Instruction Manual of Practical Biochemistry for MBBS

Compiled by

Prof. Dr. N.RAMA KRISHNA, MD

Head of the Department
Department of Biochemistry
GVPIHC & MT, Marikavalasa, Visakhapatnam.

Assisted by

Dr. C.VAMSI KRISHNA, MD Dr. J.SUDHA RANI, M.Sc,Phd.

Assistant Professor Assistant Professor
Department of Biochemistry Department of Biochemistry
GVPIHC &MT, Marikavalasa, GVPIHC &MT, Marikavalasa,
Visakhapatnam Visakhapatnam

Dr. N.ASHA KIRAN, M.Sc,Phd. PSP TEJASWI, M.Sc,

Assistant Professor Tutor
Department of Biochemistry Department of Biochemistry
GVPIHC &MT, Marikavalasa, GVPIHC&MT, Marikavalasa,
Visakhapatnam Visakhapatnam
 CONTENTS
I. INTRODUCTION
1. Commonly Used Laboratory Apparatus & Equipments

2. Good and Safe Laboratory Practice

3. Biomedical Waste Disposal

4. Preparation of Buffers And Estimation of pH

II. QUALITATIVE EXPERIMENTS

1. REACTIONS OF CARBOHYDRATES
A. Reactions of Glucose
B. Reactions of Fructose
C. Reactions of Maltose
D. Reactions of Lactose
E. Reactions of Sucrose
F. Osazones
G. Special Reactions of Carbohydrates

2. Identification of Unknown Carbohydrate - I

Identification of Unknown Carbohydrate – II

3. REACTIONS OF PROTEINS
A. Precipitation Reactions of Proteins
B. General Colour Reactions of Proteins
C. Reactions of Albumins & Globulins
D. Reactions of Casein
E. Reactions of Gelatin
F. Reactions of Peptone
4. Identification of Unknown Protein - I
Identification of Unknown Protein – II

5. NORMAL CONSTITUENTS OF URINE

A. Inorganic B. Organic
6. Identification Of Unknown Physiological Substance

7. ANALYSIS OF ABNORMAL CONSTITUENTS OF URINE

8. Identification Of Abnormal Constituents Of Urine

III. QUANTITATIVE EXPERIMENTS

1. ESTIMATION OF BLOOD GLUCOSE
2. ESTIMATION OF BLOOD UREA
3. ESTIMATION OF SERUM TOTAL PROTEINS
4. ESTIMATION OF SERUM CREATININE
5. ESTIMATION OF URINE CREATININE & CLEARANCE VALUES
6. ESTIMATION OF CSF PROTEINS
7. ESTIMATION OF CSF GLUCOSE
8. ESTIMATION OF SERUM CHOLESTEROL

9. ESTIMATION OF SERUM HDL CHOLESTEROL

10. ESTIMATION OF SERUM TRIGLYCERIDES

11. ESTIMATION OF SERUM TOTAL BILIRUBIN

12. ESTIMATION OF SGOT/AST

13. ESTIMATION OF SGPT/ALT

14. ESTIMATION OF ALKALINE PHOSPHATASE

15. ESTIMATION OF SERUM ALBUMIN & A/G RATIO

16. ESTIMATION OF SERUM CALIUM

17. ESTIMATION OF SERUM PHOSPHORUS

IV. DEMONSTRATIONS

1. Colorimetry & Spectrophotometry

2. Auto analysers for Biochemistry
3. Flame Photometry
4. Electrolyte Analysis By ISE
5. ABG Analyser
6. Chromatography
7. Electrophoresis
8. TLC & PAGE
9. GTT (Glucose Tolerance Test)
10. Immuno Diffusion
11. ELISA
12. DNA Isolation from Blood/Tissue

V. SPOTTERS
1. Crystals 2. Charts 3. Instruments

VI. CLINICAL DATA INTERPRETATIONS

QUALITATIVE
EXPERIMENTS
REACTIONS OF
CARBOHYDRATES
 REACTIONS OF CARBOHYDRATES

Introduction:
 Carbohydrates are hydrophilic substances with a potential free
Aldehyde or Keto group and a number of hydroxyl groups.
 Carbohydrates are body’s primary source of energy and provide
about 4 calories per gram.
 Carbohydrates play a crucial role in healthy, balanced diet.
 Carbohydrates are made up of units of sugar (also called
saccharides).
 They are classified into simple or complex carbohydrates depending
on number of sugar units they contain.
 Sugar units are bound together by glycosidic linkages.
 Simple carbohydrates contain one sugar unit (monosaccharide) or
two sugar units (disaccharides) or many sugar units
(polysaccharides).
Eg: Monosaccharides - Glucose, Fructose
Disaccharides - Maltose, Lactose, Sucrose
Polysaccharides - Starch, Glycogen
 REACTIONS OF CARBOHYDRATES

S.No EXPERIMENT OBSERVATION INFERENCE

1. Molisch Test:-
To 2ml of sugar solution, add 2 Purple/violet ring is Presence of a
drops of 1% alcoholic - observed at the carbohydrate
naphthol (Molisch reagent) & junction of the two
add 2ml of conc. H2SO4 liquids.
carefully along the sides of the
test tube.

Principle: It is the general reaction to identify carbohydrate. Carbohydrates on

acid hydrolysis produce monosaccharides. They undergo dehydration when
treated with concentrated H2SO4 to form hydroxy methyl furfural (from hexoses)
or furfural (from pentoses). The purple/violet ring is due to the condensation
product of hydroxy methyl furfural or furfural with -naphthol.

Note: This test is also given by aldehydes, formic acid, lactic acid, oxalic acid, citric
acid and certain other organic acids.

2. Iodine Test:-
To 1ml of sugar solution, add Deep blue colour Indicates the
few drops of iodine solution is formed presence of
slowly into the test tube. polysaccharide
(Starch)

Principle: It is the test to identify polysaccharide (Starch). Starch molecule

assumes a helical bundle shape exposing the hydroxyl groups and the
electronegative iodide ion interacts with the electropositive hydrogen. This results
in formation of polyiodide starch complex that gives a characteristic deep blue
colour. If deep blue colour is not formed, it confirms that starch is absent in the
given solution.

REDUCTION TESTS: (Fehling’s, Benedict’s & Methylene Blue Reduction tests)

S.No EXPERIMENT OBSERVATION INFERENCE

3. Fehling’s Test:-
To 1ml of Fehling’s A solution, Red/ Reddish Indicates presence
add 1ml of Fehling’s B solution brown precipitate is of reducing sugar
and 1ml of Fehling’s C solution formed
and heat. Add few drops of
sugar solution and mix. Boil
the reaction mixture for 2
minutes in a water bath.

Principle: It is a test to differentiate reducing sugars from non reducing sugars. In

the alkaline reaction medium, the cupric ions (Cu++) are reduced to cuprous ions
(Cu+) and forms cuprous oxide precipitate. This is due to reducing action of free
aldehyde or ketone functional groups present in the sugars.

Composition of Fehling’s A solution: Copper sulphate

Fehling’s B solution: Sodium potassium tartrate (Rochelle salt)
Fehling’s C solution: Sodium hydroxide

4. Benedict’s Test:-
Take 5ml of Benedict’s Brick red Indicates presence
qualitative reagent in a test precipitate is of reducing sugar
tube and heat. Add 8-10 observed
drops (0.5ml) of sugar
solution. Mix well and boil for
2 minutes.

Principle: It is a test to differentiate reducing sugars from non reducing sugars.

Under alkaline conditions reducing sugars undergo tautomerization to form
1,2-enediols, which are powerful reducing agents. They reduce cupric ions to
cuprous ions and forms cuprous hydroxide. On heating cuprous hydroxide gets
converted to cuprous oxide which is brick red precipitate. This is due to reducing
action of free aldehyde or ketone functional groups present in the sugars.

Note: If Benedict’s solution change its colour in first heating, do not use the
solution. Repeat the test with freshly prepared solution.

Composition of Benedict’s Reagent: Copper Sulphate

Sodium Citrate
Sodium Carbonate

5. Methylene Blue
Reduction Test:-
To 3ml of distilled water add Initially blue colour Indicates presence of
1 drop of 1% methylene persists. After reducing sugar
blue, 1 -2 drops of 5% NaOH addition of sugar
and 1ml of sugar solution. solution and boiling
Boil gently for 2 minutes. the colour
disappears

Principle: It is a test to differentiate reducing sugars from non reducing sugars. In

alkaline medium blue coloured dye (methylene blue) is reduced to leuco
methylene blue (colourless). This is due to reducing action of free aldehyde or
ketone functional groups present in the sugars.
Note: Fehling’s test, Benedict’s test & Methylene Blue Reduction test are reducing
tests which confirms the presence of reducing sugars.

Eg: Monosaccharides - glucose, fructose

Disaccharides - maltose, lactose

S.No EXPERIMENT OBSERVATION INFERENCE

6. Barfoed’s:-
Take 2ml of sugar solution in Red scum adherent Presence of a
a test tube & add 2ml of to the sides and monosaccharide
Barfoed’s reagent. Mix & bottom of the tube
heat for 3 minutes and cool is observed
under tap water.

Principle: It is the test to distinguish monosaccharides from reducing

disaccharides. Under acidic condition, monosaccharides are strong reducing
agents and reduce cupric ions (Cu++) of copper acetate to red colour
precipitate/scum of cuprous oxide.

Composition of Barfoed’s Reagent: Cupric acetate in acetic acid

Note: However on prolonged boiling disaccharides will answer the test.

7. Seliwanoff’s Test:-
Take 3ml of Seliwanoff’s Cherry red colour Indicates presence of
reagent in a test tube & add is observed ketose sugar
1 ml of sugar solution. Boil (ketohexose i.e
for 1 minute and cool it. Fructose)

Principle: It is used to detect the presence of ketose sugars. In the presence of

hydrochloric acid (HCl) and heating, ketose sugars are dehydrated to form
hydroxy methyl furfural derivative. Hydroxy methyl furfural condenses with
Resorcinol to form a cherry red coloured complex.

Composition of Seliwanoff’s Reagent: Resorcinol in dilute hydrochloric acid (HCl)

Seliwanoff’s test, Foulger’s test, Rapid furfural test are the test to differentiate
ketose sugars from aldose sugars.

S.No EXPERIMENT OBSERVATION INFERENCE

8. Foulger’s Test:-
Take 3ml of Foulger’s Deep blue colour is Indicates presence of
reagent in a test tube & add observed ketose sugar
1 ml of sugar solution. Boil (ketohexose i.e
for 1 minute and cool it. Fructose)

Principle: It is used to detect the presence of ketose sugars. In the presence of

sulphuric acid (H2SO4) and heating, ketose sugars are dehydrated to form hydroxy
methyl furfural derivative. Hydroxy methyl furfural condenses with stannous
chloride (SnCl2) to form a deep blue coloured complex.

Composition of Foulger’s Reagent: 40% Suphuric acid,

Urea
Stannous Chloride

9. Rapid Furfural Test:-

Take 1ml of sugar solution Purple/violet Indicates presence of
in a test tube, add 2 drops colour is observed ketose sugar
of 1% Alcoholic - (ketohexose i.e
naphthol & 2ml of conc. Fructose)
HCl. Boil on the flame and
cool.

Principle: It is used to detect the presence of ketose sugar. In the presence of

concentrated acid (HCl) and heating, ketose sugars are dehydrated to form
hydroxy methyl furfural derivative. Hydroxy methyl furfural condenses with -
naphthol to form a purple coloured complex.

This is similar to Molisch test where concentrated sulphuric acid (H2SO4) is used.

Seliwanoff’s test, Foulger’s test and Rapid Furfural test are positive for ketose
sugars like fructose due to presence of ketose group. These tests are negative for
glucose as there is no ketose group. (Glucose contains aldose group).

S.No EXPERIMENT OBSERVATION INFERENCE

10. Osazone Test:-

Take 5ml of sugar solution in Yellow crystals are Reducing sugars form
a test tube, add 1 formed during corresponding
spatula of phenyl hydrazine boiling. osazones with
hydrochloride, 2 spatulas of characteristic shape
sodium acetate & 8 drops of
glacial acetic acid. Mix and
keep in boiling water bath Glucose
for 20 minutes. Allow it to
cool. Mount a small part of Needle shaped or
the precipitate on a glass bundle of hay Maltose
slide & cover them with crystals
cover slip. Examine under
or
microscope and make a
sketch. Sun flower petals
Lactose
shaped

or

Hedge hog/powder
puff shaped/over
used ball badminton
Principle: This test is answered only by reducing sugars. Three molecules of phenyl
hydrazine hydrochloride react with one molecule of reducing sugar to form
corresponding osazones (glucosazones, maltosazones, lactosazones).

REACTIONS OF MONOSACCHARIDES
 Monosaccharides are simplest form of carbohydrates.
 Monosaccharides are simple sugars which consist of one sugar unit
(single carbon chain).
 Glucose (aldohexose) and Fructose (ketohexose) are most common
monosaccharides.
 Monosaccharides are reducing agents due to presence of free
aldehyde or keto group.

Glucose:

Glucose is most important simple sugar in human metabolism.

Its molecular formula is C6H12O6 .

Glucose is present in fruits, honey, sugarcane & milk.

Glucose is common medical analyte measured in blood samples.

High glucose levels in blood may be a sign of diabetes.

Fructose:

Fructose is also known as fruit sugar.

It is present in fruits and honey.

It is a constituent of cane sugar & inulin.

 REACTIONS OF DISACCHARIDES
 Disaccharides are carbohydrates which are made up of two
monosaccharide units.
 It forms two monosaccharide units on hydrolysis.
 Maltose, Lactose & Sucrose are common disaccharides.
 Maltose and lactose are reducing disaccharides, where as sucrose
is a non-reducing disaccharide.

Maltose:

Maltose is a disaccharide made up of two glucose units joined together

by -1,4 glycosidic bond.

Since one of the aldehyde group remains free, it is a reducing sugar.

It is also called malt sugar and is present in malt & germinating seeds.

It is obtained by partial hydrolysis of starch by the enzyme amylase

(present in saliva).

Lactose:

It is a disaccharide made up of a molecule of -D-glucose united to β-D-

galactose by β-1,4 glycosidic bond.

It is also called milk sugar and present in milk.

It can be hydrolyzed to glucose and galactose by the lactase present in

intestinal juice.

Absence or reduced activity of enzyme lactase leads to lactose

intolerance and causes diarrhea in children.
Sucrose:

It is non-reducing disaccharide.

It is made up of one molecule of -D-glucose and one molecule of β-D-

fructose united by ,β-1,2 glycosidic bond (between aldehyde group of
glucose and keto group of fructose).

Hence there is no free functional group in sucrose and hence it is a non-

reducing sugar. It does not form osazones.

It is also called invert sugar or table sugar.

It is present in sugar cane, beetroot sugar and in ripen fruits.

Trehalose:

Trehalose is also a non-reducing disaccharide formed of two glucose

units joined by -1,1 glycosidic bond.

It is seen in hemolymph of insects, fungi & yeast.

 REACTIONS OF SUCROSE

S.No EXPERIMENT OBSERVATION INFERENCE

Special Test for Sucrose-

Acid Hydrolysis Test:

Take 5ml of sucrose solution
and add 5 drops of dilute
Brick red precipitate Confirms presence of
HCl. Boil for 1minute. Now sucrose
observed
neutralize the solution with
20% sodium carbonate till
the effervescence ceases.
Perform Benedict’s test
using the above neutralized
solution.

Principle: On hydrolysis of sucrose, 1,2 glycosidic linkage is broken releasing

glucose and fructose. Under alkaline conditions glucose and fructose (reducing
sugars) undergo tautomerization to form 1,2-enediols, which are powerful
reducing agents. They reduce cupric ions to cuprous ions and forms cuprous
hydroxide. On heating cuprous hydroxide gets converted to cuprous oxide which is
brick red precipitate. This is due to reducing action of free aldehyde or ketone
functional groups present in the sugars.
 OSAZONES

S.No EXPERIMENT OBSERVATION INFERENCE

1. Osazone Test:-
Take 5ml of sugar solution Yellow crystals are Reducing sugars form
in a test tube, add 1 spatula formed during corresponding
of phenyl hydrazine boiling. osazones with
hydrochloride, 2 spatulas of characteristic shape
sodium acetate & 8 drops of
glacial acetic acid. Mix and Needle shaped or
keep in boiling water bath bundles of hay Glucose
for 20 minutes. Allow it to crystals
cool. Mount a small part of
or
the precipitate on a glass
slide & cover them with Sun flower petals
Maltose
cover slip. Examine under shaped
microscope and make a
or
sketch.
Hedge hog/powder
puff shaped Lactose

Principle: This test is useful in identification of different reducing sugars. Only

reducing sugars form their corresponding osazones. Three molecules of phenyl
hydrazine hydrochloride react with one molecule of reducing sugar to form
corresponding osazones (glucosazones, maltosazones, lactosazones) which are
highly insoluble and crystalises during the reaction.
SCHEME FOR IDENTIFICATION OF UNKNOWN CARBOHYDRATE SOLUTION

Unknown Solution

Molisch Test

(Violet/Purple Ring)

Iodine Reaction

Blue Colour Red Colour No Colour

(may be starch) (may be dextrin) (except yellow colour of iodine)
↓

Benedict’s test

Brick red Precipitate No colour

change
(reducing sugar) (Non-reducing sugar, may be sucrose)

Barfoed’s Test Acid Hydrolysis Test

Red precipitate with

Red scum No Red scum Benedict’s test
(May be monosaccharide) (May be disaccharide) SUCROSE

Seliwanoff’s test & others Osazone formation Seliwanoff’s test &

others
Cherry red
colour

No red colour cherry red colour Sunflower shaped Hedge –hog shape crystals
GLUCOSE FRUCTOSE MALTOSE LACTOSE
 SPECIAL REACTIONS OF PENTOSE & LACTOSE

Reactions for Pentoses

S.No EXPERIMENT OBSERVATION INFERENCE

1 Tauber’s test for

Pentoses:-

Add 1 ml of 4% benzidine
solution in glacial acetic
A pink to red colour Indicates the
acid to 0.2 ml of pentose is given by pentoses presences of
solution. Heat and cool pentoses
under running water and
add 2 ml of distilled
water.

2 Bial’s test for Pentoses:-

Indicates the
Mix 5 ml of Bial's reagent A green colour
presences of
with 0.5 ml of pentose solution is obtained. pentoses
solution and heat until
boil.
Principle: Pentoses heated with Conc.HCl forms furfural it condenses wit
Orcinol in the presence of ferric ions to give blue-green colour.

Bial’s Reagent: Dilute Solution of Orcinol in 30% HCl. (1.5gm orcinol + 500ml
conc.HCl + 20 drops 10% FeCl3)
Reactions for Galactose and Lactose
S.No EXPERIMENT OBSERVATION INFERENCE

3 Mucic Acid test:-

To about 1 ml of sugar Formation of The primary

solution add few drops crystals can be functional group
observed and the secondary
of concentrated HNO3
alcoholic group are
and gently evaporate oxidized to
it over a boiling water carboxylic groups
bath till the acid fumes in the presence of
are removed concentrated
completely. Add few HNO3. The
resultant saccharic
drops of water to the
acid of galactose
resultant mixture and
termed as mucic
leave it over night. acid is insoluble in
water and forms
crystals.
REACTIONS OF
PROTEINS
 REACTIONS OF PROTEINS
Proteins are one of the major macronutrients. Aminoacids are the
building blocks of proteins. Proteins are biomolecules consisting of one or
more long chains of amino acid residues. Proteins are used for building
muscle tissue. Proteins form colloidal solutions of the emulsoid type. A
certain amount of water is imbibed by the protein molecule (water of
hydration) and is taken up by various free COOH & NH2 groups.

The stability of colloid solution depends on this water of hydration and

on the electric charges carried by the molecules. Removal of water of
hydration by any dehydrating agent (eg: neutral salts & alcohol) or
neutralization of the charges on the protein molecules precipitate them.

1. Precipitation by Metallic Salts

S.No EXPERIMENT OBSERVATION INFERENCE

a. With Mercuric Sulphate:-

To 3ml of protein solution, add White This is due to
few drops of mercuric sulphate precipitate is formation of
drop by drop. Mix well. formed mercuric
proteinate

Principle: The positive charge of mercury ion neutralizes the negative charge of
protein forming a precipitate of mercuric proteinate.

b. With Lead Acetate:-

To 3ml of protein solution, add White This is due to
few drops of 10% lead acetate precipitate is formation of
drop by drop. Mix well. formed lead proteinate

Principle: The positive charge of lead ion neutralizes the negative charge of
protein forming a precipitate of lead proteinate.
 2. Precipitation by Alkaloidal Reagents
S.No EXPERIMENT OBSERVATION INFERENCE

a. With Sulphosalicylic acid:- This is due to

formation of
To 2ml of protein solution, add White
protein
3 drops 20% sulphosalicylic acid. precipitate is
Mix well. sulphosalicylate
formed
complex.

Principle: In acid medium proteins exist as cations, which neutralize the

negative charge of acids such as sulphosalicylic acid resulting in the formation of
precipitate protein sulphosalicylate complex.

b. With Esbach’s Reagent

(Picric acid in Citric Acid):-
Yellow This is due to
To 3ml of protein solution, add precipitate is formation of
1ml of Esbach’s reagent drop by formed protein picrate.
drop. Mix well.
Principle: In acid medium proteins exist as cations, which neutralize the
negative charge of acids such as picric acid resulting in the formation of
precipitate protein picrate complex.

3. Precipitation by Alcohols:
S.No EXPERIMENT OBSERVATION INFERENCE

a. By Absolute Ethyl Alcohol:- White This is due to

precipitate is precipitation of
To 2ml of absolute ethyl alcohol
formed protein
add 1ml of protein sol dropwise.
Principle: Alcohol acts as dehydrating agent and removes water of hydration
resulting in precipitation of protein.
 4. Precipitation by Neutral Salts:
S.No EXPERIMENT OBSERVATION INFERENCE

a. Half Saturation Test: This indicates the

absence of
To 3ml of protein solution, add proteins in the
equal volume of
No violet colour filtrate i.e the
saturated ammonium sulphate
is seen (Biuret - given protein is
(NH4)2SO4 solution and mix. precipitated by
ve)
Allow it to stand for 5 minutes & half saturation.
filter. With the filtrate perform
Biuret test using 40% NaOH. (Eg: Globulins,
Casein, Gelatin)

b.
Full Saturation Test:
This indicates the
To 5ml of protein solution, add absence of
solid ammonium sulphate proteins in the
No violet colour
(NH4)2SO4 with mixing till the filtrate i.e the
is seen (Biuret -
solution is saturated (there given protein is
ve)
should be undissolved crystals in precipitated by full
the bottom of the test tube). saturation.
Allow it to stand for 5 minutes &
(Eg : Albumin)
filter. With the filtrate perform
Biuret test using 40% NaOH.

Principle: Solubility of protein molecule depends on charge and hydration. Polar

groups of protein interact with water molecules around them to produce shell
of hydration. Any factor which neutralizes the charge or removes the water of
hydration will cause precipitation of proteins. When a salt like ammonium
sulphate is added to protein solution, water available to protein is reduced. The
protein-protein interaction increases thus causing protein precipitation. This
process is called as salting out.
 5. Heat Coagulation Test:
S.No EXPERIMENT OBSERVATION INFERENCE
a. Heat Coagulation Test:
Fill 3/4th full of the test tube Cloudy white This indicates the
with protein solution. Hold the precipitate or a presence of heat
tube over a flame in a slanting coagulum is coagulable
position and heat the top layer formed in the proteins.
of the protein solution. Cool and heated portion
(Eg : Albumin)
add few drops of 1% Acetic acid. of solution

Principle: Proteins are easily denatured, when subjected to heat treatment.

Denatured proteins are generally less soluble than the native proteins and easily
get precipitated. In case of albumin, the denaturation is followed by coagulation
(irreversible denaturation) and it is precipitated.

b. Heat Coagulation Test at

Isoelectric pH:
Take 5ml of protein in a the test Dense coagulum Indicates the given
tube and add 2 drops of is formed protein is a heat
chlorophenol red indicator coagulable protein
(yellow at pH-4.8, red at pH- which is
6.4). pH is adjusted by adding precipitated at
1% acetic acid drop by drop. isoelectric pH.
Now heat the top layer of the
protein solution.

Principle: Proteins are easily denatured, when subjected to heat treatment.

Denatured proteins are generally less soluble than the native proteins and easily
get precipitated. In case of albumin, the denaturation is followed by coagulation
(irreversible denaturation) and it is precipitated. At isoelectric pH denaturation
and coagulation occurs easily.
 COLOUR REACTIONS OF PROTEINS
S No. EXPERIMENT OBSERVATION INFERENCE
1. Biuret Test:-
To 3ml of protein solution, Violet / rose pink Indicates the
add 1ml of 5% NaOH and 3 colour is observed presence of
drops of 1% CuSO4. Mix well. proteins

Principle: The violet or rose pink colour is due to the formation of a copper
coordinated complex. Copper reacts with peptide bonds in alkaline medium to
give violet colour. Biuret test is answered by compounds containing 2/more
peptide bonds.

2. Ninhydrin Test:- Indicates the

presence of free
To 3ml of protein solution, add Blue/purple colour
-amino group in
3drops of Ninhydrin & boil. is observed
the protein

Principle: -amino groups of protein and free aminoacids react with ninhydrin to
form ammonia & hydrindantin which reacts with another molecule of ninhydrin
to give blue/purple colour (Ruhemann’s purple).

3. Xanthoproteic Test:- Yellow colour is Indicates the

observed in acid presence of
To 2ml of protein solution,
medium which aromatic
add 1ml of concentrated
HNO3 and boil for 1 minute. changes to orange aminoacids (like
Cool under tap water. Add colour in alkaline Tyrosine or
1ml of 40% NaOH and medium Tryptophan)
observe.
Principle: On heating with concentrated nitric acid proteins containing aromatic
aminoacids form yellow colour due to nitration of benzene ring. The nitro
compounds are freely ionized in alkaline medium and thus orange colour is
formed by adding strong alkali.
4. Modified Millon’s Test
(Cole’s Test):-
To 2ml of protein solution, Red coloured Indicates the
add 1ml of Millon’s reagent solution/precipitat presence of
(10% mercuric sulphate in e is observed Tyrosine
10% H2SO4) and boil gently for
30 seconds. Cool under tap
water and add 2 drops of 1%
Sodium nitrite (NaNO2).

Principle: This test is answered by compounds containing hydroxy phenyl group

like tyrosine. Proteins containing tyrosine will give red coloured solution or
precipitate. These proteins undergo mercuration and nitration in strong acidic
medium to form red coloured mercuric phenolate.

5. Aldehyde Test:-
To 2ml of protein solution,
add 1-3 drops of dilute
Violet coloured Indicates the
formalin (1/500) & 2 drops of
ring is formed at presence of
10% mercuric sulphate in
the junction of the Tryptophan which
10% sulphuric acid. Now add
two liquids contains indole
1ml of conc. H2SO4 carefully
ring
along the sides of the test
tube.

Principle: H2SO4 and HgSO4 act as oxidizing agents. These oxidizes the indole
ring of tryptophan present in protein. The oxidized product formed, then
condenses with formaldehyde to give violet coloured compound.
6. Sakaguchi Test:-
To 2ml of protein solution, Indicates the
add 4 drops of alcoholic presence of
Bright red colour
naphthol & 5 drops of sodium is observed Arginine which
hypobromite (alkaline). Mix contains guanidine
well. group

Principle: In alkaline medium naphthol combines with the guanidine group of

Arginine to form complex. This complex is oxidized by sodium hypobromite and
produce bright red colour.

Bromine water can be used instead of sodium hypobromite

7. Sulphur Test:-
To 2ml of protein solution, Indicates the
add 1ml of 40% NaOH. Boil presence of
Black or brown
and cool. Then add 2 drops of sulphur containing
colour is observed
lead acetate. aminoacids like
Cysteine and its
oxidized product
Cystine

Principle: When heated with strong alkali, sulph hydryl group of cysteine is
converted to sulphide. This inorganic sulphide reacts with lead acetate to form
insoluble lead sulphide which is black or brown in colour. Only cysteine and
cystine answers this test. Methionine does not answer the test.
 Heat Coagulation Test of Albumin

S.No EXPERIMENT OBSERVATION INFERENCE

Heat Coagulation Test

at Isoelectric pH (5.4):
Take 5ml of protein in a Indicates albumin is a
the test tube and add 2 heat coagulable
drops of chlorophenol red Dense coagulum protein which is
indicator (yellow at pH-4.8, is formed precipitated at
red at pH-6.4). pH is isoelectric pH (5.4)
adjusted by adding 1%
acetic acid drop by drop
until light pink colour is
obtained (indicates pH 5.4).
Now heat the top layer of
the protein solution.

Principle: Proteins are easily denatured, when subjected to heat treatment.

Denatured proteins are generally less soluble than the native proteins and easily
get precipitated. In case of albumin, the denaturation is followed by coagulation
(irreversible denaturation) and it is precipitated. At isoelectric pH denaturation
and coagulation occurs easily.

Note: If the solution colour is yellow 1% sodium carbonate is used to bring pH

to 5.4 i.e light pink colour.
 Special test for Casein

S.No EXPERIMENT OBSERVATION INFERENCE

1. Isoelectric Precipitation
Test:-
Take 3ml of casein solution, add
2 drops of bromo cresol green
indicator. Blue colour indicates A curdy Indicates the
H
P is >7. Add 1% acetic acid drop precipitate is seen presence of
wise with mixing until green in green solution Casein which is
colour is obtained. PH at this precipitated at its
stage is 4.6. Observe whether isoelectric PH
any precipitate is formed.

Principle: Proteins have minimum solubility at their isoelectric PH and get

precipitated. Isoelectric PH of casein is 4.6. When PH of casein solution is adjusted
to 4.6 by addition of acetic acid, casein is precipitated. At isoelectric point the
precipitation of proteins is maximum.

2. Neumann’s Test (Organic

Phosphorus test):-
Take 5ml of casein solution, add
Indicates the
0.5ml of 40% NaOH. Boil strongly
presence of
for 2 minutes. Cool under tap Canary yellow
organic
water. Then add 0.5ml of HNO3. colour is seen
phosphorus in
Add a pinch of solid ammonium
casein
molybdate & warm gently.

Principle: The organic phosphorus present in casein (which is a phospho-protein)

is converted to inorganic phosphate on boiling with strong NaOH (40% NaOH).
Inorganic phosphate reacts with ammonium molybdate to form ammonium
phospho molybdate, which is canary yellow in colour.
SCHEME FOR IDENTIFICATION OF UNKNOWN PROTEIN SOLUTION

Unknown solution

BiuretTest
Violet/ Rose pink colour

Iso electric precipitation (at pH 4.6)

Curdy precipitate No precipitate

(May be casein ) (Other proteins)

Test for organic phosphorus Heat Coagulation Test

(Neumann’s Test)
Coagulation No Coagulation
Canary yellow colour (May be Albumin/Globulins) (May be Gelatin/Peptone)
(or) precipitate
CASEIN Half Saturation & Half Saturation &
Biuret test with Filtrate Biuret test with Filtrate
Perform
(i) Sulphur Test
(ii) All colour reactions

Violet colour Blue colour Blue color Rose-Pink colour

(May be Albumin) (Globulins) GELATIN (May be Peptone)

Full Saturation & Perform Full Saturation &

(i) Aldehyde Test Biuret test with Filtrate
Biuret test with Filtrate
(ii) All Color Reactions Rose Pink Colour
No violet color
ALBUMIN
PEPTONE
Perform all color Reactions Perform Color Reactions
 NORMAL
CONSTITUENTS
OF URINE
 NORMAL CONSTITUENTS OF URINE
Physical characteristics:

1. Volume : 1000 - 2000ml/day

2. Colour : Straw/pale yellow colour (due to pigment
urochrome)
3. Appearance : Clear & Transparent
4. Odour : Faintly Aromatic
H
5. P : 6.0 - 7.5
6. Specific gravity : 1.004 - 1.040

Chemical characteristics:
Normal urine contains both inorganic and organic constituents.
The normal inorganic constituents of urine include Na+, K+, Ca++, Mg++,
NH4+, Cl-, H2PO4--, SO4-- and traces of HCO3- ions. Normal organic
constituents of urine are urea, uric acid and creatinine.
The total non - protein nitrogen varies from 10-15 grams per day
depending mainly on the protein intake. In addition to these major
organic constituents, detoxified products like indican and ethereal
sulphates are found in urine. Routine analysis of urine includes tests
for Cl- , SO4--, PO4- , Ca++, NH4+, urea, uric acid, creatinine, ethereal
sulphates and urobilonogen.
Normal urine is pale yellow in colour because of the pigment,
urochrome. When the output of urine is low, it appears deep yellow. A
freshly voided urine is clear and transparent. On standing it may
become turbid due to precipitation of phosphates.
The average daily excretion of various organic and inorganic
constituents in normal urine is as follows:
INORGANIC ORGANIC
NaCl : 5-15 grams Urea : 15-30 grams
Phosphorous : 1-2 grams Creatinine : 1-2 grams
Calcium : 0.1-0.2 grams Ammonia : 0.5-0.9 grams
Sulphur : 2-3 grams Uric Acid : 0.6-0.7 grams
 NORMAL CONSTITUENTS OF URINE
TESTS FOR INORGANIC CONSTITUENTS OF URINE

S.No EXPERIMENT OBSERVATION INFERENCE

1. Test for Chlorides:-

To 2ml of urine, add 0.5 ml of A White curdy Indicates the
conc.HNO3 and 1ml of 3% presence of
Silver nitrate (AgNo3). precipitate is seen chlorides
Principle: Chloride is precipitated as silver chloride with AgNo3 in the presence of
HNO3.
Note:- On an average diet 24- hour urine contains 8-15 g of sodium chloride.

2. Test for Sulphates:-

To 2ml of urine, add 1ml of HCl White precipitate Indicates the
and 2ml of 10% Barium presence of
chloride (Bacl2). is seen
sulphates

Principle: Sulphate is precipitated as barium sulphate with BaCl2 in presence of HCl.

Note:- Sulphate is derived from the catabolism of sulphur containing aminoacids.
On an average about 1 gm of sulphate is excreted per day. About 85- 95% of sulphur
is excreted as inorganic suplhate.

3. Test for Inorganic

phosphates:-
Canary yellow colour Indicates the
To 5ml of urine, add few drops precipitate is seen presence of
of conc.HNO3 and a pinch of inorganic
ammonium molybdate & phosphates
warm .

Principle: Phosphate is precipitated as canary yellow ammonium phospho

molybdate. Note:- On an average 24- hours urine contains 1gm of phosphate.
S.No EXPERIMENT OBSERVATION INFERENCE

4. Test for Calcium :-

To 5 ml of urine, add 5 Trace amounts of Indicates the
drops of 1% acetic acid and
white precipitate presence of
5ml of 2% potassium oxalate.
is seen. Calcium.

Principle: Ca 2+ is precipitated as calcium oxalate in presence of acetic acid.

Note:- On average 24 – hours urine contains around 200 mg of calcium.

5. Test for Ammonia :-

To 5 ml urine, 1 drop of Tip of the glass Indicates the
phenolphthalein is added.
rod turns pink for presence of
(colour less at PH 6 and dark
pink at PH 8 ). Then 0.1N a moment ammonia
NaOH is added drop by drop
till pink colour is seen. Now
heat the solution and hold a
glass rod dipped in
phenolphthalein the mouth
of the test tube .

Principle:- Ammonia evolved turns the medium alkaline. Phenolphthalein give dark
pink colour in alkaline medium and hence turns glass rod pink for a moment.

Note:- Urinary ammonia is derived from glutamine and other amino acids in kidney .
The normal daily output of ammonia is about 0.5-0.8 gms. There is an increase in
ammonia excretion when acid forming foods are taken.
 NORMAL CONSTITUENTS OF URINE
TESTS FOR ORGANIC CONSTITUENTS (or NPN SUBSTANCES)
OF URINE

1. Test for Urea:

S.No EXPERIMENT OBSERVATION INFERENCE

a. Sodium hypobromite
Test:-
To 2ml of urine, add 4 Brisk effervescence Indicates the
to 5 drops of sodium of N2 gas is seen presence of Urea.
hypobromite solution.

Principle: When urea is treated with NaOBr it decomposes to give Nitrogen gas
and carbondioxide gas.

b. Specific Urease Test:-

Take two test tubes and label
them as test (T) and control
Pink colour is Confirms the
(C). Take 5ml urine and 5ml of
water in two test tubes observed in the presence of Urea
respectively. Add 2 drops of
test tube.
Phenolphthalein indicator and
2ml of Urease enzyme extract
into two tubes. Incubate at
37O for 15 minutes.

Principle: Urea is hydrolyzed by Urease to form ammonium carbonate which

make the solution alkaline and the colour of the solution changes to pink.

Note:- Urea is formed in the liver as the end product of protein metabolism.
About 20 to 30 grams of urea is excreted in 24 hour urine.
2. Test for Uric acid:

S.No EXPERIMENT OBSERVATION INFERENCE

a. Phosphotungstic Acid
Reduction Test:-
Take 2 ml of urine, add few Deep blue colour is Indicates the
drops of phosphotungstic
observed presence of Uric
acid reagent and few drops
of 20% Na2CO3. acid

Principle: Uric acid is a reducing agent in strong alkaline condition. It reduces

colorless phosphotungstic acid to tungsten blue.

Note:- Uric Acid is the end product of purine metabolism. The daily output of
uric acid varies from 0.6 to 1 gm.

b. Schiff’s Test:-
Wet a Piece of filter paper Black colour is Confirms the
with few drops of
formed presence of Uric
ammonical AgNO3 solution.
Add 1 or 2 drops of urine acid
solution on the same paper.

Principle: Uric acid reduces ammonical AgNO3 to metallic silver which is in

black color.

Note:- Uric Acid is the end product of purine metabolism. The daily output of
uric acid varies from 0.6 to 1 gm.
3. Test for Creatinine:
S.No EXPERIMENT OBSERVATION INFERENCE
Jaffe’s Test:-
Take two test tubes and label
them as test (T) and control Orange colour is Indicates the
(C). Take 2ml urine and 2ml of
seen in test presence of
water in two test tubes
respectively. Add 2ml of Yellow colour Creatinine
saturated Picric acid and few persists in control
drops of 10% NaOH in both
test tubes.

Principle: In presence of alkali, creatinine reacts with picric acid to form orange
colored creatinine picrate complex.
Note: Creatinine is the excretory product. It is the anhydride of creatine & is
formed from 3 amino acids (i.e gly, arg & met). The daily output of is 1-2g/day.

4. Test for Ethereal sulphate:

S.No EXPERIMENT OBSERVATION INFERENCE
Test for Ethereal sulphate:-
Take 5ml urine, add 2ml BaCl2
and 2ml of conc. HCl. Mix &
A trace turbidity Indicates the
filter. Divide the filtrate into 2
portions. Boil one (test) & over that in control presence of
compare with the other (a red colour may Ethereal
(control). also be seen)
Sulphate
Principle: On heating HCl hydrolyses ethereal sulphate to inorganic sulphate
which then gives precipitate with BaCl2.
Note: About 100mg of organic sulphate is excreted per day. Eg: Indican
 5. Test for Urobilinogen:

S.No EXPERIMENT OBSERVATION INFERENCE

Test for Urobilinogen:-

Take 5ml of freshly voided A red colour is Indicates the
urine and add 1ml of seen when viewed Presence of
Ehrlich’s Reagent. Mix well through the
and allow it to stand for mouth of the test urobilinogen
5 minutes. tube

Principle: Urobilinogen reacts with p-dimethyl amino benzaldehyde of the

reagent to give a red colour.

Note: Normal urine contains traces of urobilinogen. This is derived from

bilirubin by the action of bacterial flora in the intestine, enters into
circulation & then excreted by the kidneys. Absence of urobilinogen in urine
is suggestive of obstructive jaundice. Excess of urobilinogen in urine is
indicative of hemolytic & hepatic jaundice.

Ehrlisch’s Reagent Composition: Para dimethyl amino benzaldehyde in

concentrated HCl.
 SCHEME FOR IDENTIFICATION OF UNKNOWN PHYSIOLOGICAL
SUBSTANCE IN THE GIVEN SOLUTION

Unknown Solution

Biuret test

Violet / Rose-Pink color No Color Change

Protein is present (may be Carbohydrates/NPN substances)
Follow the scheme for
Identification of Proteins.
Molisch test

Reddish Violet Ring No Reddish Violet ring

Carbohydrate is present (may be NPN substance)
Follow the scheme for
Identification of Carbohydrates.
Sodium Hypobromite Test

Effervescence No Effervescence
(may be Urea) (may be Uric Acid/ Creatinine)

Confirm by
Specific Urease Test Phoshotungstic acid reduction test
Pink color

Blue color No Blue color

(may be Uric Acid) (may by Creatinine)

Confirm by Confirm by
Schiff’s test Jaffe’s test
Black deposit Orange color
 ABNORMAL
CONSTITUENTS
OF URINE
 ANALYSIS OF ABNORMAL CONSTITUENTS OF URINE

The abnormal constituents which are routinely analyzed in urine are

 Albumin (Protein)

 Glucose

 Ketone bodies

 Bile salts

 Bilirubin (bile pigment) &

 Blood.
 ANALYSIS OF ABNORMAL CONSTITUENTS OF URINE

1. TEST FOR PROTEINS:

S.No EXPERIMENT OBSERVATION INFERENCE

a. SULPHOSALICYLIC ACID TEST:- It indicates the

Take 5 ml clear urine White precipitate presence of
or
and add 1 ml of 20% proteins ( It may
turbidity is formed be albumin).
Sulphosalicylic Acid

Principle: 10% TCA (trichloro acetic acid) 28% sodium sulfite (Na2SO3) and 20%
sulphosalicylic acid are used as precipitating reagents. So, after the addition of
20% sulphosalicylic acid to the given urine sample, the proteins will get
precipitated and a white precipitate or turbidity is seen.

b. HEAT COAGULATION
TEST:-

Fill 3/4th of a test tube

with the urine
solution. Hold the tube A Cloudy white This test confirms
over a flame in a precipitate will be the presence of
slanting position & observed in the albumin in urine
heat the top layer of heated portion
the protein solution,
cool & add few drops
of 1% Acetic Acid.

Principle: Proteins are easily denatured, when subjected to heat treatment.

Denatured proteins are generally less soluble than the native proteins. In case
of albumin, the denaturation is followed by coagulation. Irreversible
denaturation leads to coagulation and albumin is precipitated.
CLINICAL SIGNIFICANCE
Normal adults excrete a small amount of protein, i.e. up to 150 mg / day in
urine. This small amount of protein is not detectable by routine methods.
Increased amount of protein in urine is termed as Proteinuria. Most
common type of proteinuria is due to albumin. Hence, proteinuria &
albuminuria are often used synonymously. The proteinuria may be
a.) Functional or physiological b.) Organic or pathological

FUNCTIONAL ALBUMINURIA:-
Usually it is intermittent & asymptomatic, it is seen in
1. Severe stress like exposure to severe cold (or) excessive physical activity.
2. In last weeks of pregnancy (protein levels up to 300 mg/ day) may be
excreted).

ORGANIC / PATHOLOGICAL ALBUMINURIA:-

The clinical conditions which lead to organic or pathological albuminuria
can be due to;
1. Renal diseases:
- Nephrotic Syndrome (more amount of proteinuria)
- Pyelonephritis
- Glomerulonephritis
- Renal stones
- Renal tumours
- T.B kidney

2. Other Conditions:
- Severe dehydration and vomiting.
- Hemolytic diseases
- Severe urinary tract infection
- Multiple myeloma
- Bladder schistosomiasis
- Urinary tract stones or tumours
- Inflammation / degeneration of ureter, bladder, urethra or prostate
 2. TEST FOR REDUCING SUGARS:

S.No EXPERIMENT OBSERVATION INFERENCE

a. BENEDICTS TEST:-
Take 5ml of Benedict’s Green/Yellow/ Indicates presence
qualitative reagent in a test Orange/ Brick red of reducing sugar
tube and heat. Add 8 drops of
colour is seen. (Generally glucose)
sugar solution. Mix well and
boil for 2 minutes.

Principle: Under mild alkaline conditions, reducing sugars undergo

tautomerization to form 1,2 enediols which are unstable, powerful reducing
agents. These 1,2 enediols reduce Cupric ions to Cuprous ions. Cuprous ions in
solution form cuprous hydroxide. On heating Cuprous Hydroxide gets
converted to Cuprous Oxide which is a red precipitate.

Composition of Benedict’s Reagent: Copper Sulphate

Sodium Citrate
Sodium Carbonate
Depending upon the amount of glucose that is present in urine, the
following colours will be seen.
Colour Blue Green Yellow Orange Brick Red

Amount of Glucose Nil 0.5% 1% 1.5% 2%

CLINICAL SIGNIFICANCE:
Normal urine contains small amount of reducing sugar, i.e. 1 to 1.5 gm per
24 hours. Out of this, glucose present in the concentration of 50 to 300mg
per 24 hours. They are not detectable by routine tests. Excretion of readily
detectable amounts of reducing sugar in urine is called as GLYCOSURIA and
of glucose specifically is called GLUCOSURIA. Positive benedicts test is
usually taken for glucose. Glucosuria may be either
 BENIGN or PATHOLOGICAL.
I. RENAL GLYCOSURIA is an example of benign glycosuria.
II. DIABETES MELLITUS is an example of pathological glycosuria.
Positive reaction in urine can also be seen in the following cases:
i) Due to lactosuria in pregnancy & lactation.
ii) Due to galactose in galatosuria.
iii) Due to pentose in pentosuria
iv) Due to other reducing agents like homogentistic acid, glucoronic
acid and drugs like salicylate and Isoniazid.
The identity of different sugars may be established by other relevant tests
like Seliwanoff’s, Osazone test etc.

3. TEST FOR KETONE BODIES:

S.No EXPERIMENT OBSERVATION INFERENCE

a. ROTHERA’S TEST:- (for

Acetone & Acetoacetate)

Take 10ml of urine in attest

tube and add solid ammonium
Sulphate a little at a time with A purple ring is It indicates the
mixing to saturate the observed at the presence of
solution. Then add 2 or 3 junction of the two acetone or/and
drops of freshly prepared acetoacetic acid.
layers.
sodium Nitroprusside solution.
.
Mix gently and add 1ml of
strong ammonia solution
(Liquor ammonia) drop wise
along the sides of the test
tube. Don’t mix the solution.

Principle: Nitroprusside in alkaline conditions reacts with keto group & forms a
purple ring. It is given by acetone and acetoacetate but not by β hydroxy butyric
acid which lacks the keto group.

b. GERHARDT’S TEST:- (for

Acetoacetic acid only)
Port-wine colour is It indicates the
Take 3ml of urine & add 10% seen. presence of
ferric chloride drop by drop. Acetoacetic acid
only.

Principle: Aceto-acetic acid present in urine combines with ferric chloride to

give a port-wine or red colour.

Ketone bodies are

- Acetone
- Acetoacetic acid.
- β hydroxy butyric acid

CLINICAL SIGNIFICANCE:
Presence of Ketone bodies in urine is known as ketonuria.
Presence of excess of Ketone bodies in blood is known as ketonemia.

Ketone bodies are seen in urine when fat metabolism is excessive as in

following conditions:
1. Diabetic keto acidosis.
2. Starvation.
3. Intake of high fat diet.

Acetone is present in expired air.

Both glycosuria and ketonuria are present in diabetic keto acidosis.

Rothera’s test is also positive in Maple syrup urine disease in which there is
branched chain ketoacid dehydrogenase complex deficiency.
 4. TEST FOR BILE SALTS:
S.No EXPERIMENT OBSERVATION INFERENCE

a. HAY’S TEST:-

Take 2ml of urine and water Sulphur powder It indicates the

in 2 test tubes (Test & sinks to the bottom presence of bile
Control). Sprinkle a little in test and floats in salts in urine
sulphur powder in both test control
tubes. Observe without
mixing.

Principle: Bile salts have the property of lowering the surface tension of
solution in which they are dissolved. Hence sulphur powder sinks in urine
containing bile salts. In the absence of bile salts, the surface tension of urine is
same as that of water and holds the sulphur powder floating.

5. TEST FOR BILE PIGMENTS:

S.No EXPERIMENT OBSERVATION INFERENCE

a. FOUCHETS TEST:-

Take 10 ml of urine, add 1ml

of glacial acetic acid, 5ml of Blue or green It indicates the
BaCl2 sol. &10 drops of colour is seen on
saturated (NH4)2SO4. A thick the precipitate presence
white precipitate is formed. of bile pigment
After 5 minutes filter the
solution. Unfold the filter
paper over dry filter papers
& add a drop of Fouchet’s
 reagent on the precipitate.

Principle: BaCl2 reacts with (NH4)2SO4 to form BaSO4 precipitate. This adsorbs
with bilirubin if it is present in the urine. Fouchet’s reagent oxidizes bilirubin to
green (biliverdin) and blue (bilicyanin) products.

Fouchet’s reagent Composition- 10% FeCl3 in 25% TCA

CLINICAL SIGNIFICANCE:
Bile salts are sodium and potassium salts of bile acids.
Eg: Sodium glycocholic acid
Sodium taurocholic acid
Bile acids are derived from cholesterol.
Function of bile salts is related to fat digestion due to their property to
reduce surface tension and act emulsifying agent.
Primary bile acids are cholic acid and chenodeoxy cholic acid.
Secondary bile acids are taurine and glycine conjugated with bile acids
(glycocholic acid).
Bile pigments are breakdown product of blood pigment hemoglobin.
Eg: Bilirubin
Biliverdin
Bile pigments are formed in liver and leave the body in feces and urine.
Yellow colour of urine is due to bile pigments.
It helps to differentiate between different forms of jaundice.
Urobilinogen in urine is detected by adding 5ml of urine to 1ml of Ehrlich’s
reagent. Pink colour is developed in presence of urobilinogen.

Presence of bile salts and bile pigments in urine is suggestive of

“Obstructive Jaundice” & “Hepatic Jaundice”.

Hemolytic Jaundice have excessive urobilinogen that can be identified by

Ehrlich test
 6. TEST FOR BLOOD:
S.No EXPERIMENT OBSERVATION INFERENCE
a. BENZIDINE TEST:- Blue/green colour is It indicates the
formed immediately presence of
Mix 1ml of Benzidine solution
and 1ml of H2O2. Add 1ml of and disappears with blood/ blood
urine & 1ml of water in two test in few seconds in pigment in urine.
tubes test (T) and control (C). test (T) tube.

Principle: Heme of hemoglobin decomposes H2O2 catalytically & liberates oxygen.

This oxygen oxidizes Benzidine to blue or green colour compounds which are quite
unstable. This colour changes to brown within few minutes on exposure to air.

b. PHENOLPTHALIN TEST:- Pink or Red colour It indicates the

isformed immediately presence of
To 2 ml of urine solution, add
2 drops of phenolphthalein and disappears within blood/ blood
solution and 2 drops of H2O2. few seconds. pigment in urine.

Principle: The basis of the test is that the peroxidase like activity of the
hemoglobin in blood catalyzes the oxidation of the colourless reduced
phenolpthalin into pink colour.

CLINICAL SIGNIFICANCE:
1. HEMATURIA (Presence of RBC in urine):
- Is the passing of Blood through urinary tract into the urine.
- Occurs due to bleeding in the urinary tract due to trauma
Eg: introduction of catheter through urethra.
Diseases like pyelonephritis, glomerulonephritis kidney stones and injury.
HEMOGLOBINURIA (Presence of Hemoglobin only in urine)
- Is due to hemolysis i.e rupturing of the stroma of RBC & liberation of Hb.
- Occurs in severe burns, chemical poisoning, enteric fever and
incompatible blood transfusion. Presence of protein and blood together is
seen in infection, injury or malignancy of urinary tract, bladder stones and
renal T.B.
III. QUANTITATIVE
EXPERIMENTS
 ESTIMATION OF BLOOD GLUCOSE
By Orthotoluidine Method

Aim:
To estimate the amount of glucose present in the given blood/serum
sample.

Principle:
Glucose condenses with O-Toluidine in glacial acetic acid at 1000 C and the
product formed is N-glycosylamine which is blue green in colour. The
absorbance is measured at 630nm. It is compared with standard solution of
glucose similarly treated. Since blood proteins interfere colorimetrically,
they are precipitated. The protein free filtrate is used for determination of
blood glucose concentration. Alternatively serum/plasma can be used
directly.

Procedure:
Preparation of Protein Free Filtrate (PFF):
In a dry test tube pipette 0.5ml of blood, 3.5ml of distilled water and 0.5ml
of 10% sodium Tungstate and mixed. Then 0.5ml of 2/3 N H2SO4 is added.
Allow to stand for 10 minutes and filter into a dry test tube.

Reagents in ml Blank (B) Standard (S) Test (T)

Distilled Water 0.1 ml - -

Standard Glucose - 0.1 ml -
(100mg/dl)

Plasma/ Serum - - 0.1 ml

O-Toluidine 3.0 ml 3.0 ml 3.0 ml
Keep the tubes in boiling water bath for 10 minutes. Cool and then read the
absorbance of B, S & T at 630nm.

Optical Density (O.D)

Calculation:
Mg of glucose in 100ml of blood =

OD of T - OD of B 100
Glucose Conc. (mg/dl) = x Conc. of Std x
OD of S - OD of B Vol. of sample

OD of T - OD of B 100
= x 0.1 x
OD of S - OD of B 0.1

OD of T
= x 100
OD of S

= ________ mg/dl

Result:
The amount of glucose in the given sample of blood found by
Orthotoluidine method is _________ mg/dl.

Normal Values:
Fasting Blood Sugar (FBS) - 70-110mg%
Post Prandial Blood Sugar (PPBS) - < 140 mg%
CLINICAL SIGNIFICANCE:
- Fasting blood glucose level in normal subjects is 70 to 110 mg/dl.
- Post prandial blood glucose level in normal subjects is < 140 mg/dl.

1. HYPERGLYCEMIA (Increased Blood Glucose level) is seen in :

A. Uncontrolled diabetes mellitus.
B. Cushing’s Syndrome (Adreno Cortical Hyperactivity)
C. Hyperthyroidism
D. Hyperpituitarism –Acromegaly , Gigantism
E. Intra Cranial Diseases – Meningitis, tumors etc.
F. Administration of drugs like----Cortico steroids,estrogens etc.

- In hyperthyroidism, fasting glucose level may be normal but there is

elevation of blood sugar in fed state.
- Fear, anger, anxiety & other emotional states also cause increase in
blood sugar.
- This is due to increased secretion of adrenaline which has
hyperglycemic action.

2. HYPOGLYCEMIA (Blood glucose < 45mg/dl) is seen in

1. Starvation
2. Hyperinsulinism
a) Increased dose of insulin while treating Diabetes Mellitus
b) Insulin secreting tumors of β-cells of pancreas.
3. Hypothyroidism (Myxoedema, Cretinism)
4. Hypopituitarism (eg: Simmonds disease)
5. Hypoadrenalism (Addison’s disease)
6. In Children – Glycogen storage diseases
Eg: Von Gierke’s disease.
OTHER METHODS USED FOR ESTIMATION OF BLOOD GLUCOSE

CHEMICAL METHODS ENZYMATIC METHODS

1. Folin – Wu method 1. Glucose Oxidase (GOD) & Peroxidase

(POD) method

2. Nelson-Somagyi method 2. Hexokinase method.

Disadvantages of Chemical Methods:

1. Nonreducing substances present in blood are also measured they are,
a) Uric acid
b) Glutathione
c) Vitamin-C
2. O-Toluidine is carcinogenic.
3. Time taking.
4. So, in older methods we used to get high values for blood glucose.
 ESTIMATION OF BLOOD/SERUM UREA
By Diacetyl Monoxime (DAM-TSC) Method

Aim:
To estimate the amount of urea present in the given blood/serum sample.

Principle:
Urea present in blood sample react with diacetyl monoxime under strong
acidic medium in presence of catalyst ferric chloride & Thiosemicarbazide
(TSC). Diacetyl monoxime decomposes into hydroxylamine and diacetyl.
Diacetyl condenses with urea to give pink coloured complex (Diazine).
Serum can be used directly instead of blood.

MECHANISM OF REACTION:
When urea is treated with diacetyl (compound containing two adjacent
carboxyl groups) in acidic medium coloured products are formed

1. Decomposition of diacetyl monoxime:

DAM decomposes on heating into diacetyl and hydroxylamine

CH3CO CONH CH3 + H2O CH3COCOH3 + NH2OH

DAM Diacetyl hydroxylamine

2. This diacetyl reacts with urea in acidic medium to form a pink colored
complex called DIAZINE.
CH3 – CO – CO – CH3 + NH2 CO NH2 H3C – C – C -CH3
Diacetyl urea N N
C

Diazine (Pink Colored)

Procedure:
Take 3 test tubes and label them as B (Blank), S (Standard) & T (Test)

Reagents Blank (B) Standard (S) Test (T)

Distilled Water
20 µL - -

Standard Urea
- 20 µL -
(50mg/dl)

Serum
- - 20 µL

2.0 ml 2.0 ml 2.0 ml

DAM-TSC

2.0 ml 2.0 ml 2.0 ml

Acid Reagent

Mix well. Keep the tubes in boiling water bath for 15 minutes. Cool and
then read the absorbance of B, S & T at 530nm.

Optical Density (O.D)

Calculation:
Mg of urea in 100ml of blood =

OD of T - OD of B 100
Urea Conc. (mg/dl) = x Conc. of Std x
OD of S - OD of B Vol. of sample

OD of T - OD of B 100
= x 0.01 x
OD of S - OD of B 0.02

T
= x 50
S

= ________ mg/dl

RESULT:
The amount of urea in the given sample of blood found by Diacetyl
monoxime method is _________ mg/dl.

Normal Values:-
Blood Urea - 15 - 40 mg/dl

CLINICAL SIGNIFICANCE:
Normal blood urea level is 15 to 40 mg / dl.

A. HYPOUREMIA:
Low blood urea levels are seen under the following conditions.
Low protein diet,
Increased protein catabolism,
Malabsorption of proteins (sprue),
 Severe Liver disease,
Late pregnancy,
Longterm replacement of blood with dextrose

B. UREMIA (Increased urea)

High blood urea levels-uremia are seen under the following conditions.
1. Pre – renal condition:
Dehydration due to severe vomiting & diarrhea,
Bleeding in GIT,
Increased protein breakdown,
Cardiac Failure
2. Renal conditions:
Acute and chronic Glomerulonephritis, Nephrotic syndrome,
Hydronephrosis, Congenital Cystic Kidneys,
Malignant Hypertension,
Renal Tuberculosis,
Calcium deposits as in Hyperparathyroidism & Hyper Vitaminosis D,
Stones and Tumors in kidneys.
3. Post renal conditions:
Stones in the urinary tract,
Tumors of urinary bladder,
Enlargement of prostate (in men)
Urinary Urea:
Normal daily urea excretion in urine is 15 – 30 gm/ day.
Decreased level of urea in urine is found in - - Acute Renal impairment.
Increased urea in urine is found in pre Renal Causes eg: Massive GI
Bleeding. Urinary urea excretion is helpful in management of cases of Acute
Renal Failure and in assessing. The ability of transplanted kidney to handle
an increasing protein intake.

OTHER METHODS USED FOR ESTIMATION OF BLOOD UREA

1. Nesslerisation Method 2. Berthelot Method
3. Urease Method 4. Stick Test
 ESTIMATION OF SERUM TOTAL PROTEINS
By Henry Method (Using Biuret Principle)

Aim:
To estimate the amount of total proteins present in the given serum
sample.

Principle:
The Peptide bonds in the proteins form a violet colored complex when they
react with cupric ions in alkaline conditions. The intensity of violet color
(O.D) is directly proportional to the amount of proteins present in the given
serum sample.

Procedure:
Take 3 test tubes and label them as B (Blank), S (Standard) & T (Test).

Reagents in ml Blank (B) Standard (S) Test (T)

Distilled Water 0.05 ml - -

Standard protein (6gm/dl)

- 0.05 ml -

Serum
- - 0.05 ml

3% Sodium Hydroxide
5.0 ml 5.0 ml 5.0 ml

Benedict’s Qualitative
1.0 ml 1.0 ml 1.0 ml
Reagent
Mix and allow to stand for 10 minutes. Read the absorbance of B, S & T at
540nm.

Optical Density (O.D)

Calculation:
Total protein in grams/ 100ml of blood =

OD of T - OD of B 100
Total Protein Conc.(mg/dl) = x Conc. of Std x
OD of S - OD of B Vol. of sample

OD of T - OD of B 100
= x 0.003 x
OD of S - OD of B 0.05

T
= x 6
S

= ________ gm/dl

Result:
The amount of total protein in the given sample of serum found by Henry
method is _________ gm/dl.

Normal Values:
Serum Total Protein - 6 - 8 gm/dl
CLINICAL SIGNIFICANCE:-
Total protein concentration in serum is in between 6 to 8 gm /dl in normal
subjects.
The albumin – globulin ratio is close to 2:1 by this method.

HYPERPROTEINEMIA
Increase in total serum proteins may occur in dehydration and diarrhoea
with the ration remaining unaltered.
There are several conditions where increase in total proteins is mainly due
to increase of one of the globulins. In many of these conditions, albumin
level remains same or is reduced slightly. Multiple myeloma is a typical
example where total protein is increased with normal or decreased
albumin.

HYPOPROTEINEMIA
Decrease in total proteins is invariably due to a fall in albumin level which
may be accompanied by; an increase in globulin concentration. The ratio is
decreased in these cases.

Hypoalbuminemia can be due to either loss of albumin in kidney diseases,

impaired synthesis in some liver diseases, inadequate supply of dietary
protein or excessive protein catabolism.
Albumin is lost in urine in nephritis, nephrosis and nephritic syndrome.
Large amount of albumin is also lost in severe burns and hemorrhage. In all
types of chronic liver diseases, particularly cirrhosis.
Excessive breakdown of body proteins together with inadequate supply or
defective utilization of proteins is seen in uncontrolled diabetes,
thyrotoxicosis and trauma.
Decrease in plasma protein levels is seen in certain disorders like
protein calorie malnutrition (kwashiorkor & marasmus),
sprue and
malignant disorders.
 ESTIMATION OF SERUM CREATININE
By Jaffe’s Method

Aim:
To estimate the Serum Creatinine by Jaffe’s Method.

Reagents: 1. Picric acid

2. NaoH
3. Stock Standard solution of creatinine (1mg/1ml)
4.Working standard solution (prepared by diluting 1ml of
stock standard solution => 1mg/100ml)
5. Sodium Tung state
6. 2/3N H2SO4

Principle: (Jaffe’s Principle)

Creatinine reacts with picric acid in alkaline medium to give creatinine
picraate at room temperature. The solution obtained is orange coloured
compound (Creatinine picrate) which is seen within 15 minutes and the
colour is measured in colorimeter at 520 nm

Procedure:
Part-1.
Preparation of protein free filtrate(PPF): Dilute 2cc of serum with 2cc of
distilled water and precipitate proteins by adding 2cc of 5% Sodium Tung
state and 2cc of 2/3N H2SO4 (total volume 8cc) mix and keep 10 minutes
and filter in a dry test tube to obtain a clear solution of ppf and take 3ml.
Part-2
Take 3 test tubes and label them as B (Blank), S (Standard) & T (Test)
Reagents in ml Blank (B) Standard (S) Test (T)

Distilled Water 3.0 ml 1.5 ml -

Standard solution (1mg/dl) - 1.5 ml -

PPF(protein free filtrate) - - 3.0 ml

Saturated Picric acid 1.0 ml 1.0 ml 1.0 ml

0.75 N NaOH 1.0 ml 1.0 ml 1.0 ml

Mix well & allow it to stand for 15 min at room temperature. Read the
absorbance of B, S & T at 520 nm.

Optical Density (O.D)

Calculation:
OD of T - OD of B Conc. of Std
Creatinine Conc. (mg/dl) = x x 100
OD of S - OD of B Vol. of sample

OD of T - OD of B 0.015
= x x 100
OD of S - OD of B 0.75

T
= x 2
S

= ________ mg/dl

Report: The given sample contain ________ mg/dl of serum creatinine

Normal range : 0.4-1.2 mg/dl

Clinical significance: the value may increase during kidney diseases like
acute or chronic insufficiency, urinary track obstruction and impairment of
renal functions induced by some drugs, nephritis, early stage of muscle
wasting diseases
 ESTIMATION OF URINE CREATININE
By Jaffe’s Alkaline Picrate Method

Aim:
To estimate the concentration of Creatinine in a given sample of urine by
Jaffe’s Method.

Principle:
Creatinine reacts with picric acid in alkaline medium to form a orange
coloured compound (Creatinine picrate). The colour is measured in
colorimeter.

Procedure:
Dilute 5 ml of urine to 500 ml in volumetric flask. So 1 ml of diluted urine
contains 0.01 ml of urine.

Take 3 test tubes and label them as B (Blank), S (Standard) & T (Test)
Reagents in ml Blank (B) Standard (S) Test (T)

Distilled Water
3.0 ml - -

Standard Creatinine
- 3.0 ml -
(1mg/dl)

- - 3.0 ml
Urine (1:100 diluted)

1.0 ml 1.0 ml 1.0 ml

Saturated Picric acid

1.0 ml 1.0 ml 1.0 ml

0.75 N NaOH
Mix well & allow it to stand for 15 min at room temperature. Read the
absorbance of B, S & T at 540 nm.

Optical Density (O.D)

Calculation:
Creatinine in grams / 1 Lt of urine =

OD of T - OD of B 1
Creatinine Conc. (mg/dl) = x Conc. of Std x
OD of S - OD of B Vol. of sample

OD of T - OD of B 1
= x 0.03 x
OD of S - OD of B 0.03

T
= x 1
S

= ________ mg/dl

If 1500 ml is the day output of urine then the amount of creatinine

excreted in a day is equal to
Creatinine in 100ml of urine = X

In 1500ml of urine = ? 1500 x X mg/day

100

Result:
The amount of Creatinine excreted per one day is equal to
________gm/day
Normal range:
Urine Creatinine:
Men : 1-2 gm/day
Women : 1-1.8 gm/day

Serum Creatinine : 0.4-1.2 mg/dl

Creatinine clearance:
It is the volume of blood plasma that is cleared of creatinine per unit time
(a minute) and is a useful measure for the estimation of GFR
(approximately).

Creatinine clearance = UV/B(P)

where U is concentration of creatinine in urine (mg/dl)

V is volume of urine excreted per minute (ml/min)
B(P) is concentration of creatinine in blood/plasma (mg/dl)

Normal range:
Men : 95 - 140 ml/min
Women : 85 - 125 ml/min

Urine clearance values are expressed as --- ml/min

Other types of clearances are inulin clearance and urea clearance.

Creatinine clearance is the best marker for assessing renal function (RFT).
CLINICAL SIGNIFICANCE:-
Creatinine is an endogenous non-protein nitrogenous waste product
formed in muscle from high energy compound Creatine phosphate.
Creatinine is the anhydride of Creatine formed by non-enzymatic reaction.

Creatinine is synthesized from 3 amino acids. they are,

1. Methionine
2. Glycine
3. Arginine

The normal daily excretion of Creatinine ranges from 1-2 gm. This is little
influenced by the diet, age, sex and exercise. As the amount of creatinine
depends on muscle tissue, its excretion in urine normally remains constant
in an individual, for which reason it can be used to check the reliability of 24
hour urine collection.

Urine Creatinine is increased in the following conditions.

A. Muscular Dystrophy
B. Thyrotoxicosis
C. Uncontrolled Diabetes Mellitus

Urine Creatinine value is decreased in Acute/chronic Renal Failure.

Creatinine is removed from plasma by glomerular filtration and then is

excreted.
Elevated levels of serum creatinine is seen in renal failure.
 ESTIMATION OF CSF PROTEINS
By Sulphosalicylic Acid Method

CSF is a clear, colorless fluid that contains small quantities of glucose and
protein. The biochemical testing of cerebrospinal fluid (C.S.F) is performed
to assist in the diagnosis of meningitis and other disorders of the central
nervous system.

Aim:
To estimate the amount of total proteins present in the given CSF sample.
Principle:
The proteins present in CSF are precipitated by sulphosalicylic acid reagent.
The resulting turbidity of CSF sample is compared with turbidity of standard
using colorimeter at 540nm.

Procedure:
Take 3 test tubes and label them as B (Blank), S (Standard) & T (Test)
Reagents in ml Blank (B) Standard (S) Test (T)

Distilled Water
0.5 ml - -

Standard
- 0.5 ml -
(100mg/dl)

- - 0.5 ml
CSF

3.5 ml 3.5 ml 3.5 ml

3% Sulphosalicylic acid
Mix well & stand for 10 minutes. Read the absorbance of B, S & T at 540nm.

Optical Density (O.D)

Calculation:
Mg of total proteins per 100 ml C.S.F =

OD of T - OD of B 100
Total Protein conc. (mg/dl) = x Conc. of Std x
OD of S - OD of B Vol. of sample

OD of T - OD of B 100
= x 0.5 x
OD of S - OD of B 0.5

T
= x 100
S

= ________ mg/dl

RESULT:
The amount of total proteins in the given CSF sample found by
Sulphosalicylic acid method is _________ mg/dl.

Normal Values:
CSF Total Proteins - 15-45 mg/dl
CLINICAL SIGNIFICANCE:
The C.S.F total protein is normally 15-40 mg/dl (0.15 – 0.40 gm/L)

1. CSF is collected by lumbar puncture (LP)

2. It is used to diagnose inflammation, infection and tumours in several
groups of nerve cells.
3. An increase in total protein in CSF is most common abnormality found.
4. This results from breakdown of blood brain barrier, usually due to
inflammatory reaction.
5. The CSF total protein is commonly increased in:
Pyogenic meningitis
Viral meningitis
Encephalitis
Polyneuritis
Acoustic neuroma & meningioma
6. Very high values are occasionally seen in spinal tumours in which there is
a complete block.
7. When the total protein exceeds 200 mg/dl (200 mg %), the fibrinogen
level is usually increased & is sufficient enough for the C.S.F to clot.
 ESTIMATION OF CSF GLUCOSE
By Orthotoluidine Method

Aim:
To estimate the amount of glucose present in the given sample of CSF.

Principle:
0-Toludine condenses with glucose in the presence of glacial acetic to
produce green color and the color produced is proportionate to the glucose
concentration and is measured at 630 nm (or) Red filter.

Procedure:
Take 3 test tubes and label them as B (Blank), S (Standard) & T (Test)

Reagents in ml Blank (B) Standard (S) Test (T)

Distilled Water
0.1 ml - -

Standard
- 0.1 ml -
(100 mg%)

- - 0.1 ml
CSF

3.0 ml 3.0 ml 3.0 ml

O-Toluidine
Keep the tubes in boiling water bath for 10 minutes. Cool and then read the
absorbance of B, S & T at 630nm.

Optical Density (O.D)

Calculation:
Mg of glucose in 100ml of CSF =

OD of T - OD of B 100
Glucose Conc. (mg/dl) = x Conc. of Std x
OD of S - OD of B Vol. of sample

OD of T - OD of B 100
= x 0.1 x
OD of S - OD of B 0.1

T
= x 100
S

= ________ mg/dl

RESULT:
The amount of glucose in the given sample of CSF found by Orthotoluidine
method is _________ mg/dl.

Normal Values:-
CSF Glucose : 50 – 70 mg/dl
CLINICAL SIGNIFICANCE:
Normal C.S.F. glucose level is 50-70 mg./dl.

CSF glucose is always slightly lower than blood glucose.

1. A raised C.S.F. glucose is found when there is a raised blood glucose.

o Diabetic hyperglycemia
o Following damages to cerebral capillaries.

2. Decrease in CSF glucose is characteristics of infection, most forms of

meningitis,
o Pyogenic meningitis
o Tuberculous meningitis
o
The glucose concentration falls to 0-20 mg % in pyogenic meninigitis while
it falls but will be above 20 mg % in tuberculous meningitis.

3. In viral meningitis however, the C.S.F. glucose is often normal.

CSF analysis in different conditions:

1. Pyogenic meningitis or acute bacterial meningitis

Protein - very much increased (100-400mg/dl)

Glucose - very much decreased or absent

Chloride - may fall (110-115mmol/lt)

Cell count - increases (Polymorphs)

2. Acute viral meningitis

Protein - slightly increased (50-100mg/dl)

Glucose - Normal

Chloride - Normal

Cell count - Lymphocytosis

3. TB Meningitis

Protein - moderately increased (50-200mg/dl)

Glucose - moderately decreased (<50mg/dl)

Cell count - Mixed cellular response

4. Poliomyelitis

Protein - moderately increased (100-200mg/dl)

Glucose - Normal

Chloride - Normal

5. Guillain Barre Syndrome

Protein - very much increased (200-400mg/dl)

Glucose - Normal

Chloride - Normal
 ESTIMATION OF SERUM CHOLESTEROL
Enzymatic Coupled Trinder’s Method

Aim:
To Estimate the amount of Cholesterol present in the given serum/Plasma

Principle:

Cholesteryl ester hydrolase hydrolyses the fatty acid residue from

cholesteryl esters, converting them to free cholesterol. The free cholesterol is
reacted by the second enzyme, cholesterol oxidase, producing hydrogen
peroxide. Peroxidase enzyme acts on this hydrogen peroxide to produce
nascent oxygen.This acts on PAP (Paraaminoanti pyrine) to produce pink
color.this can be measured in colorimeter at 540 nm. Cholesterol can be
estimated

Cholesterol esterase
Cholesterol ester Cholesterol + Fatty acid
Cholesterol oxidase (CHOD)
Cholesterol O2 Cholest-4-en-3one +H2O2

Peroxidase (POD)
2H2O2 + 4AAP + Phenol Quinoneimine dye +H2O
Procedure:

Take 3 test tubes and label them as B (Blank), S (Standard) & T (Test)
Reagents in ml
Blank (B) Standard (S) Test (T)

Distilled water 10µl - -

Standard(200mg/dl) - 10µl -

Serum/Plasma - - 10µl

Cholesterol Reagent 1ml 1ml 1ml

Mix, incubate for 5 minutes at 370C. Read absorbance of standard and

samples against reagent blank at 540 nm (Green filter).

Calculation

Cholesterol conc. (mg/dl) = OD of Test  Conc. of standard (mg/dL)

OD of standard
Result
The amount of total Cholesterol in the given sample found by Enzymatic
Coupled Trinder’s method is _________ mg/dl.

Normal Values

Total cholesterol : 150 -200 mg/dl

CLINICAL SIGNIFICANCE
`Hypercholesteremia: - Elevated levels of cholesterol are observed in
 Atherosclerosis
 Nephrotic Syndrome
 Diabetes Mellitus
 Biliary obstruction
 Hypothyroidism
 Some hyperlipoproteinemias,administration of insulin –
thyroid hormones, high blood pressure,smoking,abdominal
obesity ,lack of exercise .these factors which increases free
fatty acids like diet,stress,coffee,various diseases .

Hypocholesteremia: Decreased levels of cholesterol are observed in

 Hypothyroidism
 Hepatitis
 Cirrhosis of liver
 Malabsorption
 Anemia
 Hemolytic jaundice

OTHER METHODS

Enzymatic methods:

 Liebermann –Buchard method

 Modified Liebermann –Buchard method for free and esterified
cholesterol method
 ESTIMATION OF SERUM HDL CHOLESTEROL
Phosphotungistic acid/Precipitation/colorimetric method

Aim

To Estimate the amount of HDL cholesterol present in the given serum/Plasma

Principle

In Presence of Phosphotungistic acid and magnesium chloride LDL, VLDL and

chylomicrons are precipitated on centrifuge HDL is collected as supernatant and
cholesterol content can be detected.

Procedure

Precipitation of LDL,VLDL and chylomicrons

Pipette Volumes

Test 250 µl

Precipitating Reagent 500 µl

Take 3 test tubes and label them as B (Blank), S (Standard) & T (Test)
Reagents in ml
Blank (B) Standard (S) Test (T)

Distilled water 10µl - -

Standard(mg/dl) - 10µl -

Serum/Plasma - - 10µl

Cholesterol working
1ml 1ml 1ml
Reagent

Supernatant - - 50 µl

Mix, incubate for 10 minutes at 370C. Read absorbance of standard and

samples against reagent blank at 540 nm (Green filter).
Calculation

HDL Cholesterol (mg/dL): OD of Test  Conc. of standard (mg/dL)

OD of standard

Result

The amount of HDL Cholesterol in the given sample found by

Phosphotungstic acid method is _________ mg/dl.

Normal Range
Males: > 40 mg/dL
Females: > 50 mg/dL

OTHER METHODS

Dextran sulphate Method

LDL-CHOLESTEROL

low density lipoprotein (LDL) cholesterol was estimated by simple calculation using

Friedewald formula:

VLDL Cholesterol = Triglycerides

5

LDL Cholesterol = Total cholesterol – [VLDL + HDL]

Normal Range: VLDL: 35-40 mg/dL

LDL: < 130 mg/dL

 ESTIMATION OF SERUM TRIGLYCERIDES
Enzymatic Coupled Trinder’s method
Aim:
To Estimate the amount of Triglycerides present in the given serum/Plasma
Principle:
This procedure involves hydrolysis of triglycerides by lipase is converted by glycerol
kinase to glycerol 3-Phosphate, again oxidised by glycerol Phosphate oxidase to
dihydroxy acetone phosphate and hydrogen peroxide in presence of peroxidase
aminoantipyrine finally produce a quinoneimine, whose concentration is proportional to
the triglyceride concentration.

Lipase
Triglycerides + H2O glycerol + fatty acids

Glycerol kinase
Glycerol + ATP glycerol-3-P + ADP

G3P-oxidase
Glycerol-3-P +O2 dihydroxyacetone –P + H2O2

Peroxidase
2H2O2 + 4-aminoantipyrine + 4-chlorophenol quinoneimine + 4
H2O

Procedure: Take 3 test tubes and label them as B (Blank), S (Standard) & T (Test)

Reagents in ml
Blank (B) Standard (S) Test (T)

Distilled water 10µl - -

Standard(200mg/dl) - 10µl -

Serum/Plasma - - 10µl

Triglycerides Reagent 1ml 1ml 1ml

Mix, incubate for 5 minutes at 370C. Read absorbance of standard and samples against
reagent blank at 540 nm (Green filter).

Calculation

Triglycerides (mg/dL): OD of Test  Conc. of standard (mg/dL)

OD of standard

RESULT:
The amount of triglyceride in the given sample found by Enzymatic Coupled Trinder’s
method is _________ mg/dl.

Normal Range: <200 mg/dL

CLINICAL SIGNIFICANCE
Liver is the major source of plasma lipoprotein metabolism

Hypertriglyceridemia: Primary hypertriglyceridemia

Secondary hypertriglyceridemia

Hypertriglyceridemia: - Elevated levels of triglycerides are observed in

 Diabetes Mellitus
 Nephrotic Syndrome
 Excessive alcohol intake
Decreased levels of triglycerides are observed in

 Congenital β –lipoprotenemia
 Malnutrition
Other Methods:

 Pyruvate kinase method

 Glycerol Phosphate oxidase Method
 Glycerol Phosphate Dehydrogenase Method
 ESTIMATION OF SERUM TOTAL BILIRUBIN
Malloy and Evelyn method
Aim:
To Estimate the amount of bilirubin present in the given serum
Principle:
Bilirubin reacts with diazotized sulphanilic acid to form purple colored azobilirubin. The
water soluble bilirubin glucuronides (conjugated bilirubin) reacts fast with diazo reagent.
The free bilirubin (unconjugated bilirubin) which is present in serum complexed with
albuminreacts very slowly and requires an accelerator (indirect reaction)
Procedure:

Take 2 test tubes and label them as B (Blank),) & T (Test)

Reagents in ml Blank (B) Test (T)

Distilled water 1.8 ml 1.8 ml

Serum/Plasma 0.2 ml 0.2 ml

Diazo Reagent - 0.5 ml

Diazo blank 0.5 ml -

Accelerator (methanol) 2.5 ml 2.5 ml

Mix, incubate for 30 minutes at 370C. Read absorbance of blank and test at 540 nm
(Green filter).

Calculation

Total Bilirubin (mg/dL) = OD of Test  Conc. of standard (mg/dL)

OD of standard

RESULT:
The amount of Bilirubin in the given sample found by Malloy and Evelyn method
method is _________ mg/dl.
Normal Range

Total bilirubin : 0.2 – 1.2 mg/dl

Direct bilirubin : 0.1 – 0.4 mg/dl

CLINICAL SIGNIFICANCE
Jaundice is a condition presenting with yellowish discoloration of skin, scelera and
mucous membrane due to increase in concentration of bilirubin circulating in blood.

Types of jaundice
1. Hemolytic – malaria, hemolytic diseases
2. Hepatic - Hepatitis A
3. Obstructive – Stones and tumours in biliary tract
 ESTIMATION OF ASPARTATE TRANSAMINASE (AST/SGOT)
By IFCC Kinetic Method

Aim:
To estimate the amount of AST present in the given serum sample.

Principle:
Aspartate amino transferase catalyses the transamination of L-Aspartate
and Alpha ketoglutarate to L-Glutamate and oxaloacetate in subsequent
reaction malate dehydrogenase reduces oxaloacetate to malate along with
simultaneous oxidation of NADH. The reaction of oxidation of NADH
measured by monitoring decrease in absorbance 340nm and is directly
proportional to aspartate transaminase activity in sample.

Alpha keto glutarate + L-Aspartate AST L-Glutamine + Oxaloacetate

Oxaloacetate + NADH malate dehydrogenase Malate + NAD+

Procedure:
Take 3 test tubes and label them as B (Blank), S (Standard) & T (Test).

Reagents in ml Blank (B) Standard (S) Test (T)

Distilled Water 50 µl - -

Standard SGOT - 50 µl -

Serum - - 50 µl
SGOT Reagent 0.5 ml 0.5 ml 0.5 ml
Mix and incubate for 60seconds at 37oc. Read the first reading of
absorbance of B, S & T at 340nm. Perform other 2 reading at 60 sec
intervals. Calculate ΔA/min

Calculation:

SGOT U/ I in blood = ΔA/min X 1768

Result:
The amount of SGOT in the given sample of serum found by IFCC kinetic
method is _________ U/I.

Normal Values:
SGOT - < 45 U/I

CLINICAL SIGNIFICANCE:-
Elevated AST/SGOT levels are observed in
viral hepatitis
Liver cirrhosis and necrosis
Pulmonary embolism,
acute nephritis,
gangrene,
hemolytic diseases,
myocardial infarction

OTHER METHODS USED FOR ESTIMATION OF SGOT

1.Dinitrophenyl hydrazone method
2. Enzymatic Leucocytic Method
 ESTIMATION OF ALANINE TRANSAMINASE (ALT/SGPT)
By IFCC Kinetic Method

Aim:
To estimate the amount of AST present in the given serum sample.

Principle:
Aspartate amino transferase catalyses the transamination of L-Aspartate
and Alpha ketoglutarate to L-Glutamate and oxaloacetate in subsequent
reaction malate dehydrogenase reduces oxaloacetate to malate along with
simultaneous oxidation of NADH. The reaction of oxidation of NADH
measured by monitoring decrease in absorbance 340nm and is directly
proportional to aspartate transaminase activity in sample.

Alpha keto glutarate + L-Alanine ALT L-Glutamate + Pyruvate

Pyruvate + NADH lactate dehydrogenase L-Lactate + NAD+

Procedure:
Take 3 test tubes and label them as B (Blank), S (Standard) & T (Test).

Reagents in ml Blank (B) Standard (S) Test (T)

Distilled Water 50 µl - -

Standard SGPT - 50 µl -

Serum - - 50 µl
SGPT Reagent 0.5 ml 0.5 ml 0.5 ml
Mix and incubate for 60 seconds at 37oc. Read the first reading of
absorbance of B, S & T at 340nm. Perform other 2 reading at 60 sec
intervals. Calculate ΔA/min.

Calculation:
SGPT U/ I in blood = ΔA/min X 1768

Result:
The amount of SGPT in the given sample of serum found by IFCC kinetic
method is _________ U/I.

Normal Values:
SGPT - < 45 U/I

CLINICAL SIGNIFICANCE:-
Elevated ALT/SGPT levels are observed in
viral hepatitis
toxic hepatitis
Alcoholic hepatitis
infectious mononucleosis
Poliomyelitis
Leukemia and heparij therapy in children

Decreased ALT/SGPT levels seen in

End stage liver disease
Renal insufficiency
 ESTIMATION OF ALKALINE PHOSPHATASE (ALP)
By IFCC Kinetic Method

Aim:
To estimate the amount of Alkaline phosphatase present in the given
serum sample.

Principle:
The enzyme alkaline phosphatase hydrolizes the p-
nitrophenylophosphate to release 4 nitrophenol, under alkaline
conditions. The nitrophenol formed is detected spectrophotometrically
at 405nm to give a measurement of alkaline phosphate.

2-amino 2-methyl 1-propanol + p-nitrophenylophosphate + H2O

ALP 4 nitrophenol + 2-amino 2-methyl 1-propanol phosphate

Procedure:
Take 3 test tubes and label them as B (Blank), S (Standard) & T (Test).

Reagents in ml Blank (B) Standard (S) Test (T)

Distilled Water 20 µl - -

Standard ALP - 20 µl -

Serum - - 20 µl
ALP Reagent 1.0 ml 1.0 ml 1.0 ml
Mix and incubate for 60 seconds at 37oc. Read the first reading of
absorbance of B, S & T at 405nm. Perform other 2 reading at 60 sec
intervals. Calculate ΔA/min.

Calculation:
ALP U/ I in Serum ΔA/min X 2764

Result:
The amount of ALP in the given sample of serum found by IFCC kinetic
method is _________ U/I.

Normal Values:
Males - 41 -137 U/I
Females - 39 -118 U/I

CLINICAL SIGNIFICANCE
ALP is present in liver, bone, intestine & placenta

Elevated ALP levels is observed in

Hepatobiliary disease
Bone disease
Pregnant women & growing children
 ESTIMATION OF TOTAL SERUM CALCIUM
By O-Cresolphthalein complexone Method

Aim:
To estimate the amount of Calcium present in the given serum sample.

Principle:
In alkaline pH, O-cresolphhthalein complexone (CPC) forms a complex
(purple color) with calcium. The intensity of the color is a measure of
concentration of calcium in the serum. The serum sample is diluted with
acid to release protein bound and anion bound calcium.

Procedure:

Reagents Blank (B) Standard (S) Test (T)

Colour reagent
1 ml 1 ml
1 ml

Distilled Water
20 ul -

Standard calcium
- 20 ul -
(2.5 mmol/L)

- - 20 ul
Serum
Mix well and keep for 5 minutes. Read the absorbance of B, S & T at 578nm
in a colorimeter using yellow filter.

Optical Density (O.D)

Calculation:
Mg of serum calcium in 100ml of blood =
OD of T - OD of B 100
Calcium Conc. (mg/dl) = x Conc. of Std x
OD of S - OD of B Vol. of sample

OD of T - OD of B 100
= x 0.00005 x
OD of S - OD of B 0.02

OD of T
= x 2.5
OD of S

= ________ mg/dl

Result:
The amount of calcium in the given sample of blood found by o-
Cresolphhthalein complexone method is _________ mg/dl.

Normal Values:
Total Calcium - 8.6-10.2 mg/dl
Free Calcium - 4.6-5.3 mg/dl
CLINICAL SIGNIFICANCE:

1. HYPERCALCEMIA (Increased Blood Calcium level) is seen in :

 Alkalosis: At higher pH of alkalosis, most of the proteins
acquire more negative charge which causes binding of more
free calcium to proteins resulting in lowering of functional
free calcium. Here most often total calcium remains normal
but the free calcium is decreased.
 Hypoparathyroidism: It occurs more commonly after
thyroidectomy due to the removal of a parathyroid tissue
along with thyroid gland.
 Rickets: Due to defective calcium absorption. Associated
finding in rickets is low phosphorous levels.

2. HYPOCALCEMIA (Reduced Blood Calcium level) is seen in

i. Primary hyperparathyroidism.
ii. Malignancies
iii. Hyperthyroidism
iv. Acute adrenal insufficiency
v. Renal failure
vi. Increased serum proteins.
 ESTIMATION OF TOTAL SERUM PHOSPHORUS
By Fiske and Subbarow Method

Aim:
To estimate the amount of Phosphorus present in the given serum sample.

Principle:
Serum is treated with trichloracetic acid to get protein free filtrate. Protein
Free filtrate is then treated with acid ammonium molybdate to form
phoshomolybdic acid. The hexavalent molybdenum of phosphomolybdic
acid is reduced by 1,2,4 amino-naphthol-sulphonic acid (ANSA) to give a
blue compound, absorbance of read at 680 nm in a spectrophotometer or
using red filter in a colorimeter.

Procedure:
Reagents Blank (B) Standard (S) Test (T)

Distilled Water 5 ml - -

Standard
Phosphorus
- 5 ml -
(0.04 mg)

Protein free
filtrate(PFF) - - 5 ml

Molybdate II 1 ml - 1 ml
Molybdate I - 1 ml -

ANSA 0.4 ml 0.4 ml 0.4 ml

Make up to 10 ml with distilled water

Mix well and keep for 5 minutes. Read the absorbance of B, S & T at 680 nm
in a colorimeter using red filter.

Optical Density (O.D)

Calculation:
Mg of phosphorus in 100ml of blood =

OD of T - OD of B 100
Phos Conc. (mg/dl) = x Conc. of Std x
OD of S - OD of B Vol. of sample

OD of T - OD of B 100
= x 0.04 x
OD of S - OD of B 1

OD of T
= x 4
OD of S

= ________ mg/dl
Result:
The amount of glucose in the given sample of blood found by Fiske and
Subbarow method is _________ mg/dl.

Normal Values: reference ranges of serum inorganic phosphate

Adults - 2.5 - 4.5 mg/dL
Children - 4.0 - 7.0 mg/Dl

CLINICAL SIGNIFICANCE:
1. HYPOPHOSPHATEMIA :
The term hypophosphatemia is used when the serum inorganic
phosphate concentration is less than 2.5 mg%. Clinical
manifestations depend on duration and extent of the deficiency.
Since phosphate is a component of energy currency of the –ATP,
cellular functions are impaired in hypophosphatemia.
It leads to muscle weakness, respiratory failure decreased cardiac
output. At very low concentrations like, below 0.5
mg%,rhabdomyolysis, hemolysis, mental confusion and even coma
may occur. Chronic hypophisphatemia causes rickets in children
and osteomalacia in adults.

Causes:
 Oral or intravenous hyperalimentation and insulin:
Carbohydrates induce insulin secretion which enhances
transport of phosphate from extracellular fluid into the cells
leading to a fall in serum phosphate.
 Respiratory alkalosis: Promote an intracellular shift of phosphate
from extracellular fluid leading to a fall in its level in the serum.
  Lowered rental threshold for phosphate
- Primary and secondary hyperparathyroidism
- Fanconi’s syndrome
- X linked hypophosphatemia.
 Intestinal loss
- Malabsorption
- Ingestion of antacid containing aluminium and magnesium
which bind with phosphate making it unsuitable for
absorption.
 Acidosis, e.g. ketoacidosis, lactic acidosis. Acidosis leads to
catabolism of organic phosphates to form inorganic phosphates
which then pass into plasma and excreted in urine leading to
depletion of intracellular phosphates.

2. HYPERPHOSPHATEMIA:
 Renal failure: A decrease in GFR decreases phosphate excretion in
urine leading to hyperphosphatemia.
 Hypoparathyroidisim and acromegaly: Enhance tubular
reabsorption of phosphates.