CONTENTS

PRACTICALS IN BIOCHEMISTRY 40 HRS

A) QUALITATIVE
1) Reactions of Carbohydrates N0. Of. Practicals

a) REACTIONS OF MONOSACCHARIDES(Glucose and Fructose) 1

b) REACTIONS OF DISACCHARIDES(Maltose, Lactose and Sucrose) 1

c) IDENTIFICATION OF UNKNOWN CARBOHYDRATES 2

2) Reactions of Proteins
a) PRECIPITATION REACTIONS OF PROTEINS 1

b) GENERAL COLOR REACTIONS OF PROTEINS 1

c) REACTIONS OF ALBUMINS 1

d) REACTIONS OF GELATIN AND PEPTONES 1

e) IDENTIFICATION OF UNKNOWN PROTEIN 2

3) Normal constituents of urine (NPN substances) 2

4) Identification of unknown physiological substance 2

5) Abnormal constituents of urine 2

6) Identification of unknown abnormal constituents

of urine 2

B) QUANTITATIVE

1) ESTIMATION OF BLOOD GLUCOSE BY GOD-POD METHOD 1

2) ESTIMATION OF BLOOD UREA 1

3) ESTIMATIONS OF SERUM CREATININE BY JAFFES’S METHOD 1

5) ESTIMATION OF SERUM TOTAL PROTEINS BY 1

BIURET METHOD

5) CSF ANALYSIS 1
a) Proteins i) Sulphosalicylic acid test ii) Pandy’s test
b) Glucose
C) DEMONSTRATIONS 10 Hrs.

1. Chromatography
2. Colorimeter
3. Centrifuge
4. Flame Photometer
5. Urinometer
6. pH meter
7. Electrophoresis
8. GTT
9. S. Uric acid estimation
10. S. Bilirubin estimation

D) SPOTTERS

Revision and conduct of Tests = 10 Practicals 20 Hrs.

Tutorials and group discussions = 25 Practicals 50 Hrs

Note: Each practical carries two hours.

 PART- I
QUALITATIVE
 Reactions of Carbohydrates
Introduction;
 Carbohydrates are hydrophilic substances with a potential free aldehyde or keto group and a
number of hydroxyl groups.
 Monosaccharides, the simple sugars in this series, have one sugar carbon chain.
 Glucose (an aldo hexose) & Fructose (a keto hexose) are the most common monosaccharides.
 Glucose is present in fruits, honey, sugarcane, milk and other biologically important
carbohydrates.
 Fructose is present in fruit juices & honey (richest source). It is also a constituent of cane sugar
and inulin.

GENERAL REACTIONS OF CARBOHYDRATES

EXPERIMENT OBSERVATION INFERENCE

1. Molisch’s Test:
To 2ml of sugar solution, add 2 drops of α–
naphthol& 2ml of conc. H2SO4 carefully
along the sides of the tube.
A purple colored ring is Indicates the presence of
formed at the junction of carbohydrates in the
the interface of the two given solution.
layers of the test tube.

Principle: Carbohydrates are dehydrated in the presence of conc. sulphuric acid to form furfural
derivatives (hydroxyl methyl furfural for hexoses and furfurals for pentoses). The ring is due to the
condensation product of furfural derivatives with α- naphthol.

2. Iodine Test:
To 2 ml of sugar solution add 2-3 drops of Blue color solution is Indicates the presence of
iodine solution. formed. starch in the given
solution.
Principle: Iodine forms a colored adsorption complex on the surface of the polysaccharides like starch,
dextrin and glycogen.

3. Benedict’s Test:
5 ml of Benedict’s reagent is taken in a Brick Red colored Indicates the presence of
testtube and boiled for 1 minute. Then add 8 precipitate is formed. reducing sugars in the
drops (0.5ml) of sugar solution & boil again given solution.
for 1 minute.
(Na2 Co3; CuSo4& Sodium citrate)

Principle: Carbohydrates with free aldehyde or keto groups in alkaline medium form enediols.
Enediols are powerful reducing agents which reduce cupric ions to cuprous ions. The red color is due to
the formation of cuprous oxide (Cu2O).

4. Barfoed’s Test:
To 3 ml of Barfoed’s reagent add 1 ml of
sugar solution & boil for 1 minute. Allow
the test tube to cool in the test tube stand.
(Copper acetate in glacial acetic Acid)
Scanty red scum is formed Indicates the presence of
at the bottom and sides of monosaccharaides in the
the tube. given solution.

Principle: Monosaccharides act as reducing agents in weak acidic conditions. Barfoed’s reagent is
weakly acidic and is only reduced by monosaccharides (cupric ions to cuprous oxide).
4. Foulger’s test:
To 3ml of Foulger’s reagent add 0.5 ml of Blue color is observed. Indicates the presence of
sugar solution & boil for 1 minute and ketose sugar in the given
allowed to cool. solution

(2% stannous chloride in 40%urea in 40%

H2SO4)
Principle: Keto hexoses on treatment with H2SO4 forms furfural derivatives. Blue color is due to the
condensation of furfural derivatives with urea in the presence of stannous chloride.

6. Seliwanoff’s Test:
To 3ml of seliwanoff’s reagent add 0.5 ml Cherry red color is formed. Indicates the presence of
of sugar solution & boil for 1 minute and ketose sugar in the given
allowed to cool. solution.

(Resorcinol in HCl)
Principle: Ketohexoses on treatment with HCl forms furfural derivatives. Cherry red color is due to the
condensation of furfural derivatives with resorcinol.

7. Rapid furfural test:

To 3ml of sugar solution add 2-3 drops of Deep purple color is Indicates the presence of
1% alcoholic α-naphthol and add 3ml of observed. ketose sugar in the given
concentrated HCl is added and boiled for solution.
few seconds and allowed to cool.

(alcoholic α-naphthol in HCl)

Principle: Keto hexoses on treatment with HCl forms furfural derivatives. Deep purple color is due to
the condensation of furfural derivatives with alcoholic α-naphthol.

8.Benedict’s test after acid

hydrolysis(Special test for sucrose):

To 5ml of sucrose solution, add 5 drops of A brick red precipitate is Indicates the presence of
conc. HCl. Boil for 2 min. Cool. Neutralize formed. reducing sugars after
with 20% Na2Co3 till the effervescence acid hydrolysis.
ceases.
Perform Benedict’s test with this
neutralized solution as follows: 0.5ml
Solution + 5ml Benedict’s reagent. Boil for
2 min over a low flame.

Principle: Sucrose gives a negative Benedict’s test as it is a non-reducing sugar. But when Benedict’s
test is repeated with the hydrolysate, after acid hydrolysis it gives a positive Benedict’s test, indicating
the hydrolysis of sucrose to its constituent monosaccharides glucose and fructose. Free glucose and
fructose are reducing in nature and thus give a positive Benedict’s reagent.
9.Osazone Test:
Take 1 spatula of phenyl hydrazine Needle shaped – Confirms the presence of
hydrochloride, 2 spatula of sodium Glucosazones/Fructosazones. respective sugars in the
acetate & 8 drops of glacial acetic given solution.
acid. Add 5 ml of sugar Solution. Sunflower shaped –
Mix, label &keep in boiling water Maltosazones.
bath for 20 minutes
(monosaccharides); 40 minutes
(disaccharides). Hedge – hog/ powder puff
Let it cool on the test tube rack. shaped – Lactosazones.
Place a drop of yellow crystal on a
clean glass slide. Cover it with
cover slip and examine under a
microscope.
Principle: Osazones are yellow crystalline compounds formed when a solution of sugar with a free
carbonyl group is heated with excess of phenyl hydrazine hydrochloride. Each sugar will give rise to
an osazone of a definite crystalline form and has its own melting point.
First, the two carbon atoms take part in the reaction. Phenyl hydrazine reacts with carbonyl group of
the sugar to form phenyl hydrazone, which then reacts with two further molecules of phenyl
hydrazine in which rearrangement between nitrogen and hydrogen molecules takes place and finally
results in the formation of osazones.
 Reactions of Disaccharides

Disaccharides are the carbohydrates, which on hydrolysis form two monosaccharide units. Maltose,
lactose and sucrose are the common disaccharides. The former two are reducing disaccharides, whereas
Sucrose is a non-reducing disaccharide.

Maltose:

 It is a disaccharide made up of two glucose units joined together by α-1, 4 glycosidic bonds.
 Since, one of the aldehyde groups remains free, it is a reducing sugar.
 It is also called Malt sugar and is seen in Malt, Germinating seeds & obtained on hydrolysis of
starch.

Lactose:

 It is also a reducing disaccharide. It is made up of a molecule of α-D-glucose united to β-D-

galactose by β-1, 4 glycosidic bonds.
 It is present in milk (Milk sugar) and can be hydrolysed to glucose & galactose by the enzyme
lactase present in intestinal juice.

Sucrose:

 It is a Non-reducing disaccharide. It is made up of one molecule of α-D-glucose and one

molecule of β-D fructose united by α, β-1, 2 glycosidic bond (between the aldehyde and keto
groups of glucose and fructose respectively).
 Hence there is no free functional group in sucrose and so it is a Non-reducing sugar. It does not
form osazones.
 It is also called Invert sugar & Table sugar.
 Sucrose occurs widely in nature as cane sugar, beet sugar & in several ripe fruits.
 Trehalose is also Non-reducing disaccharide formed of two glucose units joined by α-
1,1glycosidic bond. It is seen in hemolymph of some insects, fungi & yeasts.
 Perform the following tests with maltose, lactose and sucrose.
SCHEME FOR IDENTIFICATION OF UNKNOWN CARBOHYDRATE
UNKNOWN SOLUTION

MOLISCH’S TEST

Violet ring No Violet ring

(Presence of the Carbohydrate) (Absence of Carbohydrate)

IODINE TEST

Blue Color solution No Blue Color Solution

(Presence of Starch) (Mono/Di Saccharide)

BENEDICT’S TEST

Brick red ppt No Brick Red ppt

(Reducing sugar ) (Non – Reducing Sugar)
May be sucrose

BARFOED’S TEST
BENEDICT’S TEST
AFTER
ACID HYDROLYSIS FOR
SUCROSE

Red Scum No Red Scum Red

precipitate
(Monosaccharides) (Disaccharides) (Confirms the
presence of Sucrose)

OSAZONE TEST

FOULGER’S TEST FOULGER’S TEST

Blue Color No Blue Color
(Ketose Sugar) (Aldose Sugar)
 Sunflower crystals Powder puff crystals
SELWINOFF’S TEST S ELWINOFF’S TEST (Maltose) (Lactose)
Red color (ketosugar) No red color (aldo sugar)

RAPID FURFURAL RAPID FURFURAL

Purple color(keto sugar) No purple color (aldo sugar)

Osazone Test Osazone Test

Needle Shaped Crystals Needle Shaped Crystals

(Fructose) (Glucose)

REACTIONS OF CARBOHYDRATES AT A GLANCE

Test Glucose Fructose Maltose Lactose Sucrose

Molisch’s test +ve +ve +ve +ve +ve

Benedict’s test +ve +ve +ve +ve -ve

Barfoed’s test +ve +ve -ve -ve -ve

Foulger’s test -ve +ve - - +ve

Seliwanoff’s test -ve +ve - - +ve

Rapid furfural test -ve +ve - - +ve

Acid Hydrolysis - - - - +ve

test
Osazone test Needle Needle Sunflower Powder Nil
(Crystal Shape) Puff
 COLOUR REACTIONS OF PROTEIN

Proteins are made up of amino acid residues joined by peptide bonds. Because of the presence
of peptide bonds and different amino acid residues, proteins react with a variety of reagents to
form colored products. These tests are known as color reactions of proteins. Several of these
reactions are of importance in qualitative detection and quantitative estimation of proteins and
of their constituent amino acids.

EXPERIMENT OBSERVATION INFERENCE

1.Ninhydrin Test:
To 1ml of protein solution,
add 2-3 drops of freshly
prepared ninhydrin solution
& boil.
Bluish purple color is Indicates the presence of free
formed. amino group in the given
solution.

Principle: Free amino groups of protein react with ninhydrin to give hydrindantin, which
reacts with another molecule of ninhydrin to give the bluish purple color (Ruheman’s
purple).
2. Biuret Test:
To 2 ml of protein solution, Violet color is formed Indicates the presence of peptide
add 2ml of 5% NaOH and bonds in proteins.
1-2 drops of 1% CuSO4 .
Principle: Cupric ions react with peptide bonds in alkaline medium to give violet colored
complex. An excess of CuSO4 will obscure the test because, the characteristic color will be
masked by deep blue color of cupric hydroxide.
3. Xanthoproteic Test:
To 3ml of protein add 1ml of First yellow color is Indicates the presence of
Conc. HNO3. Boil for 1 formed which changes Aromatic amino acids Tyrosine
minute and cool. Divide the to orange on addition of or Tryptophan.
solution in to 2 halves. To 40% NaOH.
one of the tube add 40%
NaOH till it becomes alkaline

Principle: Benzene ring of amino acids is nitrated to form yellow nitro substitution product.
On addition of alkali, orange salt formation occurs.
4. Millon’s Test (Cole’s
Test): Red colored precipitate Indicates the presence of
To 1ml of protein solution is formed. Tyrosine.
add 1ml of 10% mercuric
sulphate in 10% H2SO4&
boil for 30 seconds. Cool,
add 2drops of 1% Sodium
Nitrite and warm gently.
Principle: The hydroxy phenyl group of tyrosine forms red colored mercuric phenolate
compound.
5. Aldehyde Test:
(Hopkins’s – Cole test)
 To 1ml of protein solution, Violet colored ring is Indicates the presence of
add 1 drop of 1/500 formalin formed at the junction of Tryptophan (Trp).
& 2drops of 10% mercuric the two fluids.
sulphate (HgSO4) in 10%
H2SO4. Mix well & add 2ml
of conc. H2SO4 along the
sides of the tube.
Principle: H2SO4 and HgSO4 act as an oxidizing agent and oxidizes the indole ring of Trp.
The oxidized product then condenses with formaldehyde to give violet colored compound.
6. Sakaguchi Test:
To 3 ml of protein solution, Bright Red color is Indicates the presence of
add 1 ml of 40% NaOH, 2 formed. Arginine (Arg). This reaction is
drops of alcoholic α–napthol due to the guanidino group.
& 3 drops of Sodium
hypochlorite
Principle: This is a sensitive test for detection of arginine (gaunidino group). The guanidino
group of arginine in the given protein reacts with α–napthol and sodium hypochlorite in the
alkaline medium to give red colored complex.
7. Sulphur Test:
To 2 ml of protein sol, add Black (or Brown) color Indicates the presence of Sulphur
2ml of 40% NaOH. Boil for precipitate is formed. containing amino acids cysteine
at least for 2 minutes & cool. & cystine.
Add 2 drops of lead acetate
and mix well.
Principle: When heated with strong alkali, the organic sulphur is converted to sodium
sulphide (inorganic sulphur), which reacts with lead acetate to give lead sulphide that is
black (or brown) in color. Methionine does not answer this test.

Report: The color reactions indicates the protein contains all essential amino acids and have
high biological value.
 PRECIPITATION REACTIONS OF PROTEINS

Proteins are complex nitrogen containing organic compounds that are charged and have a water of
hydration around them. Precipitation of proteins can be brought about by disturbing either the charge on
them or by removing the water of hydration by various methods.

EXPERIMENT OBSERVATION INFERENCE

1) Precipitation by Metallic
Salts (Lead/ Mercury/Ferric/Zinc):
To 3 ml of protein solution, add White precipitate is formed. Protein is precipitated by
10 % lead acetate drop by drop. metallic salts.

Principle: The + ve charge of lead ion neutralizes the – ve charge of protein forming lead proteinate
complex.
2) Precipitation by Alkaloid
Reagents* (Sulfosalicylic
acid/Esbach’s/Tannic/Potassium White precipitate is formed. Protein is precipitated by
ferrocyanate in glacial acetic alkaloid reagents.
acid):
To a 3ml protein solution, add 1-3
drops of 3% sulphosalicyclic acid.
* This method is useful in the
analysis of CSF proteins.
Principle: The + ve charge of protein is neutralized by – vely charged sulphosalicylate ions to form
protein – salicylate complex.
3.Precipitation by organic
solvents(Ether/Alcohol): A white precipitate is formed. Protein is precipitated by
To 3ml of protein solution, add alcohol.
alcohol drop by drop till
precipitation occurs.
Principle: Organic solvents like ether or alcohol remove the shell of hydration (dehydration) and thus
causes precipitation.
3) Precipitation by Neutral salts:
a) Half Saturation:
To 5ml of protein solution, add equal
volume of saturated (NH4)2SO4 White precipitate is formed. The given protein is
solution and mix. Allow to stand for precipitated by half saturation.
5 minutes. Filter the solution.

Take 1 ml of filtrate & perform

Biuret Test using 40 % NaoH. No violet color is formed. This indicates the absence of
(Biuret – ve) proteins in the filtrate.
b) Full saturation:
To 5ml of protein solution add little
by little solid (NH4)2SO4and mix till
some salt is left at the bottom of the White precipitate is formed. The given protein is
tube and wait for 5minutes. precipitated by full saturation.

Filter the solution & then take 1 ml

of filtrate & perform Biuret Test No Violet color is formed. This indicates the absence of
using 40 % NaoH. (Biuret – ve) proteins in the filtrate.

Principle: The precipitation of a protein depends on the shell of water of hydration around the protein
molecule. Smaller the molecule, more the surface area and hence requires more salt to remove the water
of hydration. Albumin being smaller molecule needs more salt to precipitate as compared with globulin,
a large molecule which is precipitatable with just half-saturation.

4) Heat Coagulation Test:

Cloudy white precipitate is Indicates the presence of
To 2/3rd of testtube add protein formed in the upper part of albumin.
solution in a test tube, 1-2 drops of solution.
chlorophenol red indicator is added.
The pH is adjusted by adding a drop
or two of 1% acetic acid and 1%
sodium carbonate.(i.e., the solution
is yellow colour at pH 4.8 and
below; red at pH 6.4 and above).
A solution with light pink colour
with the indicator indicates a pH of
5.4.
Heat the upper portion of the test
test tube and the bottom portion
acts as a control.

Principle: Albumin undergoes denaturation by denaturing agents such as heat. When albumin solution
is heated, it gets denatured irreversibly to produce thick coagulum. Upon adding acetic acid, the
coagulation is more pronounced only if there is presence of albumin. Phosphates can also give the
cloudy appearance on heating, but will disappear after the addition of acetic acid.
This test is commonly employed to detect the presence of albumin in urine in renal pathology.
5) Iso electric precipitation:
To 3ml protein solution, add 2 drops Green precipitate is formed. Indicates the presence of
of bromocresol green (BCG) Casein.
indicator. Then add 1% acetic acid
drop by drop till a precipitate is
seen.
Principle: Proteins are precipitated at isoelectric pH (pI). Acetic acid lowers the pH of casein to its pI
4.6. The change in color of BCG indicates the pI of casein.
 REACTIONS OF ALBUMINS

EXPERIMENT OBSERVATION INFERENCE

1.Biuret Test:
To 2 ml of protein solution, Violet color is observed. Indicates the presence of
add 2ml of 5% NaOH and 1-2 protein.
drops of 1% CuSO4 .

2. Heat Coagulation Test:

To 2/3rd of testtube add protein Cloudy white precipitate is seen Indicates the presence of
solution in a test tube, 1-2 drops Albumins.
of chlorophenol red indicator is
added. The pH is adjusted by
adding a drop or two of 1% acetic
acid and 1% sodium carbonate.
(i.e., the solution is yellow colour
at pH 4.8 and below; red at pH
6.4 and above).
A solution with light pink colour
with the indicator indicates a pH
of 5.4.
Heat the upper portion of the test
test tube and the bottom portion
acts as a control.

3.Salt fractionation
(Precipitation by Neutral Salts):
a) Half Saturation:
To 5ml of protein solution, add an
equal volume of Saturated White precipitate is observed
(NH4)2SO4 solution & mix well.
Stand for 5min & filter.

To 1ml of filtrate add twice the Violet color is observed. Albumin is not precipitated by
volume of 40% NaOH & add 1 or (Biuret +ve) half saturation.
2 drops of CuSO4 solution (Biuret
test).

b) Full Saturation:
To 3ml of filtrate add little by
little solid (NH4)2SO4 &mix well White precipitate is observed. Albumin is precipitated by Full
till some amount of salt is left saturation.
undissolved at the bottom (the
solution is Saturated). Stand for
five minutes & Filter.

Take 1ml of filtrate & perform No violet color is observed. It indicates that no protein is
Biuret Test using 40% NaOH. present in the filtrate
.
4. Heller’s test:
To 2ml of Conc. HNO3 add 2ml A white ring is observed at the Confirms the presence of
of solution along the sides of test junction of two liquids. Albumin.
tube. Nitric acid converts proteins to
acid meta protein insoluble in
Conc. HNO3.
5. Xanthoproteic Test:
To 3ml of protein add 1ml of First yellow color is formed Indicates the presence of
Conc. HNO3. Boil for 1 minute which changes to orange on Aromatic amino acids Tyrosine
and cool. Add 40% NaOH till it addition of 40% NaOH. or Tryptophan
becomes alkaline.

6. Millon’s Test:
To 1ml of protein solution
add 1ml of 10% mercuric Red colored precipitate is Indicates the presence of
sulphate in 10% H2S04& boil for formed Tyrosine.
30 seconds. Cool, add 2drops of
1% Sodium Nitrite and warm
gently.

7. Aldehyde Test:
To 1ml of protein solution,
add 1 drop of 1/500 formalin & Violet colored ring is formed at Indicates the presence of
2drops of 10% mercuric sulphate the junction of the two fluids. Tryptophan (Trp).
(HgSO4). Mix well & add 2ml of
conc. H2SO4 along the sides of the
tube.
8. Sakaguchi Test:
To 3 ml of protein solution, add 1 Bright Red color is formed. Indicates the presence of
ml of 40% NaOH, 2 drops of Arginine (Arg). This reaction is
alcoholic α–napthol & 3 drops of due to the guanidino group.
Sodium hypochlorite.
9. Sulphur Test:
To 2 ml of protein sol, add 2ml of
40% NaOH. Boil for at least for 2 Black (or Brown) color Indicates the presence of
minutes & cool. Add 2 drops of precipitate is formed. Sulphur containing amino acids
lead acetate and mix well. cysteine & cystine.

Report: Albumin is a heat coagulable protein precipitated by full saturation and it contains all essential
amino acids and thus has a high biological value.
 REACTIONS OF GELATIN
Introduction:
1) It is derived protein from collagen (mainly excreted from tendons)
2) It is insoluble in cold water but soluble in hot water.
3) If the solution is concentrated & cooled, a gel is formed.
4) It is not heat coagulable & precipitated by half saturation with NH 4SO4
5) It is a rich source of glycine but contains only traces of Tyrosine and lacks Tryptophan, Cysteine
& cystine.

EXPERIMENT OBSERVATION INFERENCE

1.Biuret Test:
To 2 ml of protein solution, Violet color is observed. Indicates the presence of protein.
add 2ml of 5% NaOH and 1-2
drops of 1% CuSO4 .

2. Heat Coagulation Test:

To 2/3rd of testtube add protein No Cloudy precipitate is seen. Gelatin is not precipitated by heat
solution in a test tube, 1-2 drops coagulation.
of chlorophenol red indicator is
added. The pH is adjusted by
adding a drop or two of 1% acetic
acid and 1% sodium carbonate.
(i.e., the solution is yellow colour
at pH 4.8 and below; red at pH
6.4 and above).
A solution with light pink colour
with the indicator indicates a pH
of 5.4.
Heat the upper portion of the test
test tube and the bottom portion
acts as a control.

3.Salt fractionation
(Precipitation by Neutral Salts):
b) Half Saturation:
To 3ml of protein solution, add an
equal volume of Saturated White precipitate is observed Gelatin is precipitated by Half
(NH4)2SO4 solution & mix well. saturation
Stand for 5 min & filter. .

To 1ml of filtrate add twice the

volume of 40% NaOH & add 1 or Violet color is not observed. No protein is present in the filtrate.
2 drops of CuSO4 solution (Biuret (Biuret -ve)
test).

4. Xanthoproteic Test:
To 3ml of protein add 1ml of
Conc. HNO3. Boil for 1 minute
and cool. Add 40% NaOH till it
becomes alkaline.
First faint yellow color is Indicates the presence of Aromatic
formed which changes to faint amino acids Tyrosine or Tryptophan
orange on addition of 40%
NaOH.
5.Millon’s Test:
To 1ml of protein solution
add 1ml of 10% mercuric Red colored precipitate is Indicates the presence of Tyrosine.
sulphate in 10%H2SO4& boil for formed
30 seconds. Cool, add 2drops of
1% Sodium Nitrite and warm
gently.

6. Aldehyde Test:
To 1ml of protein solution,
add 1 drop of 1/500 formalin & No Violet colored ring is Indicates the absence of Tryptophan
2drops of 10% mercuric sulphate formed at the junction of the (Trp).
(HgSO4)in 10%H2SO4. Mix well two fluids.
& add 2ml of conc. H2SO4 along
the sides of the tube.
7. Sakaguchi Test:
To 3 ml of protein solution, add 1 Bright Red color is formed. Indicates the presence of Arginine
ml of 40% NaOH, 2 drops of (Arg). This reaction is due to the
alcoholic α–naphthol & 3 drops guanidino group.
of Sodium hypochlorite.

8. Sulphur Test:
To 2 ml of protein sol, add 2ml of
40% NaOH. Boil for at least for 2
minutes & cool. Add 2 drops of
lead acetate and mix well.
No Black (or Brown) color Indicates the absence of Sulphur
precipitate is formed. containing amino acids cysteine &
cystine.

Report: Gelatin is a Non- heat coagulable protein precipitated by half saturation. It contains essential
amino acids except tryptophan and sulfur containing amino acids and hence it is of low biological
value.
 REACTIONS OF PEPTONE
Introduction:
1.Peptone is a secondary derived protein.
2.They are not heat coaguable.
3.Being extremely small size,peptones are not precipitated by half saturation and not by full
saturation.
4.Peptone lack sulfur containing amino acids.

EXPERIMENT OBSERVATION INFERENCE

1.Biuret Test: Indicates the presence of
To 2 ml of protein solution, Pink/Violet color is observed. protein.
add 2ml of 5% NaOH and 1-2 (May be peptone)
drops of 1% CuSO4 .

2.. Heat Coagulation Test:

To 2/3rd of testtube add protein No Cloudy precipitate is seen Peptone is not precipitated by
solution in a test tube, 1-2 drops heat coagulation.
of chlorophenol red indicator is
added. The pH is adjusted by
adding a drop or two of 1% acetic
acid and 1% sodium carbonate.
(i.e., the solution is yellow colour
at pH 4.8 and below; red at pH
6.4 and above).
A solution with light pink colour
with the indicator indicates a pH
of 5.4.
Heat the upper portion of the test
test tube and the bottom portion
acts as a control.

3.Salt fractionation
(Precipitation by Neutral Salts):
c) Half Saturation:
To 3ml of protein solution, add an No White precipitate is Peptone is not precipitated by
equal volume of Saturated observed Half saturation
(NH4)2SO4 solution & mix well. .
Stand for 5 min & filter.
To 1ml of filtrate add twice the
volume of 40% NaOH & add 1 or Pink/Violet color is observed. Protein is present in the
2 drops of CuSO4 solution (Biuret (Biuret +ve) filterate.
test).

b) Full Saturation:
To 3ml of filterate, add little by No White precipitate is Peptone is not precipitated by
little solid (NH4)2SO4 &mix well observed. Full saturation.
till some amount of salt is left
undissolved at the bottom (the
solution is Saturated). Stand for
five minutes & Filter.
Pink/violet color is observed. Protein is present in filterate in
Take 1ml of filtrate & perform Biuret(+ve) the filtrate
Biuret Test using 40% NaOH.
4. Xanthoproteic Test:
To 3ml of protein add 1ml of
Conc. HNO3. Boil for 1 minute
and cool. Add 40% NaOH till it
becomes alkaline.
First yellow color is formed Indicates the presence of
which changes to orange on Aromatic amino acids Tyrosine
addition of 40% NaOH. or Tryptophan

5. Millon’s Test:
To 1ml of protein solution
add 1ml of 10% mercuric Red colored precipitate is Indicates the presence of
sulphate in10% H2SO4& boil for formed Tyrosine.
30 seconds. Cool, add 2drops of
1% Sodium Nitrite and warm
gently.

6. Aldehyde Test:
To 1ml of protein solution,
add 1 drop of 1/500 formalin & Violet colored ring is formed at Indicates the presence of
2drops of 10% mercuric sulphate the junction of the two fluids. Tryptophan (Trp).
(HgSO4) in10% H2SO4. Mix well
& add 2ml of conc. H2SO4 along
the sides of the tube.

7. Sakaguchi Test:
To 3 ml of protein solution, add 1
ml of 40% NaOH, 2 drops of
alcoholic α–napthol & 3 drops of
Sodium hypochlorite.
Bright Red color is formed. Indicates the presence of
Arginine (Arg). This reaction is
due to the guanidino group.

8. Sulphur Test:
To 2 ml of protein sol, add 2ml of
40% NaOH. Boil for at least for 2
minutes & cool. Add 2 drops of
lead acetate and mix well.
Faint Black (or Brown) color Indicates the absence of
precipitate is formed. Sulphur containing amino acids
cysteine & cystine.

Report: Peptone is a non- heat coagulable protein, not precipitated by half and full saturation
and it contains all essential amino acids and thus have a high biological value.
 IDENTIFICATION OF UNKNOWN PROTEIN

Biuret test

+ve
(Presence of protein)

Isoelectric precipitation with BCG Indicator

+ve -ve
(Presence of Casein) (Absence of Casein)

Heat coagulation test

+ve -ve
Presence of Albumins (Presence of Gelatin or peptone )

Half saturation Half saturation

Biuret test +ve with filterate Gelatin is pptd

Peptone is not pptd

Full saturation Full saturation

(Albumins are Ppted) Peptone is not pptd

Confirms the presence
of Albumins.
Biuret –ve with filterate

Heller’s test

(Indicates presence of heat Perform color

coagulable proteins albumin) reactions for
Gelatin & Peptone.
 Perform color reactions
of Albumin

REACTIONS OF PROTEINS AT A GLANCE

Name of the test Albumin Gelatin Peptone Casein

1. Biuret test +ve +ve +ve +ve

2.Isoelectric -ve -ve -ve +ve

precipitation

3.Heat coagulation +ve -ve -ve ----

test

4. Half Saturation

( Biuret test) +ve(Albumins) -ve +ve

Full saturation ----

(Biuret test) -ve(No Protein) ---- +ve

3. Ninhydrin Test +ve +ve +ve +ve

4. Xanthoproteic +ve +ve +ve +ve

test

5. Million’s test +ve +ve +ve +ve

6. Aldehyde test +ve -ve +ve +ve

7. Sakaguchi test +ve +ve +ve +ve

 8. Sulphur Test +ve -ve -ve +ve

ANALYSIS OF NORMAL URINE

A study of constituents of urine helps the doctor to understand the health status of an
individual. The increased amounts of normal constituents and the presence of abnormal
constituents in urine point towards a diseased state. The daily urinary output is greatly
influenced by diet, fluid intake, and climatic conditions.
For qualitative analysis of urine, a mid stream collection of the first morning sample is
preferred as it has the maximum concentration of the urinary constituents. For quantitative
analysis of urine, a 24 hour urine collection is preferred.

PHYSICAL CHARACTERISTICS

1) VOLUME:
Normal: 1 – 2 litres /day
i) Polyuria: (Increased urine output; > 3 litres/ day)
It is seen in…
- Increased water intake, high Protein diet & high salt intake.
- Diabetes mellitus, Diabetes insipidus
- Drugs
ii) Oliguria: (Decreased urine output; < 500 ml/ day)
It is seen in excessive sweating, exercise, dehydration, BP, nephritis, cardiac failure,
hypovolemic shock, diarrhea & vomiting.
iii) Anuria: (Urine output < 100 ml/ day)
It is seen in severe shock, acute nephritis, blood transfusion reactions, mercury poisoning,
and bilateral renal stones.

2) COLOUR:
Normally it is in straw colored due to the presence of a pigment urochrome.
 i) Honey colour – Presence of Bilirubin glucuronide (Jaundice).
ii) Upper portion is black – Presence of Melanin (Alkaptonuria).
iii) Black – Melanuria (Malignant melanoma)
iv) Reddish brown – Hematuria (Nephritis, Nephrosis, Hemolytic conditions).
v) Smoky color – Hemoglobinuria.

3) APPEARANCE:
Urine is normally clear & transparent.
Turbid – due to the presence of more phosphates & pus (UTI)
Milky – Chyluria (Obstruction of lymphatics)

4) ODOUR:
Normal – Faintly aromatic due to the presence of volatile organic acids.
Pungent / Ammonical smell – Urea is converted to ammonium carbonate on long
standing.
Fruity odour – Diabetic ketoacidosis (Ketosis due to acetone).

5) pH:
Normal: The pH of urine is close to neutral (7.0), but can vary between 4.4 – 8.0.
Acidic: Fever & Rich Protein diet and metabolic acidosis.
Alkaline: After heavy meal (Alkaline tide) and Metabolic alkalosis.

6) SPECIFIC GRAVITY:
Normal: 1.012 – 1.024
Increased: Diabetes mellitus, acute nephritis & increased sweating.
Decreased: Increased intake of water, chronic nephritis, Diabetes insipidus
Fixed Sp. Gr. at 1.010 – Chronic renal insufficiency.
(Specific gravity is directly proportional to the concentration of solutes excreted and inversely
proportional to the water content of urine.)

CHEMICAL CHARACTERISTICS

Normal urine contains both inorganic and organic constituents. The inorganic constituents
include Na+, K+, Ca2+, Mg2+, NH4+, Cl -, PO42 -, SO42 - and traces of HCO3- ions.
Normal organic constituents are Urea, Uric acid and Creatinine commonly grouped as non-
protein nitrogenous (NPN) substances.

A) Specific gravity test

Specific gravity (Sp.Gr) of urine is measured using urinometer which has a bulb, weighed
with mercury and a narrow stem graduated from 1000 – 1060 (corresponding to specific
gravities 1.000 – 1.060). Sp.Gr can be directly read from the stem with the level of the
meniscus. Urinometer is calibrated at 150C and therefore a temperature correction should
be made. For every 30C increase in temperature 0.001 should be added (and vice versa).
Precautions:
i) There should not be froth in the urine.
ii) Urinometer should not touch sides of the container.

B) Tests for Non Protein Nitrogenous (NPN) Constituents:

1)Urea:
a) Sodium hypochlorite Test:
2ml of urine + 4 to 5 drops of Brisk effervescence of N2 Indicates the presence of
sodium hypochlorite solution. gas is seen. Urea.

Principle: When urea is treated with NaOCl it decomposes to give N2 gas.

b) Specific Urease Test:
To 10ml of Urease enzyme(Horse Pink color is formed. Indicates the presence of
gram extract) add 5 drops of phenol Urea.
indicator (yellow at pH to 6.7 and
red at pH 8.3and adjust the pH to 7
where the red color just turns to
yellow by adding just enough of
0.1 % HCl or 1% Na2CO3.Divide
the above solution into two aliquots
(two equal parts) and mark them as
‘T’(test) and ‘C”(control) tubes.
In ‘T’ tube add 2ml of urine
solution and in “C” tube add equal
amount of distilled water , mix, and
keep both the tubes in the boiling
water which is adjusted to a
temperature of 60oC for few
minutes.

Principle: Urease enzyme splits urea into ammonia and carbonic acid. Ammonia combines
with carbonic acid to form ammonium carbonate which is alkaline. In alkaline medium
phenolpthalein gives pink color.

2)Uric Acid:-
Phosphotungstic Acid reduction
Test:-
To 2ml of urine add 5 - 6 drops of Deep Blue color is formed. Indicates the presence of
phosphotungstic acid reagent & Uric acid.
5 - 6 drops of 20% Na2Co3.

Principle: Uric acid is a reducing agent in strong alkaline condition. It reduces colorless
phosphotungstic acid to tungsten blue.

3)Creatinine:-
Jaffe’s Test:-
Label 2 test tubes as ‘Test’ &
‘Control’

Test:-
To 2ml urine add 2ml saturated Orange color with Urine. Indicates the presence of
Picric acid &5 -6 drops of 10% Creatinine.
NaOH.

Control:- Yellow color with H2O

To 2ml H2O add 2ml saturated
Picric acid & 5-6 drops of
10%NaOH
Principle: Creatinine reacts with alkaline Picrate solution to form an orange colored
creatinine picrate complex.

IDENTIFICATION OF UNKNOWN
PHYSIOLOGICAL SUBSTANCE
Biuret test

+ve -ve
(Presence of proteins) (Absence of Proteins)

Proceed for the table

given for identification Molisch’s test
of proteins.

+ve -ve
(Presence of Carbohydrates) (May be NPN
Substances)
Proceed for the table given Proceed for identification of
for identification of a) Urea ( Hypochlorite test)
Carbohydrates. b) Creatinine (Jaffe’s test)
c) Uric acid (Phospho tungstic acid test)
 ANALYSIS OF ABNORMAL CONSTITUENTS OF URINE

I) URINE SAMPLE FROM A PATIENT SUFFERING FROM DIABETES MELLITUS:

EXPERIMENT OBSERVATION INFERENCE

1) Test for Glucose:-

Benedict’s Test: Green/Yellow/Orange/ Indicates the presence of
To 5 ml of Benedict’s reagent add 8 drops Brick Red (GYOR) reducing sugars in the given
(0.5ml) of Urine & boil for 2 minutes. colored precipitate is Urine sample.
formed.
2) Test for Ketone Bodies:-
a) Rothera’s test:
Saturate 5ml Urine with solid Ammonium Indicates the presence of
Sulphate crystals (until a small amount of Purple coloured ring is Acetone or / and Aceto acetic
crystals remains at the bottom of the test seen at junction of two acid.
tube). Add 2 drops of freshly prepared liquids.
Sodium Nitroprusside solution. Mix and
add 2 ml of liquor ammonia slowly along
the sides of the tube to form a layer on the
top.
Principle: Nitroprusside reacts with ketone group in the alkaline medium to form a purple colored
complex.

II) URINE SAMPLE FROM A PATIENT SUFFERING FROM KIDNEY DISEASE:

3) Test for Albumin:

a) Heat Coagulation Test:
Take 10 ml of Urine sample in a test tube. A Cloudy white Indicates the presence of
Heat the upper 1/3’rd of the test tube & Precipitate is seen. Albumin in Urine.
add 1 or 2 drops of 1 % Acetic Acid.
Only the upper 1/3rd of the test tube is
heated to have the cooler lower portion as
control. A white turbidity is Indicates the presence of
b)Sulphosalicylic acid test: Seen. Albumin in Urine.
To 2ml of urine add a 5- 6 drops of 20%
Sulphosalicylic acid.
4) Test for Blood:-
a) Benzidine test:-
Test: 5ml of urine and 5ml of distilled Immediately Blue or Indicates the presence of blood
water are taken in two different test tubes green color appears, in urine.
marked as ‘T’ (test) and ‘C’ (control). and slowly disappears
One drop of benzidine reagent (benzidine in few minutes.
dissolved in glacial acetic acid) and 10
volumes of H2O2 are added in each test
tube
Principle: Haemoglobin exhibits peroxidase activity on hydrogen peroxide (H2O2) to yield nascent
oxygen, which acts on benzidine to produce green or blue color product which is unstable.
III) URINE SAMPLE FROM A JAUNDICED PATIENT:

5) Test for Bile Salts:-

a)Hay’s Test:- Indicates the presence of Bile

Test:To 3 ml urine in a test tube, sprinkle Sulphur powder salts.
a pinch of Sulphur powder on the surface. sinks to the bottom.
Control: 3 ml water in a test tube,
sprinkle a pinch of Sulphur powder on
the surface.

Principle: Bile salts have the property of lowering the surface tension of the solution because of
which the sulphur powder sinks to the bottom of test tube.

6) Test for Bile pigments:

a)Fouchet’s Test:-
To 5ml urine add few drops of glacial Yellow / White
acetic acid ,1 ml of10% BariumChloride Precipitate is seen.
and 2 drops of saturated ammonium
sulphate. Allow it to stand for 5 minutes.. Green or Blue color is Indicates the presence of Bile
Filter. Take the ppt. and dry it under the seen. pigments.
folds of the filter paper & add few drops
of Fouchet’s Reagent.
(Ferric chloride 10% in 25%
trichloroacetic acid)
Principle: BaCl2 reacts with Ammonium Sulphate to form BaSO4 ppt. Fouchet’s reagent oxidizes
bilirubin to green (biliverdin) and blue (bilicyanin) products.

b) Gmelin’s test: to 2 ml of conc.HNO3 play of colours is obs- Indicates the presence of of Bile
add 2 ml of urine. erved at the interface. Pigments.
Coloured rings like
Green,blue and brown
are seen.

ALBUMINURIA (Proteins in Urine)

Albuminuria is divided into two main groups.
I. Organic Albuminuria
II. Physiological Albuminuria

I. Organic Albuminuria:-
It is further subdivided into…
1) Pre-renal Albuminuria:
- In this condition kidneys are only affected secondarily to some other disease.
- The albuminuria usually disappears when the primary disease is cured.
 - In most cases the albuminuria is due to some impairment of the renal
circulation.
Eg:- Dehydration (vomiting/ severe diarrhea), Diabetic coma.
Addison’s disease, Heart disease (passive congestion of kidneys),
- Intra abdominal pressure (ascites& intra abdominal tumors) &drugs that may
have a toxic effect on the kidneys.
- The proteins are usually plasma albumin & globulins.

2) Renal Albuminuria:
- It is seen in all forms of renal disease (inflammatory, degenerative
or destructive).
- The proteins are usually plasma Albumin & Globulins, RBC.

3) Post –renal Albuminuria:-

- Protein may be added to the urine as it passes along the urinary tract.
- Lesion of the renal pelvis, bladder, prostate & urethra can all lead to such an
addition.
- Secretions & discharges from the vagina and admixture of semen may also add
protein (False +ve).
- Urine containing pus will also give positive tests (false +ve). Blood may also be
added at this stage.
- The false +ve can be ruled out by microscopic examination of the urine deposit.
- The term false albuminuria has been applied to this group since the +ve test may
wrongly lead to a suspicion that renal disease is present.

II) Physiological Albuminuria:

- The albuminuria is usually small in amount and is accompanied by no other
symptoms of renal disease.
- Ex: Severe stress (excessive physical activity, exposure to severe cold), last
trimester of pregnancy & posture.
- The Protein in these cases is mainly plasma albumin.
 PART- II

QUANTITATIVE
 ESTIMATION OF GLUCOSE
Glucose Oxidase Peroxidase Method

Glucose is the major metabolic fuel of mammals and a universal fuel of the fetus. The
maintenance of stable levels of glucose in the blood is one of the most finely regulated of all
homeostatic mechanisms, involving liver, extrahepatic tissues, and several hormones. Diseases
associated with derangement of carbohydrate metabolism include diabetes mellitus,
galactosemia, glycogen storage diseases, and lactose intolerance.

AIM:
To estimate the amount of glucose present in the given serum/plasma sample by GOD-POD
Method ( Glucose Oxidase Peroxidase Method).

PRINCIPLE:
Glucose is oxidized by glucose oxidase (GOD) to give gluconic acid and hydrogen
peroxide. The hydrogen peroxide formed is broken down by peroxidase to water and nascent
oxygen. The latter oxidizes phenol which combines with 4-aminoantipyrene (4-AAP) to give a
red colored complex (quinoneimine dye). The intensity of the red colored complex is
proportional to the concentration of glucose in the test. The intensity of the coloured complex
is measured colorimetrically at 515nm (500 – 530).

REACTION glucose oxidase

Glucose + O2 + H2O2 gluconic acid + H2O2
peroxidase
H2O2 H2O + (O)
(O) + phenol + 4- AAP Quinoneemine dye + H2O

REAGENTS:

1. Working glucose reagent has to be stored at 2 -8o C in dark bottle.

2. Serum / plasma
3. Glucose standard – concentration in 100 mg/dl.
4. Blank- dilute working glucose reagent with distilled water.
PROCEDURE:
Take 3 test tubes and label them as Blank (B), Standard (S) and Test (T) and proceed with the
tests as follows.

BLANK (B) STANDARD (S) TEST (T)

REAGENTS
Ml ml Ml
working glucose
reagent 1 ml 1 ml 1 ml

Serum or Plasma
– – 10 µl

Standard
– 10 µl –

Distilled water
10 µl – –

Mix the contents of the test tubes thoroughly, and incubate at room temperature for 15minutes
and read at 515 nm.

CALCULATION:
Glucose present in OD of test (T) – OD of Blank (B) Conc. Of Std
100 ml of plasma = X X 100
or serum OD of Std. (S) – OD of Blank (B) Volume of the Sample

REPORT: Glucose present in 100 ml of serum/plasma = – – – – – mg/dl

Note:
Usually plasma/serum is used for glucose estimation. Glycolysis decreases blood glucose by
approximately 5 – 7% in an hour. This can be prevented by adding sodium fluoride (NaF),
which inhibits the enzyme enolase. This method is linear up to 500 mg%.
Normal levels:
Fasting plasma / serum: 70 – 110 mg /dl
Post prandial: <140 mg /dl
Random blood glucose: < 200 mg/dl
Clinical Interpretations:

1. Hyperglycemia: (Increased Blood Sugar) Found in:

1. Diabetes mellitus
2. Cushing’s syndrome (Adreno cortical hyperactivity)
3. Hyperthyroidism
4. Hyperpituitarism – Acromegaly, gigantism
5. Emotional stress (small increase)
6. Infections
7. Intra cranial diseases – Meningitis, tumours etc.
8. Effect of drugs – corticosteroids, Oestrogens, Alcohol, etc.

2. Hypoglycemia (Decreased blood sugar values) Found in:

1. Starvation
2. Hyperinsulinism
a) Increased dose of Insulin while treating Diabetes mellitus
b) Insulin secreting tumours of pancreas
3. Hypothyroidism (myxodema, cretinism)
4. Hypopituitarism (e.g. Simmond’s disease)
5. Hypoadrenalism (Addison’s disease)
6. Severe exertion
7. In children – glycogen storage diseases e.g. Von Gierke’s disease (Congenital inherited
diseases)
 ESTIMATION OF UREA
DAM METHOD

Urea is formed in the liver and excreted by the kidneys. Urea is the end product of protein
metabolism in our body. Synthesis of urea occurs in the liver cells from ammonia which is
liberated from the breakdown of amino acids.

AIM:
To estimate the amount of urea present in the given blood sample by Diacetyl Monoxime
Method.

PRINCIPLE:
Proteins in the blood are precipitated with Trichloroacetic acid.Urea present in the protein free
filterate reacts with Diacetyl Monoxime under strongly acidic condition and heat in presence of
Thiosemicarbazide to give pink colored complex. The intensity of the color is directly
proportional to the concentration of Urea present in the blood.

REAGENTS:
1. 10% Trichloroacetic acid
2. DAM Reagent (Diacetyl Monoxime + Thiosemi carbazide+ Distilled Water)
3. Acid Reagent (Sulphuric Acid+O-Phosphoric Acid+Ferric Chloride)
4. Standard : Urea standard 1ml=0.01mg
5. Sample : Whole blood

Procedure:
1.Preparation of Protein Free Filterate:
Into a dry test tube.
1. Blood: 0.1ml
2. Distilled Water: 3.4ml
3. 10% TCA : 1.5ml
Mix thoroughly and allow it to stand for 10 mins at room temperature . Filter into a dry test
tube.
2.Take 3 test tubes and label them as Blank (B), Standard (S) and Test (T) and proceed with the
tests as follows.

REAGENTS BLANK (B) STANDARD (S) TEST (T)

Distilled Water 1 ml - -

Urea Standard - 1ml -

Sample/ Protein
– - 1ml
Free Filterate

DAM Reagent 1ml 1ml 1ml

Acid Mixture 3ml 3ml 3ml

Mix well .place the test tubes in a boiling water bath for 15 mins .cool and read the
optical density at 540nm or green filter in a colorimeter
Measure OD of Blank, Standard and Test against Distilled water. Set at Zero OD using a
yellow filter at 540 nm .
CALCULATION:

Urea present in OD of test (T) – OD of Blank (B) Conc. Of Std

100 ml of blood = X X 100
OD of Std. (S) – OD of Blank (B) Volume of the Sample

OD (T) 0.005
= X X 100
OD (S) 0.01

OD (T)
= X 50 mg%
 OD (S)

REPORT:
Urea present in 100 ml of whole blood = mg%

NORMAL VALUE: 15 – 40 mg/dl

Blood urea Nitrogen (BUN):

Serum Urea is expressed in terms of its nitrogen content, such expression known as Blood
Urea Nitrogen (BUN). Molecular weight of Urea is 60. It contains 2 nitrogen atoms (M.W is
28). So each gram of Urea contains 28 gram of nitrogen. Thus serum concentration of 28 mg/dl
of BUN is equivalent to 60mg/dl of Urea. BUN can be converted into Urea by multiplying
figure by 2.14(60/28).

Hence BUN has to be calculated as follows:

BUN = Amount of urea for 100ml of Blood X Mol.Wt. of Nitrogen

Mol.Wt.of Urea

= Amount of urea for 100ml of Blood X 28

60

= Amount of urea for 100ml of Blood X 0.467

Amount of Blood Urea = BUN X 60

28
= BUN X 2.143
Ex: Suppose if Blood Urea Nitrogen is 14 mg/dl, the amount of Urea in Blood is

= 14 X 2.143 = 30 mg/dl.
Normal range of BUN: 8 – 20 mg/dl

Clinical Interpretation:
Urea diffuses readily into all the body fluids. The ratio of urea in plasma to cells is about 5:4.
Increase in blood urea:

1) Pre-Renal causes:
Decreased blood volume leads to Decreased cardiac output. This in turn leads to decreased
Renal Plasma Flow, which causes Decreased GFR. Thus less amount of Urea is filtered in the
kidneys and hence blood urea levels are elevated.
i) Dehydration due to salt & water depletion. This condition can occur in untreated Cases:
a) Severe & Protracted vomiting.
b) Pyloric & Intestinal obstruction.
c) Heavy loss of intestinal contents as in prolonged diarrhea.
ii) Diabetic Coma, Diabetic Ketoacidosis
iii) Shock (Severe Burns)

2) Renal causes:
The damage to the kidney causes reduced glomerular filtration rate leading to urea retention.
The blood urea level can be naturally increased in all forms of kidney disease.
i) Acute glomerulonephritis
iii) Malignant hypertension
iv) Chronic pyelonephritis
v) Mercurial poisoning
vi) Hydronephrosis
vii) Congenital cystic kidneys
viii) Renal tuberculosis

3) Post-Renal causes:
When there is an obstruction to the flow of urine, there is backpressure on the glomerulus’s.
This causes retention of urine & so reduces the effective filtration which decreases GFR. Thus
there is an increase in the blood urea levels.
i) Enlargement of prostate
ii) Stones in the urinary tract
iii) Stricture of the urethra
iv) Tumors of the bladder affecting the ureters.

ESTIMATION OF SERUM CREATININE

(JAFFE’S METHOD)

Creatinine is the anhydride of creatine. It is formed from creatine-phosphate which is high

energy compound present in the muscle. The synthesis of creatinine occurs relatively at a
constant rate. The production of creatinine is directly proportional to the muscle mass and
almost all the creatinine is endogenously synthesized. Therefore the levels of creatinine and
the amount of creatinine excreted are constant and hence creatinine is an important
biochemical marker for kidney function.

AIM:
To estimate the amount of creatinine present in the given serum sample.

PRINCIPLE:
Creatinine reacts with picric acid in presence of sodium hydroxide i.e. alkaline medium to give
orange color complex (creatinine picrate) – Jaffe’s reaction. This is read at 520 nm.

MATERIALS:
1) Protein-free Filtrate
2) 0.04 M Picric acid (10.16gms of re– crystallized picric acid in water and make up to
1 litre.
3) 0.75 N NaOH (30gms of NaOH / litre)
4) Creatinine stock standard: 100mg of creatinine in 100ml of 0.1N Hcl(1mg/1ml)
5) Creatinine working standard: 0.01 mg/ml (1ml makes up to 100ml with distilled water).

PROCEDURE:
Take 3 test tubes and mark them as B, S & T.
Contents B S T

Distilled water 3ml - -

Creatinine working standard - 3ml -
 Protein-free Filtrate - - 3ml

0.04 M Picric acid 1ml 1ml 1ml

0.75N NaOH 1ml 1ml 1ml

Mix well and stand for 15min. for color development and read at 520nm.

Calculations:
Concentration of creatinine in = O.D of T – O.D of B X conc. of the Std X 2
O.D of S – O.D of B Volume of sample

=…………………mg/dl

Clinical Significance:
Reference Value: Male: 0.7 – 1.4mg/dl
Female: 0.6-1– 3mg/dl
Serum Creatinine concentrations can be elevated in some of the following conditions:
1. Renal Failure
2. Muscular Injury and dystrophies
3. Hyperthyroidism
4. Increased Body temperature
Low values can be due to Hypothyroidism
There is no significance for decreased values
 ESTIMATION OF URINE CREATININE
(JAFFE’S METHOD)

AIM:
To estimate the amount of creatinine present in the given urine sample.

PRINCIPLE:
Creatinine in urine reacts with picric acid in presence of sodium hydroxide i.e. alkaline
medium to give orange color complex creatinine picrate.The intensity of the color is directly
proportional to the concentration of creatinine in urine. The Optical density is read at 520 nm.

REQUIREMENTS:
1) Urine
2) 0.04 M Picric acid (10.16gms of re-crystallized picric acid in water and make up to
1 liter).
3) 0.75 N NaOH (30gms of NaOH / litre).
4) Working standard of Creatinine: 0.01mg/ml

PROCEDURE:
Dilute 1ml of urine to 100ml with water in a standard flask, 3ml of diluted urine contains
0.03ml of urine. Take 3 test tubes and mark them as B, S & T.
Contents B S T

Distilled water 3ml - -

Creatinine working standard - 3ml -

Diluted urine - - 3ml Stand for

0.04 M Picric acid 1ml 1ml 1ml 15min. for
0.75N NaOH 1ml 1ml 1ml color

development and read at 520nm.

CALCULATIONS:
Concentration of creatinine in gm/lit of urine

= O.D of T – O.D of B X conc. of Std. gm/lit

O.D of S – O.D of B Vol. of test

= T–B gm/lit
S–B

If 1500ml is the day’s output of urine then the amount of creatinine excreted in g/day

= T–B X 1500 g/day

S-B 1000

Clinical Significance:

Normal Value: 1– 2 g/day

Creatinine clearance:
It is the volume of plasma in ml that would be completely cleared of creatinine, each minute by
kidney in order to account for its rate of excretion.
Creatinine clearance is widely used as creatinine is freely filtered at glomerulus and creatinine
level is constant throughout and not affected by diet.

Creatinine clearance is most widely used method to measure Glomerular Filtration Rate (GFR).

Glomerular Filtration Rate: Defined as volume of filtrate made by kidneys per minute.

Measuring GFR-
1. helps in diagnosis and monitoring renal diseases
2. GFR is guide to dosage of renal excreted drugs
3. Renal replacement.
Procedure:
For Creatinine clearance 24 hour urine specimen is collected and blood taken during the day
and creatinine in both blood and urine estimated. Urine volume in ml/min is calculated by
dividing the urine volume (ml) by the time of collection (min).
Normal GFR range: male 95-135 ml/min
Female 85-125 ml/min

Creatinine clearance C= U x V/P

U = mg of creatinine in 100 ml of urine

P = mg of creatinine in 100ml of plasma
V = volume of urine formed / min

A value below 75% of the normal creatinine clearance indicates impairedrenal function.
Impaired renal function:
1. Acute tubular necrosis
2. glomerulonephritis
3. acute renal failure
4. chronic renal failure
5. end stage renal diseases
6. hypovolemic shock
7. dehydration
8. congestive cardiac failure

ESTIMATION OF SERUM TOTAL PROTEINS

 (BIURET METHOD)

Estimation of protein and albumin in the serum is one of the most important routine tests
performed in the clinical laboratory for the evaluation of liver function.

SIGNIFICANCE:
Most of the plasma proteins expect globulins are synthesized in the liver. Several diseases lead
to characteristic alteration in the plasma proteins, e.g. advanced liver disease leads to a
decrease in the level of several plasma proteins, particularly albumin. Albumin is the most
abundant plasma protein. It helps in the maintenance of colloid osmotic pressure and transport
of compounds like free fatty acids, hormones, bilirubin, drug etc.

AIM: To estimate the serum total proteins by Biuret method.

PRINCIPLE:
This method is based on the formation of violet Colored Complex when the peptide bonds in
proteins react with cupric ions in alkaline medium. The intensity of the violet color is
proportional to the protein concentration and the color is read at 540nm.

REAGENTS:
1. Working Biuret reagent: contains NaOH, Na-K tartarate ,CuSo4. It is ready to use and
stable at room temperature.
2. Protein standard: 7 gms/dl
3. serum/plasma
4. Blank: dilute working reagent with distilled water.

PROCEDURE:
Mark three tubes as Blank (B), Standard (S) & Test (T) & proceed as follows.
S.No. Contents B S T
1 Distilled water (ml) 1.025ml 1ml 1ml
2 Standard solution (ml) - 0.025 ml -
3 Serum (ml) - - 0.025 ml
4 Working biuret reagent 0.5ml 0.5 ml 0.5ml
Mix & let stand for 15min.Read O.D at540nm.

CALCULATION:
Concentration of serum Total proteins = O.D of T – O.D of B X conc of the Std. X 7
O.D of S – O.D of B Volume of the Sample

=…………………g/dl

RESULT:
The amount of total protein present in the given serum sample is ___ g/dl.

CLINICAL SIGNIFICANCE:-
Total protein concentration in Serum is 6 – 8 g/dl in normal subjects.
Albumin normal range: 3.5 – 5 g/dl.
 The albumin globulin ratio is 2:1
 Hypoalbuminemia can be due to either loss of albumin in kidney disease, impaired
synthesis in some liver diseases, and inadequate supply of dietary protein (or) excessive
protein catabolism.

Causes of hypoproteinemia:
1. dietary: malnutrition
2. Intestinal: malabsorption, protein losing enteropathy.
3. hepatic: cirrhosis
4. renal: nephritic syndrome
5. Severe burns.

Causes of hyperproteinemia:
1. dehydration
2. Multiple myeloma
 ESTIMATION OF CSF GLUCOSE
Glucose Oxidase Peroxidase Method

Cerebrospinal fluid (CSF) is found in the sub-arachnoid space ,ventricles of the brain and
around the spinal cord. The total volume of CSF is about 125 ml in a healthy adult and is
renewed every 3-4hrs. CSF is obtained by passing a sterile needle between the L 3 and L4
vertebrae(Lumbar puncture)

AIM:
To estimate the amount of glucose present in the given CSF sample by GOD-POD Method
( Glucose Oxidase Peroxidase Method).

PRINCIPLE:
Glucose is oxidized by glucose oxidase (GOD) to give gluconic acid and hydrogen
peroxide. The hydrogen peroxide formed is broken down by peroxidase to water and nascent
oxygen. The latter oxidizes phenol which combines with 4-aminoantipyrene (4-AAP) to give a
red colored complex (quinoneimine dye). The intensity of the red colored complex is
proportional to the concentration of glucose in the test. The intensity of the coloured complex
is measured colorimetrically at 515nm (500 – 530).

REACTION glucose oxidase

Glucose + O2 + H2O2 gluconic acid + H2O2
peroxidase
H2O2 H2O + (O)
(O) + phenol + 4- AAP Quinoneemine dye + H2O

REAGENTS:

1) Working glucose reagent has to be stored at 2 -8o C in dark bottle.

2) CSF Sample
3) Glucose standard – concentration in 100 mg/dl.
PROCEDURE:
Take 3 test tubes and label them as Blank (B), Standard (S) and Test (T) and proceed with the
tests as follows.

BLANK (B) STANDARD (S) TEST (T)

REAGENTS
ml ml ml
working glucose
reagent 1 ml 1 ml 1 ml

CSF – – 10 µl

Standard
– 10 µl –

Distilled water
10 µl – –

Mix the contents of the test tubes thoroughly, and incubate at room temperature for 15minutes
and read at 515 nm.

CALCULATION:
Glucose present in OD of test (T) – OD of Blank (B) Conc. Of Std
100 ml of CSF = X X 100
OD of Std. (S) – OD of Blank (B) Volume of the Sample

REPORT: Glucose present in 100 ml of CSF = – – – – – mg/dl

Normal Value: 50-80 mg/dl( approx. 60% of blood Glucose)

 ESTIMATION OF PROTEINS IN CSF

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.

1. Estimation of C.S.F. Total Proteins by Sulphosalicylic Acid Method

AIM:
To estimate the amount of protein in C.S.F by sulphosalicylic acid precipitation method.

PRINCIPLE:
Proteins in C.S.F are precipitated using Sulphosalicylic acid. The turbidity produced is
measured in spectrophotometer at 540 nm. The extent of turbidity is proportional to the
protein content. This method is very sensitive & rapid.

REAGENTS:
1) 3% Sulphosalicylic acid: , 3g of Sulphosalicylic acid in 100 ml of distilled water.
2) Standard solution: (0.6 mg/ml) (This is prepared by diluting stock protein std 6gm/dl to
1:100 dilution).

PROCEDURE:
Take three tubes B, S & T
Contents Blank (B) Standard Test (T)
(S)
Standard (mL) – 1ml –
Test (mL) – – 1ml
Distilled water 1ml - -
3% sulphosalicylic acid 4ml 4ml 4ml

Mix & take the readings after 10 mins at 640nm.

CALCULATION:
Mg of total proteins per 100 ml C.S.F. = T –B × Conc of std. X 100 (mg//dl)
S–B Volume of the sample

Mg of total proteins per 100 ml C.S.F. = T –B X 60 (mg//dl)

S–B

2. PANDY’S TEST:
REAGENT :Prepared by dissolving 10 gms of phenol in 150 ml of distilled water. The
solution should be clear and colourless.

PROCEDURE: Add 2 drops of C.S.F to 2 ml of pandy’s solution. Most normal fluids

show no opalescence at all.

Turbidity indicates an increase of globulin in C.S.F.

CLINICAL INTERPRETATION:

Normal value of CSF Protein:15-40 mg %

1) An increase in Total Proteins occurs in all forms of Meningitis - Amoebic &

Trypanosomiasis, Meningoencephalitis, Cerebral malaria, Brain tumors, cerebral
injury, Spinal cord compression, Poliomyelits, Gullian Barre’s syndrome and
Polyneuritis.

2) Increase in C.S.F protein also occurs in diseases, which cause changes in plasma
proteins such as Multiple myeloma.

3) When the total protein exceeds 200 gm or (200mg %), the fibrinogen level is usually
increased sufficient enough for the C.S.F to clot.
THE CEREBROSPINAL FLUID IN HEALTH AND DISEASE

Appearance Pressure Cells Protein Other

(mm CSF) (per mm3) (mg/100 ml)
Normal Clear 100-180 0-5 15-45mg/dl Glucose
Colorless Lymphocytes (50-80mg/dl)

Traumatic Blood at first 100 – 180 Red blood cells Elevated 4 Supernatant clear/
mg per 5000 sometimes with
red blood blood stained.
cells
Subarachnoid Bloody Raised Red blood cells As above Supernatant
hemorrhage Throughout Xanthochromic

Purulent Turbid Raised Polymorphs 100 – 400 low Glucose conc.,

meningitis 100 - 5000 organisms on smear
Viral Clear to Normal Lymphocytes 150 Raised neutrophil
meningitis Opalescent 20 - 2000 count.

Tuberculous Clear to Raised Lymphocytes 80 - 400 low Glucose conc.,

meningitis cloudy Up to 500 organisms on smear
or culture
Neuro Clear Normal Lymphocytes Up to 100 Positive antibody
syphilis Up to 500 titres

Multiple Clear Normal Normal or up Normal up Raised γ-globulins,

sclerosis to 20 to 120 Oligoclonal bands
Lymphocytes