiling with strong sodium hydroxide, the organic phosphate present in phosphoproteins is released as inorganic phosphate. Inorganic phosphate reacts with ammonium molybdate in the presence of nitric acid (acidic media) to form a canary yellow precipitate of ammonium phosphomolybdate. Experiment

Observation

Inference

Take 5 ml protein solution in a test tube. Add 0.5 ml of 40% sodium hydroxide. Heat strongly and cool under tap water. Add 0.5 ml of conc. Nitric acid. Filter. To the filtrate add a pinch of solid ammonium molybdate and warm gently.

Canary yellow precipitate is formed.

Indicates the presence of phosphoprotein.

Points to Remember: • This test detects the presence of organic phosphorus in casein. • Casein is a phosphoprotein. • Ammonia is added to remove the sulfate ions. • Nitric acid provides the acidic medium.

Qualitative Analysis of Proteins 73 IDENTIFICATION OF UNKNOWN PROTEIN

Flow chart 6.1: Scheme for identification of an unknown protein

74 An Easy Guide for Practical Biochemistry

7

Nonprotein Nitrogenous Substances

Nonprotein nitrogenous substances include all nitrogenous substances other than proteins. Most important NPN substances present in urine are uric acid, urea, creatinine and ammonia. Urea • • • • •

It is the end product of protein catabolism. Synthesized in the liver. Excreted mainly in the urine. Normal blood urea level is 15- 45 mg/dl. Normal level of urea excreted in urine is 25- 30 gm/day.

Tests for Urea Urea tests are as follows: Physical Properties 1. 2. 3. 4.

Color: Colorless Clarity: Clear Odor: Odorless Reaction to litmus: Neutral.

Chemical Tests Chemical tests are as follows: Biuret Formation Principle: When heated above its melting point, two molecules of Urea condense to form biuret and ammonia.

Nonprotein Nitrogenous Substances 75 Biuret reacts with alkaline copper sulfate to form a violet color. Experiment

Observation

Inference

Take a pinch of Urea crystals in a dry test tube and heat it in a low flame. Urea melts with the liberation of ammonia. On further heating it solidifies. Cool the test tube. Dissolve the residue in 1 ml of 10% sodium hydroxide and add one drop of copper sulfate.

Violet color is formed.

Indicates the presence of urea.

Point to Remember: • Excess of copper sulfate should not be added; otherwise copper sulfate will form cupric hydroxide with sodium hydroxide forming a blue color. This is sometimes mistaken for a positive biuret test. Sodium Hypobromite Test Principle: When urea is treated with Sodium hypobromite, it decomposes to give nitrogen, carbon dioxide and water. Liberation of nitrogen gas produces brisk effervescence. Experiment

Observation

Inference

Take 2 ml Urea solution in a test tube. Add 5 drops of freshly prepared alkaline sodium hypobromite solution and mix.

Brisk effervescence of Nitrogen gas is seen.

Indicates the presence of urea.

76 An Easy Guide for Practical Biochemistry Point to Remember: • This principle is the basis for the quantitative estimation of urea in urine. Specific Urease Test Principle: Under optimum pH and temperature, the enzyme urease decomposes urea into ammonia and carbon dioxide which together form ammonium carbonate (alkaline substance) which changes the solution to pink color in the presence of the indicator. Indicator phenolphthalein changes to pink color in alkaline medium. Experiment

Observation

Inference

Take 3 ml of urea solution in a test tube. Add 3 ml of urease suspension and add 1-2 drops of phenolphthalein indicator. Warm the tube for a few seconds with the hands and keep for 10 minutes.

Pink color is formed

Urea is confirmed.

Points to Remember: • This test is specific for urea because the enzyme Urease shows its specificity for the substrate urea. • Urease suspension contains 10 gm of horse gram powder mixed with 100 ml of 30% ethanol. • pH range of phenolphthalein indicator is 8.3 to 10. • Urease present in horse gram powder acts on urea to form ammonium carbonate which raises the pH of the solution above 9 which is indicated by the change of color by phenolphthalein indicator.

Nonprotein Nitrogenous Substances 77 • Over heating should be avoided. Otherwise the enzyme will be destroyed. 60°C temperature is maintained by touch only. This 60°C temperature is the optimum temperature for Urease for its maximum activity. Tests for Uric Acid Uric Acid • It is the end product of catabolism of purines, in human body. • It is synthesized in the liver and excreted through urine as urates. • Normal uric acid level in blood is, Males: 3.5-7 mg/dl Females: 3-6 mg/dl • Normal level of uric acid excreted in urine is 250-750 mg/day. • Uric acid is sparingly soluble in water but soluble in alkaline solution. Physical Properties 1. 2. 3. 4.

Color: Colorless Clarity: Clear Odor: Odorless Reaction to litmus: Alkaline.

Chemical Tests Chemical tests are as follows. Phosphotungstic Acid Reduction Test/ Benedict’s Uric Acid Test Principle: Uric acid in alkaline condition reduces phosphotungstic acid to tungsten blue.

78 An Easy Guide for Practical Biochemistry Benedict’s uric acid reagent: Composed of sodium tungstate, orthophosphoric acid, concentrated sulfuric acid and solid sodium carbonate. Experiment

Observation

Inference

Take 3 ml uric acid solution in a test tube; add 1 ml of Benedict’s uric acid reagent and 1 ml of 20% sodium carbonate. Mix.

Deep blue color is formed.

Indicates the presence of uric acid.

Schiff’s Test Principle: Uric acid reduces salts of silver nitrate to metallic silver. Experiment

Observation

Inference

Moisten a piece of filter paper with few drops of ammoniacal silver nitrate solution. Add 2-3 drops of uric acid on the silver nitrate drops.

Black spots on the filter paper are seen.

Indicates the presence of Uric acid.

Murexide Test Principle: When uric acid is treated with conc. nitric acid, it undergoes oxidation. The imidazole ring is cleaved and the derivatives condense to give reddish yellow purpuric acid.

Nonprotein Nitrogenous Substances 79 This combines with ammonia to form ammonium purpurate or murexide which is purple red in color. Experiment

Observation

Inference

Take 5 ml of uric acid in a china dish and add 5 drops of conc. Nitric acid. Warm gently over a low flame. A reddish yellow residue is obtained. Allow the dish to cool. Add two drops of dilute ammonia solution.

A purplish red color is formed.

Indicates the presence of Uric acid.

Test for Creatinine Creatinine • Creatine is found in muscle as creatine phosphate, plays an important role in muscular contraction. • Creatinine is the anhydride form of creatine phosphate. • Creatine is synthesized from three amino acids, glycine, arginine and methionine. Glycine + Arginine –→ Guanidoacetic acid + Ornithine (Kidney) Guanidoacetate + S adenosyl methionine –→ Creatine + S adenosyl homocystine (Liver) ATP + Creatine –→ Creatine phosphate + ADP Creatine phosphate –→ Creatinine + H2O + Pi

• Normal serum creatinine is 0.5-1.2 mg/dl • Normal urine creatinine is 0.8-1.2 g/day • Creatinine clearance: Male : 94-140 ml/min Female: 70-110 ml/min

80 An Easy Guide for Practical Biochemistry Physical Properties 1. 2. 3. 4.

Color: Colorless Clarity: Clear Odor: Odorless Reaction to litmus: Neutral.

Jaffe’s Test Principle: Creatinine reacts with picric acid in the presence of alkaline medium to form reddish orange colored creatinine picrate. Experiment

Observation

Inference

Take 3 ml of creatinine solution in a test tube. Add 1 ml saturated picric acid solution, and 3-4 drops of 10% sodium hydroxide.

Reddish orange color is formed.

Indicates the presence of Creatinine.

Report: The given NPN substance is —————.

Flow chart 7.1: Schematic representation for identification of an unknown substance of physiological importance

IDENTIFICATION OF UNKNOWN SUBSTANCE OF PHYSIOLOGICAL IMPORTANCE

Nonprotein Nitrogenous Substances 81

82 An Easy Guide for Practical Biochemistry

8

Qualitative Analysis of Normal Urine

Urine is an ultrafiltrate formed by the kidneys carrying the waste and toxic substances from the blood. The composition of urine is a mirror not only of renal function but also of many physiological and metabolic processes occurring in the body. Thus, examination of urine may lead to the diagnosis of many metabolic and systemic diseases.

EXAMINATION OF URINE Examination of urine includes: 1. Physical examination 2. Chemical examination 3. Microscopic examination. SPECIMEN COLLECTION 1. Fresh mid-stream specimen of 10- 20 ml is collected in a clean dry container. 2. For most of the qualitative tests, a random urine sample is satisfactory. 3. Morning specimen is desirable for normal analysis. 4. Repeated urine samples are necessary for orthostatic proteinuria. 5. 24 hours urine is collected for total urinary proteins, calcium, uric acid, ketosteroids and certain hormonal assays, as the concentrations vary at different times of the day. The patient is instructed to collect the urine

Qualitative Analysis of Normal Urine 83 sample from morning 8 o’ clock to the next day morning 8 o’ clock.

PRESERVATION OF URINE SAMPLES 1. Several changes like urinary decomposition, precipitation of phosphates, crystallization of uric acid and bacterial action may alter the urinary composition if it is kept for long periods, especially in the collection of 24 hours urine samples. Also urine may become alkaline, due to precipitation of uric acid and urates. 2. This requires addition of preservatives (to prevent the growth of bacteria and moulds) such as 2N hydrochloric acid, conc. sulfuric acid, toluene, liquid petroleum crystals of thymol or 10% acetic acid, etc. depending on the analysis of parameters in urine. 3. Before carrying out any estimation in urine, the urinary deposits must be well mixed. The total volume is measured which is required to calculate the amount of constituents of urine excreted/day and to calculate output per unit time in clearance tests.

GENERAL AND PHYSICAL CHARACTERISTICS Volume • Normal adult excretes around 800 to 2500 ml/ day with an average of 1500 ml/day. • Day output is greater than night output. • Factors influencing the volume are : – Quantity of fluid intake. – Quality of food taken. – Climate- output is low in hot climate due to excessive sweating. – Physical exercise.

84 An Easy Guide for Practical Biochemistry • A high protein diet causes a physiological polyuria due to the diuretic effect of urea, the end product of protein metabolism. Appearance • Freshly voided normal urine is clear and transparent. • On standing it may become turbid due to the bacterial action that converts urea to ammonium carbonate. This makes urine alkaline and causes precipitation of phosphates/oxalates/urates. • It may also become turbid due to the precipitation of nucleoproteins and mucoproteins. Color • Fresh normal urine is straw or amber yellow due to the presence of the pigment urochrome, a compound of urobilin or urobilinogen. • The color may be light or dark depending on the volume of urine. • Yellow colored urine will be present in people who consume vitamin B complex. Odor • Fresh urine has an aromatic odor due to the presence of volatile organic acids. • On standing urine undergoes decomposition converting urea into ammonium carbonate giving an unpleasant ammoniacal odor. Specific Gravity • The specific gravity of normal urine varies in the range of 1.012 to 1.024.

Qualitative Analysis of Normal Urine 85 • Physiologically, the specific gravity may decrease with high fluid intake where the urine volume is increased and may rise with restricted water intake where the urine volume is low. • It can be as low as 1.001 when water intake is high and as high as 1.04 when water intake is restricted. • The specific gravity is directly proportional to the concentration of solutes excreted. • Specific gravity is measured with Urinometer.

CHEMICAL CHARACTERISTICS Reaction • Fresh urine is normally acidic with a mean pH of 6 (4.8- 7.5) • pH of urine is influenced by the nature of the diet. • In people on high protein diets the urine is more acidic because more sulfates and phosphates are eliminated from the protein catabolism. • Diet rich in fruits and vegetables makes the urine alkaline. • Urine on standing becomes alkaline by the bacterial action on urea and formation of ammonia. • After meals, due to hydrochloric acid secretion in the stomach, the urine becomes alkaline. This is known as the ‘alkaline tide’. Constituents of Normal Urine • Normal urine contains both inorganic and organic constituents. • The inorganic constituents include sodium, potassium, magnesium, chloride, calcium, phosphorus, inorganic sulfates and ammonia. • The normal organic contents are urea, uric acid, creatinine, amino acids and ethereal sulfates (also urobilinogen, hippuric acid, indican).

86 An Easy Guide for Practical Biochemistry • The normal non-protein nitrogenous contents are urea, uric acid, creatinine. • The total non-protein nitrogen varies from 10 to 15 mg per day depending mainly on the protein intake. • In addition to these major organic constituents, detoxified products like indican and ethereal sulfates are found in urine.

ANALYSIS OF NORMAL URINE Physical Examination Physical examination of urine Experiment

Observation

Inference

Appearance

Clear

Given sample of urine is normal.

Volume

1000 to 1500 ml Normal volume.

Color

Amber yellow

Given sample of urine is normal.

Odor

Aromatic smell

Given sample of urine is normal.

Reaction to litmus

Blue litmus turns red

Normal urine is acidic.

Specific gravity 1.016 to 1.025

Normal.

Determination of Specific Gravity Specific gravity of urine is measured by an apparatus known as Urinometer. Urinometer consists of a thin stem graduated from 1000 to 1060 corresponding to Specific Gravity of 1.0 to 1.06. Urinometer is calibrated at 60°F (15°C). Procedure: Take sufficient urine in a urine Jar. Allow the urinometer to float in it without touching the sides. Observe the reading at the meniscus. This gives the observed specific gravity at the temperature at which the urinometer is calibrated. Note the urine temperature (room temperature).

Qualitative Analysis of Normal Urine 87 Calculation: Suppose the meniscus of the urine coincides with the reading, 1010 and the room temperature is 37°C. Urinometer is calibrated at 15°C. Since the room temperature is higher, a temperature correction has to be applied. For every 3°C rise over the temperature of calibration (15°C), a

correction factor of 0.001 is added to the last digit of the observed reading. The difference between 37°C and 15°C is 21°C. This when divided by 3 gives 7. Thus, the corrected specific gravity = 1.010 + 0.00 7 = 1.017 If the room temperature is below 15°C, one unit should be subtracted from the last digit for every 3°C difference in temperature. Long’s Coefficient The total solids normally excreted in the urine may be calculated using Long’s coefficient that is 2.6. The solid content of 1000 ml of urine is calculated by multiplying last two digits of specific gravity by 2.6 and is expressed in g/ L.

88 An Easy Guide for Practical Biochemistry Total solid in g/liter = last 2 digits of corrected specific gravity × 2.6 = 17 × 2.6 = 44.2 gm/liter Points to Remember: • Specific gravity of a sample decreases with increase of temperature. • Specific gravity of a sample is directly proportional to the concentration of the solid contents. Specific gravity increases with increase in solid content. • It is inversely proportional to the volume of the urine. As the volume increases the specific gravity decreases. Chemical Tests Inorganic Constituents Tests of inorganic constituents are as follows. Test for Chloride Principle: A white precipitate of silver chloride is formed when acidified urine reacts with silver nitrate. Experiment

Observation

Inference

Take 3 ml of urine in a test tube. Add 0.5 ml of conc. Nitric acid and 1 ml of 3% silver nitrate.

A white precipitate is formed.

Indicates the presence of chloride.

Qualitative Analysis of Normal Urine 89 Points to Remember: • Chloride ion is the chief anion in urine. • Excreted as sodium chloride. • On an average diet, 10- 12 gm of chloride is excreted per day. • Urates and phosphates can interfere with this test by forming silver urates and silver phosphates. Hence, nitric acid is added to prevent such interference. • Decreased urinary chloride is seen in: – Excessive sweating – Fasting – Diarrhea and vomiting – Diabetes insipidus – Cushing’s syndrome – Infections • Increased urinary chloride is seen in: • Excessive intake of fluids • Addison’s disease Test for Inorganic Sulfates Principle: Urine being acidified with hydrochloric acid forms a white precipitate of barium sulfate by the reaction with barium chloride solution. Experiment Take 3 ml of urine in a test tube. Add 1 ml of conc. Hydrochloric acid. Mix well and add 2 ml of 10% barium chloride.

Observation

Inference

A white precipitate is formed.

Indicates the presence of inorganic sulfates.

90 An Easy Guide for Practical Biochemistry Points to Remember: • There are three forms of sulfates: – Inorganic sulfates of sodium and potassium (80-85%) – Organic sulfates- ethereal sulfates (5%) – Neutral sulfur (15-50%) • Sulfates are derived from the metabolism of sulfur containing amino acids such as cysteine, cystine and methionine. • The presence of hydrochloric acid prevents the precipitation of other inorganic salts like phosphates. • On an average diet about 0.7-1 gram of inorganic sulfate is excreted per day. • Excretion is increased in: – High protein diet – Acute hyperthyroidism – Cystinuria • Decreased in renal dysfunction. • Neutral sulfur increases in poisoning. Test for Phosphates and Calcium Procedure: Take 10 ml of urine in a test tube. Add 3 ml of ammonium hydroxide boil and cool. A flaky precipitate of calcium phosphate is formed. Filter and discard the filtrate. Add 3 ml of hot 10% acetic acid on to the residue on the filter paper, through the sides of the paper. Collect the filtrate and divide into two parts. Test for phosphates Principle: Phosphates of calcium and magnesium are precipitated by ammonium hydroxide on boiling and these phosphates are dissolved in hot dilute acetic acid. This forms

Qualitative Analysis of Normal Urine 91 yellow precipitate of ammonium phosphomolybdate reacting with ammonium molybdate. Experiment

Observation

Inference

To one part of the filtrate, add a few drops of conc. Nitric acid and a pinch of ammonium molybdate. Warm.

Canary yellow precipitate is formed.

Indicates the presence of inorganic phosphates.

Points to Remember: • Normally 0.8-1 gm of phosphorus as phosphate is excreted per day. • Phosphates are present in urine as salts of sodium, potassium, ammonium, calcium and magnesium. These are crystallized out in alkaline urine. • Excretion is increased in bone diseases like rickets, osteomalacia, and parathyroid dysfunction. • Excretion is decreased in: – Diarrhea – Infections – Nephritis – Hypoparathyroidism – Pregnancy Test for calcium: Principle: Calcium is precipitated as calcium oxalate with potassium oxalate in acidic condition.

92 An Easy Guide for Practical Biochemistry Experiment

Observation

Inference

To the second part of the filtrate, add 2 ml of 2% potassium oxalate solution.

White precipitate is formed.

Indicates the presence of calcium.

Points to Remember: • The excretion of calcium is 100- 200 mg/day. • Excretion increases in: – Hyperparathyroidism – Hyperthyroidism – Hypervitaminosis D – Multiple myeloma • This test is known as Sulkowaski’s test and is useful in evaluating parathyroid abnormalities and cases of kidney stones. • Urinary calcium level is related to serum calcium level. • When serum calcium level is less than 7.5 mg/ dl there may be no detectable calcium in urine. • When serum calcium level is 7.5- 9 mg/ dl, urine shows slight cloudiness in this test. • A heavy precipitate indicates high serum calcium. Test for Ammonia Principle: Ammonia present in urine is liberated by heat. The evolution of alkaline ammonium vapors changes the color of red litmus to blue.

Qualitative Analysis of Normal Urine 93 Experiment

Observation

Inference

Take 2 ml of urine in a test tube. Add 1-2 drops of phenolphthalein indicator. Mix. Add 2% sodium carbonate drop by drop with constant mixing till the color of the solution turns faint pink. Boil. Hold a piece of red litmus paper at the mouth of the test tube.

Red litmus changes to blue.

Indicates the presence of ammonia.

Points to Remember: • Urinary ammonia is derived from glutamine and other amino acids in kidney. • The average excretion of ammonia is about 0.7 gm/ day. • There is an increase in ammonia excretion when acid forming foods are taken. • Ammonia is excreted as ammonium salts. • The kidneys manufacture ammonia in proportion to the amount of acid radicals excreted in urine. • In alkaline urine, ammonium salts are absent. • Excretion of ammonia is increased in acidosis. • Excretion of ammonia is decreased in alkalosis • Impaired protein metabolism increases the output of ammonia in urine. • To enhance the conversion of NH4 into NH3, the solution is made alkaline before boiling. • If the solution is made strongly alkaline, urea will interfere with the reaction.

94 An Easy Guide for Practical Biochemistry Organic Constituents Tests for organic constituents are as follows: Test for Urea Sodium Hypobromite Test: Principle: When urea is treated with Sodium hypobromite, it decomposes to give nitrogen, carbon dioxide and water. Liberation of nitrogen gas produces brisk effervescence. Experiment

Observation

Inference

Take 3 ml of urine in a test tube. Add 5 drops of freshly prepared alkaline sodium hypobromite solution and mix.

Brisk effervescence of Nitrogen gas is seen.

Indicates the presence of urea.

Specific Urease Test: Principle: The enzyme urease under optimum pH and temperature decomposes urea into ammonia and carbon dioxide which together form ammonium carbonate (alkaline substance) which changes the solution to pink color in the presence of the indicator. Experiment Take 3 ml of urine in a test tube. Add 3 ml of Urease suspension and add 1-2 drops of phenolphthalein indicator. Warm the tube for a few seconds with the hands and keep for 10 minutes.

Observation

Inference

Pink color is formed. Urea is confirmed.

Qualitative Analysis of Normal Urine 95 Points to Remember: • Urea is the major nitrogenous constituent of urine. • Urea is formed in liver as the end product of protein metabolism and so its excretion depends on protein intake. • About 20-40 grams of urea is excreted in 24 hours. • Excretion is increased in: – High protein diet – Fever – Diabetes mellitus • Excretion is decreased in: – Liver diseases – Nephritis – Acidosis Test for Uric Acid Phosphotungstic Acid Reduction Test/ Benedict’s Uric Acid Test: Principle: Uric acid in alkaline condition reduces phosphotungstic acid to tungsten blue. Benedict’s uric acid reagent: Composed of sodium tungstate, orthophosphoric acid, concentrated Sulfuric acid and solid sodium carbonate. Experiment

Observation

Inference

Take 3 ml of urine in a test tube; add 1 ml of Benedict’s uric acid reagent and 1 ml of 20% sodium carbonate. Mix.

Deep blue color is formed.

Indicates the presence of Uric acid

96 An Easy Guide for Practical Biochemistry ii. Schiff’s Test: Principle: Uric acid reduces salts of silver nitrate to metallic silver. Experiment

Observation

Inference

Moisten a piece of filter paper with few drops of ammoniacal silver nitrate solution. Add 2-3 drops of urine on the silver nitrate drops.

Black spots are seen on the filter paper.

Indicates the presence of Uric acid.

Points to Remember: • Uric acid is the end product of purine metabolism. • The daily output of uric acid varies in the range of 0.6 to 1 gm. • Excretion is increased in: – Leukemias especially during cytotoxic drug therapy – Wilson’s disease – Administration of cortisone/ ACTH • Excretion decreases in renal failure. Test for Creatinine (Jaffe’s Test) Principle: Creatinine reacts with picric acid in alkaline medium to form reddish orange colored creatinine picrate.

Qualitative Analysis of Normal Urine 97 Experiment

Observation

Inference

Take 3 ml of urine in a test tube. Add 1 ml saturated picric acid solution, and 3-4 drops of 10% sodium hydroxide.

Reddish orange color is formed.

Indicates the presence of creatinine.

Points to Remember: • Creatinine is the anhydride of creatine. • Urinary creatinine is derived from muscle creatine. • It is not influenced by the protein intake. • Excretion in adults ranges from 1-2 gm/day. • In women and in elderly people the values are lower due to lesser muscular mass. • Excretion is increased in: – High intake of meat, fish – Fever – Myopathy/wasting diseases • Excretion is decreased in: – Renal failure – Anemia – Paralysis Test for Ethereal Sulfate (Organic Sulfate) Principle: This test is done after removing the inorganic sulfate. Hot hydrochloric acid hydrolyses ethereal sulfate to inorganic sulfate, which then gives precipitate with barium chloride.

98 An Easy Guide for Practical Biochemistry Experiment

Observation

Inference

Take 5 ml of urine in a test tube. Add 2 ml of 10% barium chloride and 2 ml concentrated hydrochloric acid. Mix and filter. Divide the filtrate into two portions. Boil one and compare with the control.

Trace turbidity is seen over that in control.

Indicates the presence of organic sulfate.

Points to Remember: • Ethereal sulfates in urine are the conjugated sulfates, phenol- sulfuric acid and indoxyl sulfuric acid. • These ethereal sulfates result from phenols produced during putrefaction of protein material (amino acids) in the intestine. • About 100 mg of organic sulfate are excreted per day. • Indican (Potassium salt of indoxyl sulfate) is a typical example. • Bacterial decomposition of body protein as in gangrene and putrid pus formation, etc. result in the increased excretion of indican. • Excretion is increased in: – Inherited disorders—cystenuria, homocystinuria – Cyanide poisoning—thiocyanate. Report: 1. Organic constituents present in the given sample of normal urine are urea, uric acid, creatinine and ethereal sulfates. 2. Inorganic constituents present in the given sample of normal urine are chloride, calcium, phosphorus, inorganic sulfates, ammonia, sodium, potassium and magnesium.

9

Analysis of Abnormal Constituents in Urine

Substances which are not present in easily detectable amounts in urine of normal healthy individuals but are present in urine under certain diseased conditions are said to be ‘abnormal’ or ‘pathological’ constituents of urine. Analysis of these abnormal constituents aids in the diagnosis of many diseases. Many of these pathological constituents are present in trace amounts in normal urine but they escape detection due to the low sensitivity of the methods employed. The concentrations of these constituents in urine are increased markedly in different pathological conditions. Usually the analysis is carried out in properly preserved 24 hours urine specimens. When this is not possible the early morning specimens can be used. On standing, urine undergoes bacterial fermentation and degradation of some compounds. It can be preserved under refrigeration or using chemicals such as toluene or chloroform. The nature of the preservative will depend on the compound to be tested. Physical Characteristics in Pathological Conditions Volume Polyuria: An increase in urinary output. Occurs in: • Diabetes mellitus • Diabetes insipidus

100 An Easy Guide for Practical Biochemistry • After administration of drugs like diuretics, digitalis, salicylate, etc. • Certain nervous disorders • Later stages of chronic renal failure. Oliguria: A diminished urinary excretion (< 500 ml). Occurs in: • Acute nephritis • Fever • Diarrhea and vomiting. Anuria: A total suppression of urine formation. Occurs in: • Shock • Acute tubular necrosis • Incompatible blood transfusion • Mercury poisoning • Bilateral renal stones. Appearance Abnormal urine is turbid due to • Presence of pus cells in urinary tract infections • Increased excretion of phosphates in alkaline urine • Chyluria- milky white urine- presence of fat globulins due to obstruction in the lymphatics of urinary tract as in filariasis. Color Color

Condition

Pale

Dilute urine (Diabetes insipidus, polyuria)

Dark amber

Concentrated urine/due to the presence of pigments coproporphyrin, uroporphyrin Contd...

Analysis of Abnormal Constituents in Urine 101 Contd... Color

Condition

Reddish

Hematuria due to stones in the urinary tract, carcinoma of urinary bladder, injury to the urinary passage, stricture of the urethra Reddish brown/ smoky brown hemoglobinuria

Deep yellow, foaming

Bile pigments

Yellow fluorescent, non-foaming

Riboflavin

Black

melanin

Black on standing

Alkaptonuria- due to presence of homogentisic acid

Milky white

Chyluria- filariasis, due to the presence of pus, bacterial or epithelial cells and lipids.

Odor Odor

Cause

Putrid or ammonical odor Bacterial decomposition Fruity odor

Diabetic ketoacidosis, chronic starvation

Mousy odor

Phenylketonuria

Maple syrup

Maple syrup urine disease

Specific Gravity • Increased in acute nephritis and fever. • Decreased in diabetes insipidus. pH • • • •

Significantly acidic urine is voided in fever and diabetes. Alkali therapy and urinary retention make urine alkaline. Decrease in urinary pH: metabolic acidosis Increase in urinary pH: metabolic alkalosis

102 An Easy Guide for Practical Biochemistry Chemical Constituents The commonly encountered pathological chemical constituents of urine are: • Proteins (may be albumin, globulin, Bence Jones protein) • Blood (hemoglobin, erythrocytes) • Reducing sugar (usually glucose and in special cases lactose, galactose, pentose and rarely fructose) • Ketone bodies (acetone, acetoacetic acid) • Bile salts and bile pigments • Porphobilinogen • Urobilinogen (increased or decreased).

ANALYSIS OF ABNORMAL URINE Physical Characteristics Physical Characteristic

Observation

Inference

Appearance Color Odor Reaction to litmus Specific gravity

Chemical Constituents Test for Proteins Test for proteins are as follows: Heat and Acetic Acid Test Principle: On heating the protein loses its structure and becomes denatured to form a coagulum. It is precipitated after the addition of acetic acid, which provides the suitable pH to get the maximum precipitate.

Analysis of Abnormal Constituents in Urine 103 Experiment

Observation

Inference

Take 10 ml of urine in a test tube. Hold the tube over a flame in a slanting position and boil the upper 5 ml of the albumin solution. The lower half serves as control. Add 1% acetic acid drop by drop.

A white coagulum is seen at the upper heated portion, which gets intensified after the addition of acetic acid.

Indicates the presence of heat coagulable protein like albumin.

Points to Remember: • The amount of protein excreted normally in 24 hours urine is insignificant and it is less than 150 mg/day. • When proteins appear in detectable quantities in urine, it is called proteinuria/albuminuria. • The presence of detectable amount of protein is characteristic of kidney diseases. • The normal glomeruli of kidneys are not permeable to substances with molecular weight of 70 kD. The plasma proteins of molecular weight of more than 70 kD, hence are absent in normal urine. • When glomeruli are damaged or diseased, they become more permeable and plasma proteins appear in urine. • The smaller molecules of albumin pass through damaged glomeruli more readily than the heavier globulin and so, when the proteins appear in urine, the albumin fraction predominates.

104 An Easy Guide for Practical Biochemistry • Bence Jones protein, an immunoglobulin appears in urine in cases of multiple myeloma. Protein precipitates between 40- 60ºC, disappears at 100ºC and reappears on cooling. • Types of proteinuria: 1. Functional proteinuria 2. Organic proteinuria a. Prerenal

b. Renal

c. Postrenal

• • • • • • • • • •

Violent exercise Cold bath Pregnancy Cardiac diseases Abdominal tumors Cancer Collagen diseases Fever Anemia Acute and chronic glomerulonephritis • TB kidneys • Inflammatory conditions of kidney, ureter, bladder, prostate • Bleeding in genitourinary tract

• Rating of proteinuria: Proteinuria can be rated as +, ++, +++ depending upon the visibility of newspaper held at the other side of the test tube after the coagulation test is performed. Visibility of news print

Rating of proteinuria

Visible with difficulty

+

Visible but letters cannot be distinguished

++

Not visible

+++

Analysis of Abnormal Constituents in Urine 105 • Acetic acid is added: – To provide the suitable pH to get maximum precipitate. – To differentiate between protein and interfering substances mainly phosphates. – If the precipitate persists and deepens after the addition of acetic acid it is due to proteins. – If it disappears it indicates the presence of phosphates. • Sometimes urine may be alkaline; in that case heating alone may not precipitate. Acetic acid is to be added to make it acidic. Heller’s Nitric Acid Ring Test Principle: Nitric acid causes precipitation of proteins. Experiment

Observation

Inference

Take 3 ml of nitric acid in a test tube. Add 3 ml of urine along the sides of test tube.

A white ring is formed at the junction of the two liquids.

Indicates the presence of protein.

Points to Remember: • This is a highly sensitive test and can be taken as confirmatory test for protein. • If urine has a high concentration of urea, urea nitrate may be formed and it gives a false positive test for proteins.

106 An Easy Guide for Practical Biochemistry Sulfosalicylic Acid Test Principle: Sulfosalicylic acid is an alkaloidal reagent and so it neutralizes the positively charged protein to produce precipitation.

Experiment

Observation

Inference

Take 3 ml of urine in a test tube. Add 20% sulfosalicylic acid drop by drop.

White precipitate is formed.

Indicates the presence of protein.

Point to Remember: • This test is used as a routine test for protein. Test for Reducing Sugar (Benedict’s Test) Principle: In mild alkaline medium reducing sugars undergo tautomerization to form enediols which reduce cupric ions to cuprous ions. Cuprous hydroxide is formed. During the process of heating cuprous hydroxide is converted to cuprous oxide which gives different shades of color precipitate depending upon the concentration of the sugar.

Analysis of Abnormal Constituents in Urine 107 Experiment

Observation

Inference

To 5 ml of Benedict’s Depending on the reagent in a test tube amount of Glucose add 8 drops of Urine. present the following Mix and boil for 2 min. colors will be obtained. over a small flame. Cool and observe the contents. Blue

Nil

Green

0.5% (trace) +

Yellow

1% ++

Orange

1.5% +++

Red

2 % ++++

Brick red

> 2%

Points to Remember: • The presence of detectable amounts of sugar in urine is called glycosuria.

108 An Easy Guide for Practical Biochemistry • Positive Benedict’s test is usually suggestive of presence of glucose in urine. • Common causes of glycosuria are: – Diabetes mellitus – Endocrinal disorders such as hyperpituitarism, hyperthyroidism, hyperadrenalism. – Emotional glycosuria: It is a benign condition seen in anger, fear, etc. due to hypersecretion of adrenaline in stress. – Renal glycosuria in which glucose reabsorption by kidney tubules is defective. – Alimentary glycosuria: It is a benign condition which is seen after excessive intake of carbohydrate or patient is on glucose infusion. Reducing sugar

Condition

Glucose

Diabetes mellitus, Renal glycosuria

Fructose

Disorders of fructose metabolism, essential fructosuria, hereditary fructose intolerance

Galactose

Galactosemia

Lactose

Pregnancy, lactating woman

Pentose

Disorder of uronic acid pathway (essential pentosuria)

• Non-sugars such as ascorbic acid, glutathione, salicylates, uric acid, glucuronides and homogentisic acid will also give positive result with Benedict’s reagent.

Analysis of Abnormal Constituents in Urine 109 Test for Ketone Bodies Test for ketone bodies are as follows: Rothera’s Test for Acetone and Acetoacetic Acid Principle: Acetone and acetoacetic acid form permanganate colored complex with sodium nitroprusside in presence of ammonia. Experiment

Observation

Inference

Take 5 ml of urine in a test tube. Add solid ammonium sulfate a little at a time with mixing to saturate the solution. Add 2 or 3 drops of freshly prepared 5% sodium nitroprusside solution. Mix well and add 1 ml of strong ammonium hydroxide drop-wise along the side of the test tube.

Permanganate colored ring is formed.

Indicates the presence of ketone bodies.

Gerhardt’s Test for Acetoacetic Acid Principle: Acetoacetic acid gives a red color with ferric chloride. Experiment

Observation

Inference

Take 3 ml of urine in a test tube and add 10% ferric chloride solution drop by drop till maximum precipitate of ferric phosphate is formed. Filter. To the filtrate add further quantities of 10% ferric chloride.

Port-wine color is obtained

Indicates the presence of acetoacetic acid.

110 An Easy Guide for Practical Biochemistry Precaution: • A large number of substances such as aspirin, antipyrin, salicylates, etc. may develop similar port-wine color. If the urine is boiled, acetoacetic acid is converted into acetone; but the other substances remain unchanged. Now, if the urine gives negative test, it indicates the presence of acetoacetic acid. • Fresh urine is necessary for this test as acetoacetic acid is quickly decomposed into acetone and carbon dioxide. Points to Remember: • Ketone bodies are acetone, acetoacetic acid and β-hydroxy butyric acid. • Ketone bodies do not appear in urine because acetoacetic acid, which is produced normally in the liver, is completely oxidized in tissues. Ketone bodies are formed in excess when the glucose metabolism is impaired as in diabetes mellitus or when fat is used exclusively to give energy as in starvation (starvation ketosis). This condition is called as ketosis. • The tissues are unable to oxidize the excess amount of acetoacetic acid with the limited supply of oxygen. A part of excess acetoacetic is decarboxylated to acetone and remaining circulates in blood as acetoacetic acid and β-hydroxy butyric acid. • Rothera’s test is very sensitive. It is answered even by small amounts of acetone and acetoacetic acid. • β-hydroxy butyrate does not answer Rothera’s or Gerhardt’s test because it does not have a ketone group. It gives positive when converted to acetoacetic acid and then to acetone by oxidation. • The excretion of ketone bodies in urine is called ketonuria. This occurs in ketosis where there will be ketonemia and ketonuria.

Analysis of Abnormal Constituents in Urine 111 • Total ketone bodies are found in normal urine to the extent of about 20 mg/day. • Ketonuria may also be seen in conditions like intake of high fat and low carbohydrates diet and toxemia of pregnancy. • Whenever glucosuria is more than 0.5 mg% (++) the patient should be tested for ketone bodies also. • If Gerhardt’s test is negative and Rothera’s is positive, acetone is present. • Gerhardt’s test or ferric chloride test is useful in detecting a large number of abnormal constituents in urine, in rare disorders. In addition to metabolites, drugs excreted can be detected by this test. Some of the compounds detected are listed below. Compound

Color in the test

Phenyl pyruvic acid in phenylketonuria

Stable blue/bluish green color

Homogentisic acid in alkaptonuria

Rapidly fading blue or green color

β-hydroxyphenyl pyruvic acid in tyrosinosis

Rapidly fading green color

Branched chain amino acids in Maple syrup urine disease

Blue color

Imidazole pyruvic acid in histidinemia

Green color

Melanin

Green to black

Indican in Hartnup disease

Violet or blue color

Salicylates

Stable red color/ port-wine color

Phenothiazine derivatives

Purple pink color

p-Aminobenzaldehyde

Reddish brown

Phenols

Violet color

112 An Easy Guide for Practical Biochemistry Test for Bile Salts (Hay’s Test) Principle: Hay’s test is based on the fact that bile salts lower the surface tension of urine allowing the sulfur to sink. Experiment

Observation

Inference

Take 2 ml of urine in a test tube. Gently sprinkle a little fine sulfur powder over the surface of urine. Observe without mixing.

Sulfur powder sinks to the bottom.

Indicates the presence of bile salts.

Points to Remember: • Bile salts are sodium and potassium salts of glycocholates and taurocholates. • Normally bile salts and bile pigments do not enter the general circulation and therefore, they are absent in the normal urine. • But, if there is intrahepatic or posthepatic obstruction to the flow of bile, regurgitation occurs in the general circulation and bile salts appear in urine. • Bile salts are present in urine along with bile pigments in obstructive jaundice. • This is not a specific test for bile salts but is usually done to detect bile salts. • Alcohol and salicylates give a false positive test.

Analysis of Abnormal Constituents in Urine 113 Test for Bile Pigments Tests for bile pigments are as follows: Gmelin’s Test Principle: Bile pigments are oxidized by nitric acid to various colored products, e.g. biliverdin (green), bilicyanin (blue), bilifuscin (red) and choletelin (yellow) Experiment

Observation

Inference

Take 5 ml of conc. Nitric acid in a test tube. Add 5 ml of urine carefully to form a separate layer.

Various colored rings will be formed at the point of contact of the two liquids. (play of colors).

Indicates the presence of bile pigments.

Fouchet’s Test Principle: Bile pigments adsorbed on barium sulfate precipitate are oxidized to colored products by Fouchet’s reagent. Fouchet’s reagent: 10% ferric chloride in 25% trichloroacetic acid. Experiment

Observation

Inference

Take 5 ml of urine in a test tube. Add few crystals of magnesium sulfate. Then add 3 ml of 10% barium chloride solution. Mix. Filter. Unfold the filter paper. Add a few drops of Fouchet’s reagent on the precipitate.

A white precipitate of barium sulfate is formed. A green color develops on the filter paper.

Indicates the presence of bile pigments.

114 An Easy Guide for Practical Biochemistry Points to Remember: • Bile pigments are bilirubin and biliverdin. • They are produced by the breakdown of heme in the reticuloendothelial system. • Bilirubin is in unconjugated form soon after it is produced from heme. It gets conjugated with UDP glucuronic acid in liver to form mono/di-glucuronide. Bile contains conjugated bilirubin which is excreted into the intestine. • In normal persons bile pigments are not present in urine. • Fouchet’s test is a highly sensitive test for bilirubin. • Ferric chloride, present in the Fouchet’s reagent acts as an oxidizing agent. It oxidizes bilirubin to biliverdin (green) or bilicyanin (blue). Test for Blood Principle: Hemoglobin (peroxidase) of blood decomposes hydrogen peroxide catalytically and liberates nascent oxygen. This oxygen oxidizes benzidine to a blue or green compound. This color changes to brown within a few minutes on exposure to air. Benzidine reagent: It contains benzidine and glacial acetic acid. Experiment

Observation

Inference

Take 2-3 drops of A blue or green color Indicates the benzidine solution is formed, which is stable presence in a test tube. only for a few minutes of blood. Add 2-3 drops of and changes to brown. hydrogen peroxide solution. Add 1 or 2 drops of this mixture to 2 ml of urine.

Analysis of Abnormal Constituents in Urine 115 Points to Remember: • This is a very sensitive test but not specific for blood. • Presence of blood in urine is called hematuria. • Causes: – Injury to urinary tract or kidney. – Infection of urinary tract. – Benign or malignant carcinoma of kidney or urinary tract. – Enlargement of prostrate due to rupture of engorged venous plexus. – Obstruction due to urinary stones. – Nephritis. – Nephrotic syndrome. – Due to trauma, caused by introduction of catheter through the urethra. – Tuberculosis. – Acute glomerulonephritis. • Hematuria can be frank when urine appears red (due to blood) or it can be microscopic when it is not visible to naked eye (occult blood) • Microscopic hematuria may be seen in: – Malignant hypertension – Sickle cell anemia – Coagulation abnormalities – Polycystic kidney diseases. • Excretion of free hemoglobin in urine is called Hemoglobinuria. • This occurs in severe burns, chemical poisoning, incompatible blood transfusion, malaria, typhoid and hemolytic jaundice. • This test is also positive when pus cells are present in urine. These cells contain a peroxidase, which is responsible for the positive reaction. However, if urine is

116 An Easy Guide for Practical Biochemistry subjected to heat treatment (95-100°C), the enzyme is inactivated and the test becomes negative. • Heme, is stable to heat. • When high concentration of ascorbic acid is present in urine, it is oxidized more readily than benzidine by oxygen liberated from hydrogen peroxide. The benzidine reaction then becomes negative although sufficient blood is present in urine. Report: The abnormal constituents present in the given sample of urine are…

Hemoglobin and its Derivatives 117

10

Hemoglobin and its Derivatives

Hemoglobin is a conjugated protein, consisting of the protein part called globin and the prosthetic part called heme. The hemoglobins differ depending on the type of polypeptide chains they are composed of. The normal hemoglobins are Hb-A (hemoglobin of adult), Hb-F (hemoglobin of fetal life) and Hb-A2 (hemoglobin of postnatal). The following are the derivatives of hemoglobin. a. Native hemoglobin: It serves as oxygen carrier in the blood. b. Oxyhemoglobin: Hemoglobin in combination with 4 molecules of oxygen. c. Carboxy-hemoglobin: Hemoglobin in combination with 4 molecules of carbon monoxide. d. Methemoglobin: It is oxidized non-functional form of hemoglobin is which iron is in the ferric state. e. Hemochromogen: Denatured hemoglobin in which iron is in the ferrous (Fe++) form. During heating with alkali, globin portions get denatured and heme is oxidized to hematin (ferrous). On treatment with reducing agent, the hematin is converted to heme which combines with denatured globin to form hemochromogen. f. Hematin: It is the oxidized form of heme in which iron is in ferric state (Fe3+). g. Hemin: Chloride form of hematin.

118 An Easy Guide for Practical Biochemistry

DETECTION OF HEMOGLOBIN AND ITS DERIVATIVES Hemoglobin derivatives are prepared from oxalate blood and the study of their absorption spectra is done with direct vision spectroscope. Direct Vision Spectroscope Spectroscope is a simple device that resolves light into its seven component colors. It consists of narrow slit through which light enters. A set of prisms resolves the light that can be viewed through the eyepiece. The wavelength scale is superimposed upon the spectrum by means of a small telescope containing wavelength scale.

Fig. 10.1: Spectroscope

Principle Sunlight is made up of seven components. When a beam of light is passed through a prism the light is resolved into its components VIBGYOR. This phenomenon is known as dispersion.

Hemoglobin and its Derivatives 119 When sunlight passes through the atmosphere consequently, light of certain wavelength is absorbed by atmosphere. Consequently, the corresponding areas in the visible spectrum appear as dark lines known as Fraunhofer’s lines. For example, two prominent lines are seen at 589 nm and 518 nm due to absorption of light by sodium and magnesium respectively in solar atmosphere. Colored solutions have the property of absorbing the light of certain specific wavelengths in the visible region of the spectrum. Thus when a colored solution is placed between the source of light and the prism, certain regions appear dark known as absorption band and they are constant under all circumstances. The different derivatives of hemoglobin produce characteristic absorption spectra and can be easily identified by spectroscopy. Precaution: It is essential that solutions used in spectroscopic studies are not concentrated. At high concentration two adjacent bands will merge and appear as a single, broad band.

PREPARATION OF HEMOGLOBIN AND DERIVATIVES Oxyhemoglobin (HbO2) Add one drop of blood to 5 ml of water in a test tube (1 in 100 dilution). Mix till a clear solution is obtained. This is HbO2. Note the color. Hold the solution of HbO2 against the slit of the spectroscope and expose to indirect sunlight (from reflecting walls) and view through the eyepiece. Two dark absorption bands (α at 577 nm, β at 541 nm) will be seen in the green portion of the spectrum. Note the readings on the

120 An Easy Guide for Practical Biochemistry scale at the center of the bands which give the wavelength of these bands. Reduced Hemoglobin To 5 ml of 1 in 100 dilution of blood, add a pinch of sodium hydrosulfite (sodium dithionate Na2S2O4 ) and gently mix. Note the bright crimson red color of the oxyhemoglobin is changed to purple due to reduced hemoglobin. Examine with the spectroscope. You will observe that the two bands of oxyhemoglobin are replaced by a single broad faint band with maximum absorption at 565 nm. Now shake the tube vigorously. Sodium dithionite is air oxidized. Note again the change of color from purple to crimson red due to reoxygenation of hemoglobin and formation of oxyhemoglobin (HbO2). Carboxy Hemoglobin Bubble through the diluted blood sample, coal gas or a mixture of carbon monoxide and carbon dioxide obtained by treating oxalic acid with concentrated Sulfuric acid. Add a drop of caprylic alcohol during bubbling of gas to prevent frothing. Observe the absorption spectrum. Two bands will be seen much like those of oxyhemoglobin. But there is subtle difference (α at 570 and β at 535 nm) which may be determined by Hatridge Reversion (high resolution) spectroscope. Note the color. It is light pink as against the crimson red color of oxyhemoglobin. Add a little sodium hydrosulfite and mix. The color does not change. Carboxyhemoglobin cannot be reduced, i.e. it is stable. Methemoglobin Add 5 ml of water to 4 drops of blood. Mix. Add a pinch of potassium ferricyanide and mix gently. The solution

Hemoglobin and its Derivatives 121 turns brown. Ferricyanide oxidizes ferrous iron in heme to ferric form. Examine with the spectroscope. A band is seen in the red region with its center at 630 nm.

Fig. 10.2

Points to Note • Normally 1-3% of hemoglobin in blood is in the form of methemoglobin. • About 1-4% of hemoglobin exists as carboxyhemoglobin also called carbonyl hemoglobin. • Automobile exhaust and burning of coal increases carboxy hemoglobin level. When its concentration reaches 30%, severe headache, dizziness, nausea and dim vision develop. • When the concentration is 60%, unconsciousness, coma and respiratory failure result. • Breathing of fresh air or hyperbaric oxygen therapy is useful in treating carbon monoxide poisoning.

122 An Easy Guide for Practical Biochemistry Preparation of Hemin Crystals Principle: Upon heating with Nippe’s fluid hemoglobin is denatured and heme is oxidized to hematin. Hematin is finally converted to hematin chloride, also called as hemin. Nippe’s fluid: Composed of 0.1 g each of potassium chloride, potassium bromide and potassium iodide dissolved in 100 ml of glacial acetic acid. Procedure: Place a drop of blood on a clean glass slide, spread it with a glass slide uniformly so as to form a thin smear not exceeding the area of a cover slip. Add one drop of Nippe’s fluid from each side of the cover slip, which is placed over the smear. The fluid enters under the cover slip, by capillary action. Warm gently the area of blood and reagent near the flame so that bubbles appear. As the fluid is getting evaporated, cool and add further quantities of Nippe’s fluid in the same way and heat again, till the fluid is just evaporated. Do not overheat. Examine the crystals of hemin under the low power as well as the high power of the microscope. The hemin crystals are rhombic and brown colored. Draw the crystals in the same color. Clinical application/ medicolegal importance: Used in forensic medicine to detect traces of blood. Fig. 10.3: Hemin crystals (Label)

Spot Tests 123

11

Spot Tests

PHENYLKETONURIA • Phenylketonuria is an inherited metabolic disorder in amino acid metabolism. • It is due to the deficiency of the enzyme, phenylalanine hydroxylase. Therefore, the conversion of phenylalanine to tyrosine is deficient, i.e. phenylalanine accumulates in the blood giving a concentration of 10 to 80 mg/dl compared with < 2 mg/dl in normal infants. • Among the derivatives of phenylalanine present in urine, the largest amounts are phenylpyruvic acid and phenyl lactic acid. The tests used for screening phenylketonuria are: Guthrie’s Bacterial Inhibition Test Bacteria Bacillus subtilis requires phenylalanine for growth in culture media. In minimal culture media when B2 thienylalanine, an analog of phenylalanine is added, the bacterial growth is inhibited. When blood from a normal infant is added to such a media no growth is noticed because the phenylalanine concentration is not adequate to reverse the effect of an analogue, whereas blood from the PKU patient is added bacterial growth is observed, because of the reversed effect of analogue by the accumulated metabolic products.

124 An Easy Guide for Practical Biochemistry Ferric Chloride Test Experiment

Observation

Inference

Take 3 ml of urine in a test tube and add 10% ferric chloride solution drop by drop till maximum precipitate of ferric phosphate is formed. Filter. To the filtrate add 2 ml of 10% ferric chloride.

Bluish green color is obtained

Indicates the presence of phenylpyruvate

Points to Remember: • This test is not specific since many other compounds give a false positive test. • Nowadays prenatal diagnosis is possible using DNA based test. Alkaptonuria • It is an autosomal recessive disorder. • Prevalence is 1 in 25,000. • The defective enzyme in alkaptonuria is homogentisate oxidase in tyrosine metabolism. • Homogentisate accumulates in blood and is excreted in urine. • Homogentisate on standing gets oxidized to the corresponding quinines, which polymerize to give black or brown color. Because of this reason, urine of alkaptonuric patients is black in color (coke in color) on standing.

Spot Tests 125 Spot Test Change in color of the urine on standing to brown or dark is a simple method to identify alkaptonuria. The other test to diagnose alkaptonuria is given by K. Valmikinathan and Ninan Verghese, (J Clinical Path 19, 200. 1996). Procedure • Freshly voided urine about 10 to 15 ml from suspected alkaptonuric patients is concentrated over a boiling water bath. • The concentrates are extracted with 2 ml of N butanol. • The butanol layer is then partitioned with 5 ml water and the aqueous layer is separated. • Take 2 drops of the aqueous layer, add 2 drops of 0.01% copper sulfate followed by 5 ml of water and 2 drops of 0.1 N NaOH. • The contents of the tubes are mixed immediately and left aside for 20 minutes. • A pinkish brown color develops confirms the presence of homogentisic acid in urine.

HOMOCYSTINURIA • • • •

It is an inborn error of metabolism. Autosomal recessive disorder. Incidence is 1 in 200,000 births. The deficient enzyme in homocystinuria is cystathionine synthetase which converts homocysteine into cystathionine. • Normal homocysteine level in blood is 5- 15 micromol/L. In diseases, it may increase to 50 to 100 times.

126 An Easy Guide for Practical Biochemistry • Moderate increase is seen in aged persons, vitamin B12 or B 6 deficiency, tobacco smokers, alcoholics and in hypothyroidism. • If homocysteine level in blood is increased, there is increased risk for coronary artery diseases. Screening Test for Homocystinuria (Spaeth and Barber) This test depends on the marked difference in reactivity shown by homocysteine and cystine to the silver diamine ion. Under the conditions used homocysteine is effectively reduced to the thiol form whereas cystine is unaffected. Reagents 1. 2. 3. 4. 5.

Solid sodium chloride Ammonia Silver nitrate Sodium nitroprusside solution Sodium cyanide.

Procedure • Saturate the urine with sodium chloride. • Add 0.5 ml silver nitrate solution to 5 ml saturated specimen and to 5 ml dilute ammonia. • Allow to stand for 1 minute, and then add to each 0.5 ml sodium cyanide. • Terminal addition of cyanide is needed to bind the silver ion and allow reaction of homocysteine with the nitroprusside. • At this point excess cyanide begins to react with any cysteine present to give a slowly developing positive test. • The test is positive for homocysteine if pink or purple color develops in the test sample immediately.

Section 3

Quantitative Tests

12

Principles of Colorimetry

Many biochemical experiments involve the measurement of a compound present in a complex mixture. The most widely used method for determining the concentration of biochemical compounds is colorimetry.

PHOTOMETRY Photometry means measurement of light. The color of light is a function of its wavelength. As the wavelength is changed within the visible range, an alteration in color is detected. Principle When white light passes through a colored solution, some light is absorbed and some light is transmitted. The absorbed light is measured as optical density (OD). This absorbed light is made to fall on the photo cell, which converts light energy into electrical energy which is measured by a galvanometer. Many compounds which are colorless can be colored on reacting with suitable reagents. The color intensity of the unknown is compared with a standard (solution of known concentration) and is measured, which is proportional to the concentration of the substance.

130 An Easy Guide for Practical Biochemistry Wavelength (in nm)

Color of light absorbed

Color of light reflected

400-435 435-500 500-570 570-600 600-630 630-700

Violet Blue Green Yellow Orange Red

Green-yellow Yellow Red Blue Green-blue Green

COLORIMETER • The quantum of light absorbed by a colored solution may be determined by certain optical instrument called colorimeter. • Colorimeter has been the traditional name for an instrument that isolates specific wavelengths of light with interchangeable filters for the visible portions of the spectrum. In contrast to this, spectrophotometers have a continuously adjustable monochromatic prism (or grating) and can often measure the intensity of light from the UV range, visible and infra red regions. Components of the Colorimeter • Source of light: A lamp provides light in visible region of the spectrum. Usually tungsten lamp is the source of light. • Adjustable slit: The light emerging from tungsten lamp is allowed to pass through a narrow adjustable slit. • Condensing lens: Provides parallel beam of light. • Filter: It provides the desired monochromatic light (of single wavelength) by filtering other wavelengths. The color of the filter is complementary to the color of the solution. This allows only appropriate wavelength of light to pass through the colored solution.

Principles of Colorimetry 131 • Cuvette (sample holder): Cuvette is a special glass tube with some absorptive capacity. It holds the solution to be analyzed in a colorimeter. Cuvette should have uniform thickness, inner diameter and refractive index. Cuvettes usually have 1 cm light path. • A photocell/detector: It is a photosensitive element usually made of selenium. It is activated when light falls on it. It emits electrons proportional to the amount of light falling on it. It converts the light energy into electrical energy. • Galvanometer: To measure the output electrical energy.

PREPARATION OF SOLUTION FOR INVESTIGATION In colorimetric estimation it is necessary to prepare three solutions: • Blank (B) • Standard (S) and • Test (T)

132 An Easy Guide for Practical Biochemistry Blank • Blank is done to delete the color due to reagents. Since some reagents are colored, they add on to the color produced by the substance which is to be estimated. This increases the color intensity which in turn gives high concentration of the substance to be estimated. • Alternately the blank solution is used to set the meter of the instrument to 100% transmittance (T) or zero absorbance. • The values of blank are subtracted from tests and standard. • A blank is prepared by using all the reagents except the biological material to be estimated. Type of Blank Two types of blank are used. • Water blank: It is used to adjust the OD to zero and T to 100%. • Reagent blank: It is prepared by adding all reagents except the substance to be estimated. Standard Solution It is a solution of known concentration of the substance in pure form which is to be estimated. As both concentration and OD of the standard solution are known, the concentration of unknown can be calculated by using the formula. Test Solution The test solution is made by treating a specific volume of the test sample with reagents as specified in the procedure.

Principles of Colorimetry 133

TECHNIQUE • The light passes through an adjustable slit and then through a condenser lens which gives a parallel beam of light. This beam of light passes through a colored filter to give a monochromatic light. • The colored solution to be analyzed is taken in a glass cuvette. Complementary colors for selection of filters Filter

Color of Solution

Blue Purple Yellow Orange

Red Green Violet Blue green

• The monochromatic light is incident (Io) on the solution, a part of it is absorbed by the solution and the rest is transmitted (I). • The transmitted light is detected by a photocell. It converts light energy into electrical energy, the strength of which is calibrated in percentage transmitted light. • A galvanometer connected to the photocell measures the output electrical energy. • The measurement of color intensity of a colored solution by photometry is governed by two laws 1. Beer’s law 2. Lambert’s law Beer’s Law The amount of light absorbed by a colored solution is proportional to the concentration of the solution. If A is the light absorbed (Absorbance) and C is the concentration of the color in the solution then, A α C.

134 An Easy Guide for Practical Biochemistry Lambert’s Law The amount of light absorbed by a colored solution is proportional to the depth through which the light passes in the solution. If L is the depth through which the light passes in the solution then, A α L Combining the two laws, A α C × L or A= K × C × L Where K = the constant for the colored solution AT = absorbance of the test solution CT = concentration of test solution AS = absorbance of the standard solution CS = concentration of standard solution

AT = AS Since in the colorimetric measurements, optically similar cuvettes having the same length of light path are used for blank, test and standard the below formula can be used, = CT = Concentration of test solution = Absorbance of test × Concentration of standard Absorbance of standard

If this concentration is present in × ml of the test sample taken then.

Principles of Colorimetry 135 Concentration in 100 ml of test sample (Percent concentration) =

A T CS   100 AS X

=

OD of test × conc. of std.  100 OD of std × effective volume

RELATIONSHIP BETWEEN ABSORBANCE AND TRANSMITTANCE • When light passes through a colored solution, some amount of light is absorbed by the solution depending on the concentration of the light absorbing compound, while remaining light is transmitted. • The amount of light absorbed is termed as Absorbance (A) or optical density (OD) and the amount of light transmitted is termed as transmittance (T). • Transmittance is defined as the ratio of the intensity of light emerging (I) to the intensity of light incident (Io) , i.e. T=

Intensity of light emerging (I) Intensity of light incidence (I o )

T=

I Io

% T = 100 

I Io

• When Io is 100 (adjusted with blank on the galvanometer scale), I gives % transmittance.

136 An Easy Guide for Practical Biochemistry

SELECTION OF FILTER IN COLORIMETRIC ESTIMATION The color of the filter should be complementary to the color of the solution under investigation to give maximum. Steps in the Operation of the Colorimeter 1. Place glass filter recommended in the procedure in the filter slot. 2. Fill the cuvette to about 3/4th with the distilled water and place it in the cuvette slot. 3. Switch on the instrument and allow it to warm up 4-5 minutes. 4. Pressing the button adjust the “coarse” and “fine” knobs to give zero optical activity in the galvanometer. Release the button. 5. Take blank solution in an identical cuvette and place it in the cuvette slot, press the button and read the optical density (OD), without disturbing the previous adjusted ‘Coarse’ and fine knobs. Release the button. Let the OD be ‘B’ 6. Transfer the ‘blank’ solution to the original test tube. 7. Take ‘standard’ solution in the same cuvette and record the OD. Let it be ‘S’ 8. Transfer the ‘standard’ solution back to the original test tube. 9. Next take ‘test’ solution in the same cuvette and read the OD. Let it be ‘T’ 10. Transfer the test solution back to the original test tube and wash the cuvette. Satisfactory results are obtained when the OD values are in the range of 0.1-0.7.

Principles of Colorimetry 137

CALCULATIONS Percent concentration of the test =

APPLICATION OF COLORIMETER • Colorimetric procedure is widely used in laboratories for the estimation of various biochemical compounds in various biological samples like blood, plasma, serum, CSF, urine and other body fluids. • Some of the routinely estimated biochemical compounds by colorimeter are glucose, urea, creatinine, uric acid, bilirubin, lipids, total proteins, and enzymes like AST, ALT, ATP, minerals like calcium, phosphorus, etc. OD of test  OD of blank Concentration of std.  × 100 OD of standard  OD of blank Volume of test sample

138 An Easy Guide for Practical Biochemistry

13

Estimation of Blood Sugar

The main sugar of blood is glucose along with other carbohydrate constituents. Hence, ‘blood glucose’ is commonly referred as ‘blood sugar’. Blood glucose estimation is a common test done in all laboratories because it helps in diagnosis, management of diabetes mellitus and is a common prerequisite for any surgery. Methods of Estimation • Folin -Wu’s method • Glucose oxidase method (God- Pod autoanalyzer method) • O- toluidine method • Nelson- Somogyi method. Choice of Blood Specimen Blood sugar can be measured in whole blood, plasma, serum or in capillary blood. But the modern trend is to use mainly plasma or serum.

Estimation of Blood Sugar 139 Preservation of Blood Due to the presence of glycolytic enzymes in RBC, glucose disappears quite rapidly from the whole blood (at a rate of 2-10 mg/dl/hour). So the blood must be collected into an anticoagulant and anti-glycolytic preservative. Fluoride and oxalate mixture is used in the ratio of 1:3. Sodium fluoride acts as anti-glycolytic agent and potassium oxalate as an anticoagulant. When this is done glucose content will remain unchanged for 2-3 days. Estimation of Blood Sugar by Folin-Wu’s Method Aim of the Test To estimate the amount of blood sugar. Method Folin-Wu’s method. Principle Blood is deproteinized by Tungstic acid formed by the reaction of sodium tungstate and sulfuric acid. The protein-free filtrate containing glucose is treated with alkaline copper reagent. Glucose in the protein- free filtrate at higher temperature in alkaline medium reduces cupric oxide to cuprous oxide .The cuprous oxide formed is treated with Phosphomolybdic acid which is reduced to phosphomolybdous acid (molybdenum blue), a blue solution. The intensity of this blue solution is a measure of the amount of glucose present.

140 An Easy Guide for Practical Biochemistry Reagents 1. 2. 3. 4. 5.

10% sodium tungstate 2/3 N Sulfuric acid Alkaline copper reagent Phosphomolybdic acid Standard glucose: Contains known amount of glucose (0.2 mg/ml).

Procedure a. Preparation of protein-free filtrate: Into a dry 100 ml conical flask, pipette 7 ml distilled water and 1 ml blood. Rotate the flask. Add one ml sodium tungstate and mix. Add 1 ml of H2SO4 drop by drop with shaking. Thus, the dilution is 1 in 10. Let it stand for 10 minutes. Filter into a dry test-tube. The filtrate should be clear and colorless. b. Development of color: Set up 3 Folin-Wu tubes, marked B, S, and T for blank, standard and test respectively. Pipette 2 ml of distilled water in ‘B’, 2 ml standard glucose into ‘S’ and 2 ml of protein free filtrate into ‘T’. Pipette 2 ml alkaline copper reagent into each. Mix the contents. Keep the tubes in a boiling water bath for exactly 8 minutes. Then remove them and cool in a beaker containing cold water. After cooling add 2 ml Phosphomolybdic acid reagent to each. Rotate to mix and make up the volume to 25 ml mark with distilled water. Mix the contents by inverting the tube by placing your palm tightly over the mouth of Folin -Wu tube. Read the Optical density values using a blue filter.

Estimation of Blood Sugar 141 Protocol Reagents

Blank

Standard Test

Distilled water

2 ml

-

Standard Glucose

-

2 ml

-

Protein-free filtrate

-

-

2 ml

Alkaline copper reagent

2 ml

2 ml

2 ml

-

Mix the contents-keep the tubes in a boiling water bath for exactly 8 minutes. Phosphomolybdic acid reagent

2 ml

2 ml

2 ml

Rotate to mix and make up the volume to 25 ml mark with distilled water. Mix the contests by inverting the tube placing your palm tightly over the mouth. Read the optical densities using a blue filter. Optical Density at 490 nm.

Calculation Concentration of glucose in mg/100 ml blood = OD of test – OD of blankT – B ×Conc. 100 of standard

= OD of standard – OD of blank S – B× Volume of sample × 100 =

OD of T – OD of B 10 100 × Concentration of standard ×  OD of S – OD of B 2 1

=

T–B 10 100 × 0.2 × × S–B 2 1

= = –––––––––– mg/dl.

Report Amount of Glucose present in 100 ml of blood is ________ mg/dl.

142 An Easy Guide for Practical Biochemistry Points to Remember: • True blood glucose level is 60- 90 mg/dl. • The fasting blood sugar value by Folin- Wu method amounts to 80-120 mg/100 ml in normal subjects, which is about 20% higher than the true blood glucose level. This is due to the presence of non-sugar reducing substances. • Non-sugar reducing substances are blood constituents other than glucose, which reduce alkaline copper sulfate and ferricyanide reagents. Examples: glutathione, glucuronic acid and its compounds, uric acid, ascorbic acid, threonine and other sulfydryl compounds. • The special Folin-Wu tube is designed to prevent the autooxidation of cuprous oxide formed by atmospheric oxygen by decreasing the surface area of the solution in the constricted area exposed to the atmosphere, and providing a larger area in the bulb for reaction to take place. • Oxalate precipitates Ca2+ of blood to prevent coagulation. • Fluoride inhibits glycolytic enzymes of RBC to prevent glycolysis before estimation. • The Folin-Wu filtrate still contains some polypeptides, which escape precipitation by tungstate. These polypeptides bind Cu2+ at their peptide bonds to form colored complexes and consequently produce some errors in the estimated blood glucose value. So it is important to precipitate the proteins. • Both O-toluidine and glucose oxidase method are highly specific for glucose compared to Folin-Wu method. • Any increase in blood glucose level is called hyperglycemia and any decrease in blood glucose level is called hypoglycemia.

Estimation of Blood Sugar 143 • Hyperglycemia is seen in: – Diabetes mellitus – Hyperthyroidism – Hyperpituitarism – Pancreatitis – Cushing syndrome – Pheochromocytoma – Sepsis and infectious diseases and administration of general anesthetics. • Transient rise may occur in emotional status such as anger, anxiety and fear due to excessive secretion of epinephrine, which favors glycogenolysis. • Value below 40 mg/dl by glucose oxidase method is known as hypoglycemia. • Commonest causes of hypoglycemia are: – Increased level of insulin: accidental over administration of insulin in diabetes or certain tumors of the β-cells of islets of Langerhans which leads to hypersecretion of insulin. – Hypothyroidism – Addison’s disease and severe hepatic disease can produce hypoglycemia. • Other rare causes are: – Severe exercise (due to depletion of liver glycogen store) – Glycogen storage disorder (deficiency of glucose-6phosphatase) – Steatorrhea (due to impaired glucose absorption) – Starvation – Alcohol ingestion.

14

Estimation of Blood Urea

Urea is the metabolic end product derived from the catabolism of proteins. It is synthesized in the liver from ammonia and carbon dioxide and is excreted by kidney. Aim To estimate the serum urea. Method Diacetyl monoxime method (DAM method). Principle Urea reacts with Diacetly monoxime under strong acidic condition in presence of ferric ions and thiosemicarbazide to give a pink colored complex. Intensity of color is a measure of amount of urea present in blood. Color intensity is compared with standard and is measured using green filter (540 nm). Procedure Label three test tubes as B (Blank), T (Test) and S (Standard). Pipette 1 ml distilled water into B, 1 ml of diluted serum

Estimation of Blood Urea 145 (1:100) into T and 1ml of standard urea solution (1 mg in 100 ml of distilled water) into S. Add 2 ml of diacetly monoxime (DAM) solution and 2 ml of acid reagent into each tube. Mix well and keep in a boiling water bath for 20 minutes. Cool at room temperature and take OD at 540 nm. Protocol Reagents

Blank

Distilled water Standard Serum

Standard

Test

1 ml

-

-

-

1 ml

-

-

-

1 ml

Color reagent (DAM)

2 ml

2 ml

2 ml

Acid reagent

2 ml

2 ml

2 ml

Mix well and Boil for 20 min and cool Optical Density at 540 nm

Calculation Concentration of urea in mg/100 ml of blood, =

OD of Test  OD of Blank Conc. of Standard × × 100 OD of Standard  OD of Blank Volume of Sample

=

OD of T 0.01   100 OD of S 0.01

=

OD of T  100 OD of S

= __________ mg/dl.

Report The amount of urea present in the given blood sample is ………..mg/dl.

146 An Easy Guide for Practical Biochemistry Clinical Significance • Normal blood urea level ranges 15-45 mg/dl. • Causes of increased blood urea level: 1. Pre-renal causes: Since blood supply to the kidney is reduced, filtration and excretion of urea is minimum. a. High protein diet. b. Increases with age c. Dehydration as in diarrhea, vomiting. d. Increased cardiac output/ failure e. Increased catabolism of proteins as in fever and wasting diseases. 2. Renal causes: Synthesis of urea is normal. But damage in the renal tissue leads to poor filtration and excretion of urea. a. Nephritis b. Nephrotic syndrome c. Acute renal failure d. Chronic renal failure e. Polycystic kidney f. Hydronephrosis g. Malignant hypertension. 3. Postrenal causes: Synthesis and filtration are normal, but renal passage is blocked. Hence, minimum excretion of urea occurs. a. Obstruction in the renal tract b. Enlargement of prostate c. Stones in bladder. • Blood urea level decreases in liver diseases due to decreased synthesis. • Blood urea level is commonly monitored to evaluate kidney diseases.

Estimation of Blood Urea 147 • A high urea concentration in the urine reflects the concentrating power of the kidney. • Relationship between blood urea nitrogen (BUN) and urea is Blood urea nitrogen (mg/dl) = urea (mg/dl)/ 2.14 • Urea level is generally studied in conjunction with creatinine level to identify renal dysfunction. Urea/ creatinine ratio is sometimes used to discriminate between prerenal and postrenal uremia.

15

Estimation of Urine Creatinine

Creatinine is a waste product derived from endogenous sources by tissue creatine breakdown and is excreted by the kidney. Creatinine is the anhydride of creatine. The reaction occurs non-enzymatically. Creatine ——— → creatinine  H2O 98% of the total creatine is in the muscle as creatine phosphate. About 2% of the total creatine is converted daily to creatinine so that the amount of creatinine produced is related to the total muscle mass. As muscle mass remains approximately same, creatinine also remains same in plasma and urine. Aim To estimate the amount of creatinine in urine. Method Jaffe’s alkaline picrate method. Principle Creatinine present in urine reacts with picric acid in the presence of sodium hydroxide to give an orange color. The intensity of the color developed is directly proportional to

Estimation of Urine Creatinine 149 the amount of creatinine present. The color intensity is compared with standard and is measured at 520 nm (green filter). Procedure Dilute 5 ml of urine to the mark in a 50 ml volumetric flask (dilution is 1 in 10). Label three test tubes as test (T), standard (S) and blank (B). Into T, pipette 5 ml diluted urine. Into S, pipette 5 ml standard creatinine solution (0.5 mg). Take 5 ml water into B. To each tube, add 2 ml of saturated picric acid solution and 2 ml of 0.75 N sodium hydroxide. Mix. Read optical density values (OD) after 15 minutes using green filter (520 nm). Protocol Reagents

Blank

Standard

Test

Diluted urine

-

-

5 ml

Std. creatinine solution

-

5 ml

-

5 ml

-

-

Water Saturated picric acid

2 ml

2 ml

2 ml

0.75 N sodium hydroxide

2 ml

2 ml

2 ml

Mix. Read the OD values after 15 minutes using green filter. OD

Calculation Creatinine in mg per 100 ml urine =

OD of Test – OD of Blank Conc. of Standard × × 100 OD of Standard – OD of Blank Volume of Sample

=

OD of T – OD of B 0.5 100   OD of S – OD of B 0.5 1

150 An Easy Guide for Practical Biochemistry =

OD of T – OD of B × 100 OD of S – OD of B

= ––––––––– mg/dl.

Assuming the 24 hours urine output is about 150 ml/ day, the amount of creatinine excreted in g/day OD of T – OD of B 1500 = ———————–— × –—— × 100 OD of S – OD of B 1000 T - B 1500 = —— × ——— g/day S - B 1000 = ————— g/day

Report Amount of creatinine excreted is ———— g/day. Points to Remember • Creatinine is the end product of creatine metabolism. • Creatine is found as creatine phosphate in muscle, plays an important role in muscular contraction. • Normal excretion of creatinine in urine is in the range of 1-2 g per day. • Excretion is more in males because of more muscle mass. • Creatinine excretion is useful to check the reliability of 24 hours urine samples in assaying other biochemical parameters. • Urine creatinine is largely endogenous and is little influenced by diet. The excretion is therefore remarkably constant. • Excretion of other metabolites in random samples of urine may be expressed in terms of creatinine in the same sample.

Estimation of Urine Creatinine 151 • Creatinine coefficient is milligram of creatinine excreted in urine per kg body weight in 24 hours. Normally it is 2026 mg/kg/day in men and 14- 20 mg/kg/day in women. • Creatinine coefficient is more precise and is used to assess the functional muscle mass in the body. • Very little creatine is normally found in adult urine except in women during pregnancy and early postpartum. • The excretion of creatinine increases in fevers and wasting diseases. • The excretion of creatinine decreases in myopathies and renal failure. • A variety of compounds, like proteins, glucose, pyruvate, ascorbate and ketones interfere in Jaffe’s method for creatinine estimation. • In serum the interfering compounds contribute to about 20% of the color, whereas in urine, the interference is only to the extent of 5% or less. Creatinine Clearance Test Definition Creatinine clearance is defined as volume of plasma completely cleared of creatinine per minute by the kidney. Creatinine clearance measures GFR and is used as a renal function test. Procedure The test is performed in the morning. The patient is given 600 ml glasses of water to drink. The bladder is emptied completely and the urine is discarded. The time is noted.

152 An Easy Guide for Practical Biochemistry Urine is collected for the next 5 hours in a container. A sample of blood is drawn for creatinine estimation in serum. Measure the total urine volume. Estimate the creatinine in the urine. Calculation Creatinine Clearance (ml/min) = Where U = P = V = A = 1.73 =

U × V × 1.73 P×A

mg of creatinine/dl in urine mg of creatinine/dl in serum or plasma Volume of urine in ml/min Body surface area of the patient Standard average surface area of normal individual

Clinical Significance • Normal value of creatinine clearance in Males: 95 – 140 ml/min/1.73 sq. mt mean: 120 ml/min Females: 85 – 125 ml/min/1.73 sq. mt mean: 110 ml/min • The values are close to GFR as measured by inulin clearance. The ideal test is, however, inulin clearance test which precisely measures GFR. • Creatinine clearance is the suitable assay over urea clearance and inulin clearance. • It is an endogenous product almost with stable values and does not depend on protein intake. It is neither absorbed nor secreted. Diagnostic Importance • A decrease in creatinine clearance value (< 75% of normal) serves as sensitive indicator of a decreased GFR due to renal damage. This test is useful for an early detection of

Estimation of Urine Creatinine 153 impairment in kidney function, often before the clinical symptoms are seen. Precautions: 1. First source of error is improper hydration of the patient by giving very little drinking water before the start of test. 2. Improper collection of urine is the second source of error 3. The patient should rest during the test period since any muscular exercise may affect the results.

154 An Easy Guide for Practical Biochemistry

16

Estimation of Serum Inorganic Phosphate

Phosphorus in the body is mainly present in the bones; small portion is present in cells and soft tissue. It is a component of many important biological compounds, e.g. some proteins, lipids, nucleic acid and coenzymes. It plays an important role in acid base regulation particularly by the kidneys. The serum phosphate may exist as free ions (40%) or in a complex form (50%) with cations such as calcium, magnesium, sodium, potassium, etc. About 10% of serum phosphate is bound to proteins. Aim To estimate serum inorganic phosphate. Method Fiske and Subbarow method. Principle A quantitative method for the estimation of inorganic phosphate was first developed by Fiske and Subbarow. In this method serum proteins are precipitated by

Estimation of Serum Inorganic Phosphate 155 trichloroacetic acid. The protein-free filtrate is treated with molybdic acid reagent to form phosphomolybdate. It is reduced by 1-amino 2-naphthol 4-sulfonic acid (ANSA) to form molybdenum blue. Intensity of the color is a measure of inorganic phosphate. Reagents 1. 10% trichloroacetic acid 2. Molybdic acid reagent: 2.5% ammonium molybdate in 3N Sulfuric Acid 3. ANSA reagent: 1-amino 2-naphthol 4-sulfonic acid, sodium bisulfate and sodium sulfate 4. Standard phosphorus – 0.04 mg/5 ml. Procedure Preparation of Protein-free Filtrate Pipette 2.0 ml of serum into a test tube. Add 8.0 ml of 10% trichloroacetic acid with constant shaking (dilution is 1 in 5). Allow to stand for 10 minutes. Filter through a dry Whatman No.1 filter paper. Color Development Label three test tubes as test (T) standard (S) and blank (B). Into T, pipette 5 ml of protein free filtrate. Into S, pipette 5 ml of standard phosphate solution (0.04 mg equivalent of phosphorus) into B, pipette 5 ml of water. To each tube add 1 ml of molybdic acid reagent and mix. Add to each tube 0.4 ml of ANSA solution. Mix and allow to stand for 10 minutes. Add 3 ml of distilled water to each tube. Mix. Read the optical density (OD) values exactly after 10 minutes using a red filter (660 nm).

156 An Easy Guide for Practical Biochemistry Protocol Reagents

Blank Standard

Water

Test

5 ml

-

Standard phosphate solution

-

5 ml

-

Protein-free filtrate

-

-

5 ml

1 ml

1 ml

1 ml

0.4 ml

0.4 ml

0.4 ml

Molybdic acid reagent Mix ANSA Solution

Mix and allow to stand for 10 minutes Distilled water

3 ml

3 ml

3 ml

Mix and read OD values exactly after 10 minutes using a red filter (660) Optical Density at 660 nm.

Calculation Phosphate expressed as phosphorus (P) in mg per 100 ml serum =

OD of Test  OD of Blank Conc. of Standard × × 100 OD of Standard  OD of Blank Volume of Sample

=

OD of T  OD of B 0.04 × × 100 OD of S  OD of B 1

= __________ mg/100 ml of serum.

Report The amount of Inorganic phosphate present in the given sample of serum is –—— mg/dl. Clinical Significance • Phosphorus is present in nearly all foods; so dietary deficiency is not known in human being.

Estimation of Serum Inorganic Phosphate 157 • Normal value in adults is in the range 2.5-4.5 mg%. • In children, the value is 4-6 mg%. • The amount of phosphorus excreted in urine is about 1 gm. This is in the form of inorganic phosphate only. • Excretion depends upon the dietary phosphorus. • Serum phosphate level is higher in fasting state and lower in post-prandial period. This is due to the fact that after ingestion of carbohydrates the phosphate is drawn by the cells for metabolism (phosphorylation reactions). • Inorganic phosphate determination should be performed on serum or plasma separated from the cells soon after withdrawing the blood as ester phosphates in red cells are hydrolyzed with formation of inorganic phosphate causing its concentration in the serum to rise. • Since tap water contains substantial amounts of phosphorus, care should be taken to rinse all equipment with deionized water and dried before use. • Total phosphorus in blood includes: – Inorganic phosphorus: 2-5 mg/100 ml. – Organic or ester phosphorus: glycerophosphatesnucleotide phosphates, etc. 15- 30 mg/100 ml. – Phospholipids: lecithin, cephalin, sphingomyelin 1016 mg/100 ml. – Residual phosphorus: 87% of phosphorus in the body is present in bones and the rest is present in cells and tissues. • Hyperphosphatemia (increase in serum phosphate) is seen in hypoparathyroidism, hypervitaminosis D and renal failure. • Decreased level is seen in hyperparathyroidism, rickets, osteomalacia, vitamin D deficiency and due to decreased

158 An Easy Guide for Practical Biochemistry reabsorption of phosphate by kidney tubules as in Fanconi’s syndrome. • In diabetes mellitus organic phosphorus is lower but inorganic phosphorus is higher. • There is a reciprocal relationship between serum calcium and phosphorus. • Physiological fall of phosphorus occurs when there is increased carbohydrate utilization. Insulin therapy also has a similar effect.

17

Estimation of Serum Total Proteins

Serum proteins represent a complex mixture containing a number of components which differ in properties and functions. Major components of serum proteins are, 1. Albumin 2. Globulins 3. Conjugated proteins—serum mucoid and lipoproteins. Aim To estimate the serum total proteins. Method Biuret method (Autoanalyzer method). Principle The peptide bonds (-CO-NH) present in the protein react with copper sulfate in an alkaline medium to form a purple/violet colored complex. The intensity of this color is proportional to the number of peptide linkages present and thus is a measure of the concentration of proteins.

160 An Easy Guide for Practical Biochemistry Reagents 1. 28% sodium sulfite 2. Standard protein solution (6 g/dl) 3. Dilute Biuret reagent. Procedure Precipitation of Globulins Pipette 0.2 ml of serum into a test tube. Add 5.8 ml of 28% sodium sulfite solution. Mix. Allow to stand for 5 minutes. Filter through Whatman No.44 dry filter paper. Use the clear filtrate for estimation of albumin. Globulins in serum are selectively precipitated by 28% sodium sulfite. Albumin is estimated in the filtrate of processed serum. It reacts with copper sulfate in an alkaline medium to give a purple/violet color. The difference in protein content of whole serum and the serum filtrate (albumin) after sodium sulfite treatment is the value for globulins. Color Development Set up four test tubes marked B for blank, S for standard, A for albumin and TP for total protein. To B add 3 ml of water. To S add 3 ml of standard protein solution. To A add 3 ml of globulin-free filtrate and to TP add 0.1 ml of serum and 2.9 ml of water. To all the four tubes, add 3 ml of Biuret reagent. Mix. After 10 minutes read the optical densities using a green filter (540 nm).

Estimation of Serum Total Proteins 161 Protocol Reagent

Blank

Standard

Water

3 ml

-

-

2.9 ml

-

3 ml

-

-

Standard protein solution

Albumin Total Protein

Globulin-free filtrate

-

-

3 ml

-

Serum

-

-

-

0.1 ml

3 ml

3 ml

3 ml

3 ml

Biuret reagent

Mix. After 10 min read the O.D. using green filter (540 nm) Optical density at 540 nm

Calculation Total protein in 100 ml of serum =

OD of Test  OD of Blank Conc. of Standard × × 100 OD of Standard  OD of Blank Volume of Sample

=

TP  B 6 × × 100 mg% S  B 0.1

=

TP  B 6 100 g% × × S  B 1000 0.1

=

TP  B ×6 S B

= __________ g%

Albumin in 100 ml of serum =

OD of Test  OD of Blank Conc. of Standard × × 100 OD of Standard  OD of Blank Volume of Sample

=

AB 6 × × 100 mg% S  B 0.1

162 An Easy Guide for Practical Biochemistry =

AB 6 100 g% × × S  B 1000 0.1

=

TP  B ×6 SB

= __________ g%

Globulin in 100 ml of serum = Total protein – Albumin = __________ g% Determination of Serum Albumin by Bromocresol Green Method Principle Albumin present in serum binds specifically with bromocresol green at pH 4.1 to form a green colored complex. Intensity can be measured by colorimeter at 640 nm (red filter). Albumin standard = 4 g/ dl in normal saline containing 0.1 g/dl sodium azide. Procedure Pipette in three tubes as follows: Reagents

Blank

Standard

Bromocresol green reagent

Test

5 ml

5 ml

5 ml

Serum

-

-

0.05 ml

Albumin standard

-

0.05 ml

-

0.05 ml

-

-

Distilled water

Mix thoroughly and keep at room temperature for 10 minutes measuring the intensity of the test and standard by setting blank at 100% T using 640 nm.

Estimation of Serum Total Proteins 163 Calculations Serum albumin g/ dl =

OD of Test  OD of Blank ×4 OD of Standard  OD of Blank

Reference Value Total serum protein Normal Albumin Normal Globulin Normal A:G Ratio

= = = =

6-8 g/100 ml. 3.5-5.5 g/100 ml 2-3.5 g/100 ml 1.2 : 1 to 1.5 : 1

Report 1. The amount of Total Protein in the given sample of serum is _______ g/dl. 2. The amount of Albumin in 100 ml serum _______ g/dl. 3. Globulin _______ g//dl. 4. A:G Ratio _______. Clinical Significance • Biuret method is the most common method used in the student’s practical lab as well as clinical laboratories because it is simple and one step process. • The rate of color development varies with time and temperature. • The presence of lipid and carbohydrate in test solution reduces the amount of color given by the protein. • Hemolyzed blood should be discarded as it will increase the color intensity. • Minimum requirement for a positive reaction is the presence of 2 peptide/amide bonds . The reaction is so

164 An Easy Guide for Practical Biochemistry

• •

• •

•

•

• •

•

named because the compound biuret (H2N-CO-NH-CONH2) also answers the test. Increase in serum protein can occur in dehydration with A:G ratio remaining constant. In multiple myeloma, increase in total protein is mainly due to increased level of globulins. Albumin concentration remains same or is slightly reduced. Overall increase in total protein is rare. Changes occur in globulin fraction. Decrease in serum protein level is invariably due to decrease in albumin. A:G ratio also decreases due to either reduction of albumin and or elevation of globulin. The measurement of total proteins in plasma is of limited value as it may be altered by changes in plasma volume. An increase of plasma protein is caused by dehydration and decrease by overhydration. Total protein concentration is higher when a person is in standing position than recumbent position (due to shift of water from vascular compartment into interstitial compartment). Exercise increase total protein concentration (5-10%). Significant increase in total protein concentration arises with an increase in total globulin (usually gamma globulin). Decrease in albumin in serum can be due to the following conditions: 1. Loss of albumin due to: a. Nephrotic syndrome b. Protein loosing enteropathy c. Wide spread burns d. Severe hemorrhage

Estimation of Serum Total Proteins 165 2. Defective anabolism which may be due to: a. Liver disease due to reduced synthesis b. Malnutrition ex: Kwashiorkor c. Carcinoma of stomach or pancreas • Although the concentration of serum albumin is reduced in severe liver diseases, globulin is increased so that total protein concentration is high. • Serum albumin level can be as low as 1.5 g per 100 ml in kwashiorkor. Defective absorption from intestine in pancreatic diseases, malignancy of gastrointestinal tract, intestinal fistula, congenital malabsorption syndrome and severe tuberculosis can result in low serum albumin level. • Albumin is lost in severe burns and hemorrhage. Excessive breakdown of body proteins with inadequate supply or defective utilization of proteins is seen in uncontrolled diabetes, thyrotoxicosis, prolonged febrile diseases and trauma. A small decrease in serum protein level is seen in pregnancy. In toxemia of pregnancy, serum albumin level is lowered.

Section 4

Chromatography 167

Demonstration Practicals

18

Chromatography

Chromatography was introduced by Tswett in 1906 for separation of colored products. Chromatography is a laboratory technique used for the separation of closely related substances of macromolecules like proteins, amino acids, lipids and so on. Similar substances are separated by continuous distribution and redistribution between stationary phase and mobile phase. Separation depends on difference of size, and adsorption properties. Types of Chromatography • Paper chromatography. • Thin layer chromatography. • Gel chromatography. • Ion exchange chromatography. • Gas liquid chromatography. • High pressure liquid chromatography. Paper Chromatography Paper chromatography is a simple and widely used technique. It is based on the principle of partition between

170 An Easy Guide for Practical Biochemistry stationary phase and mobile phase. Paper serves as a stationary phase. Solvent system (organic solvent) which moves over the paper is known as mobile phase. Relative mobility of the compounds in chromatography is a function of the partition of the coefficients of the compound in two solvent phases. Solvent usually contains organic and inorganic substances and water, e.g. Butanol, Acetic acid and water in the ratio of 4:1:5. A mixture of amino acids can be separated either by ascending or descending chromatography. The solvent moves either by capillary action (ascending chromatography) or by gravitation force (descending chromatography). After stipulated period, paper is sprayed with suitable staining reagent and the solvent front (SF) is marked. (SF: the distance to which the solvent has moved on the filter paper along with the relative mobility of different compounds in cm). Rf value is the ratio of the distance moved by a compound to the distance moved by the solvent front. Example: If the solvent has moved 40 cm and the compounds have moved 30, 20, 10 cm respectively, Rf of the compound can be found using the formula, Rf =

Distance traveled by the compound Distance traveled by the solvent

i.e. 30/40 = 0.75 , 20/40 = 0.5 and 10/40 = 0.25 Rf value are 0.75, 0.5 and 0.25 respectively for a given solvent, Rf values is characteristic of a compound. It is possible to identify unknown substances by their Rf values.

Chromatography 171 Requirement • • • • • •

Whatman No 1 filter paper Solvent system Chromatography chamber Capillary tube Standard amino acids Amino acid mixture to be identified.

Procedure Solvent is taken in a shallow trough and kept in the bell jar for saturation of the chamber. Whatman No. 1 filter paper is used for paper chromatography. Cut the paper into dimensions of 15 × 56 cm. With a pencil, draw a line along the width of the paper, 5 cm from its edge. Equal points of 2 cm apart are marked from one end of the paper leaving 2 cm from both the edges.

Fig. 18.1: Paper chromatography

172 An Easy Guide for Practical Biochemistry 0.5% of standard β unknown solution (mixture of amino acids) is prepared. 10 μl (Microliter) each of the standard and unknown solution are applied on the pencil marks by a capillary tube with intermitted drying, using a drier. Standard solutions are usually applied on the last point of right side of the paper. For ascending chromatography the paper is folded width-wise sharply along a line. It is kept in place by tying it loosely with thread, taking care not to overlap the paper edges. The paper thus folded is in circular form and is placed in the trough with solvent system. The system is made airtight by closing with the bell jar. For descending chromatography system, spotting the sample and standards is done as explained above. The paper

Fig. 18.2

Chromatography 173 is passed over the thick glass rod to anchor. The paper is dipped in solvent kept in the trough of the chromatography chamber. Paper is hanged down from the solvent trough by proper anchoring. Chamber is closed and made airtight to prevent solvent evaporation. Chromatography is made to run for 18-20 hours. After the stipulated time, the paper is removed; the solvent front is marked and dried. It is sprayed with 2% Ninhydrin. After spraying, the paper is dried in a hot air oven at 100°C for 3 minutes. Distinct purple colored amino acid spots appear (Fig. 18.2). Rf values are found out for each distinct spot by using the formula as explained. The amino acids can be identified by their corresponding Rf value and also by correlating with the Rf values of known amino acids, spotted as standards. Application • Paper chromatography is used for detection of amino acids, sugars, pigments, etc. • Used in the identification of amino aciduria, e.g. cystinuria, phenylketonuria, etc.

19

Electrophoresis

Electrophoresis is a popular technique used to separate closely related compounds. It is based on the movement of charged particles in the electric field. This technique is based on the principle that positively charged particles migrate towards cathode and negatively charged particles migrate towards the anode. Rate of migration depends on the charge of the molecule (Fig. 19.1).

Fig. 19.1: Electrophoresis

Electrophoresis 175 Applications Electrophoresis has many applications • Separation of serum proteins for diagnostic purposes. • Determination of purity and molecular weight of proteins. • Separation of isoenzymes in differential diagnosis. • Estimation of lipoprotein in health and diseases. • Finding normal and abnormal hemoglobin. • Diagnosis of Nephrotic syndrome. • Diagnosis of Hypoalbuminemia. • Diagnosis of cirrhosis of liver. • Diagnosis of multiple myeloma. • Diagnosis of chronic infections. Factors Affecting Migration of Charged Particles • • • • • • •

Structure, molecular weight and shape of the molecule. Charge on the molecule. Temperature Dilution of the sample Voltage Strength of the buffers pH of buffer

Types of Electrophoresis Depending on the nature of the supporting medium electrophoresis is classified into different types. 1. Paper electrophoresis 2. Agarose gel electrophoresis (supporting medium is agarose gel)

176 An Easy Guide for Practical Biochemistry 3. Immuno-electrophoresis 4. Cellulose acetate electrophoresis 5. Polyacrylamide gel electrophoresis (PAGE). Basic Requirements of Electrophoresis • Power pack • Buffer tank fitted with electrodes (cathode and anode) with a support to position the supporting medium with insulating transparent cover. • Buffer, the pH ionic strength and nature of the buffer may be varied according to the nature of substances to be separated. • Fixative • Staining solution • De-staining solution • Photoelectric colorimeter or densitometer. Paper Electrophoresis Whatman filter paper No. 1 is used commonly. Whatman filter paper No. 3 which is thicker and absorbs more sample is used for lipoprotein and hemoglobin electrophoresis. Separation of Plasma Proteins 1. Equal quantities of Barbitone buffer of pH 8.6 is filled in the buffer tank. 2. About 15-40 μl of the sample (serum) is carefully streaked in the middle of Whatman 3 filter paper strip (supporting medium) (dimension 30 cm × 7 cm).

Electrophoresis 177 3. The supporting medium is then connected by dipping its free ends in the buffer on either side and entire apparatus is made airtight by closing the cover. 4. Electric current is passed by means of power pack by adjusting proper voltage and current. The current is allowed to flow through the apparatus for 5 hours at a voltage of about 200 volts. 5. After the electrophoresis run, the supporting medium is dried for 10 min in the over at 100oC. 6. It is then immersed in a dye solution. Staining of the supporting medium varies depending upon the substance. Dyes usually used are bromophenol blue, naphthalene black, etc. 7. De-staining of the supporting medium is performed by dipping the paper in dilute acetic acid till the background of the supporting medium becomes clear. 8. They are kept in fixative solution. 9. The stained supporting medium is scanned by using a densitometer or alternatively each fraction of stained strip is cut and eluted by using 10% sodium hydroxide and the color intensity is measured in a colorimeter. Movement of Different Protein Fractions When serum proteins are subjected to paper electrophoresis, albumin moves rapidly and is found at the greatest distance from the streaking point followed by α1, α2 globulins, β-globulin and α-globulin. The separated proteins assume blue color after staining.

178 An Easy Guide for Practical Biochemistry

Fig. 19.2: Serum protein electrophoresis

Electrophoresis 179

Fig. 19.3: Electrophoretic patterns in different conditions

Densitometer Scanning of Cellulose Acetate Strip Conversion of bands to characteristic peaks of albumin, α1-, α2- globulins, β-globulin and γ-globulin.

180 An Easy Guide for Practical Biochemistry Normal electrophoretic separation of serum protein will have: • Albumin - 56% • α1-globulin - 3% • α2-globulin - 13.5% • β-globulin - 15.5% • γ- globulin - 12% Total - 100%.

20

Glucose Tolerance Test

The glucose tolerance signifies the ability of the body to tolerate excess load of glucose and to dispose of an additional load of glucose given. Glucose tolerance test is used to measure changes in blood glucose after glucose load. Oral GTT is most commonly used in the laboratories because it is easy to give glucose load orally. Significance • It is mainly used in the detection of diabetes mellitus. • This test is useful in distinguishing a person with a normal glucose tolerance from a person who has increased or decreased tolerance. • It is of great value in detecting renal glycosuria and endocrine malfunction. Normal Response The fasting blood glucose level will be in the range of 60-90 mg% (Enzymatic method). Blood glucose level rises to peak value of 110-140 mg%, 30-60 min after glucose administration. The peak value does not exceed the renal threshold level of 180 mg% and hence, there will be no glucose in the urine samples. The initial rise is observed

182 An Easy Guide for Practical Biochemistry

Fig. 20.1: Normal response

because the quantity of glucose absorbed from the intestine exceeds the capacity of liver and other tissues to use it. Increased glucose level in blood stimulates insulin secretion that facilitates the utilization of glucose by the peripheral tissues. As a result, the blood glucose level starts declining and may drop to a value slightly lower than the fasting level at the end of second hour (Fig. 20.1). Lag Type There is temporary rise in blood glucose. Blood glucose returns to normal limits in the usual time, but the peak of the curve is above the normal renal threshold. So glycosuria is seen. This type of curve has been termed a “lag” curve (Fig. 20.2).

Glucose Tolerance Test 183

Fig. 20.2: Lag type

Decreased Tolerance This is found in diabetes mellitus. Fasting blood glucose levels are generally above 90 mg%. There is increase in blood sugar level after glucose intake and increase is generally greater than in normal persons. In mild diabetes at least one of the urine specimens will give positive test to glucose. In severe cases all urine samples show +ve reaction to urine sugar (Fig. 20.3). Increased Tolerance The graph appears flatter than the curve for normal response. Such a profile is seen in case of starvation and malnutrition (Fig. 20.4).

184 An Easy Guide for Practical Biochemistry

Fig. 20.3: Decreased tolerance

Fig. 20.4: Increased tolerance

Glucose Tolerance Test 185 Renal Glycosuria • Renal threshold in some persons may be lowered so glycosuria occurs but blood sugar levels are normal. • Normal person should be able to remove glucose load from his blood within a specified time. This is known as normal tolerance. • If the person will have elevated blood glucose concentration for longer than the normal time the condition is called as reduced tolerance. • If the glucose concentration becomes very low or normal earlier than the normal time then the condition is called as increased tolerance. Importance of GTT This is performed to establish a diagnosis in: 1. Patients with transient or sustained glycosuria who have no clinical symptoms of diabetes and with normal fasting and post prandial blood glucose level. 2. Patients with symptoms of diabetes mellitus but with no glycosuria and normal fasting level. 3. Person with a strong family history of diabetes mellitus but with no symptoms of diabetes mellitus. 4. Patients whose glycosuria is associated with pregnancy, thyrotoxicosis, liver disease and infections. 5. Women who have characteristically large babies. Details of Performing the Test Instructions given to the patient are as follows: • The patient should be on balanced diet (containing normal daily requirement of carbohydrates) at least for 2 to 3 days prior to the test.

186 An Easy Guide for Practical Biochemistry • Patient should report to the laboratory at 9 am after over night fasting for 10 to 12 hours. • The patient should be in the laboratory for at least 2-3 hours since 5 blood samples are collected at intervals of 30 minutes. Procedure • After an overnight fasting of 12 hrs the subject is ready for the test. • At 9 am in the morning, fasting venous blood and urine samples are collected. • Glucose is administered orally to the subject. Usually 1 gm glucose/kg weight or a standard dose of 50 gm glucose dissolved in about 200 ml of water is given and the time is noted. • At intervals of 30, 60, 90, 120 and 150 min blood samples are withdrawn and corresponding urine specimens are collected. • Blood glucose levels are determined quantitatively. • Urine glucose is detected by Benedict’s test. • GTT graph is plotted on a graph sheet with concentration of glucose on Y-axis and duration of time on X-axis. Methods Used for Estimation of Blood Sugar • • • •

Folin-Wu’s method O- Toulidine method Hexokinase method Glucose oxidase-Peroxidase method.

Methods Used for Estimation of Urine Sugar • Benedict’s qualitative method • Glucose oxidase method (strip method).

Glucose Tolerance Test 187 Estimation of Urine Sugar by Benedict’s Qualitative Method Principle: Reducing sugar reduces copper sulfate to red cuprous oxide. Specimen: Urine Procedure: To 5 ml of Benedict’s reagent add 8 drops of urine mix boil for 2 min and cool. Color or precipitate

Amount of glucose

1

Blue color

Nil

2

Green color

Traces

3

Green PPT

0.5%

4

Yellow PPT

1%

5

Orange PPT

1.5%

6

Red PPT

2.0%

7

Brick Red PPT

>2.0%

Plotting a Graph for GTT Sl no. Type

Fasting

30 min

60 min

90 min

120 150 min min

1

Normal

85

105

130

110

100 7 5

2

Lag type

90

180

155

100

8 5 100

3

Mild diabetic

95

150

225

175

125 9 0

4

Severe diabetic

140

200

360

240

210 190

5

Hypoglycemic

65

100

120

85

75

60

188 An Easy Guide for Practical Biochemistry

21

Estimation of Serum AST and ALT

Human serum contains several transaminases of which ALT (SGPT) and AST (SGOT) are of diagnostic significance. Aim To estimate serum ALT and AST. Method Reitman and Frankel method. Principle For estimation of AST serum is incubated with aspartate and alpha ketoglutarate in phosphate buffer (pH 7.4) for 60 minutes. In the estimation of ALT, serum is incubated with alanine and alpha ketoglutarate for 30 minutes. After a measured time the reaction is stopped. Oxaloacetate is formed from AST and is spontaneously decarboxylated to pyruvate. Pyruvate is formed from ALT. Pyruvate reacts with DNPH (dinitrophenylhydrazine) to form the corresponding hydrazone which form a brown colored complex in alkaline medium. Color intensity is measured against blank and control.

Estimation of Serum AST and ALT 189 Reagents 1. SGPT substrate 2. SGOT substrate 3. 0.1 M phosphate buffer 4. DNPH reagent 5. 0.4 N NaOH 6. Standard pyruvate : 0.2 ml = 0.4 μg Procedure Estimation of ALT (SGPT) Take 4 test tubes and label them as T for test, C for control, S for standard and B for blank. Take 0.2 ml of serum into T, 0.2 ml of std into S and 0.2 ml of distilled water into B. To all the test tubes add 1 ml of buffered substrate of ALT. Incubate all the tubes in water bath at 37°C for 30 minutes. Exactly after 30 minutes add 0.2 ml of serum into control and 1 ml of DNPH solution into all the tubes. Mix and allow it to stand for 20 minutes at room temperature. After 20 minutes add 10 ml of NaOH to all the tubes and mix well. Allow it to stand for 20 minutes and read the OD at 540 nm. Protocol Reagents

Test

Serum (ml)

0.2

Control Standard Blank -

-

-

Standard (ml)

-

-

0.2

-

Distilled water (ml)

-

-

-

0.2

Buffered substrate

1

1

1

1

Incubate at 37°C for 30 minutes Serum (ml)

-

0.2

-

-

DNPH (ml)

1

1

1

1

Mix thoroughly. Keep at room temperature for 20 minutes. 0.4 N NaOH

10

10

10

10

Mix and keep at room temperature for 20 minutes. Read the intensity at 540 nm (green filter)

190 An Easy Guide for Practical Biochemistry Calculation Enzyme activity =

OD of T  OD of C Conc. of Std 1000  × OD of S  OD of B Volume of Std Incubation time

OD of T  OD of C 0.4 1000 × × OD of S  OD of B 0.2 Incubation time = __________ IU/L.

=

Estimation of SGOT (AST) Procedure Take 4 test tubes and label them as T for test, C for control, S for standard and B for blank. Take 0.2 ml of serum in T, 0.2 ml of standard in S and 0.2 ml of distilled water in B. To all the test tubes add 1 ml of buffered substrate. Incubate all the tubes in boiling water bath at 37°C for 60 minutes. Exactly after 1 hour add 0.2 ml of serum to control and 1 ml of DNPH solution to all the tubes. Mix and allow it to stand for 20 minutes at room temperature. After 20 minutes add 10 ml of NaOH to all the tubes and mix well. Allow it to stand for 20 minutes and read the OD at 505 nm. Protocol Pipette in the tubes labeled as follows: Reagents

Test

Serum (ml)

0.2

-

-

-

-

-

0.2

-

Standard

Control Standard Blank

Distilled water (ml)

-

-

-

0.2

Buffered substrate (ml)

1

1

1

1 Contd...

Estimation of Serum AST and ALT 191 Contd... Reagents

Test

Control Standard Blank

Incubate at 37° C for 60 min Serum (ml)

-

0.2

-

-

DNPH (ml)

1

1

1

1

Mix thoroughly, keep at room temperature for 20 min 0.4 N NaOH (ml)

10

10

10

10

Mix and keep at room temperature for 20 min. Read intensity at 540 nm (green filter)

Calculation Enzyme activity =

OD of T  OD of C Conc. of Std 1000 × × OD of S  OD of B Volume of Std Incubation time

OD of T  OD of C 0.4 1000 × × OD of S  OD of B 0.2 Incubation time = __________ IU/L.

=

Points to Remember: • AST is increased in cardiac diseases. • ALT is increased in liver diseases. • Normal values of SGPT (ALT) is 13-35 IU/ L • Normal values of SGOT (AST) is 8-20 IU/ L.

192 An Easy Guide for Practical Biochemistry

22

Estimation of Serum Cholesterol

Aim Estimation of serum cholesterol. Method Cholesterol Oxidase Peroxidase methodology. Principle Enzymatic colorimetric determination of total cholesterol is done according to the following reactions. Cholesterolesterase  Cholesterol + fatty acids Cholesterol ester + H 2 O 

Cholesterolesterase Cholesterol ester + O 2   4-Cholesten-3-one + H 2 O 2 Peroxidase 2H 2 O 2 + Phenol + 4 – Aminoantipyrine   Red quinine + 4H 2 O

Note: Cholesterol standard concentration : 200 mg/dl Sample Serum plasma.

Estimation of Serum Cholesterol 193 Procedure Reagents

Blank

Standard

Sample

1000 μl

1000 μl

1000 μl

Standard

-

10 μl

-

Sample

-

-

10 μl

Working reagent

Mix and incubate for 5 min. at 37°C. Measure the absorbance of sample and standard against reagent blank.

Calculation Cholesterol Conc. (mg/dL) =

Absorbance of sample × 200 Absorbance of standard

= __________ mg/dl. Precaution • To avoid contamination use clean laboratory equipments. • Avoid direct exposure of working reagent to light. Normal Range It is recommended that each laboratory establish its own reference values. The following values may be used as guide line. Normal serum/plasma cholesterol level is 150-250 mg/dl. Clinical Significance • Cholesterol is the main lipid found in the blood, bile and brain tissues. • It is also one of the most important steroids of the body and is a precursor of many steroid hormones.

194 An Easy Guide for Practical Biochemistry • Two thirds of cholesterol present in the blood is esterified. The liver metabolizes cholesterol and it is transported in the blood stream by lipoproteins. • Increased levels are found in hypercholesterolemia, hyperlipidemia, hypothyroidism, uncontrolled diabetes, nephrotic syndrome and cirrhosis. • Decreased levels are found in malabsorption, malnutrition, hyperthyroidism, anemia and liver diseases.

Estimation of Plasma Ascorbic Acid 195

23

Estimation of Plasma Ascorbic Acid

Aim To determine plasma ascorbic acid. Method 2,6-dichlorophenolindophenol titration. Principle The protein free filtrate is titrated with the dye 2,6 dichlorophenolindophenol in acid solution. The blue compound is red in acid solution, and on titration with a solution of ascorbic acid, is reduced to a colorless leucobase, the ascorbic acid being oxidized to dehydroascorbic acid. Reagents Trichloroacetic acid, 10% solution of freshly prepared 5% metaphosphoric acid. Solution of 2,6-dichlorophenolindophenol. Procedure Mix equal volumes (4 ml is convenient) of plasma separated immediately after withdrawing blood, and trichloroacetic acid or metaphosphoric acid. Filter or centrifuge. Pipette

196 An Easy Guide for Practical Biochemistry 0.2 ml of the diluted dye solution into a test tube and titrate with the filtrate until the red color has disappeared. Calculation 0.2 ml dye is equivalent to 0.008 mg ascorbic acid. Hence, mg of ascorbic acid/100 ml plasma =

100 × 2 × 0.008 ml titration

=

1.6 ml titration

Note: If the plasma cannot be separated immediately, the blood is collected into a test tube containing 1 drop each of 5% potassium cyanide and 20% potassium oxalate for 4 to 5 ml of blood. Points to Remember: • Persons on an adequate intake of vitamin C will generally be found to have a plasma level between 0.8 to 2 mg/100 ml. • Values below 0.2 mg% suggest the possibility of marked ascorbic acid deficiency. • Plasma values are of limited value in diagnosing scurvy and subclinical conditions of vitamin C undernutrition. • Vitamin C in WBC is the reliable index for the determination of ascorbic acid status.

Flame Photometer 197

24

Flame Photometer

Flame photometer is an analytical instrument used for the quantitative analysis of alkali metals like Sodium, Potassium, Calcium, Lithium and others in biological fluids. Principle Diluted standard solution of alkali metals like sodium or Potassium is sprayed as a fine mist of droplets on to the non-luminous flame of Bunsen burner. The solution evaporates and gets converted to atomic state. Flame acquires color by the characteristic emission of the metal present (Yellow color for sodium and violet color for potassium). Thermal energy of the flame excites the electrons into higher energy orbits. Excited electrons are prone to return to ground state. In doing so, they emit light of specific wavelength which is characteristic of each element. The emitted rays pass through a suitable filter, then to the light detectors. Light detective element is a photosensitive element. The photocell converts light energy into electrical energy which is measured by the Galvanometer.

198 An Easy Guide for Practical Biochemistry The amount of light emitted is proportional to the number of excited electrons, which is in turn proportional to the concentration of that alkali metal in the solution. Elements

Wavelength

Color of the Flame

Sodium

589 nm

Yellow

Potassium

407 nm

Violet

• Simple filters are used as Monochromatic device to provide specific wavelength. • Standard and test solution are sprayed into the flame as fine mist. Parts of Flame Photometer 1. Needles: To suck specific volume of the sample 2. Nebulizer: The most important part which breaks the solution into a spray of uniform sized droplets and it sprays the specimen into the burner. 3. Spray chamber: Is a chamber which has a rubber tube for drainage. There are two openings one is for gas supply and another for compressed air. The compressor produces air at constant high pressure of 1 Kilogram/cm.

Fig. 24.1

Flame Photometer 199 4. Monochromator: It is a device which yields a particular wavelength and screens out all other wavelength except the specific one emitted by the element to be analyzed. 5. Photo detectors: The emitted light is converted into electrical impulse which is measured by a galvanometer. Procedure The solution is appropriately diluted by using deionized water. After switching on the instrument, the air compressor is turned on. The gas is opened and ignited. The apparatus is set to zero by using deionized water. Under controlled conditions the diluted standard solution is inserted and the reading is adjusted to 150 for sodium and 5 for potassium. Test solution is inserted as a very fine spray to the burner which becomes colored by the characteristic emission of the metal present in the solution. Readings are noted. Using solution of different concentration of Sodium or Potassium, a calibration curve can be obtained. Calibration curve is used to find the concentrations of unknown alkali metals of biological samples. Note: Capillary tubes and Nebulizer should be properly cleaned. Care should be taken to use deionized water.

200 An Easy Guide for Practical Biochemistry

25

CSF Analysis

Examination of Cerebrospinal Fluid (CSF) is important in the diagnosis of neurological diseases. Collection of CSF • CSF is usually collected from spinal canal by lumbar puncture (LP). • The patient is made to lie on his side with the neck, thighs and knees flexed. • Under local anesthesia with full aseptic precautions, an LP needle is inserted into the subarachnoid space between the 3rd and 4th intervertebral space. • The CSF is allowed to flow out spontaneously drop by drop and about 5 ml is collected in clean sterile vials. • The sample is used for biochemical, cytological and microbiological examination. • CSF must be examined immediately within 1 hour and should not be refrigerated. Biochemical Examination of CSF • Biochemical examination of CSF usually consists of measurement of total proteins, glucose and chloride. • Estimation of enzymes like creatinine phosphokinase (CKBB), AST or lactate dehydrogenase (LDH3) is useful in case of cerebral infection.

CSF Analysis 201 Determination of Total Protein Since the content of protein is usually very low, it is determined by measuring the turbidity by adding sulfosalicylic acid. Principle Proteins are precipitated by sulfosalicylic acid. The turbidity of the resultant uniform suspension is measured by means of a colorimeter at 450 nm (blue filter) against a standard solution which is treated similarly. Reagents 1. 3% sulfosalicylic acid 2. Isotonic sodium chloride solution (8.8 g/L) 3. Protein standard 50 mg% (50 mg bovine albumin in 100 ml of isotonic NaCl). Procedure Mark three test tubes B, S and T for blank, standard and test respectively. Reagent (ml)

Blank

Standard

Standard protein

-

1

Test -

CSF

-

-

1

Isotonic NaCl

1

-

-

3% sulfosalicylic acid

5

5

5

Mix the contents of each tube and let it stand for 5 minutes. Read the optical densities at 450 nm (blue filter).

202 An Easy Guide for Practical Biochemistry Calculation Protein in 1 ml of CSF =

OD of test × concentration of standard OD of standar

=

OD of test × 0.5 OD of standard

Protein in 100 ml =

OD of test  0.5 × 100 OD of standard

=

OD of test × 50 OD of standard

= _________ mg/dl Determination of Glucose Glucose is analyzed by same method as used for blood glucose. Points to Remember: • The Brain and spinal cord are covered by three membranes. From inside to outside these are pia mater, arachnoid mater and dura mater. • The subarachnoid space (space between pia mater and arachnoid mater) is filled with cerebrospinal fluid (CSF). • The total volume of CSF in adults is about 150 ml (100200 ml). It is formed at the rate of about 0.5 ml/minute. • Earlier it was thought that CSF was formed by ultrafiltration of plasma. But now it is known that secretory activity of cell of the choroids plexus is the major factor in the production of CSF and ultrafiltration plays only a secondary role in the formation of CSF.

CSF Analysis 203 • Indications for CSF analysis are: 1. Bacterial or viral meningitis 2. Trauma-head injury 3. Degenerative disorders like multiple sclerosis 4. Tumors like benign meningioma or malignant glioma 5. Vascular disorders like rupture of vessels or obstruction of vessels by thrombosis (cerebral infarction) Artificial CSF for practical purposes is prepared by dissolving 300 mg bovine albumin, 600 mg glucose and 7.19 g sodium chloride in 1 litre of deionized water (freshly prepared). For proteins of high values, dilute the CSF suitably and take the dilute CSF and multiply the result by dilution factor. Normal CSF protein is essentially due to albumin (5575%). An increase in protein in certain diseases is contributed by globulin along with albumin. Normal range of protein in CSF is 15 to 45 mg/dl. Glucose level in spinal fluid is about 20 mg/dl less than that of blood. Normal range for adults is 50-80 mg/dl. Hence, blood must be analyzed simultaneously for comparison. CSF sugar is utilized by bacteria and blood cells. Low glucose level in CSF is seen in: 1. Bacterial meningitis since there is increased no. of leukocytes and pathogenic organism which may contribute to increased glycolysis. 2. Tubercular meningitis 3. Metastatic tumors of meninges • Increase of CSF glucose is seen in diabetes mellitus and brain tumors.

204 An Easy Guide for Practical Biochemistry

26

Estimation of Albumin in Urine

Aim To estimate the amount of albumin in urine. Apparatus Esbach’s albuminometer—the apparatus has the mark ‘U’ near the middle and the mark ‘R” near the top. The portion below ‘U’ is graduated from 0 to 12 that gives the quantity of proteins in gm/liter. Principle Albumin and other proteins can be measured by precipitating with picric acid solution in Esbach’s albuminometer. Reagent Esbach’s reagent: One gm of picric acid and 2 gm of citric acid dissolved in 100 ml of water. Procedure Check the specific gravity of urine. Fill the tube with urine up to ‘U’ (If urine has a high specific gravity it should be diluted so that it is around 1.008). Add Esbach’s reagent up to mark ‘R’. Stopper the tube (Plug). Mix by inversion

Estimation of Albumin in Urine 205 several times. Allow to stand for 24 hours. Read the calibration corresponding to the meniscus of the precipitate. Express the value as gm/liter. Points to Remember: • Normal urine contains only traces of proteins. • Benign and transient proteinuria found in young people can be due to severe exercise. • Orthostatic proteinuria is apparently due to the erect posture for prolonged periods. • Albuminuria is characteristic of kidney diseases like acute and chronic nephritis, nephrotic syndrome, renal infections, poisoning by heavy metals and polycystic kidney. • In nephrotic syndrome, 10-15 gm protein may be lost daily. Test for Bence Jones Proteins in Urine • A routine urinalysis will not detect Bence Jones proteins. • There are several methods used by laboratories to detect and measure these proteins. • The classic Bence Jones reaction involves heating urine to 60º and 100°C. At 60°C temperature, the Bence Jones proteins will clump. The clumping disappears if the urine is further heated to boiling (100°C) and reappears when it is cooled. • Other clumping procedures using salts, acids, and other chemicals are also used to detect these proteins. These types of test will reveal whether or not Bence Jones proteins are present, but not how much is present.

Appendix 1: Case Reports 207

Appendices

Appendix 1 Case Reports 1. A 12-year-old child has generalized edema of the body with puffiness of the face. His laboratory data is as follows: Serum total proteins 4.5 g/dl Albumin 1.5 g/dl Globulins 3 g/dl Serum cholesterol 500 mg/dl Blood urea 50 mg/dl Serum creatinine 1.8 mg/dl Urinary proteins 15 g/day I. Comment on the report. Nephrotic syndrome II. Normal serum protein level. 6-8 gm/dl III. Normal blood urea level. 15-40 mg/dl. IV. Name the pathological urinary proteins. Albumin, Bence Jones proteins. V. Test to detect the urinary proteins. Heat and acetic acid test, sulfosalicylic acid test, Heller’s test. 2. An apparently healthy man on a routine check-up was found to have the following lab findings. Blood sugar Urine sugar Fasting: 80 mg/dl + PPBS: 140 mg/dl ++ No symptoms of polyuria, polydypsia and polyphagia. I. Probable diagnosis. Renal glycosuria. II. Further Investigation. Glucose tolerance test III. Defect in this condition. Defect in renal tubules. Cannot reabsorb sugar. IV. Normal threshold for glucose. 180 mg/dl.

210 An Easy Guide for Practical Biochemistry V. Non-sugar reducing substances. Vitamin C, glutathione, salicylates, uric acid, glucuronides and homogentisic acid 3. A fair chubby mentally retarded boy was brought to the hospital. Blood chemistry revealed an abnormally high phenylalanine. Phenyl acetate, phenyl pyruvate and phenyl lactate were present in the urine in appreciable amounts. I. Comment on this. Phenylketonuria. II. Test to detect them. Guthrie test, urine ferric chloride test III. Amino acid involved in this condition. Phenylalanine, tyrosine IV. Any other metabolic disorder associated with this amino acid. Alkaptonuria, albinism V. Name essential amino acids. Phenylalanine, tryptophan, methionine, valine, leucine, isoleucine, threonine and lysine 4. A patient was admitted with acute abdominal pain. Investigations revealed increased levels of serum amylase, serum lipase and urine amylase. I. Probable diagnosis. Acute pancreatitis. II. Enzymes secreted by pancreas. Pancreatic amylase, pancreatic lipase III. Hormones secreted by pancreas. α-cells secrete glucagon, β-cells secrete insulin IV. Normal serum amylase level. 80-180 Somoygi units V. Name other lipolytic enzymes. Lingual lipase, gastric lipase, pancreatic lipase, phospholipase and cholesterol esterase 5. The following are some of the biochemical findings in a patient. Serum bilirubin 0.8 mg% Conjugated 0.2 mg%

Appendix 1: Case Reports 211 Unconjugated 0.6 mg% Serum alkaline phosphatase 8 KA units AST 20 units/L ALT 16 units/L Urine bile pigments negative Urine bile salts negative Urobilinogen traces Feces normal color I. Comment on this. Normal report II. Normal serum bilirubin level. 0.2 to 0.8 mg / dl III. Conjugated and unconjugated bilirubin. Conjugated bilirubin: Bilirubin is conjugated with UDP glucuronic acid to form bilirubin mono and di–glucuronide. It is water soluble, reacts directly with diazo reagent Unconjugated bilirubin: Bilirubin is not conjugated with UDP glucuronate. It is water insoluble and soluble in methanol IV. Normal AST levels. 4- 17 IU/L V. Test for bile pigments. Fouchet’s test, Gmelin’s test 6. The following are some of the patient. Serum bilirubin Conjugated Unconjugated Serum alkaline phosphatase AST ALT Urine Bile pigments Bile salts Urobilinogen Feces Blood coagulation time

biochemical findings in a -

10 mg% 8.5 mg% 1.5 mg% 140 KA units 80 units/L 90 units/L

-

++ ++ negative clay color prolonged

212 An Easy Guide for Practical Biochemistry I. II.

III. IV. V.

Probable diagnosis. Obstructive jaundice Causes: Intrahepatic: chronic active hepatitis, biliary cirrhosis, lymphoma, primary hepatoma. Extrahepatic: gall bladder stones, carcinoma of head of pancreas, enlarged lymph nodes Name the bile pigments. Bilirubin and biliverdin Name the bile salts. Sodium and potassium salts of glycocholic acid and taurocholic acid Test to detect bile salt. Hay’s sulfur powder test

7. A nine-year-old boy was brought to the hospital with the complaint of puffiness of face. On examination, the blood pressure was normal. Biochemical investigations were as follows: Blood urea 30 mg% Serum creatinine 1 mg% Serum cholesterol 580 mg% Total plasma protein 4.3 g% Albumin 1 g% Globulin 3.3 g% Urine protein 9 g/L I. Comment on this. Nephrotic syndrome II. Normal serum cholesterol level. 150 to 200 mg/ dl III. Name the pathological urinary protein. Albumin, Bence Jones proteins IV. Test to detect urinary proteins. Heat and acetic acid test, sulfosalicylic acid test, Heller’s test V. A:G Ratio and its importance. 1.2 : 1 to 1.5 : 1 Reversed in multiple myeloma Decreased in liver disease, kidney disease, cirrhosis of liver, nephrotic syndrome, malnutrition

Appendix 1: Case Reports 213 8. Following a prolonged hunger strike, a person was brought to the hospital in an unconscious state. The following were the laboratory findings: Blood sugar - 50 mg% Blood pH - 7.25 Serum bicarbonate - 15 mEq/L Rothera’s test (urine) - positive Benedict’s test (urine) - negative I. Probable diagnosis. Starvation ketoacidosis II. Methods to estimate blood sugar level. Folin-Wu method, ortho- toluidine method, Nelson somogyi method, glucose oxidase method III. Composition of Benedict’s reagent. Copper sulfate, sodium carbonate, sodium citrate IV. True blood glucose level. 60 to 90 mg/ dl V. Ketone bodies. Acetone, acetoacetic acid and β hydroxy butyric acid 9. A nine-year-old boy presented with complaints of puffiness of face and oliguria of insidious onset. Laboratory findings were as follows: Blood Urea 96 mg% Serum Creatinine 4.6 mg% Serum Cholesterol 450 mg% Total Protein 3.8 gm% Albumin 1 gm% Globulin 2.8 gm% Urine Protein 6 g/L I. Interpret the condition. Nephrotic syndrome II. Causes for edema. Hypoalbuminemia III. Normal serum cholesterol level. 150 to 200 mg/dl IV. Normal A: G ratio. 1.2 : 1 to 1.5 : 1

214 An Easy Guide for Practical Biochemistry V. Test to detect urine protein Heat and acetic acid test, sulfosalicylic acid test, Heller’s test 10. The following are some of the biochemical findings in a patient. What is your probable diagnosis? Serum Bilirubin 10 mg% Conjugated 8.5 mg% Unconjugated 1.5 mg% Serum Alkaline Phosphatase 50 KA units SGOT 80 IU/L SGPT 90 IU/L Urine Bile salts + Bile pigments ++ Urobilinogen negative Feces clay colored I. Normal serum Bilirubin level. 0.2 to 0.8 mg/ dl II. Name the bile salts. Sodium / potassium salts of glycocholic and taurocholic acid III. Test to detect bile salts in urine. Hay’s sulfur powder test IV. Account for clay colored stools. Absence of stercobilinogen in faeces V. Test to detect urine bile pigments. Fouchet’s test, Gmelin’s test 11. Oral glucose tolerance test was performed on a 38-year-old person and the results are given below: Time Hrs. Blood Glucose mg% Urine Sugar 0 (Fasting) 80 Nil ½H 120 Nil 1 Hr 140 Nil 1½ Hr 130 Nil 2 Hr 82 Nil I.

Comment on this. Normal glucose tolerance

Appendix 1: Case Reports 215 II. Hormones regulating blood glucose level. Insulin, glucagon, glucocorticoids, growth hormone III. Normal blood sugar level by Folin-Wu’s method. 80 to 120 mg/dl IV. Normal blood glucose level by enzymatic method. 60 to 90 mg/dl V. Renal glycosuria. Presence of sugar in urine when the blood sugar is below the renal threshold, i.e. 180 mg/dl 12. A 50-year-old non-diabetic male is brought in a semiconscious condition. He has generalized edema and oliguria. Laboratory findings are as follows: Blood urea 90 mg/dl Serum creatinine 5 mg/dl Serum inorganic phosphorous 6 mg/dl I. II. III. IV. V.

Comment on this – Renal failure Normal blood urea level – 15 to 40 mg/dl Normal serum creatinine level – 0.7 to 1.4 mg/dl Significance of creatinine clearance – To assess kidney function Amino acids involved in creatinine synthesis – Glycine, Arginine and Methionine

13. A 35-year-old man with narcotic overdose was admitted to the hospital in severe coma with respiratory depression. His blood sample showed: pH 7.22 Total CO2 26.3 mmol/L pCO2 61 mm/ Hg I. Probable diagnosis. – Respiratory acidosis II. Normal Blood pH – 7. 35 to 7.45 III. Blood buffers – Bicarbonate buffer, Phosphate buffer, Plasma protein buffer, Hemoglobin buffer IV. Normal pCO2 level – 35 to 45 mm Hg V. Normal HCO3 level – 22 to 26 mmol/ L or mEq/ L

216 An Easy Guide for Practical Biochemistry 14. A 9-year-old boy was brought with a history of mental retardation, stunted growth and swelling in the neck. He was diagnosed to have hypothyroidism. I. Mineral involved in this condition – Iodine II. Other clinical manifestations – Children : cretinism, adults: goiter III. Amino acid involved – Tyrosine IV. Dietary sources of the mineral – Seafood, drinking water, iodized salt, onions, vegetables V. Mention the RDA. – 150 µg /day 15. A female aged 30 years, came with complaints of weakness, fatigue and heavy menstrual bleeding. On examination, she was found to be anemic. Her Hb was 6 gm%. I. Probable diagnosis – Iron deficiency anemia II. Causes for the deficiency – Excess loss of iron, dietary deficiency III. Biochemical parameters to assess deficiency – hemoglobin estimation, serum iron level IV. Dietary sources – Liver, meat, egg yolk, green leafy vegetables, whole grains and cereals V. Mention the RDA.– Adult men and postmenopausal women: 10 mg/day Premenopausal women: 15 to 20 mg/day Pregnancy: 30 to 60 mg/day 16. A 3-year-old was brought with complaints of painful, swollen and bleeding gums. On examination, petechiae were seen. Joints were swollen and painful. I. Diagnosis – Scurvy II. Vitamin deficiency that causes this condition – Vitamin C III. Name the richest source – Amla/Indian gooseberry IV. Biochemical name of the vitamin – Ascorbic acid V. Physiological role of the vitamin – Antioxidant property, antiscorbutic property. 17. A person on hunger strike was brought to the hospital in an unconscious state. Following are the lab findings: Blood sugar 52 mg% Blood pH 7.25

Appendix 1: Case Reports 217 Serum bicarbonate Ketone bodies in urine -

16 mEq/L +

I. II. III. IV.

Probable diagnosis – Starvation ketoacidosis Normal blood pH – 7.35 to 7.45 Normal blood sugar level – 80 to 120 mg/dl Name the ketone bodies – Acetone, Acetoacetic acid, β-hydroxybutyric acid V. Test to detect ketone bodies – Rothera’s test, Gerhardt’ s test

18. A 35-year-old male came with history of increased pigmentation around the neck, bright reddish patches on the feet, ankles and face which increased on exposure to sunlight. He had history of irritability and isulfa. I. Name the above condition – Pellagra. II. Mention the cause – Niacin deficiency. III. Name its co-enzyme forms – NAD, NADP. IV. Name the dietary sources – Yeast, Liver, Legumes, Meat. V. Mention one biochemical reaction in which it is involved – Oxidation and Reduction reaction. Ex. Citric acid cycle Glycolysis, Synthesis of cholesterol. 19. Give your interpretation on the patient with the following observations: Blood urea 80 mg% Serum creatinine 4 mg% Serum cholesterol 400 mg% Total protein 4.5 g% Albumin 1.2 g% Globulin 3 g% Urine protein 6 g% I. Probable diagnosis – Renal failure. II. Normal creatinine level – 0.7 to 1.4 mg/dl. III. Functions of albumin – Maintenance of colloidal osmotic pressure and transport of bilirubinuria and non-esterified fatty acids IV. Normal blood urea level – 15-40 mg/dl.

218 An Easy Guide for Practical Biochemistry V. Test to determine protein in urine. – Heat and acetic acid test, sulfosalicylic acid test, Heller’s test. 20. Give your interpretation on the patient with the following observations. Blood sugar 80 mg% Blood urea 100 mg% Serum cholesterol 320 mg% Serum protein 5 g% Albumin 2.5 g% Urine shows presence of albumin and blood. I. Probable diagnosis – Nephrotic syndrome II. Normal serum cholesterol level – 150 to 200 mg/dl. III. Normal total serum protein level – 6 to 8 g/dl. IV. Term used to express blood in urine – Hematuria. V. Test to detect blood in urine – Benzidine test. 21. A 40-year-old obese female presents with icterus, intolerance to fatty food, pain in the right hypochondrium and clay colored stools. Following is the laboratory investigation report: Serum bilirubin 20 mg% Conjugated 16 mg% Unconjugated 4 mg% SGOT 45 IU/L SGPT 40 IU/L Alkaline phosphatase 135 KA units I. II. III. IV. V.

Probable diagnosis – Obstructive jaundice. Normal serum bilirubin level – 0.2 to 0.8 mg/dl. Test done to estimate serum bilirubin – van den Bergh test. Expected cholesterol level in this case – Cholesterol is elevated. What is urobilinogen? – Excretory product of bilirubin

22. A 55-year-old man is brought to the hospital in a semiconscious state. He has low BP and feeble pulse. His breath has fruity odor. The data of his laboratory investigations are given below: Blood pH 7.1 Plasma bicarbonate 22 mEq/L

Appendix 1: Case Reports 219 Blood glucose random Blood urea Serum creatinine Urine sugar Urine ketone bodies I. II. III. IV. V.

-

580 mg/dl 40 mg/dl 1.5 mg/dl ++++ +++

Probable diagnosis – Ketoacidosis Normal blood pH 7.35 to 7.45 Test to detect urine sugar – Benedict’s test. Test to detect urine ketone bodies – Rothera’s test. Cause for fruity odor – Presence of acetone.

23. Following are the findings in a patient brought to the hospital in the coma state. Blood sugar (fasting) 270 mg% Benedict’s test orange colored ppt Rothera’s test positive Serum bicarbonate 16 mEq/L Plasma pH 7.25 I. Probable diagnosis – Diabetic Ketoacidosis II. Normal FBS, PPBS and RBS – FBS:80-110 mg/dl. RBS: 80-140 mg/dl PPBS: 70-140 mg/dl. III. Name ketone bodies – Acetone, Acetoacetic acid and β-hydroxybutyric acid IV. Normal blood pH - 7.35 to 7.45 V. Name the blood buffers – Bicarbonate buffer, Phosphate buffer, Plasma protein buffer, Hemoglobin buffer. 24. A 40-year-old person with severe chest pain was admitted to the hospital. ECG was abnormal. Laboratory findings were as follows: RBS 140 mg% AST 60 IU//L ALT 28 IU/L LDH 410 IU/L I. Probable diagnosis – MI II. Normal values of AST – AST: 8-20 IU/L

220 An Easy Guide for Practical Biochemistry III. Normal values of ALT – ALT: 13-40 IU/L IV. Normal values of LDH – LDH: 100-200 IU/L V. What are the other markers to be investigated? CPK, cardiac troponin 25. A 36-year-old man was admitted to the hospital following episodes of nausea, vomiting, loss of appetite and generalized malaise. His urine was high colored. Upon examination, he had tender hepatomegaly. His lab findings are given below: Serum bilirubin 13.5 mg% Direct 9 mg% Indirect 4.5 mg% Serum alkaline phosphatase 20 KA units ALT 230 IU/L AST 80 IU/L Urine Bile salts negative Bile pigments ++ Urobilinogen + I. Probable diagnosis – Hepatic Jaundice II. What is direct bilirubin? – Conjugated bilirubin: bilirubin is conjugated with UDP glucuronate to form bilirubin mono and diglucuronide. It is water soluble, reacts directly with diazo reagent III. How are bile salts formed? – Bile acids derived from cholesterol react with alkali to form bile salts. IV. Test to detect bile salts – Hay’s sulfur test V. Importance of bile salts in the body – Helps in emulsification and digestion of fatty acids. 26. A one-year-old child was brought to the hospital by his mother with the complaint of blackening of urine on standing. Lab findings are as follows: Random blood sugar 120 mg% Benedict’s test positive Ferric chloride test positive I. Probable diagnosis – Alkaptonuria II. Deficient enzyme – Homogentisic acid oxidase.

Appendix 1: Case Reports 221 III. Compounds excreted in urine – Homogentisic acid. IV. Amino acid correlating in this disorder – Tyrosine, phenylalanine. V. Comment on positive Benedict’s test – Presence of Homogentisic acid. 27. The following are some of the biochemical findings in a patient. Serum bilirubin 10 mg% Conjugated 5.5 mg% Unconjugated 4.5 mg% Serum alkaline phosphatase 20 KA units SGOT 260 IU/L SGPT 290 IU/L Urine Bile salts negative Bile pigments + Urobilinogen + Feces –Stercobilinogen ++ I. II. III. IV. V.

What is the probable diagnosis? – Hepatic Jaundice. Normal serum bilirubin level – 0.2 to 0.8 mg/dl. Test to detect serum bilirubin – van den Bergh test. Normal alkaline phosphatase level - 3-13 KA units. Name the bile pigments – Bilirubin and biliverdin

28. The following are some of the biochemical findings in a patient. Serum bilirubin 0.8 mg% Direct - 0.3 mg% Indirect - 0.5 mg% Serum alkaline phosphatase 6 KA units ALT - 14 IU/L AST - 16 IU/L Urine Bile salts absent Bile pigments absent Urobilinogen traces Feces normal color

222 An Easy Guide for Practical Biochemistry I. II. III. IV. V.

Probable diagnosis – Normal. Normal serum bilirubin level – 0.2 to 0.8 mg/dl. Test to detect serum bilirubin – van den Bergh test. Normal levels of ALT – ALT: 13-40 IU/L. Conjugation of bile pigments – Bilirubin is conjugated with UDP – glucuronate to form Bilirubin mono and di-glucuronide.

29. Patient is giving history of malaria for which he has taken treatment a month ago. His lab findings are as follows: Serum bilirubin (total) 2.5 mg/dl Direct 0.4 mg/dl Indirect 2.1 mg/dl SGOT 15 IU/L SGPT 12 IU/L Alkaline phosphatase 6 KA units Urine: Urobilinogen positive Bile pigments absent I. Probable diagnosis – Hemolytic jaundice. II. What is indirect bilirubin? – Unconjugated bilirubin is not conjugated with UDP glucuronate. It is insoluble in water and soluble in methanol. III. Test to detect serum bilirubin levels – van den Bergh test. IV. Name the bile pigments – Bilirubin and Biliverdin. V. Test to detect urinary bile pigments – Fouchet’s and Gmelin’s test. 30. A person on hunger strike was brought to the hospital in an unconscious state. Following are the laboratory findings: Blood sugar 52 mg% Blood pH 7.25 Serum bicarbonate 16 mEq/L Rothera’s test positive I. Probable diagnosis – Starvation ketoacidosis. II. Normal serum bicarbonate – 22 to 26 mEq/L III. Name the ketone bodies – Acetone, Acetoacetic acid, β–hydroxybutyric acid.

Appendix 1: Case Reports 223 IV. Normal fasting blood sugar levels – 80 to 110 mg/dl. V. Normal pH of blood – 7.35 to 7.45 31. These are the values of oral glucose tolerance test performed on an individual by Folin- Wu method. Time(Hrs) Blood Glucose (mg %) Urine Sugar 0 (Fasting) 80 Nil ½ Hr 120 + 1 Hr 150 +++ 1½ Hr 100 Nil 2 Hrs 85 Nil I. II. III. IV. V.

Your interpretation – Renal glycosuria Renal threshold – 180 mg/dl Test to detect urine sugar – Benedict’s test Normal fasting blood glucose level – 80-110 mg/dl. True glucose level – 60-90 mg/dl.

32. A medical student aged 20 years presents with abdominal pain, fever, loss of appetite, nausea and high colored urine. On examination there is icterus, tenderness in right hypochondrium and hepatomegaly. I. Give your probable diagnosis – Acute hepatitis II. Suggested biochemical investigations – AST, ALT, Serum total bilirubin, Direct and Indirect bilirubin, A/G ratio III. Bile pigments – Bilirubin, Biliverdin IV. Bile salts – Na and K salts of glycocholic and taurocholic acid. V. Importance of bile salts – Emulsification and digestion of FA’s 33. An eight-year-old boy came with history of inability to read in poor light and dryness of skin. On examination the boy was malnourished, grayish-white spots were seen on the conjunctiva and the conjunctiva was dry. I. Probable diagnosis – Xerophthalmia. II. Deficiency of which vitamin causes these symptoms – Vit-A. III. Name the dietary sources – Milk, butter, egg yolk, liver, carrot, papaya, mango, green leafy vegetables. IV. How do you assess the deficiency? – Dark adaptation test, serum RBP level is decreased, serum vit-A level is decreased V. RDA – 5000 IU

224 An Easy Guide for Practical Biochemistry 34. A known diabetic was brought to the hospital in a comatose condition with deep sighing respiration (Kussmaul’s breathing) and fruity odor of breath. On examination he was cold and dehydrated. His report reads as: Blood sugar : 526 mg/dl pH : 7.1 Urine Benedict’s test : ++++ Rothera’s test : +++ I. Probable diagnosis – Diabetic ketoacidosis II. Normal blood pH – 7.35 to 7.45 III. Name the ketone bodies – Acetone, acetoacetic acid , beta hydroxybutyric acid IV. Renal threshold for glucose – 180 mg/dl V. Normal blood glucose level – 80-110 mg/dl. 35. A young man was brought to the hospital with stab injury to the chest. Investigations showed: pH : 7.24 pCO2 : 60 mmHg Plasma bicarbonate : 25 mEq/L Carbonic acid : 2.7 mEq/L I. Probable diagnosis – Respiratory acidosis. II. Normal pH of blood – 7.35 to 7.45 III. Name the blood buffer systems – Bicarbonate buffer, Phosphate buffer, Plasma protein buffer, Hemoglobin buffer IV. Normal plasma bicarbonate levels – 22-26 mEq/L V. Compensatory mechanism involved – Renal compensatory mechanism. Excretion of more of titrable acid and ammonia and retention of bi-carbonate. 36. A patient on routine check-up was found to excrete high amounts of creatinine. Serum creatine kinase was also elevated. I. Comment on the condition – Muscular dystrophy. II. Importance of creatinine – Creatinine excretion is increased in muscle and kidney diseases. III. Amino acids involved in its synthesis – Glycine, Arginine and Methionine.

Appendix 1: Case Reports 225 IV. Creatinine clearance – Volume of plasma completely cleared of creatinine/min. V. Normal serum creatinine levels – 0.7 to 1.4 mg/dl 37. An obese middle-aged person was brought to the hospital emergency room with complaints of dizziness, shortness of breath and chest pain. Blood chemistry showed an increased creatine kinase, lactate dehydrogenase and aspartate transaminase (AST) activities. Liver function parameters were normal. I. Comment on this – MI II. What are isoenzymes? – Multiple forms of the same enzyme that catalyses the same biochemical reaction III. Isoenzymes of CPK – CPK1 (BB), CPK2 (BM), CPK3 (MM) IV. Other conditions where CPK is increased – Muscular dystrophy. V. Other cardiac biomarkers – Cardiac Troponin, LDH, AST, C – reactive protein. 38. A two days old male baby presented with icterus and high colored urine. Total serum bilirubin - 18 mg/dl Unconjugated bilirubin - 16 mg/dl The pediatrician advised phototherapy for the baby. I. Name two conditions causing above changes – Neonatal Physiological jaundice / Hemolytic jaundice. II. Normal serum bilirubin level – 0.2 to 0.8 mg/dl. III. What is direct bilirubin? – Conjugated bilirubin: bilirubin is conjugated with UDP glucuronic acid to form bilirubin mono and di-glucuronide. It is water soluble, reacts directly with diazo reagent IV. Kernicterus – When the concentration of plasma bilirubin (unconjugated) exceeds 20 mg/dl it penetrates the blood brain barrier and causes hyperbilirubinemic toxic encephalopathy or kernicterus, which causes mental retardation V. Test to detect urinary bile pigments – Fouchet’s test and Gmelin’s test.

226 An Easy Guide for Practical Biochemistry 39. A middle aged chronic alcoholic male was brought to the casualty with complaints of hematemesis. On examination he had icterus and hepatomegaly. Biochemical investigations showed the following: Serum albumin 2.5 gm% Serum bilirubin 12 mg% Alkaline phosphatase 350 IU/L AST 134 IU/L ALT 360 IU/L I. Probable diagnosis – Alcoholic cirrhosis II. Normal serum albumin level – 3.5 to 5 g/dl III. Functions of albumin – Colloid osmotic pressure maintenance, transports bilirubin and nonesterified FA’s, acts as a buffer. IV. Test to detect serum bilirubin – van den Bergh test. V. Importance of A:G ratio – Reversed in multiple myeloma Decreased in liver disease, kidney disease, cirrhosis of liver, Nephrotic syndrome, malnutrition. 40. A child presented to the casualty with complaints of severe joint pains. Examination revealed severe pallor and an enlarged spleen. Biochemical findings were as follows: Serum bilirubin 10 mg% Conjugated 0.5 mg% Unconjugated 9.5 mg% Serum alkaline phosphatase 8 KA units AST 30 units ALT 26 units Urine bile salts negative Bile pigments negative Urobilinogen +++ Feces- stercobilinogen +++ I. Probable diagnosis – Hemolytic jaundice II. What is unconjugated bilirubin? Bilirubin is not conjugated with UDP glucuronic acid. It is water insoluble and soluble in methanol III. Test to detect serum bilirubin – van den Bergh test. IV. End product of hemoglobin metabolism – Bilirubin

Appendix 1: Case Reports 227 V. Causes – G-6PD deficiency, Sickle-cell anemia, Incompatible blood transfusion. 41. A hostel student presented with recurrent episodes of vomiting and pyrexia. On examination he was icteric, dehydrated and had enlarged liver. Biochemical Findings were as follows: Serum bilirubin 10 mg% Conjugated 5.5 mg% Unconjugated 4.5 mg% Serum alkaline phosphates 20 KA units AST 260 units ALT 290 units Urine bile pigments ++ Bile salts + Urobilinogen + I. Probable diagnosis – Hepatic jaundice. II. What is conjugated bilirubin? Bilirubin is conjugated with UDP glucuronic acid to form bilirubin mono-and di-glucuronide. It is water soluble, reacts directly with diazo reagent III. Name the bile salts – Sodium /potassium salts of glycocholic acid and taurocholic acid. IV. Test to detect urinary bile salts – Hay’s sulfur test. V. Formation of bile salts – Cholesterol ? bile acids conjugated with Glycine/ Taurine? Glycocholic /Taurocholic acids 42. An elderly gentleman with complaint of oliguria was brought to the hospital in a confused state. Biochemical investigations revealed the following: Blood urea - 119 mg% Serum creatinine - 6.4 mg% Serum uric acid - 8.8 mg% Serum inorganic phosphorus - 6.2 mg% I. Comment on this – Chronic Renal failure. II. Normal urea level – 15 to 45 mg/dl. III. Normal uric acid level – 3.5 to 7 mg/dl

228 An Easy Guide for Practical Biochemistry IV. Role of creatine in the body – creatine phosphate is involved in muscle contraction V. Amino acids involved in creatine formation – Glycine, Arginine, Methionine 43. A mother sought medical help for her child with the complaint that diapers used for the child stained dark. On analysis urine gave a positive Benedict’s but Glucose oxidase test was negative. Ferric chloride test with urine was positive. I. Name the disorder – Alkaptonuria. II. Enzyme deficient – Homogentisic acid oxidase. III. Why Benedict’s test is positive? – Presence of Homogentisic acid. IV. Name non sugar reducing agents – Vitamin-C, Glutathione, Salicylates, Uric acid, glucuronides. V. Mention other protein metabolic disorders – Phenylketonuria, Albinism, Maple syrup urine disease, Hartnup disease. 44. A fair 8-year-old chubby boy was brought to the hospital by the mother with the complaint that he is mentally retarded and has delayed milestones. Blood chemistry revealed an abnormally high Phenylalanine. Phenyl ketones were present in the urine in appreciable amounts. I. Probable diagnosis – Phenylketonuria. II. Compound excreted in urine – Phenylacetate/Lactate/ Pyruvate. III. Test performed to detect phenylketones – Guthrie test, urine Ferric chloride test. IV. Enzyme deficiency – Phenylalanine hydroxylase. V. Other clinical symptoms – Reflexes are hyperactive because of defective myelination of nerves 45. A child with retarded growth was brought to the hospital with complaints of diarrhea and delayed milestones. On examination he was found to have cataract. Urine examination showed reduction with Benedict’s reagent but not with glucose oxidase method. I. Probable diagnosis – Galactosemia. II. Deficient enzyme – Galactose-1- phosphate uridyl transferase

Appendix 1: Case Reports 229 III. Name the sugar excreted in urine – Galactose IV. Name the test to detect the compound – Mucic acid test V. How do you manage this case? – Galactose free diet 46. The following are some of the biochemical findings in a patient. Serum bilirubin 10 mg% Conjugated 0.5 mg% Unconjugated 9.5 mg% Serum alkaline phosphatase 8 KA units SGOT 30 IU/L SGPT 26 IU/L Urine: Bile salts negative Bile pigments negative Urobilinogen +++ (Excess) Feces-stercobilinogen +++ (Excess) I. II. III. IV. V.

Probable diagnosis – Hemolytic jaundice. Normal serum bilirubin level – 0.2-0.8 mg/dl. Test to detect serum bilirubin – van den Bergh. Normal SGOT level – 8-13 IU/L. Name the bile salts and bile pigments - Na and K salts of glycocholic and taurocholic acid / Bilirubin and Biliverdin.

47. A person aged 30 years was referred by a physician to the lab for routine tests. Reports are as follows: Random blood sugar 125 mg/100 ml Blood urea 35 mg/100 ml Serum cholesterol 180 mg/100 ml AST 25 IU/L ALT 20 IU/L CPK 30 IU/L LDH 120 IU/L I. Comment on the report – Normal. II. Importance of estimation CPK – Increased in cardiac and muscular diseases. III. Isoenzyme forms of LDH – LDH1, LDH2, LDH3, LDH4 and LDH5

230 An Easy Guide for Practical Biochemistry IV. PPBS and its normal level – PPBS = Post – Prandial – Blood Sugar. = 80-140 mg/dl. V. Renal threshold. – 180 mg/dl. 48. A school teacher had a routine medical checkup and LFT was done. The data is as follows. Total proteins 7 g/dl Albumin 4 g/dl Globulin 3 g/dl SGOT 16 IU/L SGPT 10 IU/L Serum bilirubin 0.6 mg/dl I. II. III. IV. V.

Comment on this – Normal. Normal serum protein level – 6 to 8 g/dl. Normal serum albumin level – 3.5 to 5 g/dl. Normal serum bilirubin level – 0.2 to 0.8 mg/dl Test to detect serum bilirubin – van den Bergh test.

49. A patient with acute chest pain showed the following blood values. Blood sugar 300 mg% Serum cholesterol 320 mg% HDL 20 mg% SGOT 52 IU/L SGPT 28 IU/L CPK and LDH values are also raised. I. II. III. IV. V.

Probable diagnosis – MI. Normal serum cholesterol level – 150-200 mg/dl. Isoenzymes of CPK – CPK1 ,CPK2, CPK3. Isoenzymes of LDH – LDH1 LDH2, LDH3, LDH4, LDH5 Normal serum levels of triglycerides and HDL – Tgl = 60-180 mg/dl HDL = 30-60 mg/dl.

50. The following are some of the biochemical findings in a patient. Blood urea 30 mg% Serum creatinine 1.8 mg%

Appendix 1: Case Reports 231 Serum cholesterol Total plasma protein Albumin Globulin Urine protein

-

560 mg% 4.5 g% 1.0 g% 3.5 g% 10 g/L

I. II. III. IV.

Probable diagnosis – Nephrotic syndrome. Normal cholesterol level – 150-200 mg/dl. Normal A:G ratio – 1.2 : 1 to 1.5: 1 Functions of plasma protein – Albumin : colloid osmotic pressure maintenance, transports bilirubin, Protein buffer. Globulin : Defence protein. V. Test to detect urinary proteins – Heat and Acetic acid test, sulfosalicylic acid test, Heller’s test. 51. The following are some of the biochemical findings in an 8-year-old child. Blood urea 18 mg% Serum creatinine 1.6 mg% Serum calcium 7.4 mg% Serum inorganic Phosphorous 2 mg% Serum alkaline Phosphatase 80 KA units I. Comment on this – Vitamin –D deficiency. II. Normal serum calcium levels – 9-11 mg/dl. III. Hormones associated with calcium metabolism – Calcitonin, PTH, calcitriol IV. Clinical features in the above case – Rickety rosary, bow legs, bossing of head, pigeon chest V. Normal serum phosphate level – 3-4 mg/dl. 52. A 20-year-old male presents with puffiness of the face. Investigation showed following results. Total protein 3.5 g/dl Albumin 1.5 mg/dl Globulin 2 mg/dl Serum cholesterol 500 mg/dl

232 An Easy Guide for Practical Biochemistry Urine examination showed marked proteinuria. I. Probable diagnosis – Nephrotic syndrome II. What is proteinuria? – Presence of protein in urine. III. Proteins excreted in urine – Albumin, Bence Jones protein. IV. Starting materials for cholesterol synthesis – Acetyl-CoA V. Compounds derived from cholesterol – Bile acids, Bile salts, Steroid hormones, Vitamin D. 53. The following are the results of blood gas analysis of a patient admitted in the medical ICU. Blood pH 7.2 Plasma bicarbonate 28 mEq/L pCO2 70 mm of Hg I. II. III. IV. V.

Interpret – Plasma bicarbonate is normal, but pCO2 is high. Blood pH is low. It is a case of uncompensated respiratory acidosis. Normal pH of blood – 7.35 to 7.45 Name the blood buffer systems – Bicarbonate buffer, Phosphate buffer, Plasma protein buffer, Hemoglobin buffer Normal plasma bicarbonate levels – 22 to 26 mEq/L Normal pCO2 level – 32 to 45 mm of Hg

54. The following are the results of blood gas analysis of a patient admitted in the medical ICU. Blood pH 7.23 Plasma bicarbonate 14 mEq/L pCO2 38 mm of Hg I. II. III. IV. V.

Interpret – Plasma bicarbonate is diminished, but no change in pCO2. Blood pH is low due to lowered bicarbonate level. It is a case of uncompensated metabolic acidosis. Compensatory mechanism – hyperventilation to wash out CO2 faster. Increased elimination of acid in the urine and rise in urinary ammonia. Normal pH of blood – 7.35 to 7.45 Normal plasma bicarbonate levels – 22 to 26 mEq/L Name the blood buffer systems – Bicarbonate buffer, Phosphate buffer, Plasma protein buffer, Hemoglobin buffer

Appendix 1: Case Reports 233 55. An adult man living in coastal region came with the complaint of difficulty in doing simple tasks such as bending and squatting. On general examination, chalky white patches with yellow or brown staining are found on the surface of the teeth (motled enamel). X-ray showed hypercalcification of spinal bones, pelvis and limbs. I. What is your diagnosis? – Fluorosis. II. Function of the mineral involved – Fluorine is an essential trace element. Sodium fluoride is a powerful inhibitor of glycolytic enzymes. Florine forms a protective layer of acidresistant fluoroapatite with hydroxyapatite crystals of the enamel. III. Source – Drinking water IV. RDA—Drinking wateshould contain 1 to 2 ppm. V. Prevention of fluorosis – Can be prevented by removing fluorides from the water by treatment with activated carbon or by some other evitable absorbents. 56. A female aged 28 years came to the OPD with complaints of palpitation and tremors. On examination she had neuromuscular irritability, carpopedal spasm and laryngeal spasm. She also gave history of some neck surgery two months ago. I. Give your diagnosis – Tetany II. Which mineral is involved? – Calcium III. Function of calcium – Activation of enzymes, contraction of muscle, bone formation, in blood clotting. IV. Normal serum calcium level – 9 to 11 mg/dl V. Hormones associated with calcium metabolism – Calcitonin, PTH, calcitriol

234 An Easy Guide for Practical Biochemistry

Appendix 2 Spotters 1. a. Mention the cause for the condition. Vitamin A deficiency b. RDA. 5000 IU Keratomalacia

2. X-ray showing features of bow legs a. Where do you see the above condition? Rickets b. Mention the cause. Vitamin D deficiency.

3. a. Diagnose the condition. Gout b. Mention the cause. Hyperuricemia due to defective enzymes of purine Biosynthesis (deficiency of HGPRTase)

Appendix 2: Spotters 235

Pellagra 4. a. Which vitamin deficiency causes the above condition? Niacin b. What are the other clinical features? Dermatitis, dementia and diarrhea.

5. a. What does the symbol stand for? Biomedical hazard

6. a. Identify the instrument. Spectroscope b. Mention its use. Used to study and identify hemoglobin and its derivatives.

236 An Easy Guide for Practical Biochemistry 7. a. Identify the instrument. Colorimeter b. Mention its use. To read the optical densities of colored substances in quantitative estimation.

8. a. Identify the instrument. Urinometer b. Mention its use. To determine the specific gravity of urine

9. a. Identify the instrument. Folin-Wu tube b. Mention its use. Used in the estimation of blood sugar by Folin-Wu tube.

Appendix 2: Spotters 237

10. a. What does the above graph indicate? Normal GTT b. Mention the significance of GTT curve. • GTT is most important in the investigation of asymptomatic hyperglycemia or glucosuria such as renal glucosuria and alimentary glucosuria. • This test may contribute useful information in some cases of endocrine dysfunction. • It is also helpful in recognizing milder cases of diabetes.

238 An Easy Guide for Practical Biochemistry

11. a. What does the above graph indicate? Abnormal GTT showing diminished glucose tolerance. b. Mention its significance. Diminished glucose tolerance occurs in diabetes mellitus and certain endocrine disorders like hyperthyroidism, hyperpituitarism and hyperadrenalism (Cushing syndrome).

Appendix 2: Spotters 239

12. a. What does the above graph indicate? Renal glycosuria b. Mention the significance of GTT curve. • GTT is most important in the investigation of asymptomatic hyperglycemia or glucosuria such as renal glucosuria and alimentary glucosuria. • This test may contribute useful information in some cases of endocrine dysfunction. • It is also helpful in recognizing milder cases of diabetes.

240 An Easy Guide for Practical Biochemistry

13. a. Identify the above compound Starch b. Test to identify the compound. Iodine test c. Components of starch. Amylose and amylopectin

14. a. Identify the above compound. Maltose b. What is the source? Malt sugar

Appendix 2: Spotters 241

15. a. Identify the above structure. t RNA b. Label the parts. I. 3’ end- acceptor arm II. 5’ phosphate end III. D arm IV. anticodon arm V. extra arm VI. TψC arm c. Function of t RNA Transfer of amino acids to the ribosomes for protein synthesis. d. Unusual bases in t RNA Dihydrouracil (DHU), pseudouridine (ø), and hypoxanthine

242 An Easy Guide for Practical Biochemistry

16. a. Identify the above compound. Cholesterol b. Normal serum level. 150 to 200 mg/dl c. Derivatives of cholesterol Steroid hormones, vitamin D, bile salts, bile acids.

17. a. Identify the above structure? Vitamin D (1, 25 Dihydroxy cholecalciferol) b. Sources: Sunlight, cod liver oil, egg yolk, liver c. Active form Calcitriol d. Deficiency manifestations Rickets in children, osteomalacia in adults

18. a. Identify the above slide. Maltosazone b. What is the significance of this test? Used to differentiate between reducing disaccharides

Appendix 2: Spotters 243 19. a. Identify the slide. Lactosazone b. What is the significance of this test? Used to differentiate between reducing disaccharides.

20. a. Identify the slide. Glucosazone/Fructosazone b. What is the significance of this test? Used to differentiate between reducing disaccharides.

21. a. Identify the slide. Hemin crystals b. What is the significance? In forensic medicine to detect blood stains

22. Indicators

Use

PH

Bromocresol Green

In isoelectric precipitation test for the identification of casein

4 to 4.6

Phenolphthalein

In specific urease test for the detection of urea

8 to 10

244 An Easy Guide for Practical Biochemistry 23. Tests

Significance

Molisch test

In identification of carbohydrates

Hay’s

Detect bile salts

Rothera’s

Detection of ketone bodies

Jaffe’s

Detection of creatinine

Iodine

Identification of starch

Appendix 2: Spotters 245 24. Reagent

Composition

Use

Molisch

Alpha Naphthol + ethyl Alcohol

To identify carbohydrates

Benedict’s

Copper sulfate + Sodium citrate + Sodium carbonate

To detect reducing sugars

Barfoed’s

Copper acetate + Glacial Acetic acid

To identify monosaccharide

Seliwanoff’s

Resorcinol in concentrated Hydrochloric acid

To detect ketosugar

Millon’s

Sodium nitrate + Mercuric sulfate

To detect hydroxyphenyl group-tyrosine

Osazone Mixture

Phenylhydrazine hydrochloride+ sodium acetate+ glacial acetic acid

Identification of reducing sugars which give characteristic osazone crystals.

Benzidine

Benzidine + glacial acetic acid

Detect blood in urine

Biuret

Sodium Potassium tartarate + Copper sulfate

In identification of Protein

DAM

Diacetyl Monoxime + water

Quantitative estimation of blood urea

ANSA

Aminonapthnol sulfonic acid + Sodium bisulfate Sodium sulfite

Quantitative estimation of serum phosphate

Nippe’s fluid

Potassium bromide + Potassium chloride + Potassium iodide + Glacial acetic acid

Preparation of hemin crystals

246 An Easy Guide for Practical Biochemistry

25. a. Identify the above bond. Peptide bond b. Test used to identify this linkage. Biuret test

Appendix 3: Quality Control 247

Appendix 3 Quality Control Quality control is defined as the study of source of variation of the procedures. Quality Control is used to recognize errors and to minimize the error in laboratory, from the time between the receipt of specimen and the dispatch of the report. Quality control is a statistical system for measuring the reproducibility of the degree of perception in laboratory procedures. It is an excellent means of improving laboratory efficiency and ensures quality results. Quality control helps to check the instruments, reagents, procedures and technical errors. Use of commercial reference control serum is recommended with each assay batch. NECESSITY OF QUALITY CONTROL The results of various tests provided by the laboratory are very important for the diagnosis and treatment of the disease. Even a small error could lead to serious consequences, wrong diagnosis and wrong treatment. It may be critical to the patient. This not only leads to prolonged hospitalization but also an additional financial burden on the patient. Hence, quality control is a must. It can be defined as study of errors. The responsibility of the laboratory persons is to minimize these errors. Quality control takes into account of - The cleanliness of glassware - Daily maintenance of instrument - Well-trained staff - Use of specific and sensitive methods of assay SOME IMPORTANT TERMS Precision Precision indicates how close the test measurements are to earlier, when the same test is conducted on the same sample repeatedly. It is the measure of reproducibility of the test values. It reflects the correctness of procedure.

248 An Easy Guide for Practical Biochemistry Accuracy This indicates closeness of the value to its actual value for a given sample. Closer the measurement to the actual value, greater will be the accuracy. Specificity The reagent should act on only specific component in the biological sample. To give an example- Blood glucose can be estimated both by chemical method and enzymatic method. Chemical method is nonspecific, in the sense, chemical reagent react with many other reducing substances found in the blood along with glucose giving higher value than the actual. Enzymatic methods are specific; enzyme reacts only with glucose to give true value of glucose. Sensitivity Sensitivity reflects the ability of a method to estimate even the minute quantities of the component of biological sample. Standard Standard is a substance of sufficient purity used for standardization. Control This is a sample which is chemically and physically similar to the unknown specimen. It is usually inserted into an analytical run and results are usually calculated from the same set of calibration standard readings. VARIANCE The analytical variance may be observed due to the following reasons: - Deterioration of reagents. - Inadequate mixing of reagent and sample. - Variation of temperature control.

Appendix 3: Quality Control 249 STANDARD DEVIATION (SD) Standard deviation is the statistical index of the degree of deviation from central tendency, namely, the variability within a distribution. It is the square root of the average (mean) of the squared deviations from the mean. If a specimen is analyzed several times, the result would be around the mean value. The mean difference of each value from the mean is SD. √ Σ (x¯ – x)2 SD = —————— n-1 Where Σ = Sum of total X = Any single observed value X = Average value (arithmetic mean) n = Number of observed value (no. of result) COEFFICIENT OF VARIATION (CV) CV expresses the dispersion of result and relates the SD to a level of measurement. SD × 100 CV = –———— Mean A CV of 3% is regarded as ideal result while 5% is acceptable. Values higher than 5% are wrong. QUALITY CONTROL CHART (LEVEY-JENNINGS CHART) Levey- Jennings chart is a chart which illustrates the allowable of errors in laboratory test performance. The limits being a defined deviation from the mean of control serum, most commonly ± 2 SD. QC data can be presented by plotting Levey-Jennings chart. A single batch of control serum is analyzed for 30 consecutive days. The mean and SD values are calculated. A horizontal line is drawn through the mean value, and at 1 SD, 2 SD, 3 SD values are marked above and below the line of mean value. The value obtained each day is plotted on this chart.

250 An Easy Guide for Practical Biochemistry READING THE CHART 1. If the analysis is satisfactory the points that are plotted will be scattered evenly on either side of midline within ± 1 SD limit. This pattern shows that accuracy is maintained. 2. Values falling within ± 2SD limit is acceptable while values at ± 2SD limit and above is Warning Limit, i.e. reanalysis of the control is required. 3. Values at ± 3SD limit are Action Limit. When six consecutive values fall above or below the mean line it shows that the assay is out of Control. In case the value is above or below + 2SD, it indicates that the reagent or standard is deteriorated. The assay should be repeated with fresh reagents and standard (Sometimes the control serum itself deteriorates due to improper storage. Fresh control serum is to be replaced). PREPARATION OF QUALITY CONTROL (QC) MATERIAL IN THE LABORATORY • 50 to 100 ml of pooled serum is collected, filtered through glass wool and mixed thoroughly. • pH is adjusted to 7.5 • 5 ml portion of this is distributed into several plastic vials and stored in deep freezer, so that it is stable for 3 months. • Each day 1 vial is taken and brought to room temperature. Once it liquefies the sample along with test are analyzed. Values are entered on the QC chart (Levey-Jennings chart) to find the standard deviation (SD). QUALITY CONTROL PROGRAMS For a quality control program to be set up the first thing to be done is the determination of SD for the procedure. At the end of the month there would be usually at least 20 values to work with. SD is calculated and the Levey-Jennings chart is plotted. The Quality Control chart and distribution curve would indicate that most of the values obtained on a group of control sera fall within ± 1 SD uniformly distributed on both the sides of mean value. The values observed between ± 1 SD lines indicate good control over the methodology.

Appendix 3: Quality Control 251 INTERNAL AND EXTERNAL QUALITY CONTROL Internal Quality Control It refers to the procedures undertaken in the laboratory for the continuous assessment of day to day work, by running the samples in duplicate, using standard and controls to check for the reproducibility of values and decide its reliability. Internal quality control can be maintained by the following ways. • Use of standardized, sterilized glassware. • Having well trained staff. • Having High Quality reagents and instruments. • Selection of accurate and precise methods. • Use of various primary standards, quality control sera, previously analyzed specimens and statistical methods. • Inclusion of at least one standard with each batch of unknown specimen analysis. • Occasional use of a different primary standard of high concentration to find stability and reliability of routine standard. • Acceptance of batch results, if the values of control sera are within ± 1SD limits. • Detection of random and systematic errors by Levey-Jennings charts. External Quality Control It is system for objectively and retrospectively comparing results from different labs by means of surveys organized by an external agency. In external quality control, quality control serum prepared by a recognized body is supplied to different laboratories for evaluation. Tabulated values of several labs are utilized to find accurate values. Tabulation of values and results of several labs that analyze the same specimen helps in judging the accuracy of labs and the standard of technical skill. External quality control can be maintained by the following way: • Lyophilized normal serum primary standard, normal and abnormal controls can be used to check the accuracy result.

252 An Easy Guide for Practical Biochemistry • An identical sample is distributed to several laboratories. Accuracy can be known by observing the gross differences obtained and by comparing the values. • With each batch the analyst includes a control solution whose exact composition is unknown to technician. Depending on whether the error of control result is lesser or greater than that prescribed for the method the value obtained by a batch may be accepted or rejected. • The control and the standard are treated exactly like the test specimen in all analytical stages. • The control may be varied each time to prevent the analyst/ technician from knowing the result. TYPES OF ERRORS OBSERVED AND THEIR CORRECTION Error could be: 1. Intrinsic 2. Systemic 3. Random or technical Intrinsic Errors These errors could be due to inaccurate methods or due to high blank values. They can be eliminated by: • Use of good instruments • Selecting precise and accurate methods. Systemic Errors A result which contains either only high values or low values indicates systemic error. Systemic errors can be corrected by: • Use of good instruments • Using different concentrations of primary standards. • Analyzing a control serum. Random /Technical Errors These errors are due to incorrect analytical applications. These can be set right by: • Correct collection of specimen. • Separation of serum after 30 min of blood collection.

Appendix 3: Quality Control 253 • Using appropriate methodology. • Correct calculation. Errors can also be classified as: 1. Preanalytical. 2. Analytical 3. Postanalytical Precise and accurate reports can be ensured by avoiding these errors. PREANALYTICAL ERRORS • Preanalytical errors can be avoided by proper collection of blood. Dry syringes are used to prevent heterolysis. Hemolyzed samples give erroneous values. • Labeling of sample with correct sample number is ascertained. • The requisition form must contain the patient’s name, age, sex, hospital number, Lab number, clinical data, date and time of collection of blood with a list of parameters to be analyzed. • Suitable anticoagulants should be employed depending on the type of investigation. • As per the procedure specification either serum, plasma or whole blood should be used. • Expiry date of standard and controls and stability of the reagents must be ascertained before processing. • Standardized pipettes and can clean glassware should be used. • Well trained technicians should be employed. ANALYTICAL ERRORS Variations in analytical could be multifactorial: • Deterioration of reagents. • Use of expired reagents. • Inadequate mixing of reagents and samples. • Temperature variance • Exposure to light sensitive substances. • Exposure of colored end products to light. • Improper time maintenance i.e. reading the value before or after a specific period of time. • Disorganized documents.

254 An Easy Guide for Practical Biochemistry • Staff mismanagements. • Heavy work load. Note: Prolongation in taking the reading may either darken or lighten the color intensity, which directly affects the values. POSTANALYTICAL ERRORS Some of the postanalytical errors are: • Wrong entry of names • Exchange of samples • Wrong entry of values • Exchange of reports Following precautions and measures have to be taken to minimize errors and to provide quality reports. • The blank and standard should be in the same analytical run along with tests so as to nullify errors. • Normal and abnormal controls should be run to ascertain Quality Control. • Report should be checked before entering them in log books or registers. • Report forms should be filled carefully taking proper precautions so as to avoid wrong entry in them. They should be checked before dispatching. • Laboratories can improve or expand their service by adopting more competent methods.

Appendix 4: Miscellaneous 255

Appendix 4 Miscellaneous MOLECULAR WEIGHT Molecular weight of a substance is the ratio of the mass of one molecule of the substance to 1/12th the mass of one atom of carbon 12. Mass atom of 1 molecule of the substance Molecular weight = ——————————————————— 1/12th mass of 1 atom of carbon 12 Molecular weight is equal to the sum of the atomic weights of all the atoms present in a molecule of a compound. The molecular weight expressed in grams is called gram molecular weight. Example: 1 Molecular weight of NaOH = (23 × 1) + (16 × 1) + (1 × 1) = 23 + 16 +1 = 40 {Atomic weight of Na = 23, O = 16, H = 1} 2 Molecular weight of NaCl = (23 × 1) + (35.5 × 1) = 23 + 35.5 = 58.5 {Atomic wt. of Na = 23, Cl = 35.5} 3 Molecular weight of Oxalic acid (COOH.COOH) = (12 + 16 + 16 + 1) + (12 + 16 + 16 + 1) = 45 + 45 = 90 4 Molecular weight of CuSO4 = 63.54 + 32 + (16 × 4) =159.54

256 An Easy Guide for Practical Biochemistry GRAM MOLECULAR WEIGHT The molecular weight of a substance expressed in grams, is called the gram molecular weight of that substance. Example: The molecular weight of CO2 is 44 and hence its gram molecular weight = 44 g. One mole of any substance contains one gram molecular weight of that substance. Thus, one mole of CO2 has a mass of 44 grams. MOLALITY The number of moles of solute dissolved in 1 kg of solvent or 1 gm mol wt of the substance dissolved in 100 gm of water. MOLARITY The number of moles of the substance dissolved in one liter of solvent is called the molarity of the solution. EQUIVALENT WEIGHT The equivalent weight of an element is defined as the number of parts by weight of the element that combines with or displaces from a compound 8 parts by weight of oxygen or 1.008 parts by weight of hydrogen or 35.45 parts by weight of chlorine. NORMALITY Normality of a solution is equal to the number of gram equivalent weights of the solute dissolved in 1 liter of the solution. Normality × Equivalent mass = Mass of solute/1000 ml i.e. Normality × Equivalent mass = W SOLUTIONS Solutions are obtained by dissolving a solute in a solvent. Example: Saline solution contains sodium chloride in water. Standard Solution It is a solution which contains a known amount of the substance in a definite volume of solvent. These are used in Quantitative estimations of biochemical parameters.

Appendix 4: Miscellaneous 257 Normal Solution Normal Solution contains one gram equivalent weight of the substance, in one liter of solution. 1 N NaOH = 40 gm of NaOH dissolved in one liter of water. Molar Solution Molar Solution contains one gm mol. Wt of the substance dissolved in one litre of solution. 1 M NaOH = 40 gm of NaOH dissolved in one liter of water Percent Solution A known weight of substance dissolved in 100 ml of solvent. If it is a liquid then it is volume of the liquid in 100 ml of solvent. (Solid/ liquid in solvent by volume basis) Example: 1% solution of a substance is 1 gm of the substance in 100 ml of solvent. 10% Solution is 10 gm of a substance in 100 ml of solvent 0.9% NaCl (Normal saline)-is 900 mg of NaCl in 100 ml of pyrogen free distilled water Saturated Solution Saturated solution contains maximum quantity of solute that can be dissolved in a particular volume at a given temperature. It states that it contains as much of the solute that will dissolve in the solvent. Example: Saturated NaCl – dissolve NaCl in particular volume, then go on adding salt till something is remained undissolved. Reagent Solution They are prepared as specifications meant for a particular estimation. Example: Benedict’s Reagent, Molisch reagent, Biuret Reagent. Stock Reagent Stock reagent is a solution of higher concentration than working solutions. Stock solutions are those which are prepared and stored

258 An Easy Guide for Practical Biochemistry and can be diluted depending upon the concentration required for working solutions. Example: Stock glucose solution (1 g%) 1 gm of glucose per 100 ml. PREPARATION OF NORMAL SOLUTIONS 1 N Sodium Carbonate Weigh accurately 53 gm Na2CO 3 crystals and make up to 1000 ml with Distilled Water. 0.01 N Sodium Carbonate Weigh accurately 5.3 gm of Na2CO3 crystals and make up to 1000 ml with Distilled Water. 1 N Sodium Hydroxide Weigh accurately 40 gm of NaOH crystals and make up to 1000 ml with Distilled Water. 0.1 N Sodium Hydroxide Weigh accurately 4 gm of NaOH crystals and make up to 100 ml with Distilled Water. Volumetric Flask is most accurate and convenient for preparing such solutions. It is a flat bottomed flask with a long neck and a tight glass stopper. The mark at the stem of the neck indicates a particular volume. Volumetric flasks of different volume are available (5 ml, 10 ml, 50 ml, 100 ml, 500 ml and 1000 ml) Put the weighed solute in a clean dry volumetric flask through a clean dry funnel, add the solvent and dissolve the solute then make the volume up to the mark by the solvent. Label it by denoting the name of solutions, its concentration and the date of preparation. EQUIVALENT WEIGHT OF AN ACID Equivalent weight of an acid Molecular Weight = ———————————————— Number of replaceable H+ atoms For monobasic acids like HCl and HNO3 the number of replaceable hydrogen atom (the basicity) is one.

Appendix 4: Miscellaneous 259 Equivalent weight of HCl Molecular Weight = ———————————————— Basicity (Replaceable hydrogen atoms) 1 + 35.5 = ———— 1 = 36.5 i.e. for monobasic acids, molecular weight equals equivalent weight. For dibasic acids like Sulfuric acid having two replaceable hydrogen atom, basicity is 2 and so, Equivalent weight of H2SO4 Molecular weight = ———————— Basicity = 98/2 = 49

260 An Easy Guide for Practical Biochemistry

Appendix 5 Normal Values (Reference Values) Analyte

Sample

Units

Ammonia

P/S

< 50 µg/dl

Acid phosphatase, (ACP) total

P/S

0.5- 4 KAU/dl

Alanine amino transferase (ALT/SGPT)

S

SI units 2.5-12 IU/L Male: 13-35 IU/L Female: 10-30 IU/L

Albumin

S

3.5- 5 g/dl

35-50 g/L

Alkaline phosphatase

S

3- 13 KAU/dl

40-125 IU/L

Amylase

S

80- 180 somogyi units/dl

50-120 IU/L

Aspartate aminotransferase (AST/SGOT)

S

8-20 IU/L

Bicarbonate

S

22- 26 mEq/L

Bilirubin, total

S

0.2- 1 mg/dl

4-17 µmol/L

Calcium

S

9- 11 mg/dl

2.1-2.5 mmol/L

Chloride

S/P

96- 106 mEq/L

96-106 mmol/L

Cholesterol, total

S/P

150- 200 mg/dl

4- 6 mmol/L

HDL

S

Male: 30- 60 mg/dl Female: 35- 75 mg/dl

0.75-1.58 mmol/L 0.98-1.95 mmol/L

LDL

S

20- 29 yr: 60- 150 mg/dl 30- 39 yr: 80-175 mg/dl 40- 60 yr: 90- 200 mg/dl

Copper

P

70- 150 µg/dl

C- reactive protein (CRP)

22-26 mmol/L

16- 30 µmol/L

0.5-1 mg/dl

Creatine

S

Creatine kinase

S

0.2- 0.4 mg/dl

15- 30 µmol/L

Creatinine

S U

0.7- 1.4 mg/dl 15- 25 mg/kg/day

60- 125 µmol/L 15-0.2 mmol/kg/day

Electrophoresis

S

Albumin: 55-65% α 1: 2-4% α 2: 6-12% β: 8-12% γ: 12-22%

3.5-4.7 g/100 ml 0.2-0.3 g/dl 0.4- 0.9 g/dl 0.5-1 g/dl 0.7-1.5 g/dl

Female: 10- 80 U/L Male: 15- 100 U/L

Contd...

Appendix 5: Normal Values (Reference Values) 261 Contd... Analyte

Sample

Units

SI units

Fibrinogen

P

200-400 mg/dl

Globulins

S

2.5-3.5 g/dl

25-35 g/L

P B CSF

70-110 mg/dl 65-100 mg/dl 50-70 mg/dl

4-6.1 mmol/L 3.5-5.6 mmol/L 2.8-4.2 mmol/L

Hemoglobin

B

Male: 14-16 g/dl Female: 13-15 g/dl

2.17-2.4 mmol/L

Glycated hemoglobin Hb A1c Iodine Iron

E S B

4-8% of total 5-10 µg/dl 5 mg/dl

Glucose (fasting)

5.8- 8.5 µmol/L

Lactate dehydrogenase (LDH)

S

Lipoproteins

S

Alpha: 40 mg/dl Beta: 180 mg/dl

Non esterified fatty acids (NEFA, FFA)

P

10-20 mg/dl

pCO2, arterial

B

35-45 mmHg

pH

B

7.35- 7.45

[H+] = 40 nmol/L

Phosphate

S U B

3-4 mg/dl 1 g/day 40 mg/dl

1-1.5 mmol/L 32 mmol/day

Phospholipids

100-200 IU/L

0.3-0.7 mEq/L

150-200 mg/dl

2-2.5 mmol/L

pO2, arterial

B

90-100 mmHg

150-220 ml/L

Potassium

S

3.5-5 mEq/L

3.5-5 mmol/L

Prostate specific antigen (PSA)

S

100-500 ng/dl

1-5 µg/L

S CSF

6-8 g/dl 10-30 mg/dl

60-80 g/L

Sodium

S

136-145 mEq/L

136-145 mmol/L

T3 (tri-iodothyronine)

S

120-190 ng/dl

1.8-3 nmol/L

T4 (thyroxine)

S

5-12 µg/dl

65-150 nmol/L

Thyroglobulin (Tg)

S

3- 5 µg/dl

3- 50 µg/L

TSH

S

0.5- 5 µU/ml

0.5- 5 mU/L

Transferrin

S

200- 300 mg/dl

23- 35 µmol/L

Proteins- total

Contd...

262 An Easy Guide for Practical Biochemistry Contd... Analyte Triglycerides Males Females Urea

Sample

Units

SI units

S

50- 200 mg/dl 40- 150 mg/dl

0.5- 2.3 mmol/L 0.4- 1.6 mmol/L 2.4- 4.8 mmol/L

S

20- 40 mg/dl

Urea nitrogen

S/P

8- 20 mg/dl

3- 9 mmol/L

Uric acid, male Female Children

S/P

3.5- 7 mg/dl 3- 6 mg/dl 2- 5.5 mg/ml

0.21- 0.4 mmol/L 0.18- 0.35 mmol/L 0.12- 0.32 mmol/L

Vitamin A

S

15- 50 µg/dl

0.5- 2 µmol/L

Vitamin C

P

0.4- 1.5 mg/dl

23- 85 µmol/L

Vitamin D3

S

1.5- 6 µg/dl

50- 160 pmol/L

Vitamin E

S

0.5- 1.8 mg/dl

12- 42 µmol/L

P- plasma; B- blood; S- serum; E- erythrocyte; U- urine; CSF- cerebrospinal fluid pg- picogram; ng- nanogram; µg- microgram; mg- milligram; d- day

Appendix 5: Normal Values (Reference Values) 263 CONVERSION CHART Units of length 1 megameter (M)

106

1 kilometer (km)

103

1 meter (m)

1

1 centimeter (cm)

10-2 m

1 millimeter (mm)

10-3 m

1 micrometer (µm)

10-6 m

1 nanometer (nm)

10-9 m

1 angstrom (A)

10-1° m

1 picometer (pm)

10-12 m

1 femtometer (fm)

10-15 m

Units of mass 1 megagram (Mg)

106 g

1 kilogram (kg)

103 g

1 gram (g)

1

1 centigram (cg)

10-2 g

1 milligram (mg)

10-3 g

1 microgram (µg)

10-6 g

1 nanogram (ng)

10-9 g

1 picogram (pg)

10-12 g

1 femtogram (fg)

10-15 g

264 An Easy Guide for Practical Biochemistry

Appendix 6 Scheme of Examination Karnataka Rajiv Gandhi University of Health Sciences examination—Biochemistry The allotment of marks as recommended by the university is as follows: INTERNAL ASSESSMENT FOR BIOCHEMISTRY Total Marks: 40 (Theory: 20 + 10 for records and Practical: 10) THEORY AND RECORDS Minimum of three internal assessments are recommended. The internal assessment preceding the University examination will be similar to the University examination. The total marks would be 20. Average marks secured out of two notified internal examination should be reduced to 20. For records 10 marks are allotted. The sum of the marks obtained in theory and records shall be sent to the University. PRACTICALS A minimum of two practical tests is to be conducted, one at the end of each term. Average of the two tests should be reduced to 10 marks and shall be sent to the University. UNIVERSITY EXAMINATION A. Theory : 100 Marks There shall be two sections. The total marks will be 100, with each section carrying 50 marks. The total duration would be 3 hours. There shall be three types of questions. The distribution of topics and weight age of marks in Biochemistry for University examination is as under*:

Appendix 6: Scheme of Examination 265 Type of question and distribution of marks in each paper. Paper I Type of Questions

Paper II

Number of Questions

Marks for each question

Total

Long Essay

1

10

10

1

10

Short Essay

5

5

25

5

5

25

Short Answer

5

3

15

5

3

15

Total Marks

Number Marks of for each Questions question

Total

10

50

50

Distribution of topics for each paper and weightage of marks in university examination is as follows: Paper I 1. Cell structure and function, subcellular organelles, cell membranes, Transport across the membranes. 2. Chemistry, digestion, absorption and metabolism of Carbohydrates 3. Chemistry, digestion, absorption and metabolism of lipids 4. Amino acids and protein chemistry, general reactions of amino acids, Digestion and absorption, urea cycle and metabolism of amino acids. 5. Endocrine functions and Biochemical tests. 6. Detoxification and Xenobiotics. 7. Enzymes 8. Biological oxidation, integration of metabolism, TCA cycle and regulation of metabolism. 9. Free radicals and antioxidants. 10. Biochemistry of cancer, oncogenes and tumor markers

Weightage of marks 05 10 10

10 05 05 10 10 05 05

266 An Easy Guide for Practical Biochemistry Paper II

Weightage of marks 05

1. Nucleotides and nucleic acid chemistry 2. Purine and pyrimidine nucleotide metabolism, DNA metabolism, RNA metabolism, Protein Biosynthesis. 3. Vitamins 4. Minerals 5. Molecular genetics, regulation of gene expression, recombinant DNA technology, PCR and gene therapy. 6. Electrolyte and water balance, acid base balance 7. Nutrition and energy metabolism 8. Heme metabolism, normal and abnormal hemoglobin’s, Plasma proteins and immunoglobulin. 9. Liver function tests 10. Kidney function tests 11. Clinical chemistry, quality control, interpretation and reference values and analysis

10 10 10 05 10 10 10 05 05 05

Note: a. Weightage of marks assigned to chapters/topics may add to more than 50. b. Long essay questions may be asked from topics with weight age of 10 marks. c. Short Essay and short answer question may be asked from any of the topics. * The topics assigned to the different papers are generally evaluated those sections. However, a strict division of the subject may not be possible and some overlapping of topics is inevitable. Students should be prepared to answer overlapping topics.

PRACTICAL EXAMINATION: 40 MARKS The Practical examination consists of two exercises, Practical I and II, each of 2 hours duration and each exercise carrying 20 marks.

Appendix 6: Scheme of Examination 267 Exercise I: Two hours, 20 marks 1. Quantitative estimation-Every candidate shall perform one given procedure. a. Principle and procedure for the estimation asked in the question should be written by the candidate in the first five minutes. Marks: 5 b. After collecting the papers, correct procedure for the estimation is given if asked by the student and practical examination is done. Total marks would be 15 and the distribution of marks would be: (i) results (values) 10 (ii) calculations and reporting (iii) for interpretation of results and application of the estimation c. Case studies, Graphs and Charts-Discussion 1 × 5 = 5 marks Exercise II: Two hours, 20 marks 1. Qualitative analysis–Every candidate shall perform one given procedure such as Identification of Carbohydrates, Proteins, Substance of Physiological importance, Analysis of normal Urine, Analysis of abnormal Urine. Total marks would be 20 and Distributions of marks would be: For selection of appropriate reactions 5 marks For reasoning of analysis and correct reporting 5 marks For interpretation of results and application of the estimation 5 marks 2. Five Spotters Biochemical TechniquesChromatography, Electrophoresis, Osazone preparation, Biochemical Tests and Reagents 1 x 5 = 5 marks Viva Voce Examination: 20 Marks The viva voce examination shall carry 20 marks and all the examiners will conduct the viva examination.

Index A Albumin 61 casein 62 chemical properties 62 aldehyde test for indole nucleus 67 Biuret test 62 Millon’s test 66 Molisch test for carbohydrate moiety in proteins 71 ninhydrin test 64 Pauly’s test for histidine and tyrosine 70 Sakaguchis test for guanidine group 68 sulfur test cystine and cysteine 69 test for organic phosphorus 72 xanthoproteic test 65 functions 61 physical properties 62 physical properties of casein 62 Alkaptonuria 124 procedure 125 spot test 125 Amino acid 51 Analysis of abnormal urine 102 chemical constituents 102 Gerhardt’s test for acetoacetic acid 109 heat and acetic acid test 102

Rothera’s test fro acetone and acetoacetic acid 109 sulfosalicylic acid test 106 test for bile pigments 113 test for bile salts 112 test for blood 114 test for ketone bodies 109 test for reducing sugar (Benedict’s test) 106 physical characteristics 102 Analysis of normal urine 86 chemical tests 88 test for ammonia 92 test for chloride 88 test for creatinine (Jaffe’s test) 96 test for ethereal sulfate 97 test for inorganic sulfates 89 test for phosphates and calcium 90 test for urea 94 physical examination 86 determination of specific gravity 86 Long’s coefficient 87

B Beer’s law 133 Bence Jones protein 102

270 An Easy Guide for Practical Biochemistry Benedict’s uric acid test 95 Benzidine test 218

C Carbohydrates 29 classification 29 monosaccharides 29 oligosaccharides 30 polysaccharides 31 functions 31 reactions 31 reaction with acids 32 reaction with alkalies 32 tests for carbohydrates 32 Barfoed’s test 39 Benedict’s test 35 iodine test 34 Molisch test 33 osazone test 42 Seliwanoff’s test 40 Chromatography 169 paper chromatography 169 application 173 procedure 171 requirement 171 types 169 gas liquid chromatography 169 gel chromatography 169 high pressure liquid chromatography 169 ion exchange chromatography 169 paper chromatography 169 thin layer chromatography 169 Cleaning of glassware 25 Cole’s mercuric nitrite test 66

Collection and preservation of urine specimens 18 collection of urine samples 18 preservation of urine samples 18 Collection of blood 16 anticoagulants 17 Colorimeter 130 application 137 components 130 adjustable slit 130 condensing lens 130 cuvette (sample holder) 131 filter 130 galvanometer 131 photocell/detector 131 source of light 130 selection of filter in colorimetric estimation 136 calculations 137 steps 136 solution for investigation 131 blank 132 standard solution 132 test solution 132 technique 133 Beer’s law 133 Lambert’s law 134 Constituents of urine 99 physical characteristics 99 appearance 100 chemical constituents 102 specific gravity 101 volume 99

Index 271 Creatinine 79 physical properties 80 Jaffe’s test 80 Creatinine clearance test 151 calculation 152 clinical significance 152 diagnostic importance 152 procedure 151 CSF analysis 200 biochemical examination of CSF 200 calculation 202 collection of CSF 200 determination of glucose 202 determination of total protein 201 principle 201 procedure 201 reagents 201

D Derivatives of hemoglobin 117 carboxy-hemoglobin 117 hematin 117 hemin 117 hemochromogen 117 methemoglobin 117 native hemoglobin 117 oxyhemoglobin 117 Detection of hemoglobin and its derivatives 118 direct vision spectroscope 118 principles 118 Determination of serum albumin by bromocresol green method 162 calculations 163 clinical significance 163

procedure 162 reference value 163 report 163

E Electrophoresis 174 applications 175 basic requirements 176 factors affecting migration of charged particles 175 movement of different protein fractions 177 paper electrophoresis 176 separation of plasma proteins 176 types 175 Estimation of albumin in urine 204 aim 204 apparatus 204 principle 204 procedure 204 reagent 204 test for Bence Jones proteins in urine 205 Estimation of ascorbic acid 195 aim 195 calculation 196 method 195 principle 195 procedure 195 reagents 195 Estimation of blood sugar 138 choice of blood specimen 138 estimation of blood sugar by Folin-Wu’s method 139 aim of the test 139 calculation 141 method 139

272 An Easy Guide for Practical Biochemistry principle 139 procedure 140 reagents 140 methods of estimation 138 Folin-Wu’s method 138 glucose oxidase method 138 Nelson-Somogyi method 138 O-toluidine method 138 preservation of blood 139 Estimation of blood urea 144 aim 144 calculation 145 clinical significance 145 method 144 principle 144 procedure 144 protocol 145 report 145 Estimation of serum AST and ALT 188 aim 188 estimation of ALT 189 estimation of SGOT (AST) 190 procedure 190 method 188 Reitman and Frankel method 188 principle 188 procedure 189 protocol 189, 190 calculation 190 reagents 189 Estimation of serum cholesterol 192 aim 192 calculation 193 clinical significance 193 method 192 normal range 193

precaution 193 principle 192 procedure 193 sample 192 Estimation of serum inorganic phosphate 154 aim 154 calculation 156 clinical significance 156 method 154 Fiske and Subbarow method 154 principle 154 procedure 155 color development 155 preparation of proteinfree filtrate 155 protocol 156 reagents 155 ANSA reagent 155 molybdic acid reagent 155 report 156 Estimation of serum total proteins 159 aim 159 method 159 Biuret method 159 principle 159 procedure 160 calculation 161 color development 160 precipitation of globulins 160 protocol 161 reagents 160 Estimation of urine creatinine 148 aim 148 calculation 149 method 148 principle 148

Index 273 procedure 149 protocol 149 report 150

F Fanconi’s syndrome 158 Flame photometer 197 parts 198 nebulizer 198 needles 198 spray chamber 198 principle 197 procedure 199 Folin-Wu method 223 Fouchet’s test 113 Fraunhofer’s lines 119

G Gerhardt’s test 217 Glucose tolerance test 181 decreased tolerance 183 details of performing the test 185 importance 185 increased tolerance 183 lag type 182 methods used for estimation of blood sugar 186 methods used for estimation of urine sugar 186 estimation of urine sugar by Benedict’s qualitative method 187 normal response 181 procedure 186 renal glycosuria 185 significance 181 Gmelin’s test 113, 225

H Hartnup disease 228 Hay’s test 112 Heat and acetic acid test 59 Heller’s test 218 Homocystinuria 125 procedure 126 reagents 126 screening test for homocystinuria 126 Hopkins-Cole-Adam’s test 67

I Importance of precipitation of proteins 50 Isoelectric precipitation 54

K Kussmaul’s breathing 244

L Laboratory first aid 7 in case of accidents in laboratory 9 precautions while handling chemicals 7 waste disposal 9 chemical waste 9 radioactive waste 9 Laboratory glasswares 20 beakers 20 bottles 22 drop bottles 22 reagent bottles 22 burettes 21 flasks 20 conical flasks 20

274 An Easy Guide for Practical Biochemistry flat-bottomed round flasks 20 round bottomed flasks 21 volumetric flasks 21 funnels 21 graduated (measuring) cylinder 21 Laboratory hazards 3 accidental swallow of corrosive solutions 6 burns 6 care while pipetting 5 in case of accidental fire 5 inhalation of corrosive gases 6 precautions regarding fire safety 5 safety measures 3 Laboratory safety rules 10 accidents and injuries 11 handling chemicals 12 handling glassware and equipment 12 heating substances 13 pipetting techniques 14 Lambert’s law 134

M Maple syrup urine disease 228

N Neumann’s test 72, 73 Nippe’s fluid 122 Normal urine 82 chemical characteristics 85 constituents 85 reaction 85

examination of urine 82 chemical examination 82 microscopic examination 82 physical examination 82 general and physical characteristics 83 appearance 84 color 84 odor 84 specific gravity 84 volume 83 preservation of urine samples 83 specimen collection 82

P Pauly’s test 71 Phenylketonuria 123 ferric chloride test 124 Guthrie’s bacterial inhibition test 123 Photometry 129 principle 129 Pipettes 22 auto pipettes 24 fixed volume type 24 variable volume adjustable type 24 manual pipettes 22 deliver type of pipettes 22 graduated pipettes 23 micropipettes 23 pasteur pipettes 23 volumetric pipettes 23 Precipitation by alkaloidal reagents 56 Precipitation by heavy metal ions 58

Index 275 Precipitation by organic solvents 55 Precipitation by salts 51 Preparation of hemin crystals 122 Preparation of hemoglobin and derivatives 119 carboxy hemoglobin 120 methemoglobin 120 oxyhemoglobin (HbO2) 119 reduced hemoglobin 120 Proteins 48 classification 48 conjugated proteins 48 derived proteins 48 secondary derived proteins 49 simple proteins 48 functions 49 reactions 49 color reactions of protein 49 precipitation reactions of proteins 49

Q Qualitative detection of protein 61 Quantitative estimation of proteins 61

R Relationship between absorbance and transmittance 135 Rothera’s test 217 Ruhemann’s purple 64

S Schiff’s test 96

T Test tubes 24 centrifuge tubes 25 desiccators 25 dispensers 25 Folin-Wu tubes 25 Types of colloids 50 emulsoids 50 suspensoids 50

U Unknown carbohydrate 47 Unknown protein 73 Urea 74 chemical tests 74 Biuret formation 74 sodium hypobromite test 75 specific urease test 76 physical properties 74 tests for urea 74 Uric acid 77 chemical tests 77 murexide test 78 phosphotungastic acid reduction test/ Benedict’s uric acid test 77 physical properties 77

V van den Bergh test 218