Practical Biochemistry: An Easy Guide For

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
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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 • Lifting and turning patients
• Crutch walking
Specific Gravity
• Underwater exercises • Examination of the body fluids
3
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 • Application of heat and cold
application
• Steam inhalation • Basal metabolism
• Thermography • Autoclave and sterilization
Light and Sound

• Actions of lenses • Use of mirrors in apparatus
• Microscopy • Ophthalmoscope
• Refraction • Visual fields
• Audiometery • Human audibility
Electricity
• Patient monitors, ECG, • Diathermy
EEG, EMG
• Electrosurgical procedures • Electric shock therapy
• Use of transistors in • Cardiac pacemakers
apparatus
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
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
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
 Table 1.1: Systems of units with their standards of measurement
Physical CGS system FPS system MKS system SI system

quantity
Length Centimeter (cm) Foot (f) Meter (m) Meter (m)

Mass Gram (gm) Pound (d) Kilogram (kg) Kilogram (kg)
Time Second (s) Second (s) Second (s) Second (s)
Temperature — Fahrenheit (F) Celsius (°C) Kelvin (K)
Electric current — — — Ampere (A)
Light intensity — — — Candela (Cd)
Amount of — — — Mole (mol)
substance
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 10 millimeters (mm) = 1 centimeter (cm)

3 feet = 1 yard 10 centimeter (cm) = 1 decimeter
5 ½ yard = 1 rod 10 decimeter = 1 meter (m)
1,760 Yard = 1 mile 10 meters = 1 decameter
5,280 feet = 1 mile 10 decameters = 1 hectometer
10 hectometers = 1 kilometer (km)
10 kilometers = 1 myriameter
Note: 1 feet = 12 inches = 30 cm (1 inch = 2.5 cm)
7
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 Metric system
Troy units
24 grains = 1 pennyweight 10 milligrams = 1 centigram
20 pennyweight = 1 ounce 10 centigram = 1 decigram
12 ounces = 1 pound 10 decigrams = 1 gram
Avoirdupois units 10 grams = 1 decagram
27.34 grains = 1 dram 1 10 decagrams = 1 hectogram
6 drams = 1 ounce 10 hectograms = 1 kilogram
16 0unces = 1 pound 1,000 kilograms = 1 metric ton
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
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 Fluid volume
1 ounce = 8 drams 60 minimus = 1 fluid dram

12 ounces = 1 pound 8 fluid drams = 1 fluid ounce
1000 microgram (Mcg) = 1 milligram (mg) 20 fluid ounce = 1 pint
1000 milligram (mg) = 1 gram (gm) 2 pints = 1 quart (1000ml)
1000 grams (g) = 1 kilogram (kg) 8 pints = 1 gallon
1 kilogram (kg) = 2.2 pounds 1 milliliters (ml) = 15 – 16 minims
1 grain = 60 milligram (mg) (15–16 drops)
1 dram = 4 grams (g) 1 liter = 35 fluid ounce
1 ounce = 30 grams 1 fluid ounce = 30 ml
1 pound = 375 grams 1 fluid dram = 4 ml
1 milligram = 1/60 grains (gr) 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
 Table 1.5: Prefixes and symbols used with SI units and Metric units
Prefix Symbol Power Value (in meter)
Tera T 1012 1,000,000,000,000

Giga G 109 1,000,000,000
Mega M 106 1,000,000
Kilo K 103 1,000
Hecto H 102 100
Deca Da 10 10
Meter m 1 1
Deci D 10–1 .1
Centi C 10–2 .01
Milli Mm 10–3 .001
Micro u 10–6 .000001
Nano n 10–9 .000000001
Pico p 10–12 .000000000001
Femto f 10–15 .000000000000001
Atto A 10–18 .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
 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
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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.
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(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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14. Meyer B Jackson. Molecular and cellular biophysics. New York:

Cambridge Publication, 2006.
15. Nicolas Rashevsky, Mathematical biophysics. . Rev. ed., University of
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12
Section 1
Laboratory
Rules and
Regulations
Laboratory Hazards
1 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.
• 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.
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.
From strong alkalies:

• Wash immediately with LOTS of water and later with
5% boric acid or dilute acetic acid solution.
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.
• 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.
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.
Laboratory Safety Rules

2
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.
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.
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
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.
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.
Specimen Collection
3 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 anticoagu-
lants 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
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.
Glasswares Used in
4 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.
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.
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 blow-
out 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.
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.
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
Qualitative Analysis of
5 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, oligosaccha-
rides 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 Type of Aldoses Ketoses

carbon sugar
1 Trioses Glyceraldehyde Dihydroxyacetone
2 Tetroses Erythrose Erythrulose
3 Pentoses Ribose, xylose Ribulose, xylulose
4 Hexoses Glucose, galactose, Fructose
mannose
5 Heptoses Glucoheptose Sedoheptulose
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 Example Type of

oligosaccharide monosaccharide monosaccharide present
Disaccharide Two Maltose Glucose + Glucose
Lactose Glucose + Galactose
Sucrose 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
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. Carbohydrates are the main source of energy in the body.
2. Storage form of energy (starch and glycogen)
3. Excess carbohydrate is converted to fat.
4. 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.
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
hydroxymethyl furfural. Keto- hexoses like fructose yield
greater amount of furfural derivative than aldohexoses
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.
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. Color: Colorless except Starch which is pale white
2. Clarity: Clear except Starch which is cloudy
3. Odor: Odorless
4. 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 Inference

Take 2 ml of given Violet ring at the junction Given
solution in a clean of the two liquids is solution is a
dry test tube; add formed. carbohydrate.
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.
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.
Iodine reagent: 0.5 ml of iodine diluted to 5 ml with distilled

water.

Take 2 ml of given Blue color is formed. Given solution is a
solution in a test polysaccharide.
tube. Add 2-3 drops
of Iodine solution.
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.
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 shelf-
life of the reagent by preventing an interaction between
sodium carbonate and copper sulfate, which may
otherwise get precipitated as cupric carbonate.

Take 5 ml of Brick-red precipitate Given solution is a
Benedict’s reagent is formed reducing sugar.
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.
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 Absence of reducing sugar - Nil
color: - no precipitate.
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%
• 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
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:

Take 5 ml of Benedict’s Brick-red On acid hydrolysis
reagent in a test tube; precipitate Sucrose is converted to
add 8 drops of is formed reducing
neutralized hydrolysate Monosaccharides
solution. Boil for (Glucose + Fructose)
2 min.
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,
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:

Take 5 ml of Brick-red On acid
Benedict’s reagent precipitate hydrolysis starch
in a test tube; is formed gives reducing
add 8 drops of sugars.
neutralized hydroly-
sate solution. Boil for
2 min.
Barfoed’s Test ( Reduction under acidic medium)

Principle: In mild acidic medium reducing sugars undergo
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.

Take 2 ml of Barfoed’s Floating red Given solution is
reagent in a test tube; precipitate a monosaccharide
add 1 ml of given is formed.
solution. Mix and
boil for 30 seconds.
Allow it to cool at
room temperature.
Points to Remember:
• It is a specific reduction test for reducing mono-
saccharides.
• 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.

Take 3 ml of Cherry red Given
Seliwanoff’s reagent color is formed. solution is a
in a test tube; ketosugar
add 1 ml of given
solution. Boil for
30 seconds and allow
it to cool at room
temperature.
Points to Remember:
• This test is specific for ketohexoses only.
• Useful in differentiating aldohexoses and keto-
hexoses.
• 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.
Seliwanoff’s test (for sucrose)

• In case of sucrose, the below procedure has to be followed.

Take 3 ml of Cherry red color Hydrolyzed
Seliwanoff’s reagent is formed sucrose contains a
in a test tube; add ketosugar.
1 ml of acid
hydrolysate.
Boil for 30 seconds.
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.
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 main-
tain 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.
Osazones Minimum time Appearance of crystals

for formation
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
• 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.
• 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 sum-
marized below:
Test Molisch Iodine Benedict’s Barfoed’s Seliwanoff’s

Glucose + - + + -
Fructose + - + + +
Lactose + - + - -
Maltose + - + - -
Sucrose + - - (+ after - + after acid
acid hydrolysis
hydrolysis)
Starch + + - (+ after - -
acid
hydrolysis)
IDENTIFICATION OF UNKNOWN CARBOHYDRATE
Flow chart 5.1: Scheme for identification of unknown

carbohydrate
6 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 non-
protein 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
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.
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:

Take 3 ml of protein a. White precipitate a. Protein in the
solution in a test is formed. given solution is
tube. Add equal b. No white precipitated.
volume of saturated precipitate b. Protein in the
ammonium sulfate is formed. given solution is
solution Mix and not precipitated
allow to stand by half-saturation
for 5 min. with ammonium
sulfate.
Filter it. Perform a. No violet
Biuret test with color is formed.
the filtrate by b. Violet color
adding 3 ml of is formed
40% sodium
hydroxide and 2-3
drops of 1%
copper sulfate.
Note: Filterate which contains protein gives violet color with Biuret
test.
b. Full saturation with ammonium sulfate:

Take 3 ml of protein a. White precipitate a. Protein in the
solution in a test is formed. given solution
tube. Add solid b. No white is precipitated.
ammonium sulfate precipitate b. Protein in the
in small quantities is formed. given solution is
at a time with not precipitated
mixing until the by full saturation
solution is saturated. with ammonium
Allow to stand for sulfate.
5 minutes.
Contd...
Contd...
Filter it. Perform a. No Violet color

Biuret test with the is formed.
filtrate by adding b. Violet color
3 ml of 40% sodium is formed
hydroxide and
2-3 drops of
1% copper sulfate.
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 ‘Salting-
in’.
• 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.
• 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.

Take 3 ml of casein A curdy green At pH 4.6,
solution in a test precipitate casein is
tube. Add 3 drops will be formed. precipitated.
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.
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.
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
causing aggregation, precipitation and denaturation of

proteins.

Take 1 ml of protein A white Protein is
solution in a test precipitate precipitated by
tube. Add 2 ml of is formed. organic solvents.
ethanol. Mix.
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.
a. Take 2 ml of A thick yellow Protein is
protein solution precipitate precipitated by
in a test tube. is formed. alkaloidal reagent.
Add picric acid
drop by drop.
Contd...
Contd...
b. Take 2 ml of A white Protein is
protein solution precipitate precipitated by
in a test tube. is formed alkaloidal reagent.
Add trichloro-
acetic acid drop
by drop.
c. Take 2 ml of A white Protein is

protein solution precipitate precipitated
in a test tube. is formed. by alkaloidal
Add sulfo- reagent.
salicylic acid
drop by drop.
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 protein-
tungstate, protein-picrate, etc. to form thick flocculent
precipitate.
• Tanning in leather processing is based on the protein
precipitating effect of tannic acid.
• 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.
a. Take 2 ml of White precipitate Protein is
protein solution is seen. precipitated by
in a test tube. heavy metals
Add 10% lead like lead.
acetate solution
drop by drop.
b. Take 2 ml of White precipitate Protein is

protein solution is seen. precipitated by
in a test tube. heavy metals
Add 5% mercuric like mercury.
nitrate solution
drop by drop.
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.
Take albumin A white coagulum is seen Indicates the
solution upto ¾ th at the upper heated presence of heat
test tube. Hold the portion, which gets coagulable
test tube over a flame intensified after the protein
in a slanting position addition of acetic acid. like albumin.
and boil the upper
portion of the
albumin solution.
The lower portion
serves as control.
Add 1% acetic acid
drop by drop.
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.
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.
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. Color: Pale white
2. Clarity: Cloudy
3. Odor: Odorless
4. Reaction to litmus: Neutral.
Physical Properties of Casein

1. Color: Pale white
2. Clarity: Cloudy
3. Odor: Characteristic odor
4. 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.

Take 2 ml of protein Violet color Indicates the
solution in a test is formed presence of
tube. Add 2 ml of peptide linkage.
5% sodium
hydroxide. Mix
and add one or
two drops of 1%
copper sulfate.
Points to Remember:
• It is a general test for proteins.
• The reaction is so named since Biuret (NH2-CO-NH-CO-
NH2) 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.
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.

Take 1ml of protein Bluish purple Indicates the
solution in a test color is formed. presence of free
tube. Add 10 drops α-amino acids.
of Ninhydrin.
Heat to boiling.
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.
Take 3 ml of protein In acid medium Indicates the
solution in a test tube. yellow color presence of
Add 1ml of conc. Nitric is formed. aromatic amino
acid and mix. Heat the acids. (Tyrosine
solution for one and tryptophan).
minute and cool
it under tap water.
Observe the color.
Divide the contents
into two parts.

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

Take 1ml of protein Red coagulum Indicates the
solution in a test is formed. presence of hydroxy-
tube. Add 1ml of phenyl group
10% mercuric (tyrosine).
sulfate. Boil gently
for 30 seconds.
Cool it under tap
water. Add 3 drops
of 1% sodium
nitrite solution.
Mix and
observe.
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.

Take 3 ml of protein A purple or violet Indicates the
solution in a test tube color is formed at presence of
Add 2 drops of 0.2% the junction of Indole ring
formalin and a drop the two liquids. (tryptophan).
of 10% mercuric
sulfate. Add
carefully 3 ml
of conc. Sulfuric
acid along the
sides of the test tube.
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.
Take 3 ml of protein Bright red color Indicates the
solution in a test is formed. presence of arginine.
tube. Add 5-6 drops
of 40% sodium
hydroxide and
4 drops of Molisch
reagent. Mix and
add 10 drops of
bromine water.
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

Take 2 ml of protein Black or brown color Indicates the
solution in a test precipitate is formed. presence of
tube. Add 2 ml of cystine or
40% sodium cysteine
hydroxide. Boil residues.
for one minute.
Cool it under
tap water. Then add
5 drops of lead
acetate.
Points to Remember:
• This test is specific for –SH (thiol) group of cysteine and
cystine.
• It is a test for sulfur- containing amino acids.
• 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.

Take 1 ml of 0.5% Cherry red color Histidine is
sulfanilic acid in a is formed. present.
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% Orange red color Tyrosine is
sodium carbonate is formed. present.
to make the solution
alkaline.
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.

Take 2 ml of protein Violet ring at the Indicates the
solution in a clean junction of the presence of bound
dry test tube; add two liquids carbohydrate.
1-2 drops of is formed.
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.
Point to Remember:
• Egg albumin has bound carbohydrate.
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.

Take 5 ml protein Canary yellow Indicates the
solution in a test precipitate presence of
tube. Add 0.5 ml is formed. phosphoprotein.
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.
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.
IDENTIFICATION OF UNKNOWN PROTEIN
Flow chart 6.1: Scheme for identification of

an unknown protein
Nonprotein Nitrogenous
7 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. Color: Colorless
2. Clarity: Clear
3. Odor: Odorless
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.
Take a pinch of Urea Violet color Indicates the
crystals in a dry test is formed. presence of urea.
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.
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.

Take 2 ml Urea Brisk effervescence Indicates the
solution in a test of Nitrogen gas is seen. presence of
tube. Add 5 drops urea.
of freshly prepared
alkaline sodium
hypobromite solution
and mix.
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.

Take 3 ml of urea Pink color is formed Urea is
solution in a test confirmed.
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.
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.
• 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. Color: Colorless
2. Clarity: Clear
3. Odor: Odorless
4. 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.
Benedict’s uric acid reagent: Composed of sodium tungstate,

orthophosphoric acid, concentrated sulfuric acid and solid
sodium carbonate.

Take 3 ml uric Deep blue Indicates the
acid solution in a color is formed. presence of
test tube; add 1 ml uric acid.
of Benedict’s uric
acid reagent and
1 ml of 20% sodium
carbonate. Mix.
Schiff’s Test
Principle: Uric acid reduces salts of silver nitrate to metallic
silver.

Moisten a piece of Black spots Indicates the
filter paper with on the filter presence of
few drops of paper are seen. Uric acid.
ammoniacal silver
nitrate solution.
Add 2-3 drops
of uric acid on the
silver nitrate drops.
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.
This combines with ammonia to form ammonium purpurate

or murexide which is purple red in color.

Take 5 ml of uric A purplish red Indicates the
acid in a china dish color is formed. presence of
and add 5 drops Uric acid.
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.
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
Physical Properties
1. Color: Colorless
2. Clarity: Clear
3. Odor: Odorless
Jaffe’s Test
Principle: Creatinine reacts with picric acid in the presence
of alkaline medium to form reddish orange colored
creatinine picrate.

Take 3 ml of Reddish orange Indicates the
creatinine solution color is formed. presence of
in a test tube. Creatinine.
Add 1 ml saturated
picric acid solution,
and 3-4 drops of
10% sodium
hydroxide.
Report: The given NPN substance is —————.

IDENTIFICATION OF UNKNOWN SUBSTANCE OF PHYSIOLOGICAL IMPORTANCE
Flow chart 7.1: Schematic representation for identification of an unknown substance of

physiological importance
8 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, precipita-
tion 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.
• 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.
• 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).
• 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
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 Blue litmus Normal urine is acidic.
litmus turns red
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).
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.
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.

Take 3 ml of urine A white Indicates the
in a test tube. precipitate presence
Add 0.5 ml of conc. is formed. of chloride.
Nitric acid and
1 ml of 3% silver
nitrate.
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.
Take 3 ml of urine A white Indicates the
in a test tube. precipitate presence of
Add 1 ml of conc. is formed. inorganic
Hydrochloric acid. sulfates.
Mix well and add
2 ml of 10% barium
chloride.
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
yellow precipitate of ammonium phosphomolybdate

reacting with ammonium molybdate.

To one part of the Canary yellow Indicates the
filtrate, add a few precipitate presence of
drops of conc. is formed. inorganic
Nitric acid and a phosphates.
pinch of ammonium
molybdate. Warm.
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.

To the second part White precipitate Indicates the
of the filtrate, add is formed. presence
2 ml of 2% potassium of calcium.
oxalate solution.
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.

Take 2 ml of urine Red litmus Indicates the
in a test tube. Add changes to blue. presence of
1-2 drops of ammonia.
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.
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.
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.
Take 3 ml of urine Brisk effervescence Indicates the
in a test tube. of Nitrogen gas presence of urea.
Add 5 drops of is seen.
freshly prepared
alkaline sodium
hypobromite solution
and mix.
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.
Take 3 ml of urine in a Pink color is formed. Urea is confirmed.
test tube. Add 3 ml of
Urease suspension
and add 1-2 drops of
phenolphthalein indi-
cator. Warm the tube
for a few seconds with
the hands and keep for
10 minutes.
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.
– High protein diet
– Fever
– Diabetes mellitus
– Liver diseases
– Nephritis
– Acidosis
Test for Uric Acid

Phosphotungstic Acid Reduction Test/ Benedict’s Uric Acid Test:
Principle: Uric acid in alkaline condition reduces phos-
photungstic acid to tungsten blue.
Benedict’s uric acid reagent: Composed of sodium tungstate,
orthophosphoric acid, concentrated Sulfuric acid and solid
sodium carbonate.

Take 3 ml of urine Deep blue color Indicates the
in a test tube; add is formed. presence of
1 ml of Benedict’s Uric acid
uric acid reagent
and 1 ml of 20%
sodium carbonate.
Mix.
ii. Schiff’s Test:

Principle: Uric acid reduces salts of silver nitrate to metallic
silver.

Moisten a piece of Black spots are Indicates the
filter paper with seen on the presence of
few drops of filter paper. Uric acid.
ammoniacal silver
nitrate solution.
Add 2-3 drops of
urine on the silver
nitrate drops.
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.
– 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.

Take 3 ml of urine Reddish orange Indicates the
in a test tube. color is formed. presence of
Add 1 ml saturated creatinine.
picric acid solution,
and 3-4 drops of
10% sodium
hydroxide.
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.
– High intake of meat, fish
– Fever
– Myopathy/wasting diseases
– 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.

Take 5 ml of urine Trace turbidity is Indicates the
in a test tube. Add seen over that in presence of organic
2 ml of 10% barium control. sulfate.
chloride and 2 ml
concentrated
hydrochloric acid.
Mix and filter.
Divide the filtrate
into two portions.
Boil one and compare
with the control.
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.
– 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.
Analysis of Abnormal
9 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
• 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, Bile pigments
foaming
Yellow fluorescent, Riboflavin
non-foaming
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
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.

Take 10 ml of urine A white coagulum is Indicates the
in a test tube. Hold seen at the upper presence of heat
the tube over a flame heated portion, which coagulable
in a slanting position gets intensified after protein like
and boil the upper the addition of albumin.
5 ml of the albumin acetic acid.
solution. The lower
half serves as control.
Add 1% acetic acid
drop by drop.
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.
• 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 • Violent exercise
proteinuria • Cold bath
• Pregnancy
2. Organic • Cardiac diseases
proteinuria • Abdominal tumors
a. Prerenal • Cancer
• Collagen diseases
• Fever
• Anemia
b. Renal • Acute and chronic
glomerulonephritis
• TB kidneys
c. Postrenal • 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 +++
• 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.

Take 3 ml of nitric A white ring is formed Indicates the
acid in a test tube. at the junction of the presence of
Add 3 ml of urine two liquids. protein.
along the sides of
test tube.
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.
Sulfosalicylic Acid Test

Principle: Sulfosalicylic acid is an alkaloidal reagent and so
it neutralizes the positively charged protein to produce
precipitation.

Take 3 ml of urine White precipitate Indicates the
in a test tube. Add is formed. presence of
20% sulfosalicylic protein.
acid drop by drop.
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
cuprous oxide which gives different shades of color
precipitate depending upon the concentration of the
sugar.

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.
• Positive Benedict’s test is usually suggestive of presence

of glucose in urine.
• Common causes of glycosuria are:
– 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.
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.
Take 5 ml of urine in a Permanganate Indicates the
test tube. Add solid colored ring presence of
ammonium sulfate a is formed. ketone bodies.
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.
Gerhardt’s Test for Acetoacetic Acid

Principle: Acetoacetic acid gives a red color with ferric
chloride.
Take 3 ml of urine Port-wine color Indicates the
in a test tube and is obtained presence of
add 10% ferric acetoacetic acid.
chloride solution
drop by drop till
maximum precipitate
of ferric phosphate
is formed. Filter. To
the filtrate add
further quantities
of 10% ferric chloride.
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.
• 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 Stable blue/bluish green color
phenylketonuria
Homogentisic acid in Rapidly fading blue or green
alkaptonuria color
β-hydroxyphenyl pyruvic acid Rapidly fading green color
in tyrosinosis
Branched chain amino acids in Blue color
Maple syrup urine disease
Imidazole pyruvic acid in Green color
histidinemia
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
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.

Take 2 ml of urine Sulfur powder Indicates the
in a test tube. Gently sinks to the bottom. presence of
sprinkle a little fine bile salts.
sulfur powder over
the surface of urine.
Observe without
mixing.
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.
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)

Take 5 ml of conc. Various colored rings Indicates the
Nitric acid in a will be formed at the presence of bile
test tube. Add 5 ml point of contact pigments.
of urine carefully to of the two liquids.
form a separate layer. (play of colors).
Fouchet’s Test
Principle: Bile pigments adsorbed on barium sulfate preci-
pitate are oxidized to colored products by Fouchet’s reagent.
Fouchet’s reagent: 10% ferric chloride in 25% trichloroacetic
acid.
Take 5 ml of urine A white precipitate Indicates the
in a test tube. Add of barium presence of bile
few crystals of sulfate is formed. pigments.
magnesium sulfate. A green color develops
Then add 3 ml of on the filter paper.
10% barium
chloride solution. Mix.
Filter. Unfold the
filter paper. Add a
few drops of
Fouchet’s reagent
on the precipitate.
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.

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.
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 Hemo-
globinuria.
• 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
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
Hemoglobin and its

10 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.
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.
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
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
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.
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/ medico-
legal importance: Used in
forensic medicine to detect
traces of blood.
Fig. 10.3: Hemin crystals
(Label)
Spot Tests 123
Spot Tests
11
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.
Ferric Chloride Test

Take 3 ml of urine Bluish green color Indicates the
in a test tube and is obtained presence of
add 10% ferric phenylpyruvate
chloride solution
drop by drop till
maximum precipitate
of ferric phosphate
is formed. Filter.
To the filtrate
add 2 ml of 10%
ferric chloride.
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.
• 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. Solid sodium chloride
2. Ammonia
3. Silver nitrate
4. Sodium nitroprusside solution
5. 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
Principles of
12 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.
Wavelength Color of light Color of

(in nm) absorbed light reflected
400-435 Violet Green-yellow
435-500 Blue Yellow
500-570 Green Red
570-600 Yellow Blue
600-630 Orange Green-blue
630-700 Red 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)
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 concen-
tration 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.
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 Red
Purple Green
Yellow Violet
Orange 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.
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.
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.
Intensity of light emerging (I)
T=
Intensity of light incidence (I o )
I
T=
Io
I
% T = 100 
Io
• When Io is 100 (adjusted with blank on the galvanometer
scale), I gives % transmittance.
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.
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
Estimation of
13 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 phospho-
molybdous acid (molybdenum blue), a blue solution. The
intensity of this blue solution is a measure of the amount of
glucose present.
Reagents
1. 10% sodium tungstate
2. 2/3 N Sulfuric acid
3. Alkaline copper reagent
4. Phosphomolybdic acid
5. 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.
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
S – B× Volume of sample × 100
= OD of standard – OD of blank
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.
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 auto-
oxidation 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 hyper-
glycemia and any decrease in blood glucose level is called
hypoglycemia.
• Hyperglycemia is seen in:

– 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 adminis-
tration 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-6-
phosphatase)
– Steatorrhea (due to impaired glucose absorption)
– Starvation
– Alcohol ingestion.
Estimation of
14 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
Distilled water 1 ml - -
Standard - 1 ml -
Serum - - 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.
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.
Estimation of
15 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

Diluted urine - - 5 ml
Std. creatinine solution - 5 ml -
Water 5 ml - -
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
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.
• Creatinine coefficient is milligram of creatinine excreted

in urine per kg body weight in 24 hours. Normally it is 20-
26 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.
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
U × V × 1.73
Creatinine Clearance (ml/min) =
P×A
Where U = mg of creatinine/dl in urine
P = mg of creatinine/dl in serum or plasma
V = Volume of urine in ml/min
A = Body surface area of the patient
1.73 = Standard average surface area of normal
individual
• 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
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.
Estimation of Serum
16 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).
Protocol
Water 5 ml -
Standard phosphate solution - 5 ml -
Protein-free filtrate - - 5 ml
Molybdic acid reagent 1 ml 1 ml 1 ml
Mix
ANSA Solution 0.4 ml 0.4 ml 0.4 ml
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
= × × 100
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.
• 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: glycerophosphates-
nucleotide phosphates, etc. 15- 30 mg/100 ml.
– Phospholipids: lecithin, cephalin, sphingomyelin 10-
16 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
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.
Estimation of Serum
17 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 lipo-
proteins.
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.
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 Albumin Total Protein
Water 3 ml - - 2.9 ml
Standard protein
solution - 3 ml - -
Globulin-free filtrate - - 3 ml -
Serum - - - 0.1 ml
Biuret reagent 3 ml 3 ml 3 ml 3 ml
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
= × × 100
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

= × × 100
AB 6
= × × 100 mg%
S  B 0.1
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:
Bromocresol green reagent 5 ml 5 ml 5 ml
Serum - - 0.05 ml
Albumin standard - 0.05 ml -
Distilled water 0.05 ml - -
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.
Calculations
Serum albumin g/ dl
OD of Test  OD of Blank
= ×4
OD of Standard  OD of Blank
Reference Value
Total serum protein = 6-8 g/100 ml.
Normal Albumin = 3.5-5.5 g/100 ml
Normal Globulin = 2-3.5 g/100 ml
Normal A:G Ratio = 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 _______.
• 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
named because the compound biuret (H2N-CO-NH-CO-

NH2) 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
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
Chromatography
18
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
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 chromato-
graphy) or by gravitation force (descending chromato-
graphy). 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,
Distance traveled by the compound
Rf =
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

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.
Electrophoresis
19
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)
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.
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.
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%.
Glucose Tolerance Test
20
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
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).
Fig. 20.3: Decreased tolerance
Fig. 20.4: Increased tolerance

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.
• 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).
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 60 90 120 150

min min min 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
Estimation of Serum
21 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 Control Standard Blank
Serum (ml) 0.2 - - -
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)
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:

Serum (ml) 0.2 - - -
Standard - - 0.2 -
Distilled water (ml) - - - 0.2
Buffered substrate (ml) 1 1 1 1
Contd...
Estimation of Serum AST and ALT 191
Contd...
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.
Estimation of Serum
22 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
Working reagent 1000 μl 1000 μl 1000 μl
Standard - 10 μl -
Sample - - 10 μl
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.
• 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.
• 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, malnut-
rition, hyperthyroidism, anemia and liver diseases.
Estimation of Plasma Ascorbic Acid 195
Estimation of Plasma
23 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
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
Flame Photometer
24
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.
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.
CSF Analysis
25
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 deter-
mined 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 Test

Standard protein - 1 -
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).
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 (100-
200 ml). It is formed at the rate of about 0.5 ml/minute.
• Earlier it was thought that CSF was formed by ultra-
filtration 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 obstruc-
tion 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 (55-
75%).
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.
Estimation of
26 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 precipi-
tating 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.
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. Investiga-
tions revealed increased levels of serum amylase, serum
lipase and urine amylase.
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%
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
patient.
Serum bilirubin - 10 mg%
AST - 80 units/L
ALT - 90 units/L
Urine
Bile pigments - ++
Bile salts - ++
Urobilinogen - negative
Feces - clay color
Blood coagulation time - prolonged
Obstructive jaundice
II. Causes:
Intrahepatic: chronic active hepatitis, biliary cirrhosis,
lymphoma, primary hepatoma.
Extrahepatic: gall bladder stones, carcinoma of head of
pancreas, enlarged lymph nodes
III. Name the bile pigments.
Bilirubin and biliverdin
IV. Name the bile salts.
Sodium and potassium salts of glycocholic acid and taurocholic
acid
V. 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
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
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
V. Test to detect urine protein

Heat and acetic acid test, sulfosalicylic acid test, Heller’s test
patient. What is your probable diagnosis?
Serum Bilirubin - 10 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
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:
Serum creatinine - 5 mg/dl
Serum inorganic phosphorous - 6 mg/dl
I. Comment on this – Renal failure

II. Normal blood urea level – 15 to 40 mg/dl
III. Normal serum creatinine level – 0.7 to 1.4 mg/dl
IV. Significance of creatinine clearance – To assess kidney function
V. 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
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 pH - 7.25
Ketone bodies in urine - +
I. Probable diagnosis – Starvation ketoacidosis

II.Normal blood pH – 7.35 to 7.45
III.
Normal blood sugar level – 80 to 120 mg/dl
IV.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%
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.
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 urea - 100 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:
Conjugated - 16 mg%
Unconjugated - 4 mg%
SGOT - 45 IU/L
SGPT - 40 IU/L
Alkaline phosphatase - 135 KA units
I. Probable diagnosis – Obstructive jaundice.
II. Normal serum bilirubin level – 0.2 to 0.8 mg/dl.
III. Test done to estimate serum bilirubin – van den Bergh test.
IV. Expected cholesterol level in this case – Cholesterol is elevated.
V. 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
Blood glucose random - 580 mg/dl
Serum creatinine - 1.5 mg/dl
Urine sugar - ++++
Urine ketone bodies - +++
I. Probable diagnosis – Ketoacidosis
II. Normal blood pH 7.35 to 7.45
III. Test to detect urine sugar – Benedict’s test.
IV. Test to detect urine ketone bodies – Rothera’s test.
V. 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
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
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:
Direct - 9 mg%
Indirect - 4.5 mg%
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.
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 Homo-
gentisic acid.
patient.
SGOT - 260 IU/L
SGPT - 290 IU/L
Urine
Bile pigments - +
Urobilinogen - +
Feces –Stercobilinogen - ++
I. What is the probable diagnosis? – Hepatic Jaundice.
III. Test to detect serum bilirubin – van den Bergh test.
IV. Normal alkaline phosphatase level - 3-13 KA units.
V. Name the bile pigments – Bilirubin and biliverdin
patient.
Direct - 0.3 mg%
Indirect - 0.5 mg%
ALT - 14 IU/L
AST - 16 IU/L
Urine
Bile salts - absent
Bile pigments - absent
Urobilinogen - traces
Feces - normal color
I. Probable diagnosis – Normal.

IV. Normal levels of ALT – ALT: 13-40 IU/L.
V. 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 pH - 7.25
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.
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. Your interpretation – Renal glycosuria
II. Renal threshold – 180 mg/dl
III. Test to detect urine sugar – Benedict’s test
IV. Normal fasting blood glucose level – 80-110 mg/dl.
V. 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
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.
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.
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.
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%
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:
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
IV. End product of hemoglobin metabolism – Bilirubin
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 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
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 – Phenylketo-
nuria, 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
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
patient.
SGOT - 30 IU/L
SGPT - 26 IU/L
Urine:
Bile pigments - negative
Urobilinogen - +++ (Excess)
Feces-stercobilinogen - +++ (Excess)
I. Probable diagnosis – Hemolytic jaundice.
II. Normal serum bilirubin level – 0.2-0.8 mg/dl.
III. Test to detect serum bilirubin – van den Bergh.
IV. Normal SGOT level – 8-13 IU/L.
V. 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
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. Comment on this – Normal.
II. Normal serum protein level – 6 to 8 g/dl.
III. Normal serum albumin level – 3.5 to 5 g/dl.
IV. Normal serum bilirubin level – 0.2 to 0.8 mg/dl
V. Test to detect serum bilirubin – van den Bergh test.
49. A patient with acute chest pain showed the following blood
values.
HDL - 20 mg%
SGOT - 52 IU/L
SGPT - 28 IU/L
CPK and LDH values are also raised.
I. Probable diagnosis – MI.
II. Normal serum cholesterol level – 150-200 mg/dl.
III. Isoenzymes of CPK – CPK1 ,CPK2, CPK3.
IV. Isoenzymes of LDH – LDH1 LDH2, LDH3, LDH4, LDH5
V. Normal serum levels of triglycerides and HDL – Tgl = 60-180
mg/dl
HDL = 30-60 mg/dl.
patient.
Blood urea - 30 mg%
Total plasma protein - 4.5 g%
Albumin - 1.0 g%
Globulin - 3.5 g%
Urine protein - 10 g/L
I. Probable diagnosis – Nephrotic syndrome.
II.Normal cholesterol level – 150-200 mg/dl.
III.
Normal A:G ratio – 1.2 : 1 to 1.5: 1
IV.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 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
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
pCO2 - 70 mm of Hg
I. Interpret – Plasma bicarbonate is normal, but pCO2 is high.
Blood pH is low. It is a case of uncompensated respiratory
acidosis.
II. Normal pH of blood – 7.35 to 7.45
III. Name the blood buffer systems – Bicarbonate buffer,
IV. Normal plasma bicarbonate levels – 22 to 26 mEq/L
V. 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
pCO2 - 38 mm of Hg
I. 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.
II. Compensatory mechanism – hyperventilation to wash out
CO2 faster. Increased elimination of acid in the urine and rise
in urinary ammonia.
III. Normal pH of blood – 7.35 to 7.45
IV. Normal plasma bicarbonate levels – 22 to 26 mEq/L
V. Name the blood buffer systems – Bicarbonate buffer,
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 acid-
resistant 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
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.

Colorimeter
b. Mention its use.
To read the optical densities of
colored substances in quanti-
tative estimation.

Urinometer
b. Mention its use.
To determine the specific
gravity of urine

Folin-Wu tube
b. Mention its use.
Used in the estimation of blood
sugar by Folin-Wu tube.
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 asymp-
tomatic 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.

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).

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.
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
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
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
19. a. Identify the slide.
Lactosazone
b. What is the significance of this
test?
Used to differentiate between

Glucosazone/Fructosazone
b. What is the significance of this
test?
Used to differentiate between

Hemin crystals
b. What is the significance?
In forensic medicine to detect
blood stains
22.
Indicators Use PH
Bromocresol Green In isoelectric 4 to 4.6
precipitation test for the
identification of casein
Phenolphthalein In specific urease 8 to 10
test for the detection
of urea
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

24.
Reagent Composition Use
Molisch Alpha Naphthol + To identify
ethyl Alcohol carbohydrates
Benedict’s Copper sulfate + To detect reducing
Sodium citrate + sugars
Sodium carbonate
Barfoed’s Copper acetate + To identify
Glacial Acetic acid monosaccharide
Seliwanoff’s Resorcinol in concentrated To detect ketosugar
Hydrochloric acid
Millon’s Sodium nitrate + To detect
Mercuric sulfate hydroxyphenyl
group-tyrosine
Osazone Phenylhydrazine Identification of
Mixture hydrochloride+ reducing sugars
sodium acetate+ which give
glacial acetic acid characteristic osazone
crystals.
Benzidine Benzidine + Detect blood in urine
glacial acetic acid
Biuret Sodium Potassium In identification of
tartarate + Copper Protein
sulfate
DAM Diacetyl Monoxime + Quantitative
water estimation of
blood urea
ANSA Aminonapthnol Quantitative
sulfonic acid + estimation of
Sodium bisulfate serum phosphate
Sodium sulfite
Nippe’s fluid Potassium bromide + Preparation of hemin
Potassium chloride + crystals
Potassium iodide +
Glacial acetic acid
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.
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.
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 (x̄ – 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.
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.
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.
• 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.
• 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.
• 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
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.
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
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
1 N Sodium Hydroxide
Weigh accurately 40 gm of NaOH crystals and make up to
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.
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
Appendix 5
Normal Values (Reference Values)
Analyte Sample Units SI units
Ammonia P/S < 50 µg/dl
Acid phosphatase, P/S 0.5- 4 KAU/dl 2.5-12 IU/L
(ACP) total
Alanine amino S Male: 13-35 IU/L
transferase (ALT/SGPT) 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 50-120 IU/L
units/dl
Aspartate amino- S 8-20 IU/L
transferase (AST/SGOT)
Bicarbonate S 22- 26 mEq/L 22-26 mmol/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 0.75-1.58 mmol/L
Female: 35- 75 mg/dl 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 16- 30 µmol/L
C- reactive protein (CRP) 0.5-1 mg/dl
Creatine S 0.2- 0.4 mg/dl 15- 30 µmol/L
Creatine kinase S Female: 10- 80 U/L
Male: 15- 100 U/L
Creatinine S 0.7- 1.4 mg/dl 60- 125 µmol/L
U 15- 25 mg/kg/day 15-0.2 mmol/kg/day
Electrophoresis S Albumin: 55-65% 3.5-4.7 g/100 ml
α 1: 2-4% 0.2-0.3 g/dl
α 2: 6-12% 0.4- 0.9 g/dl
β: 8-12% 0.5-1 g/dl
γ: 12-22% 0.7-1.5 g/dl
Contd...
Appendix 5: Normal Values (Reference Values) 261
Contd...
Fibrinogen P 200-400 mg/dl 5.8- 8.5 µmol/L

Globulins S 2.5-3.5 g/dl 25-35 g/L
Glucose (fasting) P 70-110 mg/dl 4-6.1 mmol/L
B 65-100 mg/dl 3.5-5.6 mmol/L
CSF 50-70 mg/dl 2.8-4.2 mmol/L
Hemoglobin B Male: 14-16 g/dl 2.17-2.4 mmol/L
Female: 13-15 g/dl
Glycated hemoglobin
Hb A1c E 4-8% of total
Iodine S 5-10 µg/dl
Iron B 5 mg/dl
Lactate dehydrogenase S 100-200 IU/L
(LDH)
Lipoproteins S Alpha: 40 mg/dl
Beta: 180 mg/dl
Non esterified fatty acids P 10-20 mg/dl 0.3-0.7 mEq/L
(NEFA, FFA)
pCO2, arterial B 35-45 mmHg
pH B 7.35- 7.45 [H+] = 40 nmol/L
Phosphate S 3-4 mg/dl 1-1.5 mmol/L
U 1 g/day 32 mmol/day
B 40 mg/dl
Phospholipids 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 S 100-500 ng/dl 1-5 µg/L
(PSA)
Proteins- total S 6-8 g/dl 60-80 g/L
CSF 10-30 mg/dl
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
Contd...
Contd...
Triglycerides S 50- 200 mg/dl 0.5- 2.3 mmol/L

Males 40- 150 mg/dl 0.4- 1.6 mmol/L
Females
Urea S 20- 40 mg/dl 2.4- 4.8 mmol/L
Urea nitrogen S/P 8- 20 mg/dl 3- 9 mmol/L
Uric acid, male S/P 3.5- 7 mg/dl 0.21- 0.4 mmol/L
Female 3- 6 mg/dl 0.18- 0.35 mmol/L
Children 2- 5.5 mg/ml 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
Appendix 6
Scheme of Examination
Karnataka Rajiv Gandhi University of Health Sciences examina-
tion—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 Paper II
Type of Number Marks Total Number Marks Total
Questions of for each of for each
Questions question Questions question
Long Essay 1 10 10 1 10 10
Short Essay 5 5 25 5 5 25
Short Answer 5 3 15 5 3 15
Total Marks 50 50
Distribution of topics for each paper and weightage of marks

in university examination is as follows:
Paper I Weightage of
marks
1. Cell structure and function, subcellular
organelles, cell membranes, Transport
across the membranes. 05
2. Chemistry, digestion, absorption and
metabolism of Carbohydrates 10
3. Chemistry, digestion, absorption and
metabolism of lipids 10
4. Amino acids and protein chemistry,
general reactions of amino acids,
Digestion and absorption, urea cycle
and metabolism of amino acids. 10
5. Endocrine functions and Biochemical tests. 05
6. Detoxification and Xenobiotics. 05
7. Enzymes 10
8. Biological oxidation, integration of
metabolism, TCA cycle and regulation
of metabolism. 10
9. Free radicals and antioxidants. 05
10. Biochemistry of cancer, oncogenes and
tumor markers 05
Paper II Weightage of
marks
1. Nucleotides and nucleic acid chemistry 05
2. Purine and pyrimidine nucleotide
metabolism, DNA metabolism,
RNA metabolism, Protein Biosynthesis. 10
3. Vitamins 10
4. Minerals 10
5. Molecular genetics, regulation of
gene expression, recombinant DNA
technology, PCR and gene therapy. 05
6. Electrolyte and water balance, acid base balance 10
7. Nutrition and energy metabolism 10
8. Heme metabolism, normal and abnormal
hemoglobin’s, Plasma proteins and
immunoglobulin. 10
9. Liver function tests 05
10. Kidney function tests 05
11. Clinical chemistry, quality control,
interpretation and reference
values and analysis 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 Techniques-
Chromatography, 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 Rothera’s test fro acetone

and acetoacetic acid
Albumin 61 109
casein 62 sulfosalicylic acid test
chemical properties 62 106
aldehyde test for indole test for bile pigments 113
nucleus 67 test for bile salts 112
Biuret test 62 test for blood 114
Millon’s test 66 test for ketone bodies
Molisch test for 109
carbohydrate moiety test for reducing sugar
in proteins 71 (Benedict’s test) 106
ninhydrin test 64 physical characteristics 102
Pauly’s test for histidine Analysis of normal urine 86
and tyrosine 70 chemical tests 88
Sakaguchis test for test for ammonia 92
guanidine group 68 test for chloride 88
sulfur test cystine and test for creatinine (Jaffe’s
cysteine 69 test) 96
test for organic test for ethereal sulfate
phosphorus 72 97
xanthoproteic test 65 test for inorganic sulfates
functions 61
89
physical properties 62
test for phosphates and
physical properties of casein
calcium 90
62
test for urea 94
Alkaptonuria 124
physical examination 86
procedure 125
determination of specific
spot test 125
gravity 86
Amino acid 51
Analysis of abnormal urine 102 Long’s coefficient 87
chemical constituents 102
Gerhardt’s test for B
acetoacetic acid 109
heat and acetic acid test Beer’s law 133
102 Bence Jones protein 102
Benedict’s uric acid test 95 Collection and preservation of
Benzidine test 218 urine specimens 18
collection of urine samples
C 18
preservation of urine
Carbohydrates 29 samples 18
classification 29 Collection of blood 16
monosaccharides 29 anticoagulants 17
oligosaccharides 30 Colorimeter 130
polysaccharides 31 application 137
functions 31 components 130
reactions 31 adjustable slit 130
reaction with acids 32 condensing lens 130
reaction with alkalies 32
cuvette (sample holder)
tests for carbohydrates 32
131
Barfoed’s test 39
filter 130
Benedict’s test 35
galvanometer 131
iodine test 34
photocell/detector 131
Molisch test 33
source of light 130
osazone test 42
selection of filter in
Seliwanoff’s test 40
colorimetric estimation
Chromatography 169
paper chromatography 169 136
application 173 calculations 137
procedure 171 steps 136
requirement 171 solution for investigation
types 169 131
gas liquid blank 132
chromatography 169 standard solution 132
gel chromatography 169 test solution 132
high pressure liquid technique 133
chromatography 169 Beer’s law 133
ion exchange Lambert’s law 134
chromatography 169 Constituents of urine 99
paper chromatography physical characteristics 99
169 appearance 100
thin layer chemical constituents
chromatography 169 102
Cleaning of glassware 25 specific gravity 101
Cole’s mercuric nitrite test 66 volume 99
Index 271
Creatinine 79 procedure 162
physical properties 80 reference value 163
Jaffe’s test 80 report 163
Creatinine clearance test 151
calculation 152 E
clinical significance 152
diagnostic importance 152 Electrophoresis 174
procedure 151 applications 175
CSF analysis 200 basic requirements 176
biochemical examination of factors affecting migration of
CSF 200 charged particles 175
calculation 202 movement of different
collection of CSF 200 protein fractions 177
determination of glucose paper electrophoresis 176
202 separation of plasma
determination of total proteins 176
protein 201 types 175
principle 201 Estimation of albumin in urine
procedure 201 204
reagents 201 aim 204
apparatus 204
D principle 204
procedure 204
Derivatives of hemoglobin 117 reagent 204
carboxy-hemoglobin 117 test for Bence Jones proteins
hematin 117 in urine 205
hemin 117 Estimation of ascorbic acid 195
hemochromogen 117 aim 195
methemoglobin 117 calculation 196
native hemoglobin 117 method 195
oxyhemoglobin 117 principle 195
Detection of hemoglobin and its procedure 195
derivatives 118 reagents 195
direct vision spectroscope Estimation of blood sugar 138
118 choice of blood specimen
principles 118 138
Determination of serum estimation of blood sugar by
albumin by bromocresol Folin-Wu’s method 139
green method 162 aim of the test 139
calculations 163 calculation 141
clinical significance 163 method 139
principle 139 precaution 193
procedure 140 principle 192
reagents 140 procedure 193
methods of estimation 138 sample 192
Folin-Wu’s method 138 Estimation of serum inorganic
glucose oxidase method phosphate 154
138 aim 154
Nelson-Somogyi method calculation 156
138 clinical significance 156
O-toluidine method 138 method 154
preservation of blood 139 Fiske and Subbarow
Estimation of blood urea 144 method 154
aim 144 principle 154
calculation 145 procedure 155
clinical significance 145 color development 155
method 144 preparation of protein-
principle 144 free filtrate 155
procedure 144 protocol 156
protocol 145 reagents 155
report 145 ANSA reagent 155
Estimation of serum AST and molybdic acid reagent
ALT 188 155
aim 188 report 156
estimation of ALT 189 Estimation of serum total
estimation of SGOT (AST) proteins 159
190 aim 159
procedure 190 method 159
method 188 Biuret method 159
Reitman and Frankel principle 159
method 188 procedure 160
principle 188 calculation 161
procedure 189 color development 160
protocol 189, 190 precipitation of globulins
calculation 190 160
reagents 189 protocol 161
Estimation of serum cholesterol reagents 160
192 Estimation of urine creatinine
aim 192 148
calculation 193 aim 148
clinical significance 193 calculation 149
method 192 method 148
normal range 193 principle 148
Index 273
procedure 149 H
protocol 149
report 150 Hartnup disease 228
Hay’s test 112
F Heat and acetic acid test 59
Heller’s test 218
Fanconi’s syndrome 158 Homocystinuria 125
Flame photometer 197 procedure 126
parts 198 reagents 126
nebulizer 198 screening test for
needles 198 homocystinuria 126
spray chamber 198 Hopkins-Cole-Adam’s test 67
principle 197
procedure 199 I
Folin-Wu method 223
Fouchet’s test 113 Importance of precipitation of
Fraunhofer’s lines 119 proteins 50
Isoelectric precipitation 54
G
K
Gerhardt’s test 217
Glucose tolerance test 181 Kussmaul’s breathing 244
decreased tolerance 183
details of performing the test L
185
importance 185 Laboratory first aid 7
increased tolerance 183 in case of accidents in
lag type 182 laboratory 9
methods used for estimation precautions while handling
of blood sugar 186 chemicals 7
methods used for estimation waste disposal 9
of urine sugar 186 chemical waste 9
estimation of urine sugar radioactive waste 9
by Benedict’s Laboratory glasswares 20
qualitative method beakers 20
187 bottles 22
normal response 181 drop bottles 22
procedure 186 reagent bottles 22
renal glycosuria 185 burettes 21
significance 181 flasks 20
Gmelin’s test 113, 225 conical flasks 20
flat-bottomed round examination of urine 82
flasks 20 chemical examination 82
round bottomed flasks microscopic examination
21 82
volumetric flasks 21 physical examination 82
funnels 21 general and physical
graduated (measuring) characteristics 83
cylinder 21 appearance 84
Laboratory hazards 3 color 84
accidental swallow of odor 84
corrosive solutions 6 specific gravity 84
burns 6 volume 83
care while pipetting 5 preservation of urine
in case of accidental fire 5 samples 83
inhalation of corrosive gases specimen collection 82
6
precautions regarding fire P
safety 5
safety measures 3 Pauly’s test 71
Laboratory safety rules 10 Phenylketonuria 123
accidents and injuries 11 ferric chloride test 124
handling chemicals 12 Guthrie’s bacterial inhibition
handling glassware and test 123
equipment 12 Photometry 129
principle 129
heating substances 13
Pipettes 22
pipetting techniques 14
auto pipettes 24
Lambert’s law 134
fixed volume type 24
variable volume
M adjustable type 24
manual pipettes 22
Maple syrup urine disease 228 deliver type of pipettes
22
N graduated pipettes 23
micropipettes 23
Neumann’s test 72, 73 pasteur pipettes 23
Nippe’s fluid 122 volumetric pipettes 23
Normal urine 82 Precipitation by alkaloidal
chemical characteristics 85 reagents 56
constituents 85 Precipitation by heavy metal
reaction 85 ions 58
Index 275
Precipitation by organic solvents S
55
Precipitation by salts 51 Schiff’s test 96
Preparation of hemin crystals
122 T
Preparation of hemoglobin and
derivatives 119 Test tubes 24
carboxy hemoglobin 120 centrifuge tubes 25
methemoglobin 120 desiccators 25
oxyhemoglobin (HbO2) 119 dispensers 25
reduced hemoglobin 120 Folin-Wu tubes 25
Proteins 48 Types of colloids 50
classification 48 emulsoids 50
conjugated proteins 48 suspensoids 50
derived proteins 48
secondary derived U
proteins 49
simple proteins 48 Unknown carbohydrate 47
functions 49 Unknown protein 73
reactions 49 Urea 74
color reactions of protein chemical tests 74
49 Biuret formation 74
precipitation reactions of sodium hypobromite test
proteins 49 75
specific urease test 76
Q physical properties 74
tests for urea 74
Qualitative detection of protein Uric acid 77
61 chemical tests 77
Quantitative estimation of murexide test 78
proteins 61 phosphotungastic acid
reduction test/
R Benedict’s uric acid
test 77
Relationship between physical properties 77
absorbance and
transmittance 135 V
Rothera’s test 217
Ruhemann’s purple 64 van den Bergh test 218

Practical Biochemistry: An Easy Guide For

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Practical Biochemistry: An Easy Guide For

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An Easy Guide for

Divya Shanthi D’Sa MBBS, DFH

Sowbhagya Lakshmi MSc, PhD

JAYPEE BROTHERS MEDICAL PUBLISHERS (P) LTD

First Edition: 2010

Biochemistry, a fascinating subject that deals with every

Our main aim is to make this book simple and attractive

Divya Santi D’Sa

The authors are indebted to student community who

SECTION 1: LABORATORY RULES AND

SECTION 2: QUALITATIVE TESTS

SECTION 3: QUANTITATIVE TESTS

SECTION 4: DEMONSTRATION PRACTICALS

Biophysics is intrinsically interdisciplinary. Biophysics takes a

IMPORTANCE OF BIOPHYSICS IN NURSING

Light and Sound

Work and Energy

FUNDAMENTAL AND DERIVED UNITS

these independent standards. These independent standards are length,

 Table 1.1: Systems of units with their standards of measurement

Physical CGS system FPS system MKS system SI system

Length Centimeter (cm) Foot (f) Meter (m) Meter (m)

FUNDAMENTAL UNITS IN VARIOUS SYSTEMS

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

English system Metric system

12 inches = 1 foot 10 millimeters (mm) = 1 centimeter (cm)

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

Unit of Mass and Weight

English system Metric system

Weight Fluid volume

1 ounce = 8 drams 60 minimus = 1 fluid dram

Prefix Symbol Power Value (in meter)

Tera T 1012 1,000,000,000,000

 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

14. Meyer B Jackson. Molecular and cellular biophysics. New York:

Biochemical parameters aids in diagnosis and prognosis of

Fig. 1.1: Signs of some laboratory hazards

• Chemical work involving irritating chemicals and

• General health should be maintained at all costs as an

CARE WHILE PIPETTING

PRECAUTIONS REGARDING FIRE SAFETY

IN CASE OF ACCIDENTAL FIRE

• Water should NOT be used on electrical fire.

ACCIDENTAL SWALLOW OF CORROSIVE

INHALATION OF CORROSIVE GASES

From strong alkalies:

LABORATORY FIRST AID

PRECAUTIONS TO BE TAKEN WHILE

• Corrosive chemicals should be opened with great care

4. Cylinder containing inflammable gases like hydrogen,

IN CASE OF ACCIDENTS IN THE LABORATORY

Laboratory Safety Rules

4. Be prepared for your work in the laboratory. Read all

ACCIDENTS AND INJURIES

14. If a chemical splashed into your eye(s) or on your skin,

HANDLING GLASSWARE AND EQUIPMENT

Biological samples like blood, saliva, CSF, pleural fluid,

Arterial and venous blood specimens differ in some

6. EDTA (ethylene diamine tetra acetate)

COLLECTION AND PRESERVATION

Preservation of Urine Samples