APPENDIX I

ESTIMATION OF ACID PHOSPHATASE (ACP)

(King ,1965)

Principle

The method used was that of King and Armstrong in which disodium
phenyl phosphate is hydrolyzed with the liberation of phenol and inorganic
phosphate. The liberated phenol is measured at 700 nm with Folin-Ciocalteau
reagent.

Reagents

 Citrate buffer: 0.1M, pH 5.

A : Citric acid (21.01 g in 1000 ml)
B: Sodium citrate (29.41 g in 1000 ml)
20.5 ml of A and 29.5 ml of B, diluted to a total of 100 ml.
 Disodium phenyl phosphate, 100 mmol/L: Dissolved 2.18 g in water,
heated to boil, cooled and made to a litre. Added 1.0 ml of chloroform and
stored in the refrigerator.
 Buffer - substrate: Prepared by mixing equal volume of the above two
solutions. This has a pH of 5.0.
 Folin-Ciocalteau reagent: Mixed 1.0 ml of reagent with 2.0 ml of water.
 Sodium carbonate solution, 15%: Dissolved 15 g of anhydrous sodium
carbonate in 100ml of water.
 Standard phenol solution, 1g/L: Dissolved 19 g pure crystalline phenol in
100 mmol/L HCl and made to a litre with the acid.
 Working standard solution: Diluted 10ml of stock standard to 100ml with
water. This contains 100μg phenol/ml.
Procedure

Pipetted out 4.0ml of the buffered substrate into a test tube and incubated at
37°C for 5 min. added 0.2 ml of sample and incubated further for exact 60 mins.
Removed and immediately added 1.8 ml of diluted phenol reagent. At the same
time a control was set up containing 4.0 ml buffered substrate and 0.2 ml of
sample which 1.8 ml of phenol reagent was added immediately. Mixed well and
centrifuged. To 4.0 ml of the supernatant added 2.0 ml of sodium carbonate. Took
4.0 ml of working standard solution and for blank taken 3.2 ml water and 0.8 ml of
phenol reagent. Then added 2.0 ml of sodium carbonate. Incubated all the tubes at
37°C for 15 min. Read the color developed at 700 nm.
The activity of serum acid phosphatase was expressed in μmoles of phenol
liberated / L. The activity in tissue homogenate was expressed as nmoles of phenol
liberated / min / mg protein.

APPENDIX II
ESTIMATION OF ALKALINE PHOSPHATASE (ALP)
(King and Armstrong, 1934)

Principle

The method used was that of King and Armstrong in which disodium
phenyl phosphate is hydrolyzed with the liberation of phenol and inorganic
phosphate. The liberated phenol is measured at 700nm with Folin -Ciocalteau
reagent.

Reagents

 Sodium carbonate - sodium bicarbonate buffer, 100mmol/L: Dissolved

6.36g anhydrous sodium carbonate and 3.36g sodium bicarbonate in water
and made to a litre.
  Disodium phenyl phosphate, 100mmol/L: Dissolved 2.18 g in water, heated
to boil, cooled and made to a litre. Added 1.0 ml of chloroform and stored
in the refrigerator.
 Buffer-substrate: Prepared by mixing equal volume of the above two
solution. This has a pH of 10.
 Folin-Ciocalteau reagent: Mixed 1.0 ml of reagent with 2.0 ml of water.
 Sodium carbonate solution, 15%: Dissolved 15 g of anhydrous sodium
carbonate in 100ml of water.
 Standard phenol solution, 1g/L: Dissolved 1.0 g pure crystalline phenol in
100 mmol/L HCl and made to a litre with the acid.
 Working standard solution: Added 100 ml diluted phenol reagent to 5.0 ml
of stock standard and diluted to 500 ml with water. This contains 10 μg of
phenol / ml.

Procedure

Pipetted out 4.0 ml of the buffer substrate into a test tube and incubated at
37°C for 5 min. added 0.2 ml of sample and incubated further for exact 15 min.
Removed and immediately added 1.8 ml of diluted phenol reagent. At the same
time a control was set up containing 4.0 ml buffer substrate and 0.2 ml of sample
to which 1.8 ml of phenol reagent was added immediately. Mixed well and
centrifuged. To 4.0 ml of the supernatant added 2.0 ml of sodium carbonate. Took
4.0 ml of working standard solution and for blank taken 3.2 ml of water and 0.8 ml
of phenol reagent. Then added 2.0ml of sodium carbonate. Incubated all the tubes
at 37°C for 15 min. Read the color developed at 700 nm.
The activity of serum alkaline phosphatase was expressed in μmoles of
phenol liberated/ L. The activity in tissue homogenate was expressed as μmoles of
phenol liberated / min / mg protein.

ALT
L – Alanine + α-ketoglutarate Pyruvate + L-Glutamate
 APPENDIX III

ESTIMATION OF ASPARTATE TRANSAMINASE (AST)

(Reitman and Frankel, 1957)

Principle

The enzyme catalyses the following reaction

AST
L – Aspartate + α-ketoglutarate Oxaloacetate + L-Glutamate

The oxaloacetate is measured by the reaction with 2,4 dinitrophenyl

hydrazine giving a brown colored hydrazone after the addition of sodium
hydroxide. The color developed is read at 520 nm.

Reagents
 Phosphate buffer: 0.1M, pH 7.5.
Solution A: 0.1M solution of monobasic sodium phosphate.
Solution B : 0.1M solution of dibasic sodium phosphate
Mixed 16ml of A and 84ml of B, diluted to a total of 200ml.
 Substrate : Dissolved 146mg of α-ketoglutarate and 13.3gm of aspartic acid
in 1N NaOH with constant stirring. Adjusted the pH to 7.4 and made upto
1000ml with phosphate buffer.
 Standard pyruvate, 2mM : Dissolved 22 mg of sodium pyruvate in 100ml
of phosphate buffer 0.2ml of standard contains 0.4mM of sodium pyruvate.
 Dinitrophenyl hydrazine reagent, 1mmol / L: 200mg/ L in 1mol / L HCl.
 0.4N NaOH : Dissolved 16g of NaOH in 1L distilled water.

Procedure

0.2ml of sample and 1.0ml of the buffer substrate was incubated for 60min
at 37°C. To the control tubes, enzyme was added after arresting the reaction with
1.0ml of DNPH and the tubes were kept at room temperature for 20min. Then
10ml of 0.4N sodium hydroxide was added. A set of standard pyruvate were also
treated in a similar manner. The color developed was read at 520nm.
 The enzyme activity in serum was expressed as μmoles of pyruvate
liberated / L and in liver homogenate as µmoles of pyruvate liberated / min / mg
protein.
APPENDIX IV
ESTIMATION OF ALANINE TRANSAMINASE (ALT)
(Reitman and Frankel, 1957)

Principle

The enzyme catalyses the following reaction:

ALT
L – alanine + α-ketoglutarate Pyruvate + L-glutamate

The pyruvate is measured by the reaction with 2, 4-dinitrophenylhydrazine

giving a brown colored hydrozone after the addition of sodium hydroxide. The
color developed is read at 520nm.

Reagents

 Phosphate buffer : 0.1m, pH 7.5.

 Substrate : Dissolved 146mg of α-ketoglutarate and 17.8 g of L-alanine in
1N NaOH with constant stirring. Adjusted the pH to 7.4 and made up to
1000ml with phosphate buffer.
 Standard pyruvate, 2mM : Dissolved 22 mg of sodium pyruvate in 100ml
of phosphate buffer. 0.2ml of standard contains 0.4µM of sodium pyruvate.
 Dinitrophenyl hydrazine reagent, 1mmol/L : 200mg/L in 1mol / L HCl.
 0.4 N NaOH : Dissolved 16 g of NaOH in 1L distilled water.

Procedure

0.2ml of sample and 1.0 ml of the buffer substrate were incubated for 30
min at 37°C. To the control tubes, enzyme was added after arresting the reaction
with 1.0 ml of DNPH and the tubes were kept at room temperature for 20 min.
Then 10 ml of 0.4 N NaOH was added. A set of standard pyruvate was also treated
in a similar manner. The color developed was read at 520nm.

The enzyme activity in serum was expressed as μmoles of pyruvate

liberated / L and in liver homogenate as μmoles of pyruvate liberated/min/mg
protein.
APPENDIX V
ESTIMATION OF LACTATE DEHYDROGENASE (LDH)
(King, 1965b)

Principle

The lactate is acted upon by lactate dehydrogenase to form pyruvate in the

presence of NAD. The pyruvate forms pyruvate phenyl hydrazone with
2,4-dinitrophenyl hydrazine. The color developed is read in a spectrophotometer at
440 nm.

Reagents

 Glycine buffer, 0.1 M, pH 10: 7.505 g of glycine and 5.85 g of

sodium chloride were dissolved in 1 litre of water.
 Buffered substrate: 125 ml of glycine buffer and 75 ml of 0.1N
NaOH were added to 4.0 g of lithium lactate and mixed well.
 Nicotinamide Adenine Dinucleotide: 10mg of NAD was dissolved in
2.0 ml of water.
 2, 4-Dinitrophenyl hydrazine: 20 mg of DNPH was dissolved in 100
ml of 1N HCl.
 0.4 N NaOH.
 Standard pyruvate, 1μmol/ml: 11 mg of sodium pyruvate was
dissolved in 100 ml of buffered substrate (1 μmole of pyruvate /ml).
 NADH solution, 1 μmol/ml: 8.5 mg/10 ml buffered substrate.
Procedure

Placed 1.0 ml buffered substrate and 0.1 ml of sample into each of two
tubes. Added 0.2 ml of water to the blank. Then to the test added 0.2 ml of NAD.
Mixed and incubated at 37°C for 15 min. exactly after 15 min. 1.0 ml of
dinitrophenyl hydrazine was added to each (test and control). Left for further
15 min. Then added 10 ml of 0.4 N Sodium hydroxide and the color developed
was read immediately at 440 nm. A standard curve with sodium pyruvate solution
with the concentration range 0.1 -1.0 μmole was taken.
LDH activity in serum was expressed as μmoles of pyruvate liberated / L
and in liver homogenate as nmoles of pyruvate liberated / min / mg protein.

APPENDIX VI

ESTIMATION OF GLYCOLIC ACID OXIDASE

(Richardson and Tolbert, 1961)

Principle

The principle of the method is based on converting the substrate, glycollate

to glyoxylate by the liver enzyme GAO. The glyoxylate is then made to react with
phenyl hydrazine to form the hydrozone which on oxidation with potassium
ferricyanide in the presence of HCl forms a formazan which is a pink coloured
compound, can be measured at 517 nm .

Glycolate Glyoxylate
Glyoxylate + Phenyl hydrazine Glyoxylate phenyl hydrazone

Reagents

 0.25 M Potassium phosphate buffer, pH 8.3: 2.72 g of potassium

dihydrogen phosphate was dissolved in 100 ml of distilled water. To this
about 10 drops 5N NaOH was added to bring the pH to 8.3. It was stored in
the cold room.
  Substrate : 10 nM Sodium glycollate
 8% TCA
 2% phenyl hydrazine
 10% potassium ferricyanide
 Chilled concentrated HCl
 0.05 M Sodium phosphate buffer pH 7.0
 Standard glyoxylate.

100 mg of Sodium glyoxylate was dissolved in 100 ml of water. The

working standard was prepared by diluting 0.1 ml the above stock solution with
5.0 ml of water. This contained 20µg of glyoxylate / ml of sodium glycolate was
added and the incubation continued for 30 min with constant shaking at 37 oC. The
reaction was stopped by adding 0.7 ml of 8% TCA. Enzyme blanks were set up in
which the enzyme was added after arresting the reaction with TCA. All the tubes
were centrifuged at 3000 rpm for 10 min and the glyoxylate content in supernatant
was estimated as below.

0.2 ml of the above supernatant was made upto 1 ml with distilled water,
followed by the addition of 1.0 ml of sodium phosphate buffer. The tubes were
kept at 25oC for 10 min. Then 0.1 ml of phenyl hydrazine was added. Exactly after
2 min, 1.2 ml of cold concentrated HCl was added slowly. Within 1 min after the
addition of acid, 0.1 ml of 10% potassium ferricyanide was added. The colour
developed was read after 1 min at 540 nm in a spectrophotometer. Standards were
also set up side by side with sodium glyoxylate. The same procedure was followed
from the addition of sodium phosphate buffer.

One unit of enzyme activity is defined as the enzyme required to produce

one nanomole of glyoxylate / mg protein / min at 37oC.
 APPENDIX VII

ESTIMATION OF PROTEIN
( Lowry et al., 1951)

Principle

The blue color developed by the reduction of the phosphomolybdic

phosphotungstic components in the Folin Ciocalteau reagent by the amino acids
tyrosine and tryptophan present in the protein plus the color developed by the
biuret reaction of the protein with the alkaline cupric tartarate are measured at 660
nm.

Reagents

 2.0 % sodium carbonate in 0.1 N NaOH (Reagent A)

 0.5 % Copper sulphate in 1.0 % potassium sodium tartarate (Reagent
B)
 Alkaline copper reagent: Mixed 50 ml of A and 1.0 ml of B prior to
use
 Folin-Ciocalteau reagent: Mixed 1 part of reagent with 2 parts of
water.
 Stock standard: Weighed 50 mg of bovine serum albumin and made
up to 50 ml in a standard flask with saline.
 Working standard: Diluted 10 ml of the stock of 50 ml with distilled
water. 1.0 ml of this solution contains 200 μg of protein.
Procedure

Pipetted out 0.2 to 1.0 ml working standard solution, 0.1 ml of the sample
was taken. The volume in all the tubes were made up to 1.0ml with distilled water.
Added 5.0 ml of alkaline copper reagent to each tube. Mixed well and allowed to
stand for 10 mins. Then added 0.5 ml of Folin-Ciocalteau reagent. Mixed well
and incubated at room temperature for 30 minutes. A reagent blank was also
prepared. After 30 minutes, the blue color developed was read at 660 nm.
The serum protein was expressed as g / dl and in liver homogenate as mg /
g tissue.
APPENDIX VIII

ESTIMATION GLUCOSE
(Sasaki et al., 1972)

Principle

Ortho toludine reacts with glucose in hot acetic acid solution to produce
blue color, which is measured at 630nm.

Reagents

 Ortho toludine boric acid reagent: This reagent consists of 2.5g of

thiourea and 2.4g of boric acid in 100ml of a mixture of water, acetic acid
(AR) and ortho toludine (distilled) in the ratio of 10:75:15.
 Standard glucose: 100mg of glucose in 0.1%benzoic acid. 10ml of
the above solution was diluted to 100ml to give 100μg of glucose per ml.
Procedure
To 0.2 ml of serum added 0.8ml of 10% TCA. Mixed well and centrifuged. 0.5
ml of the supernatant was taken. To this 2.0-ml of ortho toludine reagent was
added and heated in a boiling water bath for 15min along with standard solution
containing 20-100 μg of glucose. The blue color developed was read at 640nm.

The result was expressed as mg/dl in serum and urine.

 APPENDIX IX
ESTIMATION OF UREA

(Varley, 1976)

Principle

Diacetyl monoxime in the presence of acid, hydrolysis to produce the

unstable compound diacetyl. This reacts with urea to produce a yellow diazone
derivative. The color of this product becomes pink by addition of
thiosemicarbazide which is measured colorimetrically at 520nm.

Reagents

 TCA, 10%
 Stock diacetylmonoxime, 25g/l
 Stock thiosemicarbazide 2.5g/l
 Acid ferric chloride solution: Added 1.0 ml sulphuric acid to 100 ml of
ferric chloride solution containing 50 g/L in water.
 Acid reagent: Added 10ml of ortho phosphoric acid, 80 ml sulphuric acid
and 10 ml acid ferric chloride solution to 1 litre of water and mixed.
 Color reagent: To 300 ml acid reagent added 200 ml water, 10 ml stock
diacetylmonoxime and 2.5 ml thiosemicarbazide.
 Stock urea standard: 5, 10, 15, 20, 30, 40, and 50 mmol/l (30, 60, 90, 120,
180, 240 and 300 mg/100 ml).
Procedure

To 0.2 ml of serum added 1.0 ml water and 1.0 ml of 10% TCA. Mixed well
and centrifuged. 0.2 ml of the supernatant was taken and added 3.0 ml of color
reagent. And it is kept in the water bath for 20 min. Cooled to room temperature
and read the color developed at 520 nm within 15 min.

The result was expressed as mg/dl in serum and urine.

 APPENDIX X
ESTIMATION OF URIC ACID
(Caraway, 1963)
Principle

Uric acid is oxidized to allantoin and carbondioxide by phosphotungstic

acid reagent in alkaline solution. Phosphotungstic acid is reduced in this reaction
to tungsten blue, which is measured at 660nm.

Reagent

 Phosphotungstic acid reagent.

 10% Sodium carbonate.

 Standard uric acid: 100 mg of uric acid and 60mg of lithium carbonate were
taken in a breaker and about 50ml of water was added. This was heated to
about 60oC to dissolve the uric acid completely. After cooling, the solution
was finally made upto 100 ml with water.

 Working standard: Dilute 1.0 ml of the stock standard to 10ml with water.
1.0 ml of this solution contains 20 μg of uric acid.

Procedure

0.1ml of the sample was taken and to this 2.9 ml of water was added
followed by 0.6 ml each of phosphotungstic acid and sodium carbonate. A blank
was set up with 3.0 ml water. Standard were also treated in a same manner. The
color was read at 640 nm after 10min.

The result was expressed as mg/dl in serum and urine.

 APPENDIX XI
ESTIMATION OF CREATININE

(Owen et al., 1954)

Principle

Creatinine forms a coloured complex with picrate in alkaline medium. The

rate of formation of the complex is measured at 540nm.

Reagents

 Picric acid: 8.02g/L

 Sodium hydroxide: 12.8g/L

 Standard creatinine: Dissolved 100 mg of creatinine in 100ml with distilled

water.

 Working standard: Diluted 2.0 ml of stock solution was diluted to 100 ml

with distilled water. This contains 20μg of creatinine / ml.

 Reagent mixture: Mixed one part by volume of diluted NaOH with one part
by volume of picric acid at least 30 minutes before the assay.

Procedure

Pipetted out 0.2ml of serum and 2.0ml of the reagent mixture in to a

cuvette. Simultaneously, a blank was set up with the reagent mixture and distilled
water. Mixed well and the change in absorbance was measured after 30sec,which
was taken as A1 and exactly after 2 min, the absorbance was read as A2 at 490nm.
Sets of standards were also treated in the same manner. A1-A2 gives the change in
absorbance, which was the measure of the creatinine present in the sample.

The result was expressed as mg/dl in serum and urine. The values are
expressed as mg of creatinine / dl.
 APPENDIX XII
ESTIMATION OF SODIUM AND POTASSIUM
(Flame photometry method)
Principle
The sample in solution is introduced in form of a fine continuous spray
into a non- luminous gas flame. The emitter light characterized for the ion being
analysed is isolated and focused on a photoelectric cell and current intensity is
measured as suitable meter.

Reagents

 Stock solution: Weighed accurately 630mg of sodium chloride and

477mg of potassium chloride and dissolved in exactly 250 ml of double
distilled water.
 Working standard solution: Diluted 1:100 to get 1 mg sodium/ 100 ml
and 1 mg potassium / 100 ml which are equivalent to 10 ppm.

Procedure
Both the instrument main and compressor were switched on and the
water spray was checked using water at inlet. The gas connector was opened
and lit the flame using a gas lighter. Aspirated the standard solution of sodium /
potassium solution and sample readings were noted.

The amount of sodium and potassium in serum was expressed as mg/dl

The amount of sodium and potassium in Urine was expressed as mg/day
 APPENDIX XIII
ESTIMATION OF CHLORIDE
(Van Slyke method)
Principle
Using a solution of acidic silver nitrate, chloride is precipitated as
silver chloride and the excess silver nitrate is then titrated against standard
thiocyanate using ferric alum as the indicator.

Reagents

 Silver nitrate 0.05 N solution: Dissolved 8.495g of silver nitrate in about

250 ml of double distilled water and then made up to the litre with the
same. The solution was standardized against sodium chloride and stored
in a brown bottle.
 Thiocyanate 0.02 N solution.
 3.5% solution of ferric alum.

Procedure

Placed 0.5 ml of sample in a clean dry conical flask. Added 3.0 ml of

silver nitrate with constant shaking followed by 2.0 ml of concentrated nitric
acid. To this added 6.0 ml of ferric alum. Then titrated with 0.02 N thiocyanate
until a reddish brown colour persisting for 10-15 seconds was obtained. To
determine the standard, added 2.0 ml of nitric acid and 6.0 ml of ferric alum
solution to 3.0 ml of silver nitrate solution. Cooled and titrated with 0.02 N
thiocyanate. The difference between the two titre values, (Titre value of
standard – titre value of test solution) gives a measure of the amount of chloride
in 0.5 ml of the given test solution in terms of 0.02 N thiocyanate solution.
The amount of chloride present in serum sample was expressed as mg/dl.
The amount of chloride present in urine sample was expressed as
mg/day.
 APPENDIX XIV
ESTIMATION OF OXALATE
(Hodgkinson and Williams, 1972).
0.5 ml of sample was added into a 25 ml graduated stoppered centrifuge
tube followed by 1.5 ml of water and 1 drop of 0.04% bromothymol blue
indicator solution. Adjusted the solution to pH 7.0 (green) by the addition of
dilute sodium hydroxide or dilute acetic acid. Added 2.0 ml of saturated
aqueous solution of calcium sulphate, followed by 14 ml of ethanol, mixed
gently and allowed the solution to stand at room temperature for atleast 3 hour.
Centrifuged at 2000 rev/min for 10 min, carefully decanted the supernatant
fluid and allowed the tube to drain for a few minutes on a filter paper and
dissolved the precipitate in 2 ml of 2N sulphuric acid solution.

Reagents

 Electrolyte zinc wire, diameter 3mm was cut into short lengths
measuring approximately 250 mg. Immediately before use, zinc was
cleaned by immersing briefly in freshly prepared 10 N nitric acid. After
washing thoroughly in distilled water the zinc was ready for use.
 Chromotropic acid solution: Dissolved 1 g of 4, 5-dihydoxy naphthalene,
2, 7-disulphonic acid (disodium salt) in100 ml of water. Stored at 4ºC
and prepared fresh once a week.
 Oxalic acid standard: Dissolved 1.023 g of potassium oxalate
monohydrate in 100 ml of water. This solution contained 5.0 mg of
anhydrous oxalic acid/ml.

Procedure

0.1 ml of aliquot was taken in a boiling tube and made up to 1.0 ml with
water. One ml of 4N sulphuric acid was added. Then added a piece of freshly
cleaned zinc. Heated in a boiling water bath for 30 min. Removed zinc, washed
with 0.5 ml of 1% chromotropic acid solution. Standards in the range of 20-
200µg were taken and treated as above. Added 5.0 ml of concentrated sulphuric
acid to both standard and test solutions. Heated for 30 min in a boiling water
bath. Added 20 ml of 10N sulphuric acid to all the tubes through their slides
slowly, with constant shaking. Cooled and read at 570 nm. The colour was
stable for several hours.
The amount of oxalate present in serum sample was expressed as mg/dl.
The amount of oxalate present in urine sample was expressed as mg/day.

APPENDIX XV
ESTIMATION OF CALCIUM
(O.C.P.C Method)
Principle

At alkaline pH, calcium binds with orthocresolphthalein complexone

(OCPC) to form a bluish-purple complex. The intensity of the colour so formed
is proportional to calcium concentration and is measured at 548 nm.
Interference from magnesium is overcome by the presence of 8-
hydroxyquinoline in reagent 1, which binds free magnesium ions.

Reagents
Reagent 1 OCPC reagent OCPC, Hydrochloric Acid,
-Hydroxyquinolin,Preservativ
e.
Reagent 2 AMP buffer AMP buffer, pH 10.4
Preservative.
Reagent 3 Calcium carbonate,
10 mg/ dL hydrochloric acid
 Procedure
Pipette into tubes Blank Standard Test
marked
Serum, Plasma, - - 20µl
Urine
Calcium Standard - 20µL -
Working Calcium 1.0 ml 1.0 ml 1.0 ml
Reagent

Mix well. Incubate at 370 C or 15-250C for 5 min.

1. Blank the analyzer with the reagent blank.
2. Aspirate standard followed by tests.
3. Read absorbance against blank at 540-580nm.

The amount of oxalate present in serum sample was expressed as mg/dl.

The amount of oxalate present in urine sample was expressed as mg/day.

APPENDIX XVI
ESTIMATION OF MAGNESIUM
(Calmagite Method)
Principle

Magnesium present in sample complexes with titan yellow in alkaline medium

to give a red color which is read at 540 nm.

Reagents
 Titan yellow: Dissolved 75 mg of titan yellow in 100 ml of double
distilled water. Diluted 1 volume of solution in 4 volumes of double
distilled water before use.
 10% sodium hydroxide.
  Magnesium standard (5 mg/ dl): Dissolved 50 mg of magnesium
sulphate in 100 ml of double distilled water.
 5 % TCA.

Procedure

Pipetted out 0.2 to 1.0 ml of standard magnesium solution in to a series

of test tubes corresponding 10µg values 10 to 50. Set up a blank with 3 ml
water. Made up the volumes in all tubes to 3 ml with water. 1 ml of sample was
mixed with 4 ml of 5 % TCA. Centrifuged and 3 ml of clear supernatant was
taken. To all tubes added 1 ml of titan yellow and 1 ml of sodium hydroxide.
The colour developed was read immediately at 540 nm.

The amount of magnesium in the serum was expressed as mg/dl.

The amount of magnesium in the urine was expressed as mg/day.

APPENDIX XVII
ESTIMATION OF PHOSPHORUS
( UV Molybdate method)
Principle

Phosphorus reacts with molybdate to form phosphomolybdate. The increase

in absorbance, due to formation of this complex is measured at 340 nm and is
proportional to the concentration of phosphorus in the sample.

Reagents
Reagent 1 Molybdate reagent :Ammonium molybdate, surfactant.
Reagent 2 Sample blank reagent: Sodium chloride, preservative, surfactant
Reagent 3 Phosphorus standard 5mg/dL : Phosphate, diluent

Procedure
 Pipette into tubes marked Blank Standard Test

Sample _ _ 10µl

Phosphorus standard _ 10µl _

Molybdate reagent 1.0 ml 1.0ml 1.0ml

Mix well. Incubate at 370 C for 5 minutes. Read absorbance against reagent blank
at 340nm.
The amount of phosphorus in the serum was expressed as mg/dl.
The amount of phosphorus in the urine was expressed as mg/day.

APPENDIX XVIII

ESTIMATION OF Na+ K+ ATPase

(Bonting, 1970)
Principle

Na+K+ ATPase transports Na, K against concentration gradient at the cost of

ATP molecule liberating inorganic phosphate (Pi). The inorganic phosphorous
liberated is estimated by Fiske and Subbarow method.
Reagents
 184 mM Tris- HCl buffer, pH 7.5
 50mM MgSO4
 50mM KCl
 600mM NaCl
 1mM EDTA
 40mM ATP

Procedure
 1.0 ml of tris buffer and 0.2 ml of each of the above reagents were mixed
together. Thus the assay medium in a final volume of 2.0 ml, contained 92 mM tris
buffer, 5 mM MgSO4, 60 mM NaCl, 1 mM EDTA and 4 mM ATP. After 10min,
equilibration at 37°C in an incubator, reaction was started by the addition of 0.1 ml
of homogenate. The assay medium was incubated for 15 min. after incubation the
reaction was arrested by the addition of 1.0 ml of 10% TCA. The phosphorus
content in the supernatant was estimated by Fiske and Subbarow method.

The enzyme activity is expressed as micromoles of Pi liberated / min / mg

protein.

APPENDIX XIX
ESTIMATION OF Mg2+ ATPase
(Ohnishi et al., 1982)

Principle

The activity of enzyme was estimated by the inorganic phosphorus

liberated is estimated by Fiske and Subbarow method.

Reagents
 375 mM Tris- HCl buffer pH 7.6
 25 mM MgCl2
 10 mM ATP

Procedure

The assay was estimated by the addition of 0.1 ml of homogenate to an

incubation medium containing 0.1 ml of water and 0.1 ml of each of the above
reagents. The final concentration of tris buffer, MgCl2 and ATP were 75 mM, 5
mM and 2 mM in total incubation volume of 0.5 ml. The reaction was terminated
after 15 min by the addition of 1.0 ml of 10 % TCA. The liberated Pi was
estimated by the method of by Fiske and Subbarow.
The enzyme activity is expressed as micromoles of Pi liberated / min / mg
protein.

APPENDIX XX
ESTIMATION OF Ca2+ ATPase
(Hjertan and Pan, 1983)

Principle
The activity of enzyme was estimated by the inorganic phosphorus
liberated is estimated by Fiske and Subbarow method.

Reagents

 375 mM Tris- HCl buffer pH 7.6

 25 mM CaCl2
 10 mM ATP

Procedure

The assay was estimated by the addition of 0.1 ml of homogenate to an

incubation medium containing 0.1 ml of water and 0.1 ml of each of the above
reagents. The final concentration of Tris buffer, CaCl 2 and ATP were 75 mM,
5 mM and 2 mM in total incubation volume of 0.5 ml. The reaction was
terminated after 15 min by the addition of 1.0 ml of 10 % TCA. The liberated
Phosphorous was estimated by the method of by Fiske and Subbarow.
The enzyme activity is expressed as micromoles of Pi liberated / min / mg
protein.
 APPENDIX – XXI

ESTIMATION OF β-D-GLUCURONIDASE

(Kawai and Anno, 1971).

Reagents
 Acetate buffer 0.1 M pH 4.5
Solution A : 410.2 mg of sodium acetate was dissolved in 50 ml of distilled
water.
Solution B: 0.29 ml acetic acid was mixed with 50 ml distilled water. 4.9
ml of solution A and 5.1 ml of solution B were mixed before use.
 Glycine buffer pH 10.7. This was prepared by mixing equal volume of
0.2M glycine, 0.125 M sodium carbonate and 0.1 M sodium chloride in
distilled water.
 Substrate : p-nitrophenyl-β-D-glucuronide one mg / ml in distilled water.
 Standard : Five mg of p-nitrophenol in 100 ml of distilled water.

Procedure
0.5 ml of subrate, 0.05 ml of acetate buffer, 0.3 ml of homogenate were
incubated at 37oC for 1 hour. The reaction was arrested by the addition of 3.9 ml
of glycine buffer. Standards were also run simultaneously along with a blank. The
colour developed was read at 420 nm using a colorimeter.

The enzyme activity is expressed as µmoles of p-nitrophenol liberated / L.

 APPENDIX XXII
ESTIMATION OF DNA

(Raghuramalu et al., 1983)

Principle

Under extremely acidic conditions DNA initially depurinates quantitatively

followed by dehydration of sugar to ω-levulinyl aldehyde. This aldehyde
condenses in acidic medium with diphenylamine to produce a deep colored
condensation product with absorption maximum at 600nm.

Reagents
 DNA stock standard: 60mg of DNA was dissolved in 5mM NaoH
and made upto 100 ml with the same.
 Working standard: To 5 ml of stock standard added 5.0 ml of 1.2 N
perchloric acid heated at 90˚c for 15 min. and cooled. 1.0 ml of this solution
contains 300μg of DNA.
 Saline citrate: 0.15 M sodium chloride (8.78 g/l) and 0.015 M
sodium citrate (4.14 g/l) was mixed.
 Diphenylamine reagent: 1.5 g of diphenylamine in 100 ml of glacial
acetic acid and 1.5 ml of concentrated sulphuric acid. Warm to room
temperature and swirled to remix before use. Stable for six months at 2˚C.
 0.2N, 0.6 N and 1.2N perchloric acid
 0.3N Potassium hydroxide: Dissolved 1.71g in 100ml/water
 7.5% and10% TCA
 Extraction of the sample

10% tissue homogenate of liver and kidney in ice cold distilled water was
taken. 5.0 ml of the tissue homogenate was pipetted out into a 15 ml centrifuge
tube. Added 2.5 ml of ice cold 0.6 N perchloric acid, mixed and allowed to cool at
0˚C for 10minutes. Centrifuged and discarded the acid soluble supernatant fraction
and washed the precipitate twice with ice cold 0.2N perchloric acid. Drained off
the excess acid by inverting the tube briefly over the filter paper Added 4.0 ml of
ethanol to remove phospholipid. Drained the supernatant and added 4.0 ml of 0.3
M potassium hydroxide and incubated at 37˚C for 1 hour. After incubation cooled
in an ice and precipitated the DNA by adding 6.0 ml of 0.2N perchloric acid. After
10 minutes centrifuged the precipitate and decanted the supernatant RNA fraction.
Washed the precipitate twice with 5.0 ml of 0.2N perchloric acid and added the
washing to the RNA fraction. After the addition of 10.0 ml of 0.6 N perchloric
acid to the RNA fractions and washings, this fraction is made upto 100 ml with
water giving a solution of ribonucleotides in 1.0 N perchloric acid, which was
used for the estimation. The tissue residue is suspended in 1.3 ml of 10% TCA and
heated the mixture for 15 min. at 90˚C with occasional stirring. This splitted the
DNA from tissue proteins and decanted the supernatant DNA fraction. Washed the
precipitate with 2.5 ml of 5% TCA and cooled the washings to get DNA fraction
which was made upto 5.0 ml with standard saline citrate solution.1.0 ml of this
fraction was used for estimation of DNA. The precipitate was dissolved in saline
and 1.0 ml was taken for protein estimation.

Procedure

Pipetted out 0.2 ml to 1.0 ml of the working standard DNA solution

corresponding to μg values 60 to 300. One ml of the sample was taken. Made up
the volume in all the tubes to 2.0 ml with distilled water. Set up a blank along with
the working standard. Added 3.0ml of diphenylamine reagent to each tube and
after mixing heated the tubes in a water bath for 10 min. Removed and cooled the
tubes by immersing in tap water for 5 min. Read the absorbance of blue solution at
600 nm against the blank. The amount of DNA in the sample was expressed as
mg/g tissues.
 APPENDIX XXIII
ESTIMATION OF RNA
(Raghuramalu et al., 1983)
Principle

The RNA content is estimated by orcinal method based on the estimation of

ribose moiety of RNA, which produce a green color with oricinal reagent. The
intensity of the color developed is proportional to the ribose content and is
measured colorimetrically at 665 nm.

Reagent
 RNA stock standard:Dissolved 100 mg of purified RNA in 10 ml of
1N KOH. Incubated for 16-17 hours at 37˚C and made upto 100 ml in a
standard flask with distilled water.
 Working standard: 10ml of the stock was added to 100 ml. 1.0 ml of
this solution contains 100μg of RNA.
 Orcinol reagent: Dissolved 0.34 g of ferric chloride and 0.5 g orcinol
in a little amount of water and made upto 12.5ml with water.
 Dilute-orcinal reagent: 12.5 ml of stock reagent was added to 225ml
of conc. hydrochloric acid and diluted to 250ml with water.

Procedure

Pipetted out 0.2ml to 1.0ml of the working standard RNA solution into a
series of test tubes corresponding to μg values 20 to 100. 0.5 ml of the sample and
1.0ml of the given unknown solution was pipetted out. The volume was made to
2.0ml in all the tubes with distilled water. Set up a blank along with the working
standard. Added 2.0ml of diluted orcinol reagent to all the tubes. The top of the
tubes were covered with marbies and kept in a boiling water bath for
20 min. Removed and cooled the tubes at room temperature and the color
developed was read at 665nm in a spectrophotometer against the reagent blank.
The amount of RNA in the sample was expressed as mg/g tissues.
 APPENDIX XXIV
ESTIMATION OF SUPEROXIDE DISMUTASE (SOD)
(Das et al., 2000)

Principle

The method involves generation of superoxide radical of riboflavin and its

detection by nitrite formation from hydroxylamine hydrochloride. The nitrite
reacts with sulphanilic acid to produce a diazonium compound which subsequently
reacts with naphthylamine to produce a red azo compound whose absorbance is
measured at 543 nm.
Reagents

 50 mM phosphate buffer, pH 7.4

 20 mM L-Methionine
 1 % (v/v) Triton X-100
 10 mM hydroxylamine hydrochloride
 50 μM EDTA
 50 μM riboflavin
 Greiss reagent: 1 % sulphanilamide, 2 % phosphoric acid and 0.1 %
naphthylethylene diamine dihydrochloride.
Procedure

Pipetted out 1.4 ml aliquot of the reaction mixture in a test tube.100 µl of

the sample was added followed by a preincubation at 37°C for 5 min. 80 µl of
riboflavin was added and the tubes were exposed for 10 min to 200 W Philips
fluorescent lamps. The control tube contained equal amount of buffer instead of
sample. The sample and its respective control were run together. At the end of the
exposure time, 1.0 ml of Greiss reagent was added to each tube and the absorbance
of the color formed was measured at 543 nm.
One unit of enzyme activity was defined as the amount of SOD capable of
inhibiting 50 % of nitrite formation under assay condition.
 APPENDIX – XXV
ESTIMATION OF CATALASE (CAT)
(Sinha, 1972)

Principle

Catalase causes rapid decomposition of hydrogen peroxide to water.

Catalase
2H2O2 2H2O+O2

The method was based on the fact that dichromate in acetic acid reduced to
chromic acetate when heated in the presence of H 2O2 with the formation of
perchloric acid as an unstable intermediate. The chromic acetate thus produced
was measured colorimetrically at 610 nm. Since dichromate has to absorbance in
this region, the presence of the compound in the assay mixture did not interfere
with the colorimetric determination of chromic acetate. The catalase preparation
was allowed to split H2O2 for different periods of time. The reaction was stopped
at specific time intervals by the addition of dichromate / acetic acid mixture and
the remaining H2O2 was determined by measuring chromic acetate colorimetrically
after heating the reaction.

Reagents
 0.01 M phosphate buffer, pH 7.0
A: 0.1M monobasic sodium phosphate
B: 0.1M dibasic sodium phosphate
Mixed 39 ml of A and 61 ml of B is diluted to a total of 200 ml. 10 ml of
this solution is further diluted to 100 ml with distilled water.
 0.2 M hydrogen peroxide
 Stock dichromate / acetic acid solution: Mixed a 5 % potassium dichromate
with glacial acetic acid (1:3 by volume).
 Working dichromate/acetic acid solution: The stock was diluted to 1:5 with
water to make the working dichromate / acetic acid solution.
Procedure

The assay mixture contained 0.5 ml of H2O2, 1.0ml of buffer and 0.4 ml of
water. 0.2 ml of the enzyme was added to initiate the reaction. 2.0 ml of the
dichromate / acetic acid reagent was added after 0, 30, 60, 90 seconds of
incubation. To the control tube the enzyme was added after the addition of the acid
reagent. The tubes were then heated for 10 min. and then color developed was read
at 610 nm.
The activity of catalase was expressed as μmoles of H2O2 decomposed /
min / mg protein.

APPENDIX XXVI
ESTIMATION OF GLUTATHIONE PEROXIDASE (GPx)
(Rotruck et al., 1979)

Principle

Glutathione (GSH) was measured by its reaction with DTNB to give a

compound that absorbs at 412 nm.

Reagents

 0.4 M sod ium phosphate buffer, pH 7.0

 10 mM sodium azide
 2.5 mM hydrogen peroxide
 4 mM reduced glutathione
 10 % TCA
 0.3 M phosphate solution
 0.04 % DTNB in 1 % sodium citrate
 Reduced glutathione standard: 20 mg reduced glutathione was dissolved in
100 ml of water.
Procedure

0.4 ml of buffer, 0.1 ml of sodium azide, 0.2 ml of reduced glutathione, 0.1

ml of H2O2, 0.2 ml of enzyme and 1.0 ml of water were added to a final incubation
volume of 2.0 ml. The tubes were incubated for 0, 30, 60, 90 seconds. The
reaction was then terminated by the addition of 0.5 ml TCA. To determine the
glutathione content, 2.0 ml of the supernatant was removed by centrifugation and
added to 3.0 ml disodium hydrogen phosphate solution and 1.0 ml of DTNB
reagent. The color developed was read at 412 nm. Standards in the range of 200-
1000 μg were taken and treated in the similar manner.
The activity was expressed in terms of μg of glutathione utilized / min / mg
protein.

APPENDIX XXVII
ESTIMATION OF GLUTATHIONE REDUCTASE (GR)
(Goldberg and Spooner, 1983)

Principle

Glutathione reductase catalyses the reduction of oxidized glutathione

(GSSG) to reduced glutathione (GSH) and is assayed by measuring the decrease in
absorbance at 340nm.
GR
NADPH (NADH) + GSSG NADP+ (NAD)+ + 2GSH

Reagents

 0.3 M phosphate buffer, pH 6.8.

 25 mM EDTA.
 12.5mM oxidized glutathione.
 3mM NADPH.
Procedure

0.2 ml of sample, 1.5 ml of buffer, 0.5 ml EDTA, 0.2 ml GSSG and 0.1 ml
NADPH was added. The decrease in optical density of the enzyme was measured
against that of the blank at 340 nm.
The enzyme activity is calculated in terms of µmoles of NADPH oxidized /
min / mg protein.

APPENDIX XXVIII
ESTIMATION OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE

(Bailnksy and Bernstein ,1963)

Principle

Glucose 6-phosphate dehydrogenase is assayed by measuring the increase in

absorbance, which occurs at 340nm. When NADP reduces to glucose 6 phosphate
to NADP in the reaction catalysed by glucose 6 phosphate dehydrogenase.
Reagents

 0.1M Tris HCL buffer,Ph 8.2

A: 0.1M solutions of Tris (12.1g/1000ml water)

B: 0.1 M HCl

Mixed 50 ml of solution A and 21.9ml of B and diluted to a total of 200ml

 0.2Mm NADP

 0.1M Magnesium chloride

 6mM glucose-6-phosphate

Procedure

0.4ml of Tris –HCl buffer, 0.2ml of NADP, 0.2ml of magnesium chloride,

1.0ml water and 0.2 ml of enzyme were taken in a cuvette. The reaction was
started by the addition of 0.2ml of glucose-6-phosphate and the increase in OD
was measured at 340nm.

The activity was expressed in terms of units/mg protein, in which one unit
is equal to the amount of enzyme that brought about a change in OD of 0.01/min.

APPENDIX XXIX
ESTIMATION OF GLUTATHIONE-S- TRANSFERASE (GST)
(Habig et al., 1974)
Principle

The enzyme was assayed by its ability to conjugate GSH with CDNB, the
extent of conjugation causing a proportionate change in the absorption at 340nm.

Reagents

 1 mM-1-chloro, 2,4-dinitrobenzene (CDNB) in ethanol.

 1 mM glutathione.
 0.1 M phosphate buffer, pH 6.5.

Procedure

The assay was done at 25°C under conditions giving activities linear with
respect to incubation times and protein concentrations for atleast 3 mins.
The enzyme activity was determined by monitoring the change in
absorbance at 340 nm in a spectrophotometer. 0.1 ml of both substrates (GSH and
CDNB) was taken in 0.1 M phosphate buffer (pH 6.5) at room temperature to
make a volume of 2.9 ml. The reaction was started by adding 0.1 ml of liver
homogenate to this mixture. The readings were recorded against distilled water
blank for a minimum of 3 mins. The complete assay mixture without the enzyme
(liver homogenate) served as the control to monitor non-specific binding of the
substrates. Care was taken to ensure that the final concentration of ethanol in the
mixture was always less than 4 %.
Calculation

GST activity was calculated using the extinction co-efficient of the product
formed (9.6 mm-cm-) and the values have been expressed as mean ± SD of μmoles
of CDNB-GSH conjugated formed / min / mg protein.

APPENDIX XXX
ESTIMATION OF TOTAL REDUCED GLUTATHIONE (GSH)
(Morn et al., 1979)

Principle

The method was based on the reaction of reduced glutathione with DTNB
to give a compound that absorbs at 412 nm.

Reagents

 Metaphosphoric acid : 1.67 g of glacial metaphosphoric acid, 0.2 gm of

EDTA and 30 gm of NaCl in 100 ml of distilled water.
 0.4 M Na2HPO4.
 DTNB reagent: 40 mg of DTNB in 100 ml of 1% trisodium citrate.
 Standard glutathione: 20 mg of reduced glutathione was dissolved in 100
ml of distilled water.
Procedure

1.0 ml of 10 % tissue homogenate was precipitated with 4.0 ml of

metaphosphoric acid. The precipitate was removed by centrifugation. To 2.0 ml of
the supernatant, 2.0 ml of disodium hydrogen phosphate and 1.0 ml of DTNB
reagent was added. The absorbance was read within 2 mins at 412 nm against a
reagent blank. A set of standards was also treated in the above manner.
The amount of glutathione is expressed as μg / mg protein.
 APPENDIX XXXI
ESTIMATION OF ASCORBIC ACID
(Omaye et al., 1979)

Principle

Ascorbic acid was oxidised by copper to form dehydroascorbic acid and

diketoglutaric acid. These products were treated with 2,4-dinitrophenyl hydrazine
to form the derivative of bis 2,4-dinitrophenyl hydrazine. This compound, in
strong sulphuric acid undergoes a rearrangement to form a product with an
absorption band that is measured at 520 nm. The reaction was run in the presence
of thiourea to provide a mildly reducing medium, which helps to prevent
interference from non-ascorbic acid chromogens.

Reagents

 5 % TCA.
 65 % sulphuric acid.
 DTCS reagent: 3g of 2,4-dinitrophenyl hydrazine, 0.4 g of thiourea and
0.05 g of copper sulphate were dissolved in 9 N sulphuric acid and
made upto 100 ml with the same.
 Standard solution: standard in the range of 4-20 μg/ml were prepared in
5% oxalic acid.

Procedure

1.0 ml of 10 % homogenate was precipitated with 5 % ice-cold TCA and

centrifuged for 20 mins at 3,500 rev / min. 1.0 ml of the supernatant was mixed
with 0.2 ml of DTCS reagent and incubated for 3 hours at 37°C. Then 1.5 ml of
ice-cold 65 % sulphuric acid was added mixed well and the solutions were
allowed to stand at room temperature for an additional 30 mins. Absorbance was
determined at 520 nm.
The results are expressed as μg / mg protein.
 APPENDIX XXXII
ESTIMATION OF TOCOPHEROL

(Varley et al., 1981)

Principle
Tocopherol can be estimated using Emmerie-Engel reaction, which is based
on the ferric to ferrous ions by tocopherols, which then forms a red color with
2,2- dipyridyl. Tocopherols and carotenes are first extracted with xylene and the
extinction read at 460 nm to measure carotenes. A correlation is made for these
after adding ferric chloride and reading at 520 nm.

Reagents
 Absolute ethanol

 Xylene

 2,2-dipyridyl: 1.2 g/L of n-propanol

 Ferric chloride solution: 1.2 g of FeCl3.6H2O in one litre of ethanol.

 Standard solution D,L-α-tocopherol: 10 mg/L in absolute ethanol 0.91 mg

of α-tocopherol is equivalent to 100 mg of tocopherol acetate.

 Tissue extraction: Weighed 1.0 g the tissue and were homogenised in a

blender and transferred to a conical flask. Added 50 ml of 0.1 N sulphuric
acid slowly without shaking. Stoppered and allowed to stand overnight. The
next day, the contents of the flask were shaken vigorously and filtered
through Whatmann No.1 paper, discarding the initial 10-15 ml of the
filtrate. Aliquot of the filtrate was used for the estimation.

Procedure

Into 3 stoppered centrifuge tubes (test,standard and blank) pipetted out 1.5 ml
of each liver tissue extract, 1.5 ml of the standard and 1.5 ml of water respectively.
To the test and blank added 1.5 ml of ethanol and to the standard added 1.5 ml of
water. Added 1.5 ml of xylene to all the tubes, stoppered, mixed well and
centrifuged.

Transferred 1.0ml of xylene layer into another stoppered tube, taking care not
to include any ethanol or protein, added 1.0ml of 2,2-dipyridyl reagent to each
tube, stoppered and mixed. Pipetted out 1.5 ml of the mixtures into
spectrophotometer cuvettes and read the absorbance of test and standard against
the blank at 460 nm. Then in turn beginning wit the blank, added 0.33 ml of ferric
chloride solution. Mixed well and after exactly 15 min read test and standard
against the blank at 520 nm. The amount of vitamin E can be calculated using the
formula.

(∆A520nm-∆A460 nm × conc [s] × 0.29) × Total volume

Vitamin E (μg/g) =
(∆A520nm × Vol for experiment × wt of sample

APPENDIX XXXIII
ESTIMATION OF LIPID PEROXIDATION (LPO)
(Buege amd Aust ,1978)

Principle

Malondialdehyde has been identified as the product of lipid peroxidation

that reacts with thiobarbituric acid to give a red color absorbing at 535 nm.

Reagents

 Stock TCA-TBA-HCl reagent: 15% w/v trichloroacetic acid, 0.375

w/v thiobarbituric acid and 0.25 N HCl. The solution was heated mildly
to assist the dissolution of the TBA.
Procedure

To 1.0 ml of the sample, 2.0 ml of TCA- TBA-HCl reagent was added and
mixed thoroughly. The solution was heated for 15 min in a boiling water bath.
After cooling, the flocculent precipitate was removed by centrifugation at 1,000 g
for 10 min. The absorbance was determined at 535nm against a blank that contains
all the reagents minus the sample.
The results were expressed as nmoles of MDA formed/min/mg protein
using an extinction coefficient of the chromophore 1.56 x 10 5 Mcm and expressed
as nmoles of MDA formed/min/mg protein.

APPENDIX – XXXIV
ESTIMATION OF CHOLESTEROL
(Parekh and Jung, 1970)

Principle

Cholesterol reacts with ferric chloride in the presence of concentrated

sulphuric acid to give a pink color. The intensity of color developed is directly
proportional to the amount of cholesterol present and is read at 540nm in a
colorimeter.

Reagents

 Stock ferric chloride: 840 mg of pure dry ferric chloride was weighed and
dissolved in 100 ml of glacial acetic acid.
 Ferric chloride precipitating reagent: 10 ml of stock ferric chloride reagent
was taken in 100 ml of standard flask and made up to the mark with pure
glacial acetic acid.
 Ferric chloride diluting reagent: 8.5 ml of stock ferric chloride was diluted
to 100ml with pure glacial acetic acid.
  Standard cholesterol solution: 100 mg of cholesterol was dissolved in 100
ml with glacial acetic acid. The concentration of working standard is 100
μg /ml.
 Working standard: 10 ml of stock was dissolved in 0.85 ml of stock ferric
chloride reagent and made up to 100 ml with glacial acetic acid. The
concentration of working standard is 100 μg/ml.

Procedure

To 0.1 ml of serum added 4.9 ml of ferric chloride precipitating reagent.

Centrifuged and to 2.5 ml of supernatant added 2.5 ml of ferric chloride diluting
reagent. Added 4.0 ml of concentrated sulphuric acid. A blank was prepared
simultaneously by taking 5.0 ml of diluting reagent and 4.0 ml of concentrated
sulphuric acid. A set of standards (0.5 - 2.5 ml) were taken and made up to 5.0 ml
with FeCl2 diluting reagent. Then added 4.0 ml of con.H 2SO4. After 30 mins, the
intensity of color developed was read at 450 nm against reagent blank.
The amount of cholesterol in the serum was expressed as mg/dl.

The amount of cholesterol in tissue was expressed as mg / g tissue.

APPENDIX XXXV
ESTIMATION OF PHOSPHOLIPIDS
(Rouser et al., 1970)
Principle

The organic phospholipid phosphorus is converted to inorganic phosphorus,

which reacts with ammonium molybdate to form phosphomolybdic acid, which on
reduction and reaction with ANSA forms a stable blue color and has absorption at
660 nm.
Reagent
 TCA 10 %

 70 % Perchloric acid

 3 % Ammonium molybdate : 3 g of ammonium molybdate was

dissolved in 100 ml of distilled water.

 3 % Ascorbic acid: 3 g of ascorbic acid was dissolved in 100 ml of

distilled water.

 Standard: 35.1 mg of KH2PO4 was dissolved in 100ml of water. This

contains 80μg of phosphorous/ml.

Procedure
0.1 ml of lipid extract was diluted to 2.0 ml with distilled water. 1.0 ml of
perchloric acis was added to the tubes and digested on a sand bath till the solution
became colorless and then it was made up to 5.0 ml with distilled water. Standard
phosphate solution and blank containing distilled water were mixed with 0.8 ml of
perchloric acid and the final volume was made upto 5.0 ml with distilled water.
0.5 ml each of ammonium molybdate and ascorbic acid were added and the
mixture was kept in a boiling water bath for 6 minutes. The color developed was
read at 710 nm.

Phosphorus content was multiplied by a factor 25, which gave the weight of
phospholipids. Phospholipids are expressed as mg/100ml in serum and mg/g in
tissues.
 APPENDIX XXXVI
ESTIMATION OF TRIGLYCERDIES

(Rice, 1970)

Principle

The glycerol moiety is oxidized to formaldehyde and the later condensed

with ammonia and 2,4-pentanedione (acetyl acetone) to produce 3,5-diacetyl
1,4-dihydrotoludine, which is yellow in color and has absorption at 450 nm.

Reagents
 Chloroform-methanol mixture (2:1 v/v)

 Supersaturated sodium chloride: 0.89 g of sodium chloride was

dissolved in 100 ml of distilled water.

 Activated silicic acid: It was activated by washing silicic acid with 4 N

or 2 N HCl and then with distilled water until the pH wash neutral. After
drying, ether was added in sufficient amount, stirred well and the
supernatant was discarded. Silicic acid was then dried at 60˚C and
activated at 100˚C over night prior to use.

 0.2 N H2SO4: 0.56 ml of sulphuric acid was added to 100 ml with

distilled water.

 Alcoholic potassium hydroxide 0.4%: 400 mg of KOH was dissolved in

100 ml of 5% ethanol.

 Sodium-metaperiodate 0.1 M: 50 mg of sodium metaperiodate was

dissolved in 10 ml of distilled water

 Sodium mettaarsenite 0.5 M: 650 mg of sodium metaarsenite was

dissolved in 10 ml of distilled water.

 Chromotropic acid reagent: 1.14 g of chromotropic acid was dissolved

in 100 ml of distilled water and stored as stock solution in a brown
 bottle. Before use, 10 ml of this solution was mixed with 4.5 ml of
sulphuric acid- water mixture in the ratio of 2:1 (v/v).

 Thio urea: 7% solution in chloroform

 Standard: 100 mg of tripalmitin was dissolved in 100 ml of chloroform

which gave the concentration of 1.0 mg/ml.

Procedure

Took 0.1 ml of Folch-wash aliquot, 1 ml of chloroform-methanol mixture

was added. 50 mg of activated silicic acid was added to it, shaken vigorously and
allowed to stand for 30 mins. After centrifugation, to a 0.5 ml aliquot of
supernatant (as well as to standard and blank), 0.5 ml of alcoholic potassium
hydroxide solution was added and the mixture was saponified in a 60-70oC water
bath for 20 min. To this, 0.5 ml of 0.2 N H2SO4 was added and kept in a boiling
water bath for 10 mins. After cooling the tubes, 0.1 ml of sodium metaperriodate
was added and allowed to stand for 10 mins.

The excess periodiate was reduced by the addition of 0.1 ml of sodium

metaarsenite. Then, 5.0 ml of chromattropic acid was added, mixed thoroughly
and kept in a boiling water bath for 30 mins. After cooling, 0.5 ml of thiourea
solution was added and the color developed was read at 570 nm using a
colorimeter.
Triglycerides are expressed as mg / 100 ml in serum and mg/g in tissue

APPENDIX XXXVII
ESTIMATION OF FREE FATTY ACID
(Horn and Menahan, 1981)
Principle

The free fatty acids were extracted from lipids by CHM mixture. The free
fatty acids form a complex with cupric ions when mixed with copper reagent. The
coloured complex formed with copper is soluble in chloroform and diethyl
dithiocarbanate and is used as a color developer. The color developed was read at
660nm.

Reagent
 Chloroform –heptane-methanol mixture (CHM mixture), the mixture
was prepared in the ratio of 200:150:7(v/v)
 Activated silicic acid
 Copper nitrate-triethanolamine solution: 9 volumes of aqueous 1M
triethanolamine, 1 volume of 1 N acetic acid and 10 volumes of 6.45% Cu
(NO3) 2H2O were mixed with 33g of sodium chloride. The Ph was adjusted
to 8.1.
 0.1% diethyl dithiocarbamate in n-butanol
 Standard: A solution containing 200mg/100ml of palmitic acid was
prepared in CHM mixture. The solution was diluted in 10 times for use
(200μg/ml).

Procedure

To 0.2ml serum or lipid extract, 6.0ml of CHM mixture and 200mg of

activated silicic acid were added, mixed well and centrifuged. The suparnatant was
transferred to another tube. Standard were also made upto 6.0ml with CHM
mixture. Blank contained 6.0ml of CHM mixture.

To all these tubes, 2.0ml of copper nitrate-TEA solution was added and
mixed on a mechanical shaker for 20min. They were then centrifuged to give two
separate phases. 2.0ml of the upper phase was transferred to another tube, 1.0ml of
the color reagent was then added and shaken well. The color developed was read
at 430nm against a reagent blank.

Free fatty acids are expressed as mg/100ml in serum and mg/g in tissues.
 APPENDIX XXXVIII
HISTOPATHOLOGICAL INVESTIGATION OF
LIVER AND KIDNEY

The liver and kidney samples were preserved in 20% commercial formalin
immediately on removal from animal.

Tissue processing

The tissues were placed in 10% formal saline (10% formalin in 9% sodium
chloride) for one hour to rectify shrinkage due to higher concentration of formalin.
The tissue was dehydrated by ascending grades of isopropyl alcohol by immersing
in 80% isopropanol over night, 100% isopropyl alcohol for 1hour. The dehydrated
tissues were cleared in two changes of xylene, 1 hour each. Then the tissue were
impregnated with histology grade paraffin wax (melting point 58-60 0C) at 60 0C
for 2 changes of 1 hour each. The wax impregnated tissues were embedded in
paraffin blocks were mounted and cut with rotary microtome at 3 micron
thickness. The sections were floated on tissue floatation bath at 40 0C and taken on
glass slides and smeared with equal parts of egg albumin and glycerol. The
sections were then melted in an incubator at 60 0C and after 5 min the section were
allowed to cool.

Tissue staining

The section were deparaffinised by immersing in xylene for 10 min in

horizontal staining jar. The deparaffinised section were washed in 100% isopropyl
alcohol and stained in Ehrlish’s hematoxylin for 8 min in horizontal staining jar.
After stained in hematoxylin, the sections were washed in tap water and dipped in
acid alcohol to remove excess stain (8.3% HCl in 70% Alcohol). The section were
then placed in running tap water for 10 min for blueing (show alkalization). The
section were counter stained in 1% aqueous eosin (1 g in 100ml tap water) for one
min and the excess stain was washed in tap water and the sections were allowed to
dry. Complete dehydration of stain sections was ensured by placing the section in
the incubator at 60 0C for minitus. When the section were cooled, they were
mounted in DPX mount having the optical index of glass (the section were wetted
in xylene and inverted on to the mountant placed on cover slip).

The architecture was observed at low power objective. The liver cell injury
and other aspects were observed under high power dry objective.