An Easy Guide For Practical Biochemistry (2010) (PDF) (UnitedVRG) PDF

An Easy Guide for
Practical Biochemistry
An Easy Guide for
Practical Biochemistry
Divya Shanthi DSa
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 nonClinical, 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 DSa

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

Assessment of patients by measurement of vital signs.
Motion
Inertia in accidents
Physiological reaction to high velocity centrifuges.
Gravity
Circulation of blood
Postural drainage
Postoperative position
ESR estimation
Dependent position for edema patient
Center of Gravity
Body mechanics
Crutch walking
Lifting and turning patients
Specific Gravity
Underwater exercises
Examination of the body fluids
Force
Torques in traction
Muscle action
Vector addition and analysis in traction
Pressure
Suction
Internal and external respiration
Positive pressure
Oxygen therapy
ventilation
Administration of irrigation and parenteral fluid
Heat
Thermometry
Steam inhalation
Thermography
Light and Sound

Actions of lenses
Microscopy
Refraction
Audiometery
Electricity
Patient monitors, ECG,
EEG, EMG
Electrosurgical procedures
Use of transistors in
apparatus
Application of heat and cold

application
Basal metabolism
Autoclave and sterilization
Use of mirrors in apparatus

Ophthalmoscope
Visual fields
Human audibility
Diathermy
Electric shock therapy
Cardiac pacemakers
Work and Energy

Circulation of blood
Pulse formation
Work done by heart and skeletal muscles
Molecular Physics
Artificial kidney
Colloidal dispersions
Surface tension of antiseptics Viscosity of blood
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, patients 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
patients temperature is 38C, it means that the patients temperature
is 38 times the unit of measurement, called degree Celsius (C). Thus
measurement of any quantity has two characteristicsa 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
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.
Table 1.1: Systems of units with their standards of measurement
Physical
quantity
CGS system
FPS system
MKS system
Length
Mass
Time
Temperature
Electric current
Light intensity
Amount of
substance
Centimeter (cm) Foot (f)

Meter (m)
Gram (gm)
Pound (d)
Kilogram (kg)
Second (s)
Second (s)
Second (s)
Fahrenheit (F) Celsius (C)
SI system
Meter (m)
Kilogram (kg)
Second (s)
Kelvin (K)
Ampere (A)
Candela (Cd)
Mole (mol)
FUNDAMENTAL UNITS IN VARIOUS SYSTEMS

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

English system
Metric system
12 inches = 1 foot
3 feet = 1 yard
5 yard = 1 rod
1,760 Yard = 1 mile
5,280 feet = 1 mile
10
10
10
10
10
10
10
millimeters (mm) = 1 centimeter (cm)

centimeter (cm) = 1 decimeter
decimeter = 1 meter (m)
meters = 1 decameter
decameters = 1 hectometer
hectometers = 1 kilometer (km)
kilometers = 1 myriameter
Note: 1 feet = 12 inches = 30 cm (1 inch = 2.5 cm)
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=mg
where, W denotes the weight of the body, m is its mass and
g is the acceleration due to gravity.
In the SI system, the unit of weight is Newton. Since, the value
of the acceleration due to gravity varies with the distance of an object
Table 1.3: Multiples of units of mass in the Metric and English systems
English system
Troy units
24 grains = 1 pennyweight
20 pennyweight = 1 ounce
12 ounces = 1 pound
Avoirdupois units
27.34 grains = 1 dram 1
6 drams = 1 ounce
16 0unces = 1 pound
25 pounds = 1 quarter
4 quarters = 1 hundredweight
20 hundredweight = 1 short ton
2,240 pounds = 1 long ton
Apothecaries unit
20 grains = 1 scruple
3 scruple = 1 dram
8 drams = 1 ounce
12 ounces = 1 pound
Metric system
10 milligrams = 1 centigram
10 centigram = 1 decigram
10 decigrams = 1 gram
10 grams = 1 decagram
10 decagrams = 1 hectogram
10 hectograms = 1 kilogram
1,000 kilograms = 1 metric ton
from the center of the earth, the weight of the object changes with
its position on the earth. For example, an object at sea level weighs
more than it does on a high mountain because the value of the
acceleration due to gravity of the earth on the object is greater at
sea level. The mass, however, remains the same everywhere. The mass
of an object is measured by a physical balance whereas its weight
is measured by a spring balance.
Mass is a scalar quantity while weight is a vector quantity because
it is directed towards the center of the earth. Mass of a person is the
same on the earth as well as on the moon, but weight of the person
is different at these two places because their pulls on the person are
different. A person weighs six times more on earth than on the moon.
Whereas mass and weight have the same numerical value, it is
important in solving problems to indicate the unit specifically, as one
of force (weight) or as one of mass. one gram (gm) is a unit of mass;
one gram weight is unit of weight (Tables 1.4 and 1.5).
Units of Time
The unit of time is the second and is based on the natural clock. The
natural clock is governed by the time taken by the earth to complete
Table 1.4: Conversion of weight and measurements

Weight
1 ounce = 8 drams
12 ounces = 1 pound
1000 microgram (Mcg) = 1 milligram (mg)
1000 milligram (mg) = 1 gram (gm)
1000 grams (g) = 1 kilogram (kg)
1 kilogram (kg) = 2.2 pounds
1 grain = 60 milligram (mg)
1 dram = 4 grams (g)
1 ounce = 30 grams
1 pound = 375 grams
1 milligram = 1/60 grains (gr)
Fluid volume
60 minimus = 1 fluid dram
8 fluid drams = 1 fluid ounce
20 fluid ounce = 1 pint
2 pints = 1 quart (1000ml)
8 pints = 1 gallon
1 milliliters (ml) = 15 16 minims
(1516 drops)
1 liter = 35 fluid ounce
1 fluid ounce = 30 ml
1 fluid dram = 4 ml
1 gallon = 4.5 liter
1 minimus = 0.04 ml = 1 drop
1 pint = 500 ml
Household measurements
1 teaspoonful = 4 or
5 ml = 1 fluid dram = 60 drops
Table 1.5: Prefixes and symbols used with SI units and Metric units
Prefix
Tera
Giga
Mega
Kilo
Hecto
Deca
Meter
Deci
Centi
Milli
Micro
Nano
Pico
Femto
Atto
Symbol
T
G
M
K
H
Da
m
D
C
Mm
u
n
p
f
A
Power
1012
109
106
103
102
10
1
101
102
103
106
109
1012
1015
1018
Value (in meter)

1,000,000,000,000
1,000,000,000
1,000,000
1,000
100
10
1
.1
.01
.001
.000001
.000000001
.000000000001
.000000000000001
.000000000000000001
one revolution around the moon. According to this clock, one second
is defined as (1/86400) part of a mean solar day; a solar day is the
period between noons of two consecutive days and a mean solar day
is the average solar day over a year, which is 24 hours. Since one
hour contains 60 minutes and one minute contains 60 seconds, a mean
solar day of 24 hours would have 24 60 60 = 86400. Thus, one
second is 1/86400th part of a mean solar day.
Let us consider some of the measurements of time you make in
the course of your work. You will see that the seconds hand of your
watch is sufficiently accurate for recording a patients 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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3. Dogonadze RR, Urushadze ZD. Semi-classical method of calculation of
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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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Publishers, 2004.
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August 2007.
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(4th Edition). Springer, 2006.
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(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:
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15. Nicolas Rashevsky, Mathematical biophysics. . Rev. ed., University of
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12
Section 1
Laboratory
Rules and
Regulations
Laboratory Hazards
and First Aid
Biochemical parameters aids in diagnosis and prognosis of

diseases. The medical students should have the knowledge
of the various tests, diagnostic investigations done in
biochemistry laboratory. They should also be aware of all
potential hazards and the safety measures.
HAZARDS
The student is surrounded by many dangers (Fig. 1.1)
such as:
Broken glassware.
Corrosive reagents.
Mechanical hazards.
Poisonous fumes that could be inhaled.
Inflammable chemicals.
Gas leakages.
Electrical hazards.
SAFETY MEASURES
Lab coat has to be worn in order to protect oneself from
corrosive splashes.
One has to be careful while handling gases.
Blood, urine, CSF and other biological fluids should be
handled with great care as they are potential sources of
infections like HIV and Hepatitis.
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
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.
10 An Easy Guide for Practical Biochemistry
Laboratory Safety Rules
Some rules are NOT made to be broken. That is true for the
rules used in a biochemistry lab.
GENERAL GUIDELINES
1. Conduct yourself in a responsible manner at all times
in the laboratory.
2. Follow all written and verbal instructions carefully. If
you do not understand a direction or part of a
procedure, ASK YOUR TEACHER BEFORE
PROCEEDING WITH THE ACTIVITY.
3. Do not touch any equipment, chemicals, or other
materials in the laboratory area until you are
instructed to do so.
Laboratory Safety Rules 11

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

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

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
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 4C and if the delay is more than
6 hr, it is refrigerated at - 20C.
For extracting serum, allow the blood to clot at room
temperature for 20- 30 minutes. Loosen the clot by a stick,
and centrifuge for 10 minutes at 3000 RPM. Separate the
serum and label it. They can be used later for analysis.
Special care should be taken to avoid hemolysis.
Hemolyzed samples alter the values of many chemical
estimations because of the release of RBC contents, which
can cause color change. False high results may be obtained
because of hemolysis. Hemolyzed samples affect bilirubin
and enzyme estimations giving erroneous results.
COLLECTION OF BLOOD
Blood is collected by:
Venipuncture
Arterial puncture
Capillary puncture
Specimen Collection and Processing 17

Arterial and venous blood specimens differ in some
important aspects. Venipuncture is more commonly
performed for obtaining blood.
Disposable needles are used to eliminate the hazards of
infections. Sterilization of the puncture site is done by using
70% alcohol or ether.
Whole blood, serum or plasma can be selected depending
upon the methods by which the biochemical parameters
are to be investigated. Usually serum/plasma is preferred.
ANTICOAGULANTS
Blood starts clotting within few minutes after it is removed
from the body unless an anticoagulant is used to stop the
process of clotting. Blood with anticoagulant is known as
WHOLE BLOOD.
Serum is the fluid portion of clotted blood while plasma
is the fluid portion of unclotted blood. Various anticoagulants are used depending on the parameters to be analyzed.
Blood is collected and mixed with some chemicals that
prevent clotting. These chemicals are called anticoagulants.
Most of the anticoagulants used in the laboratory, act by
binding calcium as an insoluble salt. Oxalates, citrates and
EDTA chelate calcium ion.
The routinely used anticoagulants are:
1. Potassium oxalate
2. Double oxalate
3. Ammonium oxalate
4. Sodium citrate
5. Heparin

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 8o clock to the next day morning 8o 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
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 blowout pipettes.
c. Volumetric pipettes: These pipettes are not graduated but
designed specially to deliver a specific quantity of the
specimen. They have an open- ended bulb holding the
bulk of the liquid, a long glass tube at one end that has
the mark to describe the extent to which the pipette is to
be filled and a tapered delivery portion. These pipettes
hold and deliver only the specific volumes indicated at
the upper end of the pipette. These are used mainly to
pipette specimen and standards, and are very accurate.
Pipettes must be refilled or rinsed out with the
appropriate solvent after the initial liquid has been
drained from the pipettes.
d. Micropipettes: Micropipette can deliver volumes ranging
from 1-500 l. 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 l, 20 l,
50 l, 100 l, 200 l, 1000 l 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 l, 100- 1000 l
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
Carbohydrates
DEFINITION
Carbohydrates are defined as polyhydroxy alcohols with
an aldehyde or ketone as the functional group.
CLASSIFICATION
Carbohydrates are classified according to the number of
sugar molecules in them as monosaccharides, oligosaccharides and polysaccharides.
Monosaccharides
Monosaccharides are also called as simple sugars. They
have only one potential sugar group. They consist of a single
polyhydroxy aldehyde or ketone unit, and thus cannot be
hydrolyzed into simpler form.
They may be subdivided into different groups as follows:
1. Depending upon the number of carbon atoms they
possess, e.g.
No. of
carbon
Type of
sugar
Aldoses
Ketoses
1
2
3
4
Trioses
Tetroses
Pentoses
Hexoses
Dihydroxyacetone
Erythrulose
Ribulose, xylulose
Fructose
Heptoses
Glyceraldehyde
Erythrose
Ribose, xylose
Glucose, galactose,
mannose
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
oligosaccharide monosaccharide
Example
Type of
monosaccharide present
Disaccharide
Two
Maltose
Lactose
Sucrose
Glucose + Glucose
Glucose + Galactose
Glucose + Fructose
Trisaccharide
Three
Raffinose Glucose + Galactose +

Fructose
Tetrasaccharide
Four
Stachyose
2 molecules of
Galactose + Glucose +
Fructose
Pentasaccharide
Five
Verbascose
3 molecules of
Galactose + Glucose +
Fructose
Disaccharides are classified as:

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

Polysaccharides
Polysaccharides are carbohydrates having more than ten
monosaccharide units. They are also called glycans or
complex carbohydrates.
They are classified into two types according to the type
of monosaccharide units present.
1. Homopolysaccharides: Made up of repeated units of same
type of monosaccharide units.
e.g. Starch, glycogen, cellulose, inulin, dextrins, dextrans
2. Heteropolysaccharides: Made up of different types of
monosaccharide units and their derivatives.
e.g. Agar, gum, pectins, glycosaminoglycans such as
hyaluronic acid, heparin sulfate, keratin sulfate,
chondroitin sulfate.
FUNCTIONS OF CARBOHYDRATES
1.
2.
3.
4.
Carbohydrates are the main source of energy in the body.

Storage form of energy (starch and glycogen)
Excess carbohydrate is converted to fat.
Glycoproteins and glycolipids are components of cell
membranes and receptors.
5. They form structural basis of many organisms.
REACTIONS OF CARBOHYDRATES
Chemical properties of carbohydrates are used as principles
in identification of these substances. The functional group
of the sugar molecule takes part in most of the chemical
reactions.

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. Benedicts test: specific test for reducing substances
4. Barfoeds test: specific test for monosaccharides
5. Seliwanoffs test: specific test for ketohexoses
6. Osazone test: to differentiate the reducing sugars on
the basis of crystal formation.
Physical Properties
1.
2.
3.
4.
Color: Colorless except Starch which is pale white

Clarity: Clear except Starch which is cloudy
Odor: Odorless
Reaction to litmus: Neutral
Chemical Tests
Molisch Test
Principle: Carbohydrates, when treated with concentrated
Sulfuric acid undergo dehydration to form furfural/
hydroxymethyl furfural derivatives which on condensation
with alphanaphthol form colored products (chromogens).
Experiment
Observation
Take 2 ml of given Violet ring at the junction

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

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.
Experiment
Take 2 ml of given
solution in a test
tube. Add 2-3 drops
of Iodine solution.
Observation
Inference
Blue color is formed.
Given solution is a
polysaccharide.
Points to Remember:
This is a specific test for polysaccharides.
The amylose component of starch has a helical structure.
When it is treated with iodine solution, Iodine is trapped
inside the coil and the complex has an intense blue color.
When the amylose solution is heated the helical
conformation is disrupted and loses its capacity to bind
iodine. On cooling the original conformation is regained
and the capacity to bind iodine is also recovered.
Sometimes the color may not reappear on cooling as small
amounts of iodine added may vaporize away during
heating.
Benedicts 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.

Benedicts Qualitative Reagent contains
Copper sulfate: provides cupric ions.
Sodium carbonate: makes the medium alkaline.
Sodium citrate: chelates cupric ions and releases it slowly
for reduction. Thus prevents the precipitation of cupric
ions as cupric hydroxide by forming cupric sodium citrate
complex. It acts as a stabilizing agent. Improves the shelflife of the reagent by preventing an interaction between
sodium carbonate and copper sulfate, which may
otherwise get precipitated as cupric carbonate.
Experiment
Take 5 ml of
Benedicts reagent
in a test tube.
Add 8 drops of the
given solution. Mix
and boil for 2 min.
over a small flame.
Allow to cool
spontaneously.
Observation
Inference
Brick-red precipitate
is formed
Given solution is a
reducing sugar.
Points to Remember:
This test is also a semi-quantitative test which can be
reported as under:
Observation
Inference
Sign
Approx. sugar
No change of blue
color: - no precipitate.
Absence of reducing sugar
Nil
Green precipitate.
Presence of reducing sugar
up to 0.5 %
Yellow precipitate.
++
0.5 to 1 %
Orange precipitate.
+++
1 to 1.5 %
Red precipitate
++++
2%
Brick red precipitate.
++++
>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.
Benedicts quantitative reagent contains potassium
thiocyanate and potassium ferrocyanide in addition to
copper sulfate, sodium carbonate and sodium citrate
present in Benedicts qualitative reagent.
Benedicts tests (In case of sucrose)
Since sucrose is a non-reducing sugar, it does not give a
positive Benedicts 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.
Benedicts tests after acid hydrolysis:
Experiment
Observation
Inference
Take 5 ml of Benedicts
reagent in a test tube;
add 8 drops of
neutralized hydrolysate
solution. Boil for
2 min.
Brick-red
precipitate
is formed
On acid hydrolysis
Sucrose is converted to
reducing
Monosaccharides
(Glucose + Fructose)
Benedicts test (In case of starch)

Starch being a non- reducing sugar does not give positive
Benedicts test. Therefore, Benedicts 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.
Benedicts test after acid hydrolysis:
Experiment
Take 5 ml of
Benedicts reagent
in a test tube;
add 8 drops of
neutralized hydrolysate solution. Boil for
2 min.
Observation
Inference
Brick-red
precipitate
is formed
On acid
hydrolysis starch
gives reducing
sugars.
Barfoeds 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.
Barfoeds reagent: Copper acetate in glacial acetic acid.

Experiment
Observation
Inference
Take 2 ml of Barfoeds
reagent in a test tube;
add 1 ml of given
solution. Mix and
boil for 30 seconds.
Allow it to cool at
room temperature.
Floating red
precipitate
is formed.
Given solution is
a monosaccharide
Points to Remember:
It is a specific reduction test for reducing monosaccharides.
Time for heating is one of the important factors in this
reaction.
If the boiling period exceeds 30 seconds, disaccharides
will be hydrolyzed to monosaccharides and red colored
precipitate of cuprous oxide will be formed.
Helps to differentiate reducing monosaccharides from
reducing disaccharides.
Seliwanoffs Test
Principle: Carbohydrates are dehydrated to form furfural
derivatives by hydrochloric acid present in Seliwanoffs
reagent. Furfural derivative of ketosugar condenses with
resorcinol to form a chromogen (cherry red color).
Seliwanoffs reagent: 50 mg of resorcinol in 33 ml of
concentrated hydrochloric acid and diluted to 100 ml with
water.

Experiment
Observation
Inference
Take 3 ml of
Seliwanoffs reagent
in a test tube;
add 1 ml of given
solution. Boil for
30 seconds and allow
it to cool at room
temperature.
Cherry red
color is formed.
Given
solution is a
ketosugar
Points to Remember:
This test is specific for ketohexoses only.
Useful in differentiating aldohexoses and ketohexoses.
The test will be answered by fructose, sucrose and other
fructose containing carbohydrates.
A similar color may also develop in case of glucose or
maltose if the boiling is prolonged due to transformation
of glucose into fructose by the catalytic action of the
hydrochloric acid.
This test is very sensitive even for 0.1 % fructose. In the
presence of glucose along with fructose sensitivity
decreases.
It serves as an indirect test for sucrose because sucrose
gets hydrolyzed by the hydrochloric acid present in
the Seliwanoffs reagent to fructose and glucose; the
liberated fructose reacts with the reagent to give a positive
reaction.

Seliwanoffs test (for sucrose)
In case of sucrose, the below procedure has to be followed.
Experiment
Observation
Inference
Take 3 ml of
Seliwanoffs reagent
in a test tube; add
1 ml of acid
hydrolysate.
Boil for 30 seconds.
Cherry red color

is formed
Hydrolyzed
sucrose contains a
ketosugar.
Osazone Test
Principle: Phenyl hydrazine at 100C 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 maintain the pH 5, appropriate for the formation of osazones.
Osazones of monosaccharides are insoluble in hot water,
and will form crystals in hot condition.
Osazones of disaccharides are soluble in hot water. So
they form crystals only when the test tube is cooled.
Glucose, fructose and mannose differ structurally with
respect to 1st and 2nd carbon atoms. During osazone
formation phenyl hydrazine reacts with the 1st and 2nd
carbon atoms and hence the structural difference between
these three sugars is masked. Hence glucose, fructose and
mannose form the same needle shaped osazone crystals.
In the sucrose molecule, the active groups on 1st and
2nd carbon atoms of constituent glucose and fructose
molecules are not free (no free aldehyde/ketone group).
Hence, sucrose does not produce osazone crystals.
But it produces osazone crystals when the solution is
kept in a boiling water bath for 30 minutes because of the
hydrolysis of sucrose to glucose and fructose.
Importance and significance
To identify the reducing sugar that is excreted in the urine
especially during the period of lactation. To differentiate
glucose and lactose that is excreted in the urine.

Osazones
Minimum time
for formation
of crystals
Appearance of crystals
Glucosazone
5 minutes
Needle shaped/broom-stick
shaped/hay stack or sheaves
of corn appearance
Fructosazone
2 minutes
Needle shaped/broom-stick
shaped/hay stack or sheaves
of corn appearance
Lactosazone
30 minutes
Powder-puff shaped/cotton
ball/badminton ball shaped/
pincushion with pins/hedgehog
shaped or flower of touch-me-not
plant shaped crystals
Maltosazone 30-40 minutes
Sunflower shaped/star
shaped crystals

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 - D glucose and - D galactose.
Linked by 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 D glucose.
Linked by 1-4 glycosidic linkage.
Sources are germinating seeds and malt.
Digested by maltase present in intestinal juice.
5. Sucrose
Sucrose is composed of D glucose and D fructose.
Sources of Sucrose are cane sugar, etc.

Linked by 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
1-4 glycosidic linkages.
Amylopectins have branching points linked by 1-6
glycosidic linkages.
Starch is a non-reducing sugar.
The test results for different carbohydrates are summarized below:
Test
Molisch
Iodine
Benedicts
Glucose
Barfoeds Seliwanoffs
+
Fructose
Lactose
Maltose
Sucrose
- (+ after
acid
hydrolysis)
+ after acid
hydrolysis
Starch
- (+ after
acid
hydrolysis)

IDENTIFICATION OF UNKNOWN CARBOHYDRATE
Flow chart 5.1: Scheme for identification of unknown

carbohydrate
Proteins
DEFINITION
Proteins are defined as sequence specific polymers of L
-amino acids linked by peptide bonds. They are complex
organic substances made up of carbon, hydrogen, oxygen
and nitrogen. Some proteins also contain sulfur and
phosphorus.
CLASSIFICATION
1. Simple proteins: made up of only amino acids.
Ex: Albumin, globulin and protamines
2. Conjugated proteins: made up of amino acids and a nonprotein part which is known as the prosthetic group.
e.g. Glycoproteins: immunoglobulins
Chromoproteins: hemoglobin
Lipoproteins: occur in blood and on cell membranes- HDL,
LDL, VLDL
Metaloproteins: hemoglobin, cytochrome
Nucleoproteins: histones
Phosphoproteins: casein of milk and vitelline of egg yolk
3. Derived proteins: derived from native (naturally occurring)
proteins. They are of two types:
Primary derived proteins: formed by agents such as heat,
acids, alkalies, etc. which cause only slight changes
Qualitative Analysis of Proteins 49

in the protein molecule and its properties without
hydrolytic cleavage of peptide bond.
e.g. Proteons, metaproteins.
Secondary derived proteins: formed in the progressive
hydrolytic cleavage of the peptide bonds of proteins
into smaller molecules.
Ex: proteoses, peptides and peptones.
FUNCTIONS OF PROTEINS
1. Maintain the structural integrity of bones, tendons, hair
and teeth- collagen, elastin, keratin.
2. Acts as enzymes (catalytic function).
3. Hormones (regulatory function).
4. Antibodies- immunoglobulins (defense protein).
5. Coagulation factors.
6. Carrier proteins (e.g. albumin, thyroglobulin)
7. Contractile element in the muscle (actin, myosin)
8. Proteins act as intracellular buffer in maintaining the
acid-base balance.
REACTIONS OF PROTEINS
Reactions of proteins are studied as:
1. Precipitation reactions of proteins
2. Color reactions of proteins.
Precipitation Reactions of Proteins
Proteins are large molecules with variable sizes, shapes and
charges. Solubility of a protein depends on the proportion
and distribution of polar hydrophilic groups and non-polar
hydrophobic groups in the molecule.
Proteins form colloidal systems in aqueous medium. A
colloid is a system in which the particles have diameters in

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
groupsamino 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:

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

is formed.
given solution is
b. No white
precipitated.
precipitate
b. Protein in the
is formed.
given solution is
not precipitated
by half-saturation
with ammonium
sulfate.
a. No violet
color is formed.
b. Violet color
is formed
Note: Filterate which contains protein gives violet color with Biuret
test.
b. Full saturation with ammonium sulfate:

Experiment
solution in a test
tube. Add solid
ammonium sulfate
in small quantities
at a time with
mixing until the
solution is saturated.
Allow to stand for
5 minutes.
Observation
a. White precipitate
is formed.
b. No white
precipitate
is formed.
Inference
a. Protein in the
given solution
is precipitated.
b. Protein in the
given solution is
not precipitated
by full saturation
with ammonium
sulfate.
Contd...

Contd...
Experiment
Filter it. Perform
Biuret test with the
filtrate by adding
3 ml of 40% sodium
hydroxide and
2-3 drops of
1% copper sulfate.
Observation
Inference
a. No Violet color
is formed.
b. Violet color
is formed
Note:
Solid ammonium sulfate is added in small quantities at a time
with mixing until the solution is saturated, i.e. there should be
some undissolved salt in the bottom of the test tube.
Filtrate which is not having any trace of protein gives blue color
with Biuret reagent.
Discussion:
Solubility of a protein depends on ionic concentration of
the medium. Therefore, the presence of very small
quantities of salts will increase the solubility of a protein
by diminishing protein interaction. This is called Saltingin.
Filtrate contains high concentration of ammonium ions
which interfere in Biuret test by forming a deep blue
cuprammonium ions [Cu (NH3)4++] which obscure the
violet color produced by proteins. This can be overcome
by the use of 40% sodium hydroxide instead of 10%
sodium hydroxide.
Depending on the surface area of the protein, the amount
of salt required is variable. Higher the molecular weight
of a protein the salt required for precipitation is lesser.

Smaller molecules like albumin have relatively a large
surface area. They hold more water molecules around
them. Hence, a higher concentration of salt is required
for precipitation of albumin. Thus, albumin is
precipitated by full saturation.
Casein and gelatin are precipitated by half saturation
because they have high molecular weight.
Isoelectric Precipitation
Principle: The solubility of proteins is minimum at their
isoelectric pH as the protein molecules become electrically
neutral at this pH.
Note: Perform the test with only casein.
Experiment
Observation
Inference
Take 3 ml of casein
solution in a test
tube. Add 3 drops
of bromocresol
green indicator.
Mix. Add 1%
acetic acid drop
by drop until a
light green color
is obtained which
indicates that the
pH is close to 4.6.
A curdy green
precipitate
will be formed.
At pH 4.6,
casein is
precipitated.
Points to Remember:
The pH at which the molecule carries no net charge is
known as isoelectric point or isoelectric pH (pI)
At isoelectric pH
1. The net charge is zero.
2. No mobility in an electric field.
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.
Experiment
Observation
Inference
solution in a test
tube. Add 2 ml of
ethanol. Mix.
A white
precipitate
is formed.
Protein is
precipitated by
organic solvents.
Points to Remember:
For the precipitation of protein by alcohol, protein should
be in electrolyte form. This may be achieved by dissolving
the protein in saline.
Precipitation by Alkaloidal Reagents
Principle: The negatively charged ions of the alkaloids
neutralize the positive charge on the protein causing
denaturation which results in precipitation.
Experiment
a. Take 2 ml of
protein solution
in a test tube.
Add picric acid
drop by drop.
Observation
A thick yellow
precipitate
is formed.
Inference
Protein is
precipitated by
alkaloidal reagent.
Contd...

Contd...
Experiment
Observation
Inference
b. Take 2 ml of
protein solution
in a test tube.
Add trichloroacetic acid drop
by drop.
A white
precipitate
is formed
Protein is
precipitated by
alkaloidal reagent.
c. Take 2 ml of
protein solution
in a test tube.
Add sulfosalicylic acid
drop by drop.
A white
precipitate
is formed.
Protein is
precipitated
by alkaloidal
reagent.
Points to Remember:
Tungstic acid, phosphotungstic acid, trichloroacetic acid,
picric acid, sulfosalicylic acid and tannic acid are
powerful protein precipitating agents. These acids lower
the pH of the medium, when proteins carry net positive
charges. These protein cations are electrostatically
complexed with negatively charged ions to form proteintungstate, protein-picrate, etc. to form thick flocculent
precipitate.
Tanning in leather processing is based on the protein
precipitating effect of tannic acid.

This test using sulfosalicylic acid is commonly
employed for preliminary screening of urine for the
presence of proteins. It is also used to identify proteins
in CSF.
For estimation of blood constituents photometrically,
proteins interfere with the analysis. This is avoided by
an initial protein precipitation by alkaloidal reagents.
Precipitation by Heavy Metal Ions
Principle: Proteins exist as negatively charged ions (anions)
in pH higher than their isoelectric pH (generally in an
alkaline medium). To such a solution if salt of heavy metals
are added, positively charged metal ions can complex with
protein anion and metal proteinates are formed which get
precipitated.
Experiment
Observation
Inference
a. Take 2 ml of
protein solution
in a test tube.
Add 10% lead
acetate solution
drop by drop.
White precipitate
is seen.
Protein is
precipitated by
heavy metals
like lead.
b. Take 2 ml of
protein solution
in a test tube.
Add 5% mercuric
nitrate solution
drop by drop.
White precipitate
is seen.
Protein is
precipitated by
heavy metals
like mercury.

Points to Remember:
Salts of iron, copper, zinc, lead, cadmium and mercury
are toxic, because they tend to precipitate normal proteins
of the gastrointestinal wall.
This test principle is used in treating heavy metal
poisoning.
Based on this principle, raw egg white is used as an
antidote in mercury poisoning and then an emetic is given
to remove Hg++ ions which are held by albumin.
If the sample solution is significantly alkaline, its pH
should be adjusted to 7- 7.5 to avoid formation of metal
hydroxides, which interfere with the test.
Avoid adding excess of heavy metal ions as this may
redissolve the precipitate due to absorption by the protein
molecules, which will give them a positive charge.
Precipitation by Heat and Acid (Heat and Acetic Acid Test)
Principle: On heating the protein loses its structure and
becomes denatured to form a coagulum. It is precipitated
after the addition of acetic acid, which provides the suitable
pH to get the maximum precipitate.
Experiment
Observation
Take albumin
A white coagulum is seen
solution upto th
at the upper heated
test tube. Hold the
portion, which gets
test tube over a flame
intensified after the
in a slanting position
addition of acetic acid.
and boil the upper
portion of the
albumin solution.
The lower portion
serves as control.
Add 1% acetic acid
drop by drop.
Inference
Indicates the
presence of heat
coagulable
protein
like albumin.

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.
2.
3.
4.
Color: Pale white

Clarity: Cloudy
Odor: Odorless
Reaction to litmus: Neutral.
Physical Properties of Casein

1.
2.
3.
4.
Color: Pale white

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

Experiment
Observation
Inference
solution in a test
tube. Add 2 ml of
5% sodium
hydroxide. Mix
and add one or
two drops of 1%
copper sulfate.
Violet color
is formed
Indicates the
presence of
peptide linkage.
Points to Remember:
It is a general test for proteins.
The reaction is so named since Biuret (NH2-CO-NH-CONH2) formed by the condensation of two molecules of
urea when heated at 180 C also answers this test.
The minimum requirement for a positive test is the presence
of two peptide bonds in the molecule (three amino acids).
Individual amino acids and dipeptides do not answer
this test.
This test is positive for all compounds containing more
than one peptide linkage (- CO-NH-) e.g. proteins and
their hydrolytic products (metaproteins, proteoses,
peptones, polypeptides except dipeptides and amino
acids).
This test is also positive for substances which contain
two carbamyl (-CONH2) groups joined directly or through
a single atom of nitrogen or carbon. Hence non- proteins,
e.g. oxamide and biuret give positive test.
This reaction can be used for quantitative estimation of
proteins.
Insoluble protein like keratin gives negative Biuret test.

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 amino acids to give a
bluish purple colored complex called Ruhemanns purple.
Ninhydrin is a powerful oxidizing agent and causes
oxidative decarboxylation of amino acids producing an
aldehyde. The reduced Ninhydrin (hydrindantin) then
reacts with ammonia and another molecule of Ninhydrin
and produces bluish purple colored complex.
Alpha amino acids + Ninhydrin
Aldehyde + CO2 + NH3 +

Hydrindantin
Hydrindantin + NH3 + Ninhydrin Bluish purple colored complex
Ninhydrin reagent: 0.2% of Ninhydrin in acetone.

Experiment
Observation
Inference
Take 1ml of protein

solution in a test
tube. Add 10 drops
of Ninhydrin.
Heat to boiling.
Bluish purple
color is formed.
Indicates the
presence of free
-amino acids.

Points to Remember:
This test is positive for all - amino acids.
Proline and hydroxy proline are imino acids and they
have no 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 amino acids are
present in the given solution.
This test is used to detect amino acids in chromatography.
Xanthoproteic Test
Principle: The benzene ring system in tyrosine and
tryptophan undergo nitration on treatment with strong nitric
acid at elevated temperature forming a yellow precipitate.
The yellow precipitate turns orange due to ionization, in
alkaline medium.
Experiment
solution in a test tube.
Add 1ml of conc. Nitric
acid and mix. Heat the
solution for one
minute and cool
it under tap water.
Observe the color.
Divide the contents
into two parts.
Observation
Inference
In acid medium
yellow color
is formed.
Indicates the
presence of
aromatic amino
acids. (Tyrosine
and tryptophan).

Experiment
Observation
To one part of the

solution, add 2 ml
of 40% sodium
hydroxide till the
solution to make
it alkaline. Mix well
and observe.
In alkaline medium
orange color
is formed.
Inference
Points to Remember:
This is a specific test for aromatic amino acids.
Yellow color is due to the formation of nitro derivatives
of benzene ring containing amino acids.
This reaction is also the basis of yellow stain in skin by
nitric acid.
Nitration of phenylalanine under these conditions
normally does not take place and phenylalanine alone
gives a weak positive result.
Millons Test (Coles 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.
Millons reagent: Contains 10% mercuric sulfate (prepared
in 10% Sulfuric acid) and 1% sodium nitrite.

Experiment
Observation
Inference
Take 1ml of protein

solution in a test
tube. Add 1ml of
10% mercuric
sulfate. Boil gently
for 30 seconds.
Cool it under tap
water. Add 3 drops
of 1% sodium
nitrite solution.
Mix and
observe.
Red coagulum
is formed.
Indicates the
presence of hydroxyphenyl group
(tyrosine).
Points to Remember:
This test is specific for hydroxy phenyl group of tyrosine.
This test cannot be employed to detect tyrosine in urine
because chlorides which are normally present in urine
interfere with the reaction by forming unionized mercuric
chloride.
This test is given by phenols or phenolic substances such
as salicylic acid.
Heat coagulable proteins give red precipitate, whereas
smaller molecules of proteins like peptones give red
colored solution without precipitate.
Aldehyde Test for Indole Nucleus
(Hopkins- Cole- Adams Test)
Principle: Mercuric sulfate causes mild oxidation of indole
group of tryptophan, which condenses with an aldehyde to
give the colored complex.

Experiment
Observation
Inference
solution in a test tube
Add 2 drops of 0.2%
formalin and a drop
of 10% mercuric
sulfate. Add
carefully 3 ml
of conc. Sulfuric
acid along the
sides of the test tube.
A purple or violet
color is formed at
the junction of
the two liquids.
Indicates the
presence of
Indole ring
(tryptophan).
Points to Remember:
It is a specific test for indole nucleus/ring.
Sulfuric acid with mercuric sulfate is used as an
oxidizing agent in this reaction.
Tryptophan is an essential amino acid and its presence
indicates a good nutritive value of the protein.
Casein and egg albumin give a positive test.
Sakaguchis Test for Guanidine Group
Principle: In alkaline medium - naphthol combines with
the guanidine group of arginine to form a complex which is
oxidized by sodium hypobromite to form a bright red colored
complex.
Experiment
Observation
Inference
solution in a test
tube. Add 5-6 drops
of 40% sodium
hydroxide and
4 drops of Molisch
reagent. Mix and
add 10 drops of
bromine water.
Bright red color

is formed.
Indicates the
presence of arginine.

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 - naphthol, as it masks the
color development.
Sulfur Test for Cystine and Cysteine
Principle: On boiling with sodium hydroxide the sulfur
present in the protein is converted to inorganic sodium
sulfide. This reacts with lead acetate to form a black
precipitate of lead sulfide.
R-SH + 2 NaOH R-OH + Na2S + H2O
Na2S + (CH3COO)2Pb PbS + 2CH3+COONa
Experiment
Observation
Inference
solution in a test
tube. Add 2 ml of
40% sodium
hydroxide. Boil
for one minute.
Cool it under
tap water. Then add
5 drops of lead
acetate.
Black or brown color

precipitate is formed.
Indicates the
presence of
cystine or
cysteine
residues.
Points to Remember:
This test is specific for SH (thiol) group of cysteine and
cystine.
It is a test for sulfur- containing amino acids.

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.
Paulys Test for Histidine and Tyrosine
Principle: Diazobenzene sulfonic acid reacts with imidazole
ring of histidine to form a cherry red colored diazotized
product under alkaline condition. With the hydroxyphenyl
group of tyrosine an orange-red colored product is
obtained.
Experiment
Take 1 ml of 0.5%
sulfanilic acid in a
test tube. Add 1 ml
of 0.5% sodium
nitrite solution.
Mix. After standing
for 1 minute, add
2 ml of protein solution.
Mix. Cool under tap
water and then
add 1ml of 10%
sodium carbonate
to make the solution
alkaline.
Observation
Inference
Cherry red color

is formed.
Histidine is
present.
Orange red color

is formed.
Tyrosine is
present.

Points to Remember:
This test is specific for imidazole group of histidine.
It is also positive for phenolic hydroxyl group.
A positive Paulys test and negative Millons test indicate
the presence of histidine.
Molisch Test for Carbohydrate Moiety in Proteins
Principle: Carbohydrates, when treated with Conc. sulfuric
acid undergo dehydration to form furfural derivatives which
on condensation with alpha-naphthol forms colored
products.
Molisch reagent: A 5% solution of alpha naphthol in ethyl
alcohol.
Experiment
Observation
Inference
solution in a clean
dry test tube; add
1-2 drops of
Molisch reagent.
Mix. Incline the
test tube slightly
and add 2 ml of
conc. Sulfuric
acid along the
sides of the test
tube so as to form
two layers.
Violet ring at the

junction of the
two liquids
is formed.
Indicates the
presence of bound
carbohydrate.
Point to Remember:
Egg albumin has bound carbohydrate.

Test for Organic Phosphorus ( Neumanns Test)
(Test with casein solution)
Principle: On boiling with strong sodium hydroxide, the
organic phosphate present in phosphoproteins is released
as inorganic phosphate. Inorganic phosphate reacts with
ammonium molybdate in the presence of nitric acid (acidic
media) to form a canary yellow precipitate of ammonium
phosphomolybdate.
Experiment
Observation
Inference
Take 5 ml protein
solution in a test
tube. Add 0.5 ml
of 40% sodium
hydroxide. Heat
strongly and cool
under tap water.
Add 0.5 ml of conc.
Nitric acid. Filter.
To the filtrate add a
pinch of solid
ammonium
molybdate and
warm gently.
Canary yellow
precipitate
is formed.
Indicates the
presence of
phosphoprotein.
Points to Remember:
This test detects the presence of organic phosphorus in
casein.
Casein is a phosphoprotein.
Ammonia is added to remove the sulfate ions.
Nitric acid provides the acidic medium.

IDENTIFICATION OF UNKNOWN PROTEIN
Flow chart 6.1: Scheme for identification of

an unknown protein
Nonprotein Nitrogenous
Substances
Nonprotein nitrogenous substances include all nitrogenous

substances other than proteins. Most important NPN
substances present in urine are uric acid, urea, creatinine
and ammonia.
Urea
It is the end product of protein catabolism.

Synthesized in the liver.
Excreted mainly in the urine.
Normal blood urea level is 15- 45 mg/dl.
Normal level of urea excreted in urine is 25- 30 gm/day.
Tests for Urea

Urea tests are as follows:
Physical Properties
1.
2.
3.
4.
Color: Colorless
Clarity: Clear
Odor: Odorless
Chemical Tests
Chemical tests are as follows:
Biuret Formation
Principle: When heated above its melting point, two
molecules of Urea condense to form biuret and ammonia.
Nonprotein Nitrogenous Substances 75

Biuret reacts with alkaline copper sulfate to form a violet
color.
Experiment
Observation
Inference
Take a pinch of Urea

crystals in a dry test
tube and heat it in a low
flame. Urea melts with the
liberation of ammonia. On
further heating it solidifies.
Cool the test tube. Dissolve
the residue in 1 ml of 10%
sodium hydroxide and add
one drop of copper sulfate.
Violet color
is formed.
Indicates the
presence of urea.
Point to Remember:
Excess of copper sulfate should not be added; otherwise
copper sulfate will form cupric hydroxide with sodium
hydroxide forming a blue color. This is sometimes
mistaken for a positive biuret test.
Sodium Hypobromite Test
Principle: When urea is treated with Sodium hypobromite, it
decomposes to give nitrogen, carbon dioxide and water.
Liberation of nitrogen gas produces brisk effervescence.
Experiment
Observation
Inference
Take 2 ml Urea
solution in a test
tube. Add 5 drops
of freshly prepared
alkaline sodium
hypobromite solution
and mix.
Brisk effervescence
of Nitrogen gas is seen.
Indicates the
presence of
urea.

Point to Remember:
This principle is the basis for the quantitative estimation
of urea in urine.
Specific Urease Test
Principle: Under optimum pH and temperature, the enzyme
urease decomposes urea into ammonia and carbon dioxide
which together form ammonium carbonate (alkaline
substance) which changes the solution to pink color in the
presence of the indicator. Indicator phenolphthalein
changes to pink color in alkaline medium.
Experiment
Observation
Inference
Take 3 ml of urea
solution in a test
tube. Add 3 ml of
urease suspension
and add 1-2 drops
of phenolphthalein
indicator. Warm the
tube for a few
seconds with the
hands and keep
for 10 minutes.
Pink color is formed
Urea is
confirmed.
Points to Remember:
This test is specific for urea because the enzyme Urease
shows its specificity for the substrate urea.
Urease suspension contains 10 gm of horse gram powder
mixed with 100 ml of 30% ethanol.
pH range of phenolphthalein indicator is 8.3 to 10.
Urease present in horse gram powder acts on urea to
form ammonium carbonate which raises the pH of the
solution above 9 which is indicated by the change of
color by phenolphthalein indicator.

Over heating should be avoided. Otherwise the enzyme
will be destroyed. 60C temperature is maintained by
touch only. This 60C temperature is the optimum
temperature for Urease for its maximum activity.
Tests for Uric Acid
Uric Acid
It is the end product of catabolism of purines, in human
body.
It is synthesized in the liver and excreted through urine
as urates.
Normal uric acid level in blood is,
Males: 3.5-7 mg/dl
Females: 3-6 mg/dl
Normal level of uric acid excreted in urine is 250-750
mg/day.
Uric acid is sparingly soluble in water but soluble in
alkaline solution.
Physical Properties
1.
2.
3.
4.
Color: Colorless
Clarity: Clear
Odor: Odorless
Reaction to litmus: Alkaline.
Chemical Tests
Chemical tests are as follows.
Phosphotungstic Acid Reduction Test/ Benedicts
Uric Acid Test
Principle: Uric acid in alkaline condition reduces
phosphotungstic acid to tungsten blue.

Benedicts uric acid reagent: Composed of sodium tungstate,
orthophosphoric acid, concentrated sulfuric acid and solid
sodium carbonate.
Experiment
Observation
Inference
Take 3 ml uric
acid solution in a
test tube; add 1 ml
of Benedicts uric
acid reagent and
1 ml of 20% sodium
carbonate. Mix.
Deep blue
color is formed.
Indicates the
presence of
uric acid.
Schiffs Test
Principle: Uric acid reduces salts of silver nitrate to metallic
silver.
Experiment
Observation
Inference
Moisten a piece of
filter paper with
few drops of
ammoniacal silver
nitrate solution.
Add 2-3 drops
of uric acid on the
silver nitrate drops.
Black spots
on the filter
paper are seen.
Indicates the
presence of
Uric acid.
Murexide Test
Principle: When uric acid is treated with conc. nitric acid, it
undergoes oxidation. The imidazole ring is cleaved and the
derivatives condense to give reddish yellow purpuric acid.

This combines with ammonia to form ammonium purpurate
or murexide which is purple red in color.
Experiment
Observation
Inference
Take 5 ml of uric
acid in a china dish
and add 5 drops
of conc. Nitric acid.
Warm gently over a
low flame. A reddish
yellow residue is
obtained. Allow the
dish to cool. Add
two drops of dilute
ammonia solution.
A purplish red
color is formed.
Indicates the
presence of
Uric acid.
Test for Creatinine

Creatinine
Creatine is found in muscle as creatine phosphate, plays
an important role in muscular contraction.
Creatinine is the anhydride form of creatine phosphate.
Creatine is synthesized from three amino acids, glycine,
arginine and methionine.
Glycine + Arginine Guanidoacetic acid + Ornithine
(Kidney)
Guanidoacetate + S adenosyl methionine Creatine + S adenosyl homocystine
(Liver)
ATP + Creatine Creatine phosphate + ADP
Creatine phosphate Creatinine + H2O + Pi
Normal serum creatinine is 0.5-1.2 mg/dl

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

Physical Properties
1.
2.
3.
4.
Color: Colorless
Clarity: Clear
Odor: Odorless
Jaffes Test
Principle: Creatinine reacts with picric acid in the presence
of alkaline medium to form reddish orange colored
creatinine picrate.
Experiment
Observation
Inference
Take 3 ml of
creatinine solution
in a test tube.
Add 1 ml saturated
picric acid solution,
and 3-4 drops of
10% sodium
hydroxide.
Reddish orange
color is formed.
Indicates the
presence of
Creatinine.
Report: The given NPN substance is .
Flow chart 7.1: Schematic representation for identification of an unknown substance of

physiological importance
IDENTIFICATION OF UNKNOWN SUBSTANCE OF PHYSIOLOGICAL IMPORTANCE
Normal Urine
Urine is an ultrafiltrate formed by the kidneys carrying the

waste and toxic substances from the blood. The composition
of urine is a mirror not only of renal function but also of
many physiological and metabolic processes occurring in
the body. Thus, examination of urine may lead to the
diagnosis of many metabolic and systemic diseases.
EXAMINATION OF URINE
Examination of urine includes:
1. Physical examination
2. Chemical examination
3. Microscopic examination.
SPECIMEN COLLECTION
1. Fresh mid-stream specimen of 10- 20 ml is collected in a
clean dry container.
2. For most of the qualitative tests, a random urine sample
is satisfactory.
3. Morning specimen is desirable for normal analysis.
4. Repeated urine samples are necessary for orthostatic
proteinuria.
5. 24 hours urine is collected for total urinary proteins,
calcium, uric acid, ketosteroids and certain hormonal
assays, as the concentrations vary at different times of
the day. The patient is instructed to collect the urine
Qualitative Analysis of Normal Urine 83

sample from morning 8 o clock to the next day morning
8 o clock.
PRESERVATION OF URINE SAMPLES

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

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

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
Experiment
Observation
Inference
Appearance
Clear
Given sample of urine is normal.
Volume
1000 to 1500 ml Normal volume.
Color
Amber yellow
Odor
Aromatic smell
Reaction to
litmus
Blue litmus
turns red
Normal urine is acidic.
Specific gravity 1.016 to 1.025
Normal.
Determination of Specific Gravity

Specific gravity of urine is measured by an apparatus known
as Urinometer. Urinometer consists of a thin stem graduated
from 1000 to 1060 corresponding to Specific Gravity of 1.0
to 1.06. Urinometer is calibrated at 60F (15C).
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 37C.
Urinometer is calibrated at 15C. Since the room temperature
is higher, a temperature correction has to be applied. For
every 3C rise over the temperature of calibration (15C), a
correction factor of 0.001 is added to the last digit of the

observed reading.
The difference between 37C and 15C is 21C.
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 15C, one unit should
be subtracted from the last digit for every 3C difference in
temperature.
Longs Coefficient
The total solids normally excreted in the urine may be
calculated using Longs 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.
Experiment
Observation
Inference
Take 3 ml of urine
in a test tube.
Add 0.5 ml of conc.
Nitric acid and
1 ml of 3% silver
nitrate.
A white
precipitate
is formed.
Indicates the
presence
of chloride.

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
Cushings syndrome
Infections
Increased urinary chloride is seen in:
Excessive intake of fluids
Addisons disease
Test for Inorganic Sulfates
Principle: Urine being acidified with hydrochloric acid forms
a white precipitate of barium sulfate by the reaction with
barium chloride solution.
Experiment
Take 3 ml of urine
in a test tube.
Add 1 ml of conc.
Hydrochloric acid.
Mix well and add
2 ml of 10% barium
chloride.
Observation
Inference
A white
precipitate
is formed.
Indicates the
presence of
inorganic
sulfates.

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.
Experiment
Observation
Inference
To one part of the

filtrate, add a few
drops of conc.
Nitric acid and a
pinch of ammonium
molybdate. Warm.
Canary yellow
precipitate
is formed.
Indicates the
presence of
inorganic
phosphates.
Points to Remember:
Normally 0.8-1 gm of phosphorus as phosphate is
excreted per day.
Phosphates are present in urine as salts of sodium,
potassium, ammonium, calcium and magnesium. These
are crystallized out in alkaline urine.
Excretion is increased in bone diseases like rickets,
osteomalacia, and parathyroid dysfunction.
Excretion is decreased in:
Diarrhea
Infections
Nephritis
Hypoparathyroidism
Pregnancy
Test for calcium:
Principle: Calcium is precipitated as calcium oxalate with
potassium oxalate in acidic condition.

Experiment
Observation
Inference
To the second part

of the filtrate, add
2 ml of 2% potassium
oxalate solution.
White precipitate
is formed.
Indicates the
presence
of calcium.
Points to Remember:
The excretion of calcium is 100- 200 mg/day.
Excretion increases in:
Hyperparathyroidism
Hyperthyroidism
Hypervitaminosis D
Multiple myeloma
This test is known as Sulkowaskis 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.

Experiment
Observation
Inference
Take 2 ml of urine
in a test tube. Add
1-2 drops of
phenolphthalein
indicator. Mix.
Add 2% sodium
carbonate drop by
drop with constant
mixing till the color
of the solution turns
faint pink. Boil.
Hold a piece of
red litmus paper
at the mouth of the
test tube.
Red litmus
changes to blue.
Indicates the
presence of
ammonia.
Points to Remember:
Urinary ammonia is derived from glutamine and other
amino acids in kidney.
The average excretion of ammonia is about 0.7 gm/ day.
There is an increase in ammonia excretion when acid
forming foods are taken.
Ammonia is excreted as ammonium salts.
The kidneys manufacture ammonia in proportion to the
amount of acid radicals excreted in urine.
In alkaline urine, ammonium salts are absent.
Excretion of ammonia is increased in acidosis.
Excretion of ammonia is decreased in alkalosis
Impaired protein metabolism increases the output of
ammonia in urine.
To enhance the conversion of NH4 into NH3, the solution
is made alkaline before boiling.
If the solution is made strongly alkaline, urea will
interfere with the reaction.

Organic Constituents
Tests for organic constituents are as follows:
Test for Urea
Sodium Hypobromite Test:
Principle: When urea is treated with Sodium hypobromite, it
decomposes to give nitrogen, carbon dioxide and water.
Liberation of nitrogen gas produces brisk effervescence.
Experiment
Observation
Inference
Take 3 ml of urine
in a test tube.
Add 5 drops of
freshly prepared
alkaline sodium
hypobromite solution
and mix.
Brisk effervescence
of Nitrogen gas
is seen.
Indicates the
presence of urea.
Specific Urease Test:

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

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/ Benedicts Uric Acid Test:
Principle: Uric acid in alkaline condition reduces phosphotungstic acid to tungsten blue.
Benedicts uric acid reagent: Composed of sodium tungstate,
orthophosphoric acid, concentrated Sulfuric acid and solid
sodium carbonate.
Experiment
Observation
Inference
Take 3 ml of urine
in a test tube; add
1 ml of Benedicts
uric acid reagent
and 1 ml of 20%
sodium carbonate.
Mix.
Deep blue color

is formed.
Indicates the
presence of
Uric acid

ii. Schiffs Test:
Principle: Uric acid reduces salts of silver nitrate to metallic
silver.
Experiment
Observation
Inference
Moisten a piece of
filter paper with
few drops of
ammoniacal silver
nitrate solution.
Add 2-3 drops of
urine on the silver
nitrate drops.
Black spots are

seen on the
filter paper.
Indicates the
presence of
Uric acid.
Points to Remember:
Uric acid is the end product of purine metabolism.
The daily output of uric acid varies in the range of 0.6 to
1 gm.
Leukemias especially during cytotoxic drug therapy
Wilsons disease
Administration of cortisone/ ACTH
Excretion decreases in renal failure.
Test for Creatinine (Jaffes Test)
Principle: Creatinine reacts with picric acid in alkaline
medium to form reddish orange colored creatinine
picrate.

Experiment
Observation
Inference
Take 3 ml of urine
in a test tube.
Add 1 ml saturated
picric acid solution,
and 3-4 drops of
10% sodium
hydroxide.
Reddish orange
color is formed.
Indicates the
presence of
creatinine.
Points to Remember:
Creatinine is the anhydride of creatine.
Urinary creatinine is derived from muscle creatine.
It is not influenced by the protein intake.
Excretion in adults ranges from 1-2 gm/day.
In women and in elderly people the values are lower due
to lesser muscular mass.
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.

Experiment
Observation
Inference
Take 5 ml of urine
in a test tube. Add
2 ml of 10% barium
chloride and 2 ml
concentrated
hydrochloric acid.
Mix and filter.
Divide the filtrate
into two portions.
Boil one and compare
with the control.
Trace turbidity is
seen over that in
control.
Indicates the
presence of organic
sulfate.
Points to Remember:
Ethereal sulfates in urine are the conjugated sulfates,
phenol- sulfuric acid and indoxyl sulfuric acid.
These ethereal sulfates result from phenols produced
during putrefaction of protein material (amino acids) in
the intestine.
About 100 mg of organic sulfate are excreted per day.
Indican (Potassium salt of indoxyl sulfate) is a typical
example.
Bacterial decomposition of body protein as in gangrene
and putrid pus formation, etc. result in the increased
excretion of indican.
Inherited disorderscystenuria, homocystinuria
Cyanide poisoningthiocyanate.
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
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,
foaming
Bile pigments
Yellow fluorescent,
non-foaming
Riboflavin
Black
melanin
Black on standing
Alkaptonuria- due to presence of homogentisic

acid
Milky white
Chyluria- filariasis, due to the presence of pus,

bacterial or epithelial cells and lipids.
Odor
Odor
Cause
Putrid or ammonical odor Bacterial decomposition

Fruity odor
Diabetic ketoacidosis, chronic starvation
Mousy odor
Phenylketonuria
Maple syrup
Maple syrup urine disease
Specific Gravity
Increased in acute nephritis and fever.
Decreased in diabetes insipidus.
pH
Significantly acidic urine is voided in fever and diabetes.

Alkali therapy and urinary retention make urine alkaline.
Decrease in urinary pH: metabolic acidosis
Increase in urinary pH: metabolic alkalosis

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.

Experiment
Observation
Inference
Take 10 ml of urine
in a test tube. Hold
the tube over a flame
in a slanting position
and boil the upper
5 ml of the albumin
solution. The lower
half serves as control.
Add 1% acetic acid
drop by drop.
A white coagulum is
seen at the upper
heated portion, which
gets intensified after
the addition of
acetic acid.
Indicates the
presence of heat
coagulable
protein like
albumin.
Points to Remember:
The amount of protein excreted normally in 24 hours
urine is insignificant and it is less than 150 mg/day.
When proteins appear in detectable quantities in urine,
it is called proteinuria/albuminuria.
The presence of detectable amount of protein is
characteristic of kidney diseases.
The normal glomeruli of kidneys are not permeable to
substances with molecular weight of 70 kD. The plasma
proteins of molecular weight of more than 70 kD, hence
are absent in normal urine.
When glomeruli are damaged or diseased, they become
more permeable and plasma proteins appear in urine.
The smaller molecules of albumin pass through damaged
glomeruli more readily than the heavier globulin and so,
when the proteins appear in urine, the albumin fraction
predominates.

Bence Jones protein, an immunoglobulin appears in urine
in cases of multiple myeloma. Protein precipitates
between 40- 60C, disappears at 100C and reappears on
cooling.
Types of proteinuria:
1. Functional
proteinuria
2. Organic
proteinuria
a. Prerenal
b. Renal
c. Postrenal
Violent exercise
Cold bath
Pregnancy
Cardiac diseases
Abdominal tumors
Cancer
Collagen diseases
Fever
Anemia
Acute and chronic
glomerulonephritis
TB kidneys
Inflammatory conditions of
kidney, ureter, bladder,
prostate
Bleeding in genitourinary tract
Rating of proteinuria: Proteinuria can be rated as +, ++,

+++ depending upon the visibility of newspaper held at
the other side of the test tube after the coagulation test is
performed.
Visibility of news print
Rating of proteinuria
Visible with difficulty
Visible but letters cannot be

distinguished
++
Not visible
+++

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.
Hellers Nitric Acid Ring Test
Principle: Nitric acid causes precipitation of proteins.
Experiment
Observation
Inference
Take 3 ml of nitric
acid in a test tube.
Add 3 ml of urine
along the sides of
test tube.
A white ring is formed

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

Sulfosalicylic Acid Test
Principle: Sulfosalicylic acid is an alkaloidal reagent and so
it neutralizes the positively charged protein to produce
precipitation.
Experiment
Observation
Inference
Take 3 ml of urine
in a test tube. Add
20% sulfosalicylic
acid drop by drop.
White precipitate
is formed.
Indicates the
presence of
protein.
Point to Remember:
This test is used as a routine test for protein.
Test for Reducing Sugar (Benedicts 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.

Experiment
Observation
Inference
To 5 ml of Benedicts
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 Benedicts test is usually suggestive of presence
of glucose in urine.
Common causes of glycosuria are:
Diabetes mellitus
Endocrinal disorders such as hyperpituitarism,
hyperthyroidism, hyperadrenalism.
Emotional glycosuria: It is a benign condition seen in
anger, fear, etc. due to hypersecretion of adrenaline in
stress.
Renal glycosuria in which glucose reabsorption by
kidney tubules is defective.
Alimentary glycosuria: It is a benign condition which is
seen after excessive intake of carbohydrate or patient
is on glucose infusion.
Reducing sugar
Condition
Glucose
Diabetes mellitus, Renal glycosuria
Fructose
Disorders of fructose metabolism, essential

fructosuria, hereditary fructose intolerance
Galactose
Galactosemia
Lactose
Pregnancy, lactating woman
Pentose
Disorder of uronic acid pathway (essential

pentosuria)
Non-sugars such as ascorbic acid, glutathione,

salicylates, uric acid, glucuronides and homogentisic
acid will also give positive result with Benedicts
reagent.

Test for Ketone Bodies
Test for ketone bodies are as follows:
Rotheras Test for Acetone and Acetoacetic Acid
Principle: Acetone and acetoacetic acid form permanganate
colored complex with sodium nitroprusside in presence of
ammonia.
Experiment
Observation
Inference
Take 5 ml of urine in a
test tube. Add solid
ammonium sulfate a
little at a time with
mixing to saturate
the solution. Add 2 or
3 drops of freshly
prepared 5% sodium
nitroprusside solution.
Mix well and add
1 ml of strong ammonium
hydroxide drop-wise
along the side of
the test tube.
Permanganate
colored ring
is formed.
Indicates the
presence of
ketone bodies.
Gerhardts Test for Acetoacetic Acid

Principle: Acetoacetic acid gives a red color with ferric
chloride.
Experiment
Observation
Inference
Take 3 ml of urine
in a test tube and
add 10% ferric
chloride solution
drop by drop till
maximum precipitate
of ferric phosphate
is formed. Filter. To
the filtrate add
further quantities
of 10% ferric chloride.
Port-wine color
is obtained
Indicates the
presence of
acetoacetic acid.

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 -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 -hydroxy butyric acid.
Rotheras test is very sensitive. It is answered even by
small amounts of acetone and acetoacetic acid.
-hydroxy butyrate does not answer Rotheras or
Gerhardts 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 Gerhardts test is negative and Rotheras is positive,
acetone is present.
Gerhardts test or ferric chloride test is useful in detecting
a large number of abnormal constituents in urine, in rare
disorders. In addition to metabolites, drugs excreted can
be detected by this test. Some of the compounds detected
are listed below.
Compound
Color in the test
Phenyl pyruvic acid in

phenylketonuria
Stable blue/bluish green color
Homogentisic acid in
alkaptonuria
Rapidly fading blue or green

color
-hydroxyphenyl pyruvic acid

in tyrosinosis
Rapidly fading green color
Branched chain amino acids in

Maple syrup urine disease
Blue color
Imidazole pyruvic acid in

histidinemia
Green color
Melanin
Green to black
Indican in Hartnup disease
Violet or blue color
Salicylates
Stable red color/ port-wine color
Phenothiazine derivatives
Purple pink color
p-Aminobenzaldehyde
Reddish brown
Phenols
Violet color

Test for Bile Salts (Hays Test)
Principle: Hays test is based on the fact that bile salts
lower the surface tension of urine allowing the sulfur to
sink.
Experiment
Observation
Inference
Take 2 ml of urine
in a test tube. Gently
sprinkle a little fine
sulfur powder over
the surface of urine.
Observe without
mixing.
Sulfur powder
sinks to the bottom.
Indicates the
presence of
bile salts.
Points to Remember:
Bile salts are sodium and potassium salts of glycocholates
and taurocholates.
Normally bile salts and bile pigments do not enter the
general circulation and therefore, they are absent in the
normal urine.
But, if there is intrahepatic or posthepatic obstruction to
the flow of bile, regurgitation occurs in the general
circulation and bile salts appear in urine.
Bile salts are present in urine along with bile pigments in
obstructive jaundice.
This is not a specific test for bile salts but is usually done
to detect bile salts.
Alcohol and salicylates give a false positive test.

Test for Bile Pigments
Tests for bile pigments are as follows:
Gmelins Test
Principle: Bile pigments are oxidized by nitric acid to various
colored products, e.g. biliverdin (green), bilicyanin (blue),
bilifuscin (red) and choletelin (yellow)
Experiment
Observation
Inference
Take 5 ml of conc.
Nitric acid in a
test tube. Add 5 ml
of urine carefully to
form a separate layer.
Various colored rings

will be formed at the
point of contact
of the two liquids.
(play of colors).
Indicates the
presence of bile
pigments.
Fouchets Test
Principle: Bile pigments adsorbed on barium sulfate precipitate are oxidized to colored products by Fouchets reagent.
Fouchets reagent: 10% ferric chloride in 25% trichloroacetic
acid.
Experiment
Observation
Inference
Take 5 ml of urine
in a test tube. Add
few crystals of
magnesium sulfate.
Then add 3 ml of
10% barium
chloride solution. Mix.
Filter. Unfold the
filter paper. Add a
few drops of
Fouchets reagent
on the precipitate.
A white precipitate
of barium
sulfate is formed.
A green color develops
on the filter paper.
Indicates the
presence of bile
pigments.

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.
Fouchets test is a highly sensitive test for bilirubin.
Ferric chloride, present in the Fouchets reagent acts as
an oxidizing agent. It oxidizes bilirubin to biliverdin
(green) or bilicyanin (blue).
Test for Blood
Principle: Hemoglobin (peroxidase) of blood decomposes
hydrogen peroxide catalytically and liberates nascent
oxygen. This oxygen oxidizes benzidine to a blue or green
compound. This color changes to brown within a few
minutes on exposure to air.
Benzidine reagent: It contains benzidine and glacial acetic acid.
Experiment
Observation
Inference
Take 2-3 drops of

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

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

subjected to heat treatment (95-100C), the enzyme is
inactivated and the test becomes negative.
Heme, is stable to heat.
When high concentration of ascorbic acid is present in
urine, it is oxidized more readily than benzidine by
oxygen liberated from hydrogen peroxide. The benzidine
reaction then becomes negative although sufficient blood
is present in urine.
Report:
The abnormal constituents present in the given sample of
urine are
Hemoglobin and its Derivatives 117
10
Hemoglobin and its

Derivatives
Hemoglobin is a conjugated protein, consisting of the protein

part called globin and the prosthetic part called heme. The
hemoglobins differ depending on the type of polypeptide
chains they are composed of. The normal hemoglobins are
Hb-A (hemoglobin of adult), Hb-F (hemoglobin of fetal life)
and Hb-A2 (hemoglobin of postnatal).
The following are the derivatives of hemoglobin.
a. Native hemoglobin: It serves as oxygen carrier in the
blood.
b. Oxyhemoglobin: Hemoglobin in combination with 4
molecules of oxygen.
c. Carboxy-hemoglobin: Hemoglobin in combination with 4
molecules of carbon monoxide.
d. Methemoglobin: It is oxidized non-functional form of
hemoglobin is which iron is in the ferric state.
e. Hemochromogen: Denatured hemoglobin in which iron is
in the ferrous (Fe++) form. During heating with alkali,
globin portions get denatured and heme is oxidized to
hematin (ferrous). On treatment with reducing agent, the
hematin is converted to heme which combines with
denatured globin to form hemochromogen.
f. Hematin: It is the oxidized form of heme in which iron is
in ferric state (Fe3+).
g. Hemin: Chloride form of hematin.
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
Fraunhofers 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 ( at 577 nm, 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 ( at 570 and 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 Nippes fluid hemoglobin is
denatured and heme is oxidized to hematin. Hematin is
finally converted to hematin chloride, also called as hemin.
Nippes 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 Nippes
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 Nippes fluid
in the same way and heat again, till the fluid is just
evaporated. Do not overheat.
Examine the crystals of hemin under the low power as
well as the high power of the microscope. The hemin crystals
are rhombic and brown
colored. Draw the crystals in
the same color.
Clinical application/ medicolegal importance: Used in
forensic medicine to detect
traces of blood.
Fig. 10.3: Hemin crystals
(Label)
Spot Tests 123
11
Spot Tests
PHENYLKETONURIA
Phenylketonuria is an inherited metabolic disorder in
amino acid metabolism.
It is due to the deficiency of the enzyme, phenylalanine
hydroxylase. Therefore, the conversion of phenylalanine
to tyrosine is deficient, i.e. phenylalanine accumulates
in the blood giving a concentration of 10 to 80 mg/dl
compared with < 2 mg/dl in normal infants.
Among the derivatives of phenylalanine present in urine,
the largest amounts are phenylpyruvic acid and phenyl
lactic acid.
The tests used for screening phenylketonuria are:
Guthries 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
Experiment
Observation
Inference
Take 3 ml of urine
in a test tube and
add 10% ferric
chloride solution
drop by drop till
maximum precipitate
of ferric phosphate
is formed. Filter.
To the filtrate
add 2 ml of 10%
ferric chloride.
Bluish green color

is obtained
Indicates the
presence of
phenylpyruvate
Points to Remember:
This test is not specific since many other compounds
give a false positive test.
Nowadays prenatal diagnosis is possible using DNA
based test.
Alkaptonuria
It is an autosomal recessive disorder.
Prevalence is 1 in 25,000.
The defective enzyme in alkaptonuria is homogentisate
oxidase in tyrosine metabolism.
Homogentisate accumulates in blood and is excreted in
urine.
Homogentisate on standing gets oxidized to the
corresponding quinines, which polymerize to give black
or brown color. Because of this reason, urine of
alkaptonuric patients is black in color (coke in color) on
standing.
Spot Tests 125

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

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

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

Ammonia
Silver nitrate
Sodium nitroprusside solution
Sodium cyanide.
Procedure
Saturate the urine with sodium chloride.
Add 0.5 ml silver nitrate solution to 5 ml saturated
specimen and to 5 ml dilute ammonia.
Allow to stand for 1 minute, and then add to each 0.5 ml
sodium cyanide.
Terminal addition of cyanide is needed to bind the silver
ion and allow reaction of homocysteine with the
nitroprusside.
At this point excess cyanide begins to react with any
cysteine present to give a slowly developing positive test.
The test is positive for homocysteine if pink or purple
color develops in the test sample immediately.
Section 3
Quantitative
Tests
12
Principles of
Colorimetry
Many biochemical experiments involve the measurement

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

Wavelength
(in nm)
Color of light
absorbed
Color of
light reflected
400-435
435-500
500-570
570-600
600-630
630-700
Violet
Blue
Green
Yellow
Orange
Red
Green-yellow
Yellow
Red
Blue
Green-blue
Green
COLORIMETER
The quantum of light absorbed by a colored solution may
be determined by certain optical instrument called
colorimeter.
Colorimeter has been the traditional name for an
instrument that isolates specific wavelengths of light with
interchangeable filters for the visible portions of the
spectrum. In contrast to this, spectrophotometers have a
continuously adjustable monochromatic prism (or
grating) and can often measure the intensity of light from
the UV range, visible and infra red regions.
Components of the Colorimeter
Source of light: A lamp provides light in visible region of
the spectrum. Usually tungsten lamp is the source of light.
Adjustable slit: The light emerging from tungsten lamp is
allowed to pass through a narrow adjustable slit.
Condensing lens: Provides parallel beam of light.
Filter: It provides the desired monochromatic light (of
single wavelength) by filtering other wavelengths. The
color of the filter is complementary to the color of the
solution. This allows only appropriate wavelength of
light to pass through the colored solution.
Principles of Colorimetry 131

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

In colorimetric estimation it is necessary to prepare three
solutions:
Blank (B)
Standard (S) and
Test (T)

Blank
Blank is done to delete the color due to reagents. Since
some reagents are colored, they add on to the color
produced by the substance which is to be estimated. This
increases the color intensity which in turn gives high
concentration of the substance to be estimated.
Alternately the blank solution is used to set the meter of the
instrument to 100% transmittance (T) or zero absorbance.
The values of blank are subtracted from tests and
standard.
A blank is prepared by using all the reagents except the
biological material to be estimated.
Type of Blank
Two types of blank are used.
Water blank: It is used to adjust the OD to zero and T to
100%.
Reagent blank: It is prepared by adding all reagents except
the substance to be estimated.
Standard Solution
It is a solution of known concentration of the substance in
pure form which is to be estimated. As both concentration
and OD of the standard solution are known, the concentration of unknown can be calculated by using the formula.
Test Solution
The test solution is made by treating a specific volume of the
test sample with reagents as specified in the procedure.
TECHNIQUE
The light passes through an adjustable slit and then
through a condenser lens which gives a parallel beam of
light. This beam of light passes through a colored filter to
give a monochromatic light.
The colored solution to be analyzed is taken in a glass
cuvette.
Complementary colors for selection of filters
Filter
Color of Solution
Blue
Purple
Yellow
Orange
Red
Green
Violet
Blue green
The monochromatic light is incident (Io) on the solution,

a part of it is absorbed by the solution and the rest is
transmitted (I).
The transmitted light is detected by a photocell. It converts
light energy into electrical energy, the strength of which
is calibrated in percentage transmitted light.
A galvanometer connected to the photocell measures the
output electrical energy.
The measurement of color intensity of a colored solution
by photometry is governed by two laws
1. Beers law
2. Lamberts law
Beers 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 C.

Lamberts 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 L
Combining the two laws, A 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.
T=
Intensity of light emerging (I)

Intensity of light incidence (I o )
T=
I
Io
% T = 100
I
Io
When Io is 100 (adjusted with blank on the galvanometer

scale), I gives % transmittance.
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
13
Estimation of
Blood Sugar
The main sugar of blood is glucose along with other

carbohydrate constituents. Hence, blood glucose is
commonly referred as blood sugar.
Blood glucose estimation is a common test done in all
laboratories because it helps in diagnosis, management of
diabetes mellitus and is a common prerequisite for any
surgery.
Methods of Estimation
Folin -Wus 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-Wus Method
Aim of the Test
To estimate the amount of blood sugar.
Method
Folin-Wus method.
Principle
Blood is deproteinized by Tungstic acid formed by
the reaction of sodium tungstate and sulfuric acid. The
protein-free filtrate containing glucose is treated with
alkaline copper reagent. Glucose in the protein- free filtrate
at higher temperature in alkaline medium reduces cupric
oxide to cuprous oxide .The cuprous oxide formed is treated
with Phosphomolybdic acid which is reduced to phosphomolybdous acid (molybdenum blue), a blue solution. The
intensity of this blue solution is a measure of the amount of
glucose present.

Reagents
1.
2.
3.
4.
5.
10% sodium tungstate

2/3 N Sulfuric acid
Alkaline copper reagent
Phosphomolybdic acid
Standard glucose: Contains known amount of glucose
(0.2 mg/ml).
Procedure
a. Preparation of protein-free filtrate: Into a dry 100 ml conical
flask, pipette 7 ml distilled water and 1 ml
blood. Rotate the flask. Add one ml sodium tungstate
and mix. Add 1 ml of H2SO4 drop by drop with shaking.
Thus, the dilution is 1 in 10. Let it stand for 10 minutes.
Filter into a dry test-tube. The filtrate should be clear and
colorless.
b. Development of color: Set up 3 Folin-Wu tubes, marked B,
S, and T for blank, standard and test respectively. Pipette
2 ml of distilled water in B, 2 ml standard glucose into
S and 2 ml of protein free filtrate into T. Pipette 2 ml
alkaline copper reagent into each. Mix the contents. Keep
the tubes in a boiling water bath for exactly 8 minutes.
Then remove them and cool in a beaker containing cold
water. After cooling add 2 ml Phosphomolybdic acid
reagent to each. Rotate to mix and make up the volume to
25 ml mark with distilled water. Mix the contents by
inverting the tube by placing your palm tightly over the
mouth of Folin -Wu tube. Read the Optical density values
using a blue filter.

Protocol
Reagents
Blank
Standard Test
Distilled water
2 ml
Standard Glucose
2 ml
Protein-free filtrate
2 ml
Alkaline copper reagent
2 ml
2 ml
2 ml
Mix the contents-keep the tubes in a boiling water bath for exactly
8 minutes.
Phosphomolybdic acid reagent
2 ml
2 ml
2 ml
Rotate to mix and make up the volume to 25 ml mark with distilled

water. Mix the contests by inverting the tube placing your palm
tightly over the mouth. Read the optical densities using a blue filter.
Optical Density at 490 nm.
Calculation
Concentration of glucose in mg/100 ml blood =
OD of test OD of blankT B Conc.
100 of standard
= OD of standard OD of blank
S B Volume of sample 100
=
OD of T OD of B
10 100
Concentration of standard
OD of S OD of B
2
1
TB
10 100
0.2
SB
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 autooxidation of cuprous oxide formed by atmospheric
oxygen by decreasing the surface area of the solution in
the constricted area exposed to the atmosphere, and
providing a larger area in the bulb for reaction to take
place.
Oxalate precipitates Ca2+ of blood to prevent coagulation.
Fluoride inhibits glycolytic enzymes of RBC to prevent
glycolysis before estimation.
The Folin-Wu filtrate still contains some polypeptides,
which escape precipitation by tungstate. These
polypeptides bind Cu2+ at their peptide bonds to form
colored complexes and consequently produce some errors
in the estimated blood glucose value. So it is important to
precipitate the proteins.
Both O-toluidine and glucose oxidase method are highly
specific for glucose compared to Folin-Wu method.
Any increase in blood glucose level is called hyperglycemia and any decrease in blood glucose level is called
hypoglycemia.

Hyperglycemia is seen in:
Diabetes mellitus
Hyperthyroidism
Hyperpituitarism
Pancreatitis
Cushing syndrome
Pheochromocytoma
Sepsis and infectious diseases and administration of
general anesthetics.
Transient rise may occur in emotional status such as
anger, anxiety and fear due to excessive secretion of
epinephrine, which favors glycogenolysis.
Value below 40 mg/dl by glucose oxidase method is
known as hypoglycemia.
Commonest causes of hypoglycemia are:
Increased level of insulin: accidental over administration of insulin in diabetes or certain tumors of the
-cells of islets of Langerhans which leads to
hypersecretion of insulin.
Hypothyroidism
Addisons disease and severe hepatic disease can
produce hypoglycemia.
Other rare causes are:
Severe exercise (due to depletion of liver glycogen store)
Glycogen storage disorder (deficiency of glucose-6phosphatase)
Steatorrhea (due to impaired glucose absorption)
Starvation
Alcohol ingestion.
14
Estimation of
Blood Urea
Urea is the metabolic end product derived from the

catabolism of proteins. It is synthesized in the liver from
ammonia and carbon dioxide and is excreted by kidney.
Aim
To estimate the serum urea.
Method
Diacetyl monoxime method (DAM method).
Principle
Urea reacts with Diacetly monoxime under strong acidic
condition in presence of ferric ions and thiosemicarbazide
to give a pink colored complex. Intensity of color is a measure
of amount of urea present in blood. Color intensity is
compared with standard and is measured using green filter
(540 nm).
Procedure
Label three test tubes as B (Blank), T (Test) and S (Standard).
Pipette 1 ml distilled water into B, 1 ml of diluted serum
Estimation of Blood Urea 145

(1:100) into T and 1ml of standard urea solution (1 mg in
100 ml of distilled water) into S. Add 2 ml of diacetly
monoxime (DAM) solution and 2 ml of acid reagent into
each tube. Mix well and keep in a boiling water bath for 20
minutes. Cool at room temperature and take OD at 540 nm.
Protocol
Reagents
Blank
Distilled water
Standard
Serum
Standard
Test
1 ml
1 ml
1 ml
Color reagent (DAM)
2 ml
2 ml
2 ml
Acid reagent
2 ml
2 ml
2 ml
Mix well and Boil for 20 min and cool

Optical Density at 540 nm
Calculation
Concentration of urea in mg/100 ml of blood,
=
OD of Test OD of Blank
Conc. of Standard
100
OD of Standard OD of Blank Volume of Sample
OD of T 0.01
100
OD of S 0.01
OD of T
100
OD of S
= __________ mg/dl.
Report
The amount of urea present in the given blood sample is
..mg/dl.

Clinical Significance
Normal blood urea level ranges 15-45 mg/dl.
Causes of increased blood urea level:
1. Pre-renal causes: Since blood supply to the kidney is
reduced, filtration and excretion of urea is minimum.
a. High protein diet.
b. Increases with age
c. Dehydration as in diarrhea, vomiting.
d. Increased cardiac output/ failure
e. Increased catabolism of proteins as in fever and
wasting diseases.
2. Renal causes: Synthesis of urea is normal. But damage
in the renal tissue leads to poor filtration and excretion
of urea.
a. Nephritis
b. Nephrotic syndrome
c. Acute renal failure
d. Chronic renal failure
e. Polycystic kidney
f. Hydronephrosis
g. Malignant hypertension.
3. Postrenal causes: Synthesis and filtration are normal,
but renal passage is blocked. Hence, minimum
excretion of urea occurs.
a. Obstruction in the renal tract
b. Enlargement of prostate
c. Stones in bladder.
Blood urea level decreases in liver diseases due to
decreased synthesis.
Blood urea level is commonly monitored to evaluate
kidney diseases.
Estimation of Blood Urea 147

A high urea concentration in the urine reflects the
concentrating power of the kidney.
Relationship between blood urea nitrogen (BUN) and
urea is
Blood urea nitrogen (mg/dl) = urea (mg/dl)/ 2.14
Urea level is generally studied in conjunction with
creatinine level to identify renal dysfunction. Urea/
creatinine ratio is sometimes used to discriminate between
prerenal and postrenal uremia.
15
Estimation of
Urine Creatinine
Creatinine is a waste product derived from endogenous

sources by tissue creatine breakdown and is excreted by the
kidney. Creatinine is the anhydride of creatine. The reaction
occurs non-enzymatically.
Creatine
creatinine
H2O
98% of the total creatine is in the muscle as creatine
phosphate. About 2% of the total creatine is converted daily
to creatinine so that the amount of creatinine produced is
related to the total muscle mass. As muscle mass remains
approximately same, creatinine also remains same in
plasma and urine.
Aim
To estimate the amount of creatinine in urine.
Method
Jaffes alkaline picrate method.
Principle
Creatinine present in urine reacts with picric acid in the
presence of sodium hydroxide to give an orange color. The
intensity of the color developed is directly proportional to
Estimation of Urine Creatinine 149

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

OD
Calculation
Creatinine in mg per 100 ml urine
=
Conc. of Standard
100
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 2026 mg/kg/day in men and 14- 20 mg/kg/day in women.
Creatinine coefficient is more precise and is used to assess
the functional muscle mass in the body.
Very little creatine is normally found in adult urine except
in women during pregnancy and early postpartum.
The excretion of creatinine increases in fevers and
wasting diseases.
The excretion of creatinine decreases in myopathies and
renal failure.
A variety of compounds, like proteins, glucose, pyruvate,
ascorbate and ketones interfere in Jaffes 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
Creatinine Clearance (ml/min) =
Where U =
P =
V =
A =
1.73 =
U V 1.73
PA
mg of creatinine/dl in urine
mg of creatinine/dl in serum or plasma
Volume of urine in ml/min
Body surface area of the patient
Standard average surface area of normal
individual
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.
16
Estimation of Serum
Inorganic Phosphate
Phosphorus in the body is mainly present in the bones;

small portion is present in cells and soft tissue. It is a
component of many important biological compounds, e.g.
some proteins, lipids, nucleic acid and coenzymes.
It plays an important role in acid base regulation
particularly by the kidneys.
The serum phosphate may exist as free ions (40%) or in a
complex form (50%) with cations such as calcium,
magnesium, sodium, potassium, etc. About 10% of serum
phosphate is bound to proteins.
Aim
To estimate serum inorganic phosphate.
Method
Fiske and Subbarow method.
Principle
A quantitative method for the estimation of inorganic
phosphate was first developed by Fiske and Subbarow. In
this method serum proteins are precipitated by
Estimation of Serum Inorganic Phosphate 155

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

Protocol
Reagents
Blank Standard
Water
Test
5 ml
Standard phosphate solution
5 ml
Protein-free filtrate
5 ml
1 ml
1 ml
1 ml
0.4 ml
0.4 ml
0.4 ml
Molybdic acid reagent

Mix
ANSA Solution
Mix and allow to stand for 10 minutes

Distilled water
3 ml
3 ml
3 ml
Mix and read OD values exactly after 10 minutes using a red filter
(660)
Optical Density at 660 nm.
Calculation
Phosphate expressed as phosphorus (P) in mg per 100 ml
serum
=
Conc. of Standard
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: glycerophosphatesnucleotide phosphates, etc. 15- 30 mg/100 ml.
Phospholipids: lecithin, cephalin, sphingomyelin 1016 mg/100 ml.
Residual phosphorus: 87% of phosphorus in the body
is present in bones and the rest is present in cells and
tissues.
Hyperphosphatemia (increase in serum phosphate) is
seen in hypoparathyroidism, hypervitaminosis D and
renal failure.
Decreased level is seen in hyperparathyroidism, rickets,
osteomalacia, vitamin D deficiency and due to decreased

reabsorption of phosphate by kidney tubules as in
Fanconis syndrome.
In diabetes mellitus organic phosphorus is lower but
inorganic phosphorus is higher.
There is a reciprocal relationship between serum calcium
and phosphorus.
Physiological fall of phosphorus occurs when there is
increased carbohydrate utilization. Insulin therapy also
has a similar effect.
17
Estimation of Serum
Total Proteins
Serum proteins represent a complex mixture containing a

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

Reagents
1. 28% sodium sulfite
2. Standard protein solution (6 g/dl)
3. Dilute Biuret reagent.
Procedure
Precipitation of Globulins
Pipette 0.2 ml of serum into a test tube. Add 5.8 ml of 28%
sodium sulfite solution. Mix. Allow to stand for 5 minutes.
Filter through Whatman No.44 dry filter paper. Use the clear
filtrate for estimation of albumin.
Globulins in serum are selectively precipitated by 28%
sodium sulfite. Albumin is estimated in the filtrate of
processed serum. It reacts with copper sulfate in an alkaline
medium to give a purple/violet color. The difference in
protein content of whole serum and the serum filtrate
(albumin) after sodium sulfite treatment is the value for
globulins.
Color Development
Set up four test tubes marked B for blank, S for standard,
A for albumin and TP for total protein. To B add 3 ml of
water. To S add 3 ml of standard protein solution. To A add
3 ml of globulin-free filtrate and to TP add 0.1 ml of serum
and 2.9 ml of water.
To all the four tubes, add 3 ml of Biuret reagent. Mix.
After 10 minutes read the optical densities using a green
filter (540 nm).
Estimation of Serum Total Proteins 161

Protocol
Reagent
Blank
Standard
Water
3 ml
2.9 ml
3 ml
Standard protein
solution
Albumin Total Protein
Globulin-free filtrate
3 ml
Serum
0.1 ml
3 ml
3 ml
3 ml
3 ml
Biuret reagent
Mix. After 10 min read the O.D. using green filter (540 nm)
Optical density
at 540 nm
Calculation
Total protein in 100 ml of serum
=
Conc. of Standard
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

=
Conc. of Standard
100
AB 6
100 mg%
S B 0.1

=
AB
6
100
g%
S B 1000 0.1
TP B
6
SB
= __________ g%
Globulin in 100 ml of serum = Total protein Albumin

= __________ g%
Determination of Serum Albumin by Bromocresol
Green Method
Principle
Albumin present in serum binds specifically with
bromocresol green at pH 4.1 to form a green colored complex.
Intensity can be measured by colorimeter at 640 nm (red
filter).
Albumin standard = 4 g/ dl in normal saline containing 0.1
g/dl sodium azide.
Procedure
Pipette in three tubes as follows:
Reagents
Blank
Standard
Bromocresol green reagent
Test
5 ml
5 ml
5 ml
Serum
0.05 ml
Albumin standard
0.05 ml
0.05 ml
Distilled water
Mix thoroughly and keep at room temperature for 10

minutes measuring the intensity of the test and standard by
setting blank at 100% T using 640 nm.

Calculations
Serum albumin g/ dl
=
4
OD of Standard OD of Blank
Reference Value
Total serum protein
Normal Albumin
Normal Globulin
Normal A:G Ratio
=
=
=
=
6-8 g/100 ml.

3.5-5.5 g/100 ml
2-3.5 g/100 ml
1.2 : 1 to 1.5 : 1
Report
1. The amount of Total Protein in the given sample of serum
is _______ g/dl.
2. The amount of Albumin in 100 ml serum _______ g/dl.
3. Globulin _______ g//dl.
4. A:G Ratio _______.
Biuret method is the most common method used in the
students 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-CONH2) also answers the test.
Increase in serum protein can occur in dehydration with
A:G ratio remaining constant.
In multiple myeloma, increase in total protein is mainly
due to increased level of globulins. Albumin
concentration remains same or is slightly reduced.
Overall increase in total protein is rare. Changes occur in
globulin fraction.
Decrease in serum protein level is invariably due to
decrease in albumin. A:G ratio also decreases due to either
reduction of albumin and or elevation of globulin.
The measurement of total proteins in plasma is of limited
value as it may be altered by changes in plasma volume.
An increase of plasma protein is caused by dehydration
and decrease by overhydration.
Total protein concentration is higher when a person is in
standing position than recumbent position (due to shift
of water from vascular compartment into interstitial
compartment).
Exercise increase total protein concentration (5-10%).
Significant increase in total protein concentration arises
with an increase in total globulin (usually gamma
globulin).
Decrease in albumin in serum can be due to the following
conditions:
1. Loss of albumin due to:
a. Nephrotic syndrome
b. Protein loosing enteropathy
c. Wide spread burns
d. Severe hemorrhage

2. Defective anabolism which may be due to:
a. Liver disease due to reduced synthesis
b. Malnutrition ex: Kwashiorkor
c. Carcinoma of stomach or pancreas
Although the concentration of serum albumin is reduced
in severe liver diseases, globulin is increased so that total
protein concentration is high.
Serum albumin level can be as low as 1.5 g per 100 ml in
kwashiorkor. Defective absorption from intestine in
pancreatic diseases, malignancy of gastrointestinal tract,
intestinal fistula, congenital malabsorption syndrome and
severe tuberculosis can result in low serum albumin level.
Albumin is lost in severe burns and hemorrhage.
Excessive breakdown of body proteins with inadequate
supply or defective utilization of proteins is seen in
uncontrolled diabetes, thyrotoxicosis, prolonged febrile
diseases and trauma. A small decrease in serum protein
level is seen in pregnancy. In toxemia of pregnancy, serum
albumin level is lowered.
Section 4
Chromatography 167
Demonstration
Practicals
18
Chromatography
Chromatography was introduced by Tswett in 1906 for

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

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

Distance traveled by the solvent
i.e. 30/40 = 0.75 , 20/40 = 0.5 and 10/40 = 0.25

Rf value are 0.75, 0.5 and 0.25 respectively for a given solvent,
Rf values is characteristic of a compound. It is possible to
identify unknown substances by their Rf values.
Chromatography 171
Requirement
Whatman No 1 filter paper

Solvent system
Chromatography chamber
Capillary tube
Standard amino acids
Amino acid mixture to be identified.
Procedure
Solvent is taken in a shallow trough and kept in the bell jar
for saturation of the chamber. Whatman No. 1 filter paper is
used for paper chromatography. Cut the paper into
dimensions of 15 56 cm. With a pencil, draw a line along
the width of the paper, 5 cm from its edge. Equal points of
2 cm apart are marked from one end of the paper leaving
2 cm from both the edges.
Fig. 18.1: Paper chromatography

0.5% of standard unknown solution (mixture of amino
acids) is prepared. 10 l (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 100C
for 3 minutes. Distinct purple colored amino acid spots
appear (Fig. 18.2).
Rf values are found out for each distinct spot by
using the formula as explained. The amino acids can be
identified by their corresponding Rf value and also by
correlating with the Rf values of known amino acids, spotted
as standards.
Application
Paper chromatography is used for detection of amino
acids, sugars, pigments, etc.
Used in the identification of amino aciduria, e.g.
cystinuria, phenylketonuria, etc.
19
Electrophoresis
Electrophoresis is a popular technique used to separate

closely related compounds. It is based on the movement of
charged particles in the electric field. This technique is based
on the principle that positively charged particles migrate
towards cathode and negatively charged particles migrate
towards the anode. Rate of migration depends on the charge
of the molecule (Fig. 19.1).
Fig. 19.1: Electrophoresis
Electrophoresis 175
Applications
Electrophoresis has many applications
Separation of serum proteins for diagnostic purposes.
Determination of purity and molecular weight of
proteins.
Separation of isoenzymes in differential diagnosis.
Estimation of lipoprotein in health and diseases.
Finding normal and abnormal hemoglobin.
Diagnosis of Nephrotic syndrome.
Diagnosis of Hypoalbuminemia.
Diagnosis of cirrhosis of liver.
Diagnosis of multiple myeloma.
Diagnosis of chronic infections.
Factors Affecting Migration of Charged Particles
Structure, molecular weight and shape of the molecule.

Charge on the molecule.
Temperature
Dilution of the sample
Voltage
Strength of the buffers
pH of buffer
Types of Electrophoresis
Depending on the nature of the supporting medium
electrophoresis is classified into different types.
1. Paper electrophoresis
2. Agarose gel electrophoresis (supporting medium is
agarose gel)

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 l 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 1, 2 globulins,
-globulin and -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,
1-, 2- globulins, -globulin and -globulin.

Normal electrophoretic separation of serum protein will
have:
Albumin
- 56%
1-globulin - 3%
2-globulin - 13.5%
-globulin
- 15.5%
- globulin - 12%
Total
- 100%.
20
Glucose Tolerance Test
The glucose tolerance signifies the ability of the body to

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

Benedicts qualitative method
Glucose oxidase method (strip method).

Estimation of Urine Sugar by Benedicts Qualitative Method
Principle: Reducing sugar reduces copper sulfate to red
cuprous oxide.
Specimen: Urine
Procedure: To 5 ml of Benedicts reagent add 8 drops of urine
mix boil for 2 min and cool.
Color or precipitate
Amount of glucose
Blue color
Nil
Green color
Traces
Green PPT
0.5%
Yellow PPT
1%
Orange PPT
1.5%
Red PPT
2.0%
Brick Red PPT
>2.0%
Plotting a Graph for GTT

Sl no. Type
Fasting
30
min
60
min
90
min
120 150
min min
Normal
85
105
130
110
100 7 5
Lag type
90
180
155
100
8 5 100
Mild diabetic
95
150
225
175
125 9 0
Severe diabetic
140
200
360
240
210 190
Hypoglycemic
65
100
120
85
75
60
21
Estimation of Serum
AST and ALT
Human serum contains several transaminases of which

ALT (SGPT) and AST (SGOT) are of diagnostic significance.
Aim
To estimate serum ALT and AST.
Method
Reitman and Frankel method.
Principle
For estimation of AST serum is incubated with aspartate
and alpha ketoglutarate in phosphate buffer (pH 7.4) for 60
minutes. In the estimation of ALT, serum is incubated with
alanine and alpha ketoglutarate for 30 minutes. After a
measured time the reaction is stopped. Oxaloacetate is
formed from AST and is spontaneously decarboxylated to
pyruvate. Pyruvate is formed from ALT. Pyruvate reacts with
DNPH (dinitrophenylhydrazine) to form the corresponding
hydrazone which form a brown colored complex in alkaline
medium. Color intensity is measured against blank and
control.
Estimation of Serum AST and ALT 189

Reagents
1. SGPT substrate
2. SGOT substrate
3. 0.1 M phosphate buffer 4. DNPH reagent
5. 0.4 N NaOH
6. Standard pyruvate :
0.2 ml = 0.4 g
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 37C for 30 minutes.
Exactly after 30 minutes add 0.2 ml of serum into control
and 1 ml of DNPH solution into all the tubes. Mix and allow
it to stand for 20 minutes at room temperature. After 20
minutes add 10 ml of NaOH to all the tubes and mix well.
Allow it to stand for 20 minutes and read the OD at 540 nm.
Protocol
Reagents
Test
Serum (ml)
0.2
Control Standard Blank

-
Standard (ml)
0.2
Distilled water (ml)
0.2
Buffered substrate
Incubate at 37C for 30 minutes

Serum (ml)
0.2
DNPH (ml)
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 37C for 60
minutes. Exactly after 1 hour add 0.2 ml of serum to control
and 1 ml of DNPH solution to all the tubes. Mix and allow
it to stand for 20 minutes at room temperature. After 20
minutes add 10 ml of NaOH to all the tubes and mix well.
Allow it to stand for 20 minutes and read the OD at 505 nm.
Protocol
Pipette in the tubes labeled as follows:
Reagents
Test
Serum (ml)
0.2
0.2
Standard
Distilled water (ml)
0.2
Buffered substrate (ml)
1
Contd...
Estimation of Serum AST and ALT 191

Contd...
Reagents
Test
Incubate at 37 C for 60 min

Serum (ml)
0.2
DNPH (ml)
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.
22
Estimation of Serum
Cholesterol
Aim
Estimation of serum cholesterol.
Method
Cholesterol Oxidase Peroxidase methodology.
Principle
Enzymatic colorimetric determination of total cholesterol is
done according to the following reactions.
Cholesterolesterase Cholesterol + fatty acids
Cholesterol ester + H 2 O
Cholesterolesterase
Cholesterol ester + O 2
4-Cholesten-3-one + H 2 O 2
Peroxidase
2H 2 O 2 + Phenol + 4 Aminoantipyrine
Red quinine + 4H 2 O
Note: Cholesterol standard concentration : 200 mg/dl

Sample
Serum plasma.
Estimation of Serum Cholesterol 193

Procedure
Reagents
Blank
Standard
Sample
1000 l
1000 l
1000 l
Standard
10 l
Sample
10 l
Working reagent
Mix and incubate for 5 min. at 37C. 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, malnutrition, hyperthyroidism, anemia and liver diseases.
Estimation of Plasma Ascorbic Acid 195
23
Estimation of Plasma
Ascorbic Acid
Aim
To determine plasma ascorbic acid.
Method
2,6-dichlorophenolindophenol titration.
Principle
The protein free filtrate is titrated with the dye 2,6
dichlorophenolindophenol in acid solution. The blue
compound is red in acid solution, and on titration with a
solution of ascorbic acid, is reduced to a colorless leucobase,
the ascorbic acid being oxidized to dehydroascorbic
acid.
Reagents
Trichloroacetic acid, 10% solution of freshly prepared 5%
metaphosphoric acid.
Solution of 2,6-dichlorophenolindophenol.
Procedure
Mix equal volumes (4 ml is convenient) of plasma separated
immediately after withdrawing blood, and trichloroacetic
acid or metaphosphoric acid. Filter or centrifuge. Pipette

0.2 ml of the diluted dye solution into a test tube and titrate
with the filtrate until the red color has disappeared.
Calculation
0.2 ml dye is equivalent to 0.008 mg ascorbic acid. Hence,
mg of ascorbic acid/100 ml plasma
=
100 2 0.008
ml titration
1.6
ml titration
Note: If the plasma cannot be separated immediately, the

blood is collected into a test tube containing 1 drop each of
5% potassium cyanide and 20% potassium oxalate for 4 to
5 ml of blood.
Points to Remember:
Persons on an adequate intake of vitamin C will generally
be found to have a plasma level between 0.8 to 2 mg/100
ml.
Values below 0.2 mg% suggest the possibility of marked
ascorbic acid deficiency.
Plasma values are of limited value in diagnosing scurvy
and subclinical conditions of vitamin C undernutrition.
Vitamin C in WBC is the reliable index for the
determination of ascorbic acid status.
Flame Photometer 197
24
Flame Photometer
Flame photometer is an analytical instrument used for the

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

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.
25
CSF Analysis
Examination of Cerebrospinal Fluid (CSF) is important in

the diagnosis of neurological diseases.
Collection of CSF
CSF is usually collected from spinal canal by lumbar
puncture (LP).
The patient is made to lie on his side with the neck, thighs
and knees flexed.
Under local anesthesia with full aseptic precautions, an
LP needle is inserted into the subarachnoid space
between the 3rd and 4th intervertebral space.
The CSF is allowed to flow out spontaneously drop by
drop and about 5 ml is collected in clean sterile vials.
The sample is used for biochemical, cytological and
microbiological examination.
CSF must be examined immediately within 1 hour and
should not be refrigerated.
Biochemical Examination of CSF
Biochemical examination of CSF usually consists of
measurement of total proteins, glucose and chloride.
Estimation of enzymes like creatinine phosphokinase
(CKBB), AST or lactate dehydrogenase (LDH3) is useful
in case of cerebral infection.
CSF Analysis 201

Determination of Total Protein
Since the content of protein is usually very low, it is determined by measuring the turbidity by adding sulfosalicylic
acid.
Principle
Proteins are precipitated by sulfosalicylic acid. The turbidity
of the resultant uniform suspension is measured by means
of a colorimeter at 450 nm (blue filter) against a standard
solution which is treated similarly.
Reagents
1. 3% sulfosalicylic acid
2. Isotonic sodium chloride solution (8.8 g/L)
3. Protein standard 50 mg% (50 mg bovine albumin in
100 ml of isotonic NaCl).
Procedure
Mark three test tubes B, S and T for blank, standard and test
respectively.
Reagent (ml)
Blank
Standard
Standard protein
Test
-
CSF
Isotonic NaCl
3% sulfosalicylic acid
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 (100200 ml). It is formed at the rate of about 0.5 ml/minute.
Earlier it was thought that CSF was formed by ultrafiltration of plasma. But now it is known that secretory
activity of cell of the choroids plexus is the major factor
in the production of CSF and ultrafiltration plays only a
secondary role in the formation of CSF.
CSF Analysis 203

Indications for CSF analysis are:
1. Bacterial or viral meningitis
2. Trauma-head injury
3. Degenerative disorders like multiple sclerosis
4. Tumors like benign meningioma or malignant glioma
5. Vascular disorders like rupture of vessels or obstruction of vessels by thrombosis (cerebral infarction)
Artificial CSF for practical purposes is prepared by
dissolving 300 mg bovine albumin, 600 mg glucose and
7.19 g sodium chloride in 1 litre of deionized water
(freshly prepared).
For proteins of high values, dilute the CSF suitably
and take the dilute CSF and multiply the result by dilution
factor.
Normal CSF protein is essentially due to albumin (5575%).
An increase in protein in certain diseases is
contributed by globulin along with albumin.
Normal range of protein in CSF is 15 to 45 mg/dl.
Glucose level in spinal fluid is about 20 mg/dl less
than that of blood.
Normal range for adults is 50-80 mg/dl. Hence,
blood must be analyzed simultaneously for comparison.
CSF sugar is utilized by bacteria and blood cells.
Low glucose level in CSF is seen in:
1. Bacterial meningitis since there is increased no. of
leukocytes and pathogenic organism which may
contribute to increased glycolysis.
2. Tubercular meningitis
3. Metastatic tumors of meninges
Increase of CSF glucose is seen in diabetes mellitus and
brain tumors.
26
Estimation of
Albumin in Urine
Aim
To estimate the amount of albumin in urine.
Apparatus
Esbachs albuminometerthe apparatus has the mark U
near the middle and the mark R near the top. The portion
below U is graduated from 0 to 12 that gives the quantity
of proteins in gm/liter.
Principle
Albumin and other proteins can be measured by precipitating with picric acid solution in Esbachs albuminometer.
Reagent
Esbachs 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 Esbachs 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 60 and 100C. At 60C temperature, the Bence Jones
proteins will clump. The clumping disappears if the urine
is further heated to boiling (100C) 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, Hellers
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. Investigations 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.
-cells secrete glucagon, -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 diglucuronide.
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.
Fouchets test, Gmelins test
6. The following are some of the
patient.
Serum bilirubin
Conjugated
Unconjugated
AST
ALT
Urine
Bile pigments
Bile salts
Urobilinogen
Feces
Blood coagulation time
biochemical findings in a
-
10 mg%
8.5 mg%
1.5 mg%
140 KA units
80 units/L
90 units/L
++
++
negative
clay color
prolonged

I.
II.
III.
IV.
V.
Probable diagnosis.
Obstructive jaundice
Causes:
Intrahepatic: chronic active hepatitis, biliary cirrhosis,
lymphoma, primary hepatoma.
Extrahepatic: gall bladder stones, carcinoma of head of
pancreas, enlarged lymph nodes
Name the bile pigments.
Bilirubin and biliverdin
Name the bile salts.
Sodium and potassium salts of glycocholic acid and taurocholic
acid
Test to detect bile salt.
Hays 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, Hellers 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
Rotheras test (urine)
- positive
Benedicts 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 Benedicts reagent.
Copper sulfate, sodium carbonate, sodium citrate
IV. True blood glucose level.
60 to 90 mg/ dl
V. Ketone bodies.
Acetone, acetoacetic acid and 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, Hellers test
patient. What is your probable diagnosis?
Serum Bilirubin
10 mg%
Conjugated
8.5 mg%
Unconjugated
1.5 mg%
Serum Alkaline Phosphatase
50 KA units
SGOT
80 IU/L
SGPT
90 IU/L
Urine
Bile salts
+
Bile pigments
++
Urobilinogen
negative
Feces
clay colored
I. Normal serum Bilirubin level.
0.2 to 0.8 mg/ dl
II. Name the bile salts.
Sodium / potassium salts of glycocholic and taurocholic acid
III. Test to detect bile salts in urine.
Hays sulfur powder test
IV. Account for clay colored stools.
Absence of stercobilinogen in faeces
V. Test to detect urine bile pigments.
Fouchets test, Gmelins 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
H
120
Nil
1 Hr
140
Nil
1 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-Wus method.
80 to 120 mg/dl
IV. Normal blood glucose level by enzymatic method.
60 to 90 mg/dl
V. Renal glycosuria.
Presence of sugar in urine when the blood sugar is below the
renal threshold, i.e. 180 mg/dl
12. A 50-year-old non-diabetic male is brought in a
semiconscious condition. He has generalized edema and
oliguria. Laboratory findings are as follows:
Blood urea
90 mg/dl
Serum creatinine
5 mg/dl
Serum inorganic phosphorous
6 mg/dl
I.
II.
III.
IV.
V.
Comment on this Renal failure

Normal blood urea level 15 to 40 mg/dl
Normal serum creatinine level 0.7 to 1.4 mg/dl
Significance of creatinine clearance To assess kidney function
Amino acids involved in creatinine synthesis Glycine,
Arginine and Methionine
13. A 35-year-old man with narcotic overdose was admitted to

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

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 g /day
15. A female aged 30 years, came with complaints of weakness,
fatigue and heavy menstrual bleeding. On examination,
she was found to be anemic. Her Hb was 6 gm%.
I. Probable diagnosis Iron deficiency anemia
II. Causes for the deficiency Excess loss of iron, dietary
deficiency
III. Biochemical parameters to assess deficiency hemoglobin
estimation, serum iron level
IV. Dietary sources Liver, meat, egg yolk, green leafy vegetables,
whole grains and cereals
V. Mention the RDA. Adult men and postmenopausal women:
10 mg/day
Premenopausal women: 15 to 20 mg/day
Pregnancy: 30 to 60 mg/day
16. A 3-year-old was brought with complaints of painful,
swollen and bleeding gums. On examination, petechiae were
seen. Joints were swollen and painful.
I. Diagnosis Scurvy
II. Vitamin deficiency that causes this condition Vitamin C
III. Name the richest source Amla/Indian gooseberry
IV. Biochemical name of the vitamin Ascorbic acid
V. Physiological role of the vitamin Antioxidant property,
antiscorbutic property.
17. A person on hunger strike was brought to the hospital in an
unconscious state. Following are the lab findings:
Blood sugar
52 mg%
Blood pH
7.25

Serum bicarbonate
Ketone bodies in urine -
16 mEq/L
+
I.
II.
III.
IV.
Probable diagnosis Starvation ketoacidosis

Normal blood pH 7.35 to 7.45
Normal blood sugar level 80 to 120 mg/dl
Name the ketone bodies Acetone, Acetoacetic acid,
-hydroxybutyric acid
V. Test to detect ketone bodies Rotheras test, Gerhardt s test
18. A 35-year-old male came with history of increased

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

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

Normal serum bilirubin level 0.2 to 0.8 mg/dl.
Test done to estimate serum bilirubin van den Bergh test.
Expected cholesterol level in this case Cholesterol is elevated.
What is urobilinogen? Excretory product of bilirubin
22. A 55-year-old man is brought to the hospital in a

semiconscious state. He has low BP and feeble pulse. His
breath has fruity odor. The data of his laboratory
investigations are given below:
Blood pH
7.1
Plasma bicarbonate
22 mEq/L

Blood glucose random
Blood urea
Serum creatinine
Urine sugar
Urine ketone bodies
I.
II.
III.
IV.
V.
580 mg/dl
40 mg/dl
1.5 mg/dl
++++
+++
Probable diagnosis Ketoacidosis

Normal blood pH 7.35 to 7.45
Test to detect urine sugar Benedicts test.
Test to detect urine ketone bodies Rotheras test.
Cause for fruity odor Presence of acetone.
23. Following are the findings in a patient brought to the

hospital in the coma state.
Blood sugar (fasting)
270 mg%
Benedicts test
orange colored ppt
Rotheras test
positive
Serum bicarbonate
16 mEq/L
Plasma pH
7.25
I. Probable diagnosis Diabetic Ketoacidosis
II. Normal FBS, PPBS and RBS FBS:80-110 mg/dl.
RBS: 80-140 mg/dl
PPBS: 70-140 mg/dl.
III. Name ketone bodies Acetone, Acetoacetic acid and
-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:
Serum bilirubin
13.5 mg%
Direct
9 mg%
Indirect
4.5 mg%
20 KA units
ALT
230 IU/L
AST
80 IU/L
Urine
Bile salts
negative
Bile pigments
++
Urobilinogen
+
I. Probable diagnosis Hepatic Jaundice
II. What is direct bilirubin? Conjugated bilirubin: bilirubin is
conjugated with UDP glucuronate to form bilirubin mono
and diglucuronide. It is water soluble, reacts directly with
diazo reagent
III. How are bile salts formed? Bile acids derived from
cholesterol react with alkali to form bile salts.
IV. Test to detect bile salts Hays 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%
Benedicts 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 Benedicts test Presence of Homogentisic acid.
patient.
Serum bilirubin
10 mg%
Conjugated
5.5 mg%
Unconjugated
4.5 mg%
20 KA units
SGOT
260 IU/L
SGPT
290 IU/L
Urine
Bile salts
negative
Bile pigments
+
Urobilinogen
+
Feces Stercobilinogen
++
I.
II.
III.
IV.
V.
What is the probable diagnosis? Hepatic Jaundice.

Test to detect serum bilirubin van den Bergh test.
Normal alkaline phosphatase level - 3-13 KA units.
Name the bile pigments Bilirubin and biliverdin

patient.
Serum bilirubin
0.8 mg%
Direct - 0.3 mg%
Indirect - 0.5 mg%
6 KA units
ALT - 14 IU/L
AST - 16 IU/L
Urine
Bile salts
absent
Bile pigments
absent
Urobilinogen
traces
Feces
normal color

I.
II.
III.
IV.
V.
Probable diagnosis Normal.

Normal levels of ALT ALT: 13-40 IU/L.
Conjugation of bile pigments Bilirubin is conjugated with
UDP glucuronate to form Bilirubin mono and di-glucuronide.
29. Patient is giving history of malaria for which he has taken

treatment a month ago. His lab findings are as follows:
Serum bilirubin (total)
2.5 mg/dl
Direct
0.4 mg/dl
Indirect
2.1 mg/dl
SGOT
15 IU/L
SGPT
12 IU/L
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 Fouchets and Gmelins
test.
30. A person on hunger strike was brought to the hospital in an
unconscious state. Following are the laboratory findings:
Blood sugar
52 mg%
Blood pH
7.25
Serum bicarbonate
16 mEq/L
Rotheras test
positive
I. Probable diagnosis Starvation ketoacidosis.
II. Normal serum bicarbonate 22 to 26 mEq/L
III. Name the ketone bodies Acetone, Acetoacetic acid,
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
Hr
120
+
1 Hr
150
+++
1 Hr
100
Nil
2 Hrs
85
Nil
I.
II.
III.
IV.
V.
Your interpretation Renal glycosuria

Renal threshold 180 mg/dl
Test to detect urine sugar Benedicts test
Normal fasting blood glucose level 80-110 mg/dl.
True glucose level 60-90 mg/dl.
32. A medical student aged 20 years presents with abdominal

pain, fever, loss of appetite, nausea and high colored urine.
On examination there is icterus, tenderness in right
hypochondrium and hepatomegaly.
I. Give your probable diagnosis Acute hepatitis
II. Suggested biochemical investigations AST, ALT, Serum total
bilirubin, Direct and Indirect bilirubin, A/G ratio
III. Bile pigments Bilirubin, Biliverdin
IV. Bile salts Na and K salts of glycocholic and taurocholic acid.
V. Importance of bile salts Emulsification and digestion of
FAs
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 (Kussmauls
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 Benedicts test
:
++++
Rotheras 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.
II. Normal serum bilirubin level 0.2 to 0.8 mg/dl.
III. What is direct bilirubin? Conjugated bilirubin: bilirubin is
conjugated with UDP glucuronic acid to form bilirubin mono
and di-glucuronide. It is water soluble, reacts directly with
diazo reagent
IV. Kernicterus When the concentration of plasma bilirubin
(unconjugated) exceeds 20 mg/dl it penetrates the blood brain
barrier and causes hyperbilirubinemic toxic encephalopathy
or kernicterus, which causes mental retardation
V. Test to detect urinary bile pigments Fouchets test and
Gmelins 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%
Serum bilirubin
12 mg%
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 FAs, acts as a buffer.
IV. Test to detect serum bilirubin van den Bergh test.
V. Importance of A:G ratio Reversed in multiple myeloma
Decreased in liver disease, kidney disease, cirrhosis of liver,
Nephrotic syndrome, malnutrition.
40. A child presented to the casualty with complaints of severe
joint pains. Examination revealed severe pallor and an
enlarged spleen. Biochemical findings were as follows:
Serum bilirubin
10 mg%
Conjugated
0.5 mg%
Unconjugated
9.5 mg%
8 KA units
AST
30 units
ALT
26 units
Urine bile salts
negative
Bile pigments
negative
Urobilinogen
+++
Feces- stercobilinogen
+++
I. Probable diagnosis Hemolytic jaundice
II. What is unconjugated bilirubin? Bilirubin is not conjugated
with UDP glucuronic acid. It is water insoluble and soluble in
methanol
III. Test to detect serum bilirubin van den Bergh test.
IV. End product of hemoglobin metabolism Bilirubin

V. Causes G-6PD deficiency, Sickle-cell anemia, Incompatible
blood transfusion.
41. A hostel student presented with recurrent episodes of
vomiting and pyrexia. On examination he was icteric,
dehydrated and had enlarged liver. Biochemical Findings
were as follows:
Serum bilirubin
10 mg%
Conjugated
5.5 mg%
Unconjugated
4.5 mg%
Serum alkaline phosphates 20 KA units
AST
260 units
ALT
290 units
Urine bile pigments
++
Bile salts
+
Urobilinogen
+
I. Probable diagnosis Hepatic jaundice.
II. What is conjugated bilirubin? Bilirubin is conjugated with UDP
glucuronic acid to form bilirubin mono-and di-glucuronide.
It is water soluble, reacts directly with diazo reagent
III. Name the bile salts Sodium /potassium salts of glycocholic
acid and taurocholic acid.
IV. Test to detect urinary bile salts Hays 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 Benedicts 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 Benedicts test is positive? Presence of Homogentisic
acid.
IV. Name non sugar reducing agents Vitamin-C, Glutathione,
Salicylates, Uric acid, glucuronides.
V. Mention other protein metabolic disorders Phenylketonuria, Albinism, Maple syrup urine disease, Hartnup disease.
44. A fair 8-year-old chubby boy was brought to the hospital
by the mother with the complaint that he is mentally
retarded and has delayed milestones. Blood chemistry
revealed an abnormally high Phenylalanine. Phenyl ketones
were present in the urine in appreciable amounts.
I. Probable diagnosis Phenylketonuria.
II. Compound excreted in urine Phenylacetate/Lactate/
Pyruvate.
III. Test performed to detect phenylketones Guthrie test, urine
Ferric chloride test.
IV. Enzyme deficiency Phenylalanine hydroxylase.
V. Other clinical symptoms Reflexes are hyperactive because
of defective myelination of nerves
45. A child with retarded growth was brought to the hospital
with complaints of diarrhea and delayed milestones. On
examination he was found to have cataract. Urine
examination showed reduction with Benedicts 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.
Serum bilirubin
10 mg%
Conjugated
0.5 mg%
Unconjugated
9.5 mg%
8 KA units
SGOT
30 IU/L
SGPT
26 IU/L
Urine:
Bile salts
negative
Bile pigments
negative
Urobilinogen
+++ (Excess)
Feces-stercobilinogen
+++ (Excess)
I.
II.
III.
IV.
V.
Probable diagnosis Hemolytic jaundice.

Normal serum bilirubin level 0.2-0.8 mg/dl.
Test to detect serum bilirubin van den Bergh.
Normal SGOT level 8-13 IU/L.
Name the bile salts and bile pigments - Na and K salts of
glycocholic and taurocholic acid / Bilirubin and Biliverdin.
47. A person aged 30 years was referred by a physician to the

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

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

Normal serum protein level 6 to 8 g/dl.
Normal serum albumin level 3.5 to 5 g/dl.
Normal serum bilirubin level 0.2 to 0.8 mg/dl
49. A patient with acute chest pain showed the following blood
values.
Blood sugar
300 mg%
Serum cholesterol
320 mg%
HDL
20 mg%
SGOT
52 IU/L
SGPT
28 IU/L
CPK and LDH values are also raised.
I.
II.
III.
IV.
V.
Probable diagnosis MI.

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

patient.
Blood urea
30 mg%
Serum creatinine
1.8 mg%

Serum cholesterol
Total plasma protein
Albumin
Globulin
Urine protein
560 mg%
4.5 g%
1.0 g%
3.5 g%
10 g/L
I.
II.
III.
IV.
Probable diagnosis Nephrotic syndrome.

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

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

Blood pH is low. It is a case of uncompensated respiratory
acidosis.
Normal pH of blood 7.35 to 7.45
Name the blood buffer systems Bicarbonate buffer,
Normal plasma bicarbonate levels 22 to 26 mEq/L
Normal pCO2 level 32 to 45 mm of Hg
54. The following are the results of blood gas analysis of a

patient admitted in the medical ICU.
Blood pH
7.23
Plasma bicarbonate
14 mEq/L
pCO2
38 mm of Hg
I.
II.
III.
IV.
V.
Interpret Plasma bicarbonate is diminished, but no change

in pCO2. Blood pH is low due to lowered bicarbonate level. It
is a case of uncompensated metabolic acidosis.
Compensatory mechanism hyperventilation to wash out
CO2 faster. Increased elimination of acid in the urine and rise
in urinary ammonia.
Normal pH of blood 7.35 to 7.45
Normal plasma bicarbonate levels 22 to 26 mEq/L
Name the blood buffer systems Bicarbonate buffer,

55. An adult man living in coastal region came with the
complaint of difficulty in doing simple tasks such as bending
and squatting. On general examination, chalky white patches
with yellow or brown staining are found on the surface of
the teeth (motled enamel). X-ray showed hypercalcification
of spinal bones, pelvis and limbs.
I. What is your diagnosis? Fluorosis.
II. Function of the mineral involved Fluorine is an essential
trace element. Sodium fluoride is a powerful inhibitor of
glycolytic enzymes. Florine forms a protective layer of acidresistant fluoroapatite with hydroxyapatite crystals of the
enamel.
III. Source Drinking water
IV. RDADrinking 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 quantitative 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 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.

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. TC 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 (), 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
precipitation test for the
identification of casein
4 to 4.6
Phenolphthalein
In specific urease
test for the detection
of urea
8 to 10

23.
Tests
Significance
Molisch test
In identification of
carbohydrates
Hays
Detect bile salts
Rotheras
Detection of ketone
bodies
Jaffes
Detection of creatinine
Iodine
Identification of starch

24.
Reagent
Composition
Use
Molisch
Alpha Naphthol +
ethyl Alcohol
To identify
carbohydrates
Benedicts
Copper sulfate +
Sodium citrate +
Sodium carbonate
To detect reducing
sugars
Barfoeds
Copper acetate +
Glacial Acetic acid
To identify
monosaccharide
Seliwanoffs
Resorcinol in concentrated
Hydrochloric acid
To detect ketosugar
Millons
Sodium nitrate +
Mercuric sulfate
To detect
hydroxyphenyl
group-tyrosine
Osazone
Mixture
Phenylhydrazine
hydrochloride+
sodium acetate+
glacial acetic acid
Identification of
reducing sugars
which give
characteristic osazone
crystals.
Benzidine
Benzidine +
glacial acetic acid
Detect blood in urine
Biuret
Sodium Potassium
tartarate + Copper
sulfate
In identification of
Protein
DAM
Diacetyl Monoxime +
water
Quantitative
estimation of
blood urea
ANSA
Aminonapthnol
sulfonic acid +
Sodium bisulfate
Sodium sulfite
Quantitative
estimation of
serum phosphate
Nippes fluid
Potassium bromide +
Potassium chloride +
Potassium iodide +
Glacial acetic acid
Preparation of hemin
crystals
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.
(x x)2
SD =
n-1
Where = 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 patients 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: Benedicts 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
Ammonia
P/S
< 50 g/dl
Acid phosphatase,
(ACP) total
P/S
0.5- 4 KAU/dl
Alanine amino
transferase (ALT/SGPT)
SI units
2.5-12 IU/L
Male: 13-35 IU/L
Female: 10-30 IU/L
Albumin
3.5- 5 g/dl
35-50 g/L
3- 13 KAU/dl
40-125 IU/L
Amylase
80- 180 somogyi

units/dl
50-120 IU/L
Aspartate aminotransferase (AST/SGOT)
8-20 IU/L
Bicarbonate
22- 26 mEq/L
Bilirubin, total
0.2- 1 mg/dl
4-17 mol/L
Calcium
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
Male: 30- 60 mg/dl

Female: 35- 75 mg/dl
0.75-1.58 mmol/L
0.98-1.95 mmol/L
LDL
20- 29 yr: 60- 150 mg/dl

30- 39 yr: 80-175 mg/dl
40- 60 yr: 90- 200 mg/dl
Copper
70- 150 g/dl
C- reactive protein (CRP)
22-26 mmol/L
16- 30 mol/L
0.5-1 mg/dl
Creatine
Creatine kinase
0.2- 0.4 mg/dl
15- 30 mol/L
Creatinine
S
U
0.7- 1.4 mg/dl

15- 25 mg/kg/day
60- 125 mol/L

15-0.2 mmol/kg/day
Electrophoresis
Albumin: 55-65%
1: 2-4%
2: 6-12%
: 8-12%
: 12-22%
3.5-4.7 g/100 ml
0.2-0.3 g/dl
0.4- 0.9 g/dl
0.5-1 g/dl
0.7-1.5 g/dl
Female: 10- 80 U/L

Male: 15- 100 U/L
Contd...
Appendix 5: Normal Values (Reference Values) 261

Contd...
Analyte
Sample
Units
SI units
Fibrinogen
200-400 mg/dl
Globulins
2.5-3.5 g/dl
25-35 g/L
P
B
CSF
70-110 mg/dl
65-100 mg/dl
50-70 mg/dl
4-6.1 mmol/L
3.5-5.6 mmol/L
2.8-4.2 mmol/L
Hemoglobin
Male: 14-16 g/dl

Female: 13-15 g/dl
2.17-2.4 mmol/L
Glycated hemoglobin
Hb A1c
Iodine
Iron
E
S
B
4-8% of total
5-10 g/dl
5 mg/dl
Glucose (fasting)
5.8- 8.5 mol/L
Lactate dehydrogenase
(LDH)
Lipoproteins
Alpha: 40 mg/dl
Beta: 180 mg/dl
Non esterified fatty acids

(NEFA, FFA)
10-20 mg/dl
pCO2, arterial
35-45 mmHg
pH
7.35- 7.45
[H+] = 40 nmol/L
Phosphate
S
U
B
3-4 mg/dl
1 g/day
40 mg/dl
1-1.5 mmol/L
32 mmol/day
Phospholipids
100-200 IU/L
0.3-0.7 mEq/L
150-200 mg/dl
2-2.5 mmol/L
pO2, arterial
90-100 mmHg
150-220 ml/L
Potassium
3.5-5 mEq/L
3.5-5 mmol/L
Prostate specific antigen

(PSA)
100-500 ng/dl
1-5 g/L
S
CSF
6-8 g/dl
10-30 mg/dl
60-80 g/L
Sodium
136-145 mEq/L
136-145 mmol/L
T3 (tri-iodothyronine)
120-190 ng/dl
1.8-3 nmol/L
T4 (thyroxine)
5-12 g/dl
65-150 nmol/L
Thyroglobulin (Tg)
3- 5 g/dl
3- 50 g/L
TSH
0.5- 5 U/ml
0.5- 5 mU/L
Transferrin
200- 300 mg/dl
23- 35 mol/L
Proteins- total
Contd...

Contd...
Analyte
Triglycerides
Males
Females
Urea
Sample
Units
SI units
50- 200 mg/dl

40- 150 mg/dl
0.5- 2.3 mmol/L

0.4- 1.6 mmol/L
2.4- 4.8 mmol/L
20- 40 mg/dl
Urea nitrogen
S/P
8- 20 mg/dl
3- 9 mmol/L
Uric acid, male

Female
Children
S/P
3.5- 7 mg/dl
3- 6 mg/dl
2- 5.5 mg/ml
0.21- 0.4 mmol/L

0.18- 0.35 mmol/L
0.12- 0.32 mmol/L
Vitamin A
15- 50 g/dl
0.5- 2 mol/L
Vitamin C
0.4- 1.5 mg/dl
23- 85 mol/L
Vitamin D3
1.5- 6 g/dl
50- 160 pmol/L
Vitamin E
0.5- 1.8 mg/dl
12- 42 mol/L
P- plasma; B- blood; S- serum; E- erythrocyte; U- urine; CSF- cerebrospinal fluid

pg- picogram; ng- nanogram; g- 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 centimeter (cm)
10-2 m
1 millimeter (mm)
10-3 m
1 micrometer (m)
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 centigram (cg)
10-2 g
1 milligram (mg)
10-3 g
1 microgram (g)
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 examinationBiochemistry
The allotment of marks as recommended by the university is as
follows:
INTERNAL ASSESSMENT FOR BIOCHEMISTRY
Total Marks: 40 (Theory: 20 + 10 for records and Practical: 10)
THEORY AND RECORDS
Minimum of three internal assessments are recommended. The
internal assessment preceding the University examination will
be similar to the University examination. The total marks would
be 20. Average marks secured out of two notified internal
examination should be reduced to 20. For records 10 marks are
allotted. The sum of the marks obtained in theory and records
shall be sent to the University.
PRACTICALS
A minimum of two practical tests is to be conducted, one at the
end of each term. Average of the two tests should be reduced to
10 marks and shall be sent to the University.
UNIVERSITY EXAMINATION
A. Theory : 100 Marks
There shall be two sections. The total marks will be 100, with
each section carrying 50 marks. The total duration would be 3
hours. There shall be three types of questions. The distribution
of topics and weight age of marks in Biochemistry for University
examination is as under*:
Appendix 6: Scheme of Examination 265

Type of question and distribution of marks in each paper.
Paper I
Type of
Questions
Paper II
Number
of
Questions
Marks
for each
question
Total
Long Essay
10
10
10
Short Essay
25
25
Short Answer
15
15
Total Marks
Number
Marks
of
for each
Questions question
Total
10
50
50
Distribution of topics for each paper and weightage of marks

in university examination is as follows:
Paper I
1. Cell structure and function, subcellular
organelles, cell membranes, Transport
across the membranes.
2. Chemistry, digestion, absorption and
metabolism of Carbohydrates
3. Chemistry, digestion, absorption and
metabolism of lipids
4. Amino acids and protein chemistry,
general reactions of amino acids,
Digestion and absorption, urea cycle
and metabolism of amino acids.
5. Endocrine functions and Biochemical tests.
6. Detoxification and Xenobiotics.
7. Enzymes
8. Biological oxidation, integration of
metabolism, TCA cycle and regulation
of metabolism.
9. Free radicals and antioxidants.
10. Biochemistry of cancer, oncogenes and
tumor markers
Weightage of
marks
05
10
10
10
05
05
10
10
05
05

Paper II
Weightage of
marks
05
1. Nucleotides and nucleic acid chemistry

2. Purine and pyrimidine nucleotide
metabolism, DNA metabolism,
RNA metabolism, Protein Biosynthesis.
3. Vitamins
4. Minerals
5. Molecular genetics, regulation of
gene expression, recombinant DNA
technology, PCR and gene therapy.
6. Electrolyte and water balance, acid base balance
7. Nutrition and energy metabolism
8. Heme metabolism, normal and abnormal
hemoglobins, Plasma proteins and
immunoglobulin.
9. Liver function tests
10. Kidney function tests
11. Clinical chemistry, quality control,
interpretation and reference
values and analysis
10
10
10
05
10
10
10
05
05
05
Note:
a. Weightage of marks assigned to chapters/topics may add to
more than 50.
b. Long essay questions may be asked from topics with weight
age of 10 marks.
c. Short Essay and short answer question may be asked from
any of the topics.
* The topics assigned to the different papers are generally evaluated those
sections. However, a strict division of the subject may not be possible and
some overlapping of topics is inevitable. Students should be prepared to
answer overlapping topics.
PRACTICAL EXAMINATION: 40 MARKS

The Practical examination consists of two exercises, Practical I
and II, each of 2 hours duration and each exercise carrying 20
marks.
Appendix 6: Scheme of Examination 267

Exercise I: Two hours, 20 marks
1. Quantitative estimation-Every candidate shall perform one
given procedure.
a. Principle and procedure for the estimation asked in the
question should be written by the candidate in the first five
minutes.
Marks: 5
b. After collecting the papers, correct procedure for the
estimation is given if asked by the student and practical
examination is done. Total marks would be 15 and the
distribution of marks would be:
(i) results (values)
10
(ii) calculations and reporting
(iii) for interpretation of results and application of the
estimation
c. Case studies, Graphs and Charts-Discussion
1 5 = 5 marks
Exercise II: Two hours, 20 marks
1. Qualitative analysisEvery candidate shall perform one given
procedure such as Identification of Carbohydrates, Proteins,
Substance of Physiological importance, Analysis of normal
Urine, Analysis of abnormal Urine. Total marks would be 20
and Distributions of marks would be:
For selection of appropriate reactions
5 marks
For reasoning of analysis and correct reporting
5 marks
For interpretation of results and application of
the estimation
5 marks
2. Five Spotters Biochemical TechniquesChromatography, Electrophoresis,
Osazone preparation, Biochemical Tests and
Reagents
1 x 5 = 5 marks
Viva Voce Examination: 20 Marks
The viva voce examination shall carry 20 marks and all the
examiners will conduct the viva examination.
Index
A
Albumin 61
casein 62
chemical properties 62
aldehyde test for indole
nucleus 67
Biuret test 62
Millons test 66
Molisch test for
carbohydrate moiety
in proteins 71
ninhydrin test 64
Paulys test for histidine
and tyrosine 70
Sakaguchis test for
guanidine group 68
sulfur test cystine and
cysteine 69
test for organic
phosphorus 72
xanthoproteic test 65
functions 61
physical properties 62
physical properties of casein
62
Alkaptonuria 124
procedure 125
spot test 125
Amino acid 51
Analysis of abnormal urine 102
chemical constituents 102
Gerhardts test for
acetoacetic acid 109
heat and acetic acid test
102
Rotheras test fro acetone

and acetoacetic acid
109
sulfosalicylic acid test
106
test for bile pigments 113
test for bile salts 112
test for blood 114
test for ketone bodies
109
test for reducing sugar
(Benedicts test) 106
physical characteristics 102
Analysis of normal urine 86
chemical tests 88
test for ammonia 92
test for chloride 88
test for creatinine (Jaffes
test) 96
test for ethereal sulfate
97
test for inorganic sulfates
89
test for phosphates and
calcium 90
test for urea 94
physical examination 86
determination of specific
gravity 86
Longs coefficient 87
B
Beers law 133
Bence Jones protein 102

Benedicts uric acid test 95
Benzidine test 218
C
Carbohydrates 29
classification 29
monosaccharides 29
oligosaccharides 30
polysaccharides 31
functions 31
reactions 31
reaction with acids 32
reaction with alkalies 32
tests for carbohydrates 32
Barfoeds test 39
Benedicts test 35
iodine test 34
Molisch test 33
osazone test 42
Seliwanoffs test 40
Chromatography 169
paper chromatography 169
application 173
procedure 171
requirement 171
types 169
gas liquid
chromatography 169
gel chromatography 169
high pressure liquid
chromatography 169
ion exchange
chromatography 169
paper chromatography
169
thin layer
chromatography 169
Cleaning of glassware 25
Coles mercuric nitrite test 66
Collection and preservation of

urine specimens 18
collection of urine samples
18
preservation of urine
samples 18
Collection of blood 16
anticoagulants 17
Colorimeter 130
application 137
components 130
adjustable slit 130
condensing lens 130
cuvette (sample holder)
131
filter 130
galvanometer 131
photocell/detector 131
source of light 130
selection of filter in
colorimetric estimation
136
calculations 137
steps 136
solution for investigation
131
blank 132
standard solution 132
test solution 132
technique 133
Beers law 133
Lamberts law 134
Constituents of urine 99
physical characteristics 99
appearance 100
chemical constituents
102
specific gravity 101
volume 99
Index 271
Creatinine 79
Jaffes test 80
Creatinine clearance test 151
calculation 152
clinical significance 152
diagnostic importance 152
procedure 151
CSF analysis 200
biochemical examination of
CSF 200
calculation 202
collection of CSF 200
determination of glucose
202
determination of total
protein 201
principle 201
procedure 201
reagents 201
D
Derivatives of hemoglobin 117
carboxy-hemoglobin 117
hematin 117
hemin 117
hemochromogen 117
methemoglobin 117
native hemoglobin 117
oxyhemoglobin 117
Detection of hemoglobin and its
derivatives 118
direct vision spectroscope
118
principles 118
Determination of serum
albumin by bromocresol
green method 162
calculations 163
procedure 162
reference value 163
report 163
E
Electrophoresis 174
applications 175
basic requirements 176
factors affecting migration of
charged particles 175
movement of different
protein fractions 177
paper electrophoresis 176
separation of plasma
proteins 176
types 175
Estimation of albumin in urine
204
aim 204
apparatus 204
principle 204
procedure 204
reagent 204
test for Bence Jones proteins
in urine 205
Estimation of ascorbic acid 195
aim 195
calculation 196
method 195
principle 195
procedure 195
reagents 195
Estimation of blood sugar 138
choice of blood specimen
138
estimation of blood sugar by
Folin-Wus method 139
aim of the test 139
calculation 141
method 139

principle 139
procedure 140
reagents 140
methods of estimation 138
Folin-Wus method 138
glucose oxidase method
138
Nelson-Somogyi method
138
O-toluidine method 138
preservation of blood 139
Estimation of blood urea 144
aim 144
calculation 145
method 144
principle 144
procedure 144
protocol 145
report 145
Estimation of serum AST and
ALT 188
aim 188
estimation of ALT 189
estimation of SGOT (AST)
190
procedure 190
method 188
Reitman and Frankel
method 188
principle 188
procedure 189
protocol 189, 190
calculation 190
reagents 189
Estimation of serum cholesterol
192
aim 192
calculation 193
method 192
normal range 193
precaution 193
principle 192
procedure 193
sample 192
Estimation of serum inorganic
phosphate 154
aim 154
calculation 156
method 154
Fiske and Subbarow
method 154
principle 154
procedure 155
color development 155
preparation of proteinfree filtrate 155
protocol 156
reagents 155
ANSA reagent 155
molybdic acid reagent
155
report 156
Estimation of serum total
proteins 159
aim 159
method 159
Biuret method 159
principle 159
procedure 160
calculation 161
color development 160
precipitation of globulins
160
protocol 161
reagents 160
Estimation of urine creatinine
148
aim 148
calculation 149
method 148
principle 148
Index 273
procedure 149
protocol 149
report 150
F
Fanconis syndrome 158
Flame photometer 197
parts 198
nebulizer 198
needles 198
spray chamber 198
principle 197
procedure 199
Folin-Wu method 223
Fouchets test 113
Fraunhofers lines 119
G
Gerhardts test 217
Glucose tolerance test 181
decreased tolerance 183
details of performing the test
185
importance 185
increased tolerance 183
lag type 182
methods used for estimation
of blood sugar 186
methods used for estimation
of urine sugar 186
estimation of urine sugar
by Benedicts
qualitative method
187
normal response 181
procedure 186
renal glycosuria 185
significance 181
Gmelins test 113, 225
H
Hartnup disease 228
Hays test 112
Heat and acetic acid test 59
Hellers test 218
Homocystinuria 125
procedure 126
reagents 126
screening test for
homocystinuria 126
Hopkins-Cole-Adams test 67
I
Importance of precipitation of
proteins 50
Isoelectric precipitation 54
K
Kussmauls breathing 244
L
Laboratory first aid 7
in case of accidents in
laboratory 9
precautions while handling
chemicals 7
waste disposal 9
chemical waste 9
radioactive waste 9
Laboratory glasswares 20
beakers 20
bottles 22
drop bottles 22
reagent bottles 22
burettes 21
flasks 20
conical flasks 20

flat-bottomed round
flasks 20
round bottomed flasks
21
volumetric flasks 21
funnels 21
graduated (measuring)
cylinder 21
Laboratory hazards 3
accidental swallow of
corrosive solutions 6
burns 6
care while pipetting 5
in case of accidental fire 5
inhalation of corrosive gases
6
precautions regarding fire
safety 5
safety measures 3
Laboratory safety rules 10
accidents and injuries 11
handling chemicals 12
handling glassware and
equipment 12
heating substances 13
pipetting techniques 14
Lamberts law 134
M
Maple syrup urine disease 228
N
Neumanns test 72, 73
Nippes fluid 122
Normal urine 82
chemical characteristics 85
constituents 85
reaction 85
examination of urine 82
chemical examination 82
microscopic examination
82
physical examination 82
general and physical
characteristics 83
appearance 84
color 84
odor 84
specific gravity 84
volume 83
preservation of urine
samples 83
specimen collection 82
P
Paulys test 71
Phenylketonuria 123
ferric chloride test 124
Guthries bacterial inhibition
test 123
Photometry 129
principle 129
Pipettes 22
auto pipettes 24
fixed volume type 24
variable volume
adjustable type 24
manual pipettes 22
deliver type of pipettes
22
graduated pipettes 23
micropipettes 23
pasteur pipettes 23
volumetric pipettes 23
Precipitation by alkaloidal
reagents 56
Precipitation by heavy metal
ions 58
Index 275
Precipitation by organic solvents
55
Precipitation by salts 51
Preparation of hemin crystals
122
Preparation of hemoglobin and
derivatives 119
carboxy hemoglobin 120
methemoglobin 120
oxyhemoglobin (HbO2) 119
reduced hemoglobin 120
Proteins 48
classification 48
conjugated proteins 48
derived proteins 48
secondary derived
proteins 49
simple proteins 48
functions 49
reactions 49
color reactions of protein
49
precipitation reactions of
proteins 49
Q
Qualitative detection of protein
61
Quantitative estimation of
proteins 61
R
Relationship between
absorbance and
transmittance 135
Rotheras test 217
Ruhemanns purple 64
S
Schiffs test 96
T
Test tubes 24
centrifuge tubes 25
desiccators 25
dispensers 25
Folin-Wu tubes 25
Types of colloids 50
emulsoids 50
suspensoids 50
U
Unknown carbohydrate 47
Unknown protein 73
Urea 74
chemical tests 74
Biuret formation 74
sodium hypobromite test
75
specific urease test 76
tests for urea 74
Uric acid 77
chemical tests 77
murexide test 78
phosphotungastic acid
reduction test/
Benedicts uric acid
test 77
V
van den Bergh test 218

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An Easy Guide for

An Easy Guide for

Divya Shanthi DSa

JAYPEE BROTHERS MEDICAL PUBLISHERS (P) LTD

2/B, Akruti Society, Jodhpur Gam Road Satellite

North America Office

viii An Easy Guide for Practical Biochemistry

Divya Santi DSa

xii An Easy Guide for Practical Biochemistry

Accuracy in preparation of medications

Lifting and turning patients

Examination of the body fluids

Light and Sound

Application of heat and cold

Use of mirrors in apparatus

Work and Energy

Table 1.1: Systems of units with their standards of measurement

Centimeter (cm) Foot (f)

Fahrenheit (F) Celsius (C)

FUNDAMENTAL UNITS IN VARIOUS SYSTEMS

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

millimeters (mm) = 1 centimeter (cm)

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

Table 1.4: Conversion of weight and measurements

Value (in meter)

Table 1.6: Conversion between Metric and English systems

1 gram = 15 grains 2.54 centimeter = 1 inch 1 cubic centimeter = 15 minims

Biochemical parameters aids in diagnosis and prognosis of

An Easy Guide for Practical Biochemistry

Fig. 1.1: Signs of some laboratory hazards

Chemical work involving irritating chemicals and

Laboratory Hazards and First Aid

General health should be maintained at all costs as an

CARE WHILE PIPETTING

PRECAUTIONS REGARDING FIRE SAFETY

An Easy Guide for Practical Biochemistry

Water should NOT be used on electrical fire.

ACCIDENTAL SWALLOW OF CORROSIVE

Laboratory Hazards and First Aid

From strong alkalies:

LABORATORY FIRST AID

PRECAUTIONS TO BE TAKEN WHILE

An Easy Guide for Practical Biochemistry

Corrosive chemicals should be opened with great care

Laboratory Hazards and First Aid

4. Cylinder containing inflammable gases like hydrogen,

IN CASE OF ACCIDENTS IN THE LABORATORY

10 An Easy Guide for Practical Biochemistry

Laboratory Safety Rules

Laboratory Safety Rules 11

ACCIDENTS AND INJURIES

12 An Easy Guide for Practical Biochemistry

HANDLING GLASSWARE AND EQUIPMENT

Laboratory Safety Rules 13

14 An Easy Guide for Practical Biochemistry

Laboratory Safety Rules 15

16 An Easy Guide for Practical Biochemistry

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

Specimen Collection and Processing 17

18 An Easy Guide for Practical Biochemistry

COLLECTION AND PRESERVATION

Specimen Collection and Processing 19

20 An Easy Guide for Practical Biochemistry