APPENDIX I

ESTIMATION OF ACID PHOSPHATASE

Principle

The method was used that of king and Armstrong in which disodium phenyl
phosphate is hydrolysed with the liberation of phenol and inorganic phosphate.
The liberated phenol is measured at 700 nm with folin ciocalteau reagent.

Reagent

1.Citrate buffer: 0.1 M, pH 5

Solution A: Citric acid (21.01g in 1000ml)
Solution: Sodium citrate (29.41g in 1000ml)
20.5ml of A and 29.5 ml of B diluted to a total of 100ml.

2.Disodium phenyl phosphate, 100 mmol/l: Dissolved 2.18g in water, heated to

boil, cooled and made to a litre. Added 1.0 ml of chloroform and stored in the
refrigerator.

3.Buffer-substrate: Prepared by mixing equal volume of the above two solutions.

This has a pH of 5.0

4.Folin ciocalteau reagent: Mixed 1.0 ml of reagent with 2.0 ml of water.

5.Sodium carbonate solution, 15%: Dissolved 15g of anhydrous sodium carbonate

in 100ml of water.

6.Standard phenol solution, 1g/L: Dissolved 1g pure crystalline phenol in 100

mmol/L HCl and made to a litre with the acid.

7.Working standard solution: Diluted 10ml of stock standard to 100ml with water.
This contains 100µg phenol/ml.
Procedure

Pipetted 4.0ml of the buffer substrate into a test tube and incubated at 37ºc
for 5min.Added 0.2ml of the sample and incubated further for exact
60min.Removed and immediately added 1.8ml of diluted phenol reagent. At the
same time a control was set up containing 4.0ml buffer substrate and 0.2ml sample
to which 1.8ml phenol reagent was added immediately. Mixed well and
centrifuged .To 4.0ml of the supernatant added 2.0ml of sodium carbonate. Took
4.0ml of working standard solution and for blank taken 3.2ml water and 0.8ml of
phenol reagent. Then added 2.0ml of sodium carbonate. Incubated all the tubes at
37ºc for 15min. read the color-developed at700nm. The enzyme activity was
expressed as units/L in serum, units/protein in tissues.

APPENDIX II

ESTIMATION OF ALKALINE PHOSPHATASE

Principle

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

Reagent

1. 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.
 2. Disodium phenyl phosphate, 100mmol/l: Dissolved 2.18g in water,
heated to boil, cooled and made to a litre. Added 1.0ml of
chloroform and stored in the refrigerator.

3. Buffer substrate: Prepared by mixing equal volume of the above two

solutions. This has a pH of 10.

4. Folin – Ciocalteau reagent: Mixed 1.0ml of reagent with 2.0ml of

water.

5. Sodium carbonate solution, 15%: Dissolved 15g of anhydrous

sodium carbonate in 100ml of water.

6. Standard phenol solution, l g/L: Dissolved 1g pure crystalline phenol

in 100mmol/L HCl and made to a litre with the acid.

7. Working standard solution: Added 100ml dilute phenol reagent to

5.0 ml of stock standard and diluted to 500ml with water. This
contained 10ug phenol/ml.

Procedure

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

APPENDIX III
 ESTIMATION OF ASPARTATE TRANSAMINASE

Principle

The enzyme catalyses the following reaction:

L-Aspartate +α-oxoglutarate  Oxaloacetate + L-glutamate

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

giving a brown coloured hydrazone after the addition of sodium hydroxide. The
colour developed is read at520 nm.

Reagent

1. Phosphate buffer, 0.1M, pH 7.5

Solution A: 0.1-M solution of monobasic sodium phosphate (13.9g/1).

Solution B 0.1 M solution of dibasic sodium phosphate

26.8 of Na2 PO4.7 H 2 O g/1) 16ml of A and 84 ml of B, diluted to a total

of 200ml.

2. Substrate: Dissolved 146 mg of α –Ketoglutarate and 13.3 g of aspartic

acid in l N NaOH with constant stirring. Adjusted the pH to 7.4 and
made up to 1000 ml with phosphate buffer.

3. Standard pyruvate, 2-m mol/1: Dissolved 22 mg of sodium pyruvate in

100ml of phosphate buffer. 0.2ml of standard contained 0.4 u M of
sodium pyruvate.

4. Dinitrophenylhydrazine reagent, l mmol/l: 200 mg in l mol/l HCl.

5. 0.4 N NaOH: Dissolved 16 g of NaOH in 1000-ml water.

Procedure
 0.2 ml of sample and 1.0 ml of the buffer substrate was incubated for 60 min
0
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 colour developed was read at 520 nm.

The enzyme activity was expressed as units/L in serum, units/protein in

tissue.

APPENDIX IV

ESTIMATION OF ALANINE TRANSAMINASE

Principle

The enzyme catalyses the following reaction:

L-Alanine + α-oxoglutarate Pyruvate +L-glutamate

The oxaloacetate is measured by the reaction with

2,4 dinitrophenylhydrazine giving a brown coloured hydrazone after the addition
of sodium hydroxide. The colour developed is read at 520 nm.
Reagents

1. Phosphate buffer: 0.1M, pH 7.5

2. Substrate: Dissolved 146 mg of α-ketoglutarate and 17.8 g of L-alanine
in1 N NaOH with constant stirring. Adjusted the pH to 7.4 and made up
to 1000 ml with phosphate buffer.

3. Standard pyruvate, 2 mM: Dissolved 22 mg of sodium pyruvate in

100ml of phosphate buffer 0.2 ml of standard contained 0.4 µM of
sodium pyruvate.

4. Dinitrophenyl hydrazine reagent, l m mol/l: 200mg/l in l mol/l HC1.

5. 0.4 N NaOH: Dissolved 16 g of NaOH in 1000-ml water.

Procedure
 0.2 ml of sample and 1.0 ml of the buffer substrate were incubated for
30 min at 37oC. 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 20
min. Then 10ml of 0.4 N NaOH was added. A set of standard pyruvate was also
treated in a similar manner. The colour developed was read at520 nm.

The enzyme activities were expressed as units/L in serum, units/protein in

tissues.

APPENDIX V

ESTIMATION OF LACTATE DEHYDROGENASE

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.

Reagent

1. 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.

2. Buffered substrate: 125 ml of glycine buffer and 75 ml of 0.1 N NaOH

were added to 4 g of lithium lactate and mixed well.

3. Nicotinamide Adenine Dinucleotide: 10 mg of NAD was dissolved in 2.0

ml of water.

4. 2,4 Dinitrophenyl hydrazine: 20 mg of DNPH was dissolved in 100 ml of

1 N HCl.

5. 0.4 N NaOH.
 6. Standard, 1 µmole of pyruvate/ml: 11mg of sodium pyruvate was dissolved
in 100-ml buffered substrate (1µmole of pyruvate/ml).

7. NADH solution, 1 µmole/ml: 8.5-mg/10 ml buffered substrate.

Procedure

Placed 1.0 ml buffer substrate and 0.1 ml sample into each of two tubes.
Added 0.2-ml water to the blank. Then to the test added 0.2 ml of NAD. Mixed
and incubated at 370C for 15 min. Exactly after 15 min, 1.0 ml of dinitrophenyl
hydrazine was added to each (test and control) tubes. 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 -1.0µmole was taken.

The enzyme activity were expressed as units/L in serum, units/protein in

tissue.
APPENDIX VI

ESTIMATION OF HEXOKINASE
Principle
The enzyme was assayed by determining the concentration of glucose by
orthotoludine method.
Reagents
1. 0.005 M glucose solution
2. 0.72 M ATP
3. 0.05 M MgCl2
4. 0.0125 M KH2PO4
5. 0.1 M KCl
6. 0.5 M Sodium fluoride
7. 0.01 M Tris Hcl buffer, pH8.0
8. 10%TCA
Procedure

The incubation mixture containing 2.5 ml buffer, 1.0 ml of substrate, 0.5ml

ATP, 0.1ml each of Mgcl2 and sodium fluoride and 0.5 ml each KH2PO4and KCl
was preincubated at 37˚ c for 5min. The reaction was initiated by the addition of
1.0ml of enzyme extract .1.0ml of aliquot of the reaction mixture was removed
immediately (zero time) and added to tubes containing 1.0ml of 10%TCA. After
30min incubation at 37˚ c, 1.0ml of aliquot of the above reaction was added to
separate set of tubes and the reaction was stopped by addition of 1.0ml of 10%
TCA. After the sample were precipitated and centrifuged, the supernatants were
used for the estimation of glucose by ortho toludine method. The enzyme activity
is expressed as μ moles of glucose utilized for the formation of glucose –6-
phosphate /min/mg/protein.

APPENDIX VII

ESTIMATION OF PHOSPHOGLUCOISOMERASE

Principle

The assay is based on the estimation of fructose by the resorcinol thiourea

reagent, which is measured at 410nm.

Reagent
1. 0.1M borate buffer,pH 7.8
2. Buffered substrate: 3mg of sodium glucose-6-phosphate were dissolved in
1.0ml of buffer. This was prepared fresh before use.
3. 30%HCl
4. Resorcinol –thiourea reagent: 100mg of resorcinol and 250mg of thio urea
was dissolved in 10ml of glacial acetic acid. This stored in a brown bottle
and is discarded when the solution turned brown.
 5. Color reagent: 30% HCl, resorcinol-thiourea and water were mixed in the
proportion 7:1:1 .The solution was used in the same day as prepared.
6. Standard solution: 5.4 mg of pure fructose was dissolved in 100ml of
0.25%Benzoic acid.

Procedure

Into the tubes labeled ‘test’ and ‘blank’, 1.0ml of buffered substrate was
added. 0.2ml of enzyme extract was added to the test and tubes were incubated at
37˚c for 30min. after the period of incubation, 1.0ml of 10%TCA was added to
arrest the reaction. 0.2ml of enzyme was added to the control tubes and 9.0ml of
color reagent was immediately added to all the tubes. The tubes were heated in a
water bath maintained at 70˚c for 15min. standard containing varying
concentrations of fructose and a reagent blank containing water were similarly
treated. The tubes were cooled in running water, and the color was immediately
read at 410nm in a photoelectric colorimeter.

The enzyme activity is expressed as micromoles of fructose formed/min/mg

protein

APPENDIX VIII

ESTIMATION OF ALDOLASE

Reagents
1. 0.1M Tris-Hcl buffer, Ph 8.6
2. 0.05 M substrate: 17.05mg of fructose-1, 6-diphosphate in 10ml of Tris
Hcl buffer were prepared just before use.
3. 0.56N Hydrazine sulphate, pH 8.6
4. 10%TCA
5. Coloring reagent: 0.1%2,4 dinitrophenylhydrazine
6. 0.75 N sodium Hydrazine
 7. Standard DL- glyceraldehyde: 12.3 mg of DL–glyceraldehyde was
dissolved in 100ml of water and kept at room temperature for four days for
depolymerization.

Procedure

The incubation sample contained 0.25ml of substrate, 0.25 ml of hydrazine

sulphate, 1.0ml of Tris HCl buffer and 0.5 ml of enzyme to make it up to 2.0ml. It
was incubated at 37˚c for 15 min. The reaction was terminated by the addition of
1.0ml of 10%TCA and the tubes were centrifuged. 1.0ml of the supernatant was
transferred to the tubes containing 1.0ml of 0.75N NaOH. The tubes were left at
room temperature for 10min. 1.0ml of dinitrophenyl hydrazine reagent was added,
incubated at 37˚c for 60min. The colour developed after the addition of 7.0ml of
0.75N NaOH solution was read at 540nm.The color was developed with aliquots
of standard DL –glyceraldehyde solution by treating in a similar manner.

The enzyme activity is expressed as micromoles of gluceraldehyde

formed/min/mg protein.

APPENDIX IX
ESTIMATION OF SUCCINATE DEHYDROGENASE

Principle

The rate of oxidation reaction is followed by a coupling reaction to a redox

dye. The dye di-chloro-phenol indophenol acts as a hydrogen acceptor from FDH 2
and gets reduced. Thus by following the decrease in blue color of the dye .The rate
of oxidation of succinate of fumarate.

Reagents
1. 0.3M phosphate buffer, pH 7.6
 2. 0.03 M EDTA
3. 0.03M potassium cyanide
4. 0.4M sodium succinate, pH 7.6
5. 3% BSA (w/v)
6. 75nM pottasium ferricyanide
Procedure
Added 1.0ml of phosphate buffer, 0.1ml of EDTA, 1.0ml of KCN and
made up to 2.9ml with water. Note the extinction at 455nm,then start the reaction
by addition of enzyme and follow the change in extinction during the first two
min. Initial rates were taken as a measure of activity. A blank rate (all reagents
except succinate) must be determined separately.

In this determination, 1 mole of succinate reduces 2moles of potassium

fericyanide. Concentration of potassium ferricyanide rates can be measured by
following the reaction at 420nm(ε =1.03x 10 cm).

The enzyme activity is expressed as micromoles of succinate

produced/min/mg protein.
APPENDIX X
ESTIMATION OF MALATE DEHYDROGENASE

Principle
Malate dehydrogenase is one of the enzymes involved in TCA cycle. It
catalyses the reversible conversion of oxaloaceticacid to malic acid.
Mg2+
Oxaloaceticacid +NADH Malic acid +NAD+

The decrease in absorbance due to oxidation of NADH is measured at

340nm.
Reagents

1. 0.2M phosphate buffer, PH 7.4

2. 0.76 μm oxaloacetate (15.4 mg/5ml)
3. 0.15μm NADH (9.1mg/5ml)

Procedure

The reaction mixture contained the following reagents and enzyme in a

total volume of 3.0ml. 75μm of phosphorus buffer, 0.15μm of NADH and 0.76 μm
of oxaloacetate .The reaction were carried at 25˚c and were started by the reagents
by the addition of enzyme preparation. The control tubes contained all reagents
except NADH .The change in O.D at 340nm was measured for 2min at intravel of
15sec.

The activity of the enzyme was expressed as micromoles of NADH

oxidized/min/mg protein using the extinction coefficient of NADH as 6.22x 103

APPENDIX XI
ESTIMATION OF GLUCOSE-6-PHOSPHATASE

Principle
Glucose-6-phasephatase catalyses the reaction
Glucose-6-phosphate + H2O Glucose + PO4.
The phosphorus content was estimated by the method of Fiske and Subbarow
Reagent
1. 0.1 M citrate buffer,pH 6.5
Solution A: 0.1-M citric acid (21.01 g/L)
Solution B: 0.1M sodium citrate (28.41 g C6H5O7 Na2H2O/L)
Mixed 3.8ml of A and 46.2 ml of B and diluted to a total of 100ml.
 2. Substrate: Glucose-6-phosphate, 0.01m (20 mg in 6.5 ml distilled water)
3. 2.5% Ammonium molybdate solution
4. ANSA
5. 10% TCA

Procedure

The incubation mixture in a total volume of 1.0ml contained 0.3ml of

buffer, 0.5ml of substrate, and 0.2ml of enzyme solution. Incubation was carried
out at 37˚c for 60min. The reaction was terminated by the addition of 1.0ml of
10% TCA solution. The suspension was certified and the phosphorus content in
the supernatant was estimated by the method of Fisky and Subbarow.

The enzyme activity is expressed as micromoles of PO4 liberated/min/mg

protein.
APPENDIX XII
ESTIMATION OF FRUCTOSE-1, 6-DIPHOSPHATASE
Principle
Fructose-1, 6-diphosphatase catalyzes the reaction
Fructose-1, 6-diphosephate + H2O Fructose-6-phosphate
+PO4
The phospharus content was estimated by the method of fiske and subbarow

Reagent

1. 0.1 M Tris-HCl buffer, pH 7.0

Solution A: 0.2 m Tris (24.2g/l)
Solution B: 0.2 m HCl
Mixed 50 ml of A and 47 ml of B and diluted to 100ml. Diluted 1 part of
0.2-M solution with 1 part of water.
2. subatrate: 0.05 M fructose-1,6-diphosephate solution
3. 0.1 M MgCl2
 4. 0.1 M KCl
5. 0.01M EDTA
6. 10%TCA
7. 2.5% Ammoniun molybdate solution
8. ANSA

Procedure

The assay medium in a final volume of 2.0ml contained 1.2ml of buffer, 0.1
ml of substrate solution, 0.25 ml of mgcl 2 .0.1ml KCl solution, 0.25ml of EDTA
solution and 0.1 ml of enzyme .The incubation was carried out at 37˚c for 15min.
The reaction was terminated by addition of 1.0 ml of TCA. The suspension was
centrifuged and the phosphorus content of the supernatant was estimated by the
method of Fisky and Subbarow

The enzyme activity is expressed as micromoles of PO4 liberated/min/mg

protein.
APPENDIX XIII
ESTIMATION OF PROTEIN

Principle:

The Blue colour 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 colour developed by the
biuret reaction of the protein with the alkaline cupric tartarate are measured at
660nm.

Reagents:

1. 2% Sodium carbonate 0.1N NaOH (Reagent A) Copper

2. 0.5% Copper sulphate in 1-% potassium sodium tartarate (ReagentB)

3. Alkaline copper reagent: Mixed 50ml of A and 1.0ml of B prior to use

 4. Folin- ciocalteau reagent: Mixed 1 part of reagent with 2 part of water.

5. Stock standard: Weighed 50mg of bovine serum albumin and made up to

50ml in a standard flask with saline.

6. Working standard: Diluted 10ml of the stock to 50ml with distilled water.
1.0ml of this solution contains 200μg of protein.

Procedure

Pipetted out 0.2ml to 1.0m1 working standard solution. 0.1m1 of the

sample was taken. The volume in all the tubes was made up to 1.0m1 with
distilled water. Added 5.0ml of alkaline copper reagent to each tube. Mixed well
and allowed to stand for 10min.Then added 0.5m1 of folin-ciocalteau reagent.
Mixed well and incubated at Room temperature for 30-m .A reagent blank was
also prepared. After 30 minutes, the blue colour developed was read at 660nm.

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

APPENDIX XIV

ESTIMATION GLUCOSE

Principle

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

Reagents

1. 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.
2. 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.

APPENDIX XV
ESTIMATION OF GLYCOGEN

Principle

Glucose is dehydrated by sulphuric acid to furfural derivative which then

complexes with anthrone to give a green colored complex, which is read at 620nm.
Reagents
1. Extraction of glycogen
200 mg of liver sample was homogenized with 20ml of 5% TCA. The
precipitate of protein was filtered and the clear filtrate was used for analysis.
2. Anthrone reagent: 0.2% anthrone in concentrated sulphuric acid
3. Stock standard: 100mg of glucose were dissolved in 100ml of water.
4. Working standard: 10ml of stock standard was made up to 100ml with
water.

Procedure

2.0ml of liver sample were pipetted into a test tube. Then, 2.0ml of 10N
KOH were added and the tubes were placed in boiling water for 1h. After cooling,
1.0ml of glacial acetic acid was added to neutralize the excess of alkali and the
volume was made up to 20ml with water. From this 2.0ml of the water taken for
the experiment and slowly added 4.0ml of anthrone reagent. The tubes are placed
in boiling water for exactly 10 min for color development and cooled with tap
water. The optical density was read with in 2hr in a spectrophotometer at 650nm
against a blank with 2.0ml of5% TCA. Standard in the concentration of 20-100μg
also run along with the sample.
The amount of glycogen in the sample is expressed as mg/g tissue.

APPENDIX XVI

ESTIMATION OF UREA

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

1. TCA, 10%

2. Stock Diacetylmonoxime, 25g/l

3. Stock Thiosemicarbazide 2.5g/l

4. Acid ferric chloride solution: Added 1.0-ml sulphuric acid to 100 ml of

ferric chloride solution containing 50g/l in water.

5. 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.

6. Color reagent: To 300-ml acid reagent added 200-ml water, 10-ml stock
diacetylmonoxime and 2.5 ml thiosemicarbazide.

7. 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. At the same time took 0.2ml of 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.

APPENDIX XVII

ESTIMATION OF URIC ACID

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

1. Phosphotungstic acid reagent.

2. 10% Sodium carbonate

3. Standard uric acid: 100 mg of Uric acid and 60mg of lithium carbonate
were taken in a breaker and abou50ml 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.

4. Working standard: Dilute 1.0 ml of the stock standard to 10ml with water.
1 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 phosphotunstic acid and sodium carbonate. A blank
was set up with 3.0 ml water. Standard where also treated in a same manner. The
color was read at 640 nm after 10min. The values were expressed as mg/100ml in
serum.

APPENDIX XVIII

ESTIMATION OF CREATININE

Principle

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

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

Reagents

1. Picric acid: 8.02g/l

2. Sodium hydroxide: 12.8g/l

3. Standard creatinine: Dissolved 100 mg of Creatinine in 100ml with distilled

water.

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

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

5. 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. The values are expressed as
mg of Creatinine / dl.
 APPENDIX XIX

ESTIMATION OF DNA

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
1. DNA stock standard: 60mg of DNA was dissolved in 5mM NaoH and
made upto 100 ml with the same.
2. Working standard: To 5 ml of stock standard added 5.0 ml of 1.2 N
perchloric acid heated at 90˚c for 15 minutes and cooled.1.0 ml of this
solution contains 300μg of DNA.
3. Saline citrate: 0.15-M sodium chloride (8.78 g/l) and 0.015 M sodium
citrate (4.14 g/l) was mixed.
4. 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.
5. 0.2N, 0.6 N and 1.2N perchloric acid
6. 0.3N Potassium hydroxid: Dissolved 1.71g in 100ml/water
7. 7.5% and10% TCA
8. 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 minutes 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 300.1.0 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 XX

ESTIMATION OF RNA

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
1. 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.
2. Working standard: 10ml of the stock was added to 100ml .1.0ml of this
solution contains 100μg of RNA.
3. 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.
4. 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 .0.5ml 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
20min.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 XXI

ESTIMATION OF SUPEROXIDE DISMUTASE

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 naphtylamine to produce a red azo compound whose
absorbance is measured at 543 nm.

Reagents

1. 50 mM phosphate buffer, pH7.4

2. 20 mM L-Methionine

3. 1% (v/v) Triton X-100

4. 10 mM hydroxylamine hydrochloride

5. 50 µM EDTA

6. 50 µM Riboflavin

7. Griess reagent: 1-% sulphanilamide, 2% phosphoric acid and 0.1%

naphthylethylene diamine dihydrochloride.

Procedure

Pipetted 1.4 ml aliquot of the reaction mixture in a test tube. 100 µl of the
sample was added followed by preincubation at 37 oC for 5 min. 80 µl of
riboflavin was added and the tubes were exposed for 10min 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, 0.1 ml of Greiss reagent was added to each tube and the
absorbance of the colour 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 XXII

ESTIMATION OF CATALASE

Principle

Catalase causes rapid decomposition of hydrogen peroxide to water.

Catalase

2H2O 2 H2O + O2

The method is based on the fact that dicromate in acetic acid reduces 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 is
measured colorimetrically at 610 nm. Since dichromate has no absorbency in this
region, the presence of the compound in the assay mixture does not interfere with
the colorimetric determination of chromic acetate. The catalase preparation is
allowed to split H2O2 for different periods of time. The reaction is stopped at
specific time intervals by the addition of dichromate/acetic acid mixture and the
remaining H2O2 is determined by measuring chromic acetate colorimetrically after
heating the reaction.

Reagents

1. 0.01 M Phosphate buffer, pH 7.0

2.0.2M Hydrogen peroxide

2. Stock dichromate / acetic acid solution: Mixed a 5% potassium

dichromate with glacial acetic (1:3by volume).
 3. Working dichromate / acetic acid solution: The stock was diluted to 1:5
with water to make the working dichromate/acetic acid solution.

Procedure
10
The assay mixture contained 0.5 ml of H2O2 ,
ml of buffer and 0.4 ml
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 developed was read at 610 nm.
The activity of catalase was expressed as μmole of H 2O2 decomposed/min/mg
protein.

APPENDIX XXIII

ESTIMATION OF GLUTATHIONE PEROXIDASE

Principle

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

compound that absorbs at 412 nm.

Reagents

1. 0.4 M sodium phosphate buffer, pH 7.0

2. 10mM sodium azide

3. 2.5 mM hydrogen peroxide

4. 4mM reduced glutathione

5. 10% TCA

6. 0.3 M phosphate solution

7. 0.04% DTNB in l% sodium citrate

8. Reduced glutathione standard: 20mg reduced glutathione was dissolved in

100ml of water.
Procedure

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

ml of H2 O2, 0.2 ml of enzyme and 1.0 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 3.0 ml disodium hydrogen phosphate solution and 1.0 of DTNB reagent.
The colour 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 utilised/mg

protein.

APPENDIX XXIV

ESTIMATION OF GLUTATHIONE S-TRANSFERASE

Principle

Glutathione-S-transferase catalyses the reaction of 1-chloro 2,4

dinitrobenzene (CDNB) with the sulphydryl group of glutathione.

GST
CDNB + GSH ------------ > CDNB-S- glutathione.

The conjugate, CDNB- glutathione absorbs light at 340 nm and the activity
of the enzyme can therefore be estimated by measuring the increase in absorbance.

Reagents

1. 0.5 M phosphate buffer, ph 6.5

2. 30mMCDNB in 95% ethanol(30mg/5ml H2O)

3. 30mM reduced glutathione (14mg/1.5ml h2o)

Procedure

To 1.0ml of buffer, 0.1ml of sample, 1.7ml of water and 0.1ml of CDNB

were added and incubated at 37˚C for 5 min. After incubation, 0.1ml of reduced
glutathione were added. The increase in optical density of the enzyme was
measured against that of the blank at 340nm.

The enzyme activity is calculated in terms of μmoles of CDNB conjugate

formed/min/mg protein.

APPENDIX XXV

ESTIMATION OF GLUTATHIONE REDUCTASE

Principle

Glutathione reductase catalyses the reduction of oxidized glutathione

(GSSG) to reduced glutathione (GSH) and are assayed by measuring the decrease
in absorbance at 340nm.

NADPH (NADH)+H+ +GSSG NADP(NAD)+2GSH

Reagents

1. 0.3M phosphate buffer,Ph 6.8

2. 25 Mm EDTA (93mg/10ml H2O)

3. 12.5mM oxidized glutathione (11.5mg/1.5ml h2o)

4. 3mM NADPH (2.5mg/1ml H2O)

Procedure

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

ESTIMATION OFGLUCOSE 6 PHOSPHATE DEHYDROGENASE

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

1. 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

2. 0.2Mm NADP

3. 0.1M Magnesium chloride

4. 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 – XXVII

ESTIMATION OF TOTAL REDUCED GLUTATHIONE

Principle

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

compound that absorbs at 412 nm.

Reagent

1. Metaphosphoric acid: 1.67 g of glacial metaphosphoric acid, 0.2 g EDTA

and 30 g NaCI in 100ml water.

2. 0.4 M Na2HPO4

3. DTNB reagent: 40 mg DTNB in 100 ml of 1-% trisodium citrate

4. Standard glutathione: 20mg reduced glutathione was dissolved in 100 ml

water.

Procedure:

1.0ml of 10% tissue homogenate was with 4.0 ml of metaphosphoric acid.

The precipitate was removed by centrifugation. To 2.0 ml of the supernatant, 2.0-
ml disodium hydrogen phosphate and1.0 ml of DTNB reagent were added. The
absorbance was read within 2 min at 412 nm against a reagent blank. A set of
standards was also treated in the above manner. The amount of glutathione was
expressed as µg/mg protein.

APPENDIX – XXVIII

ESTIMATION OF ASCORBIC ACID (VITAMIN C)

Principle

Ascorbic acid is oxidized by copper to from dehyroascorbic acid and

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

Reagents

1. 5%TCA

2. 65% Sulphuric acid

3. DTCS reagent: 3g of 2,4 dinitrophenyl hydrazine, 0.4g thiourea and 0.05g

copper sulphate were dissolved in 9 N sulphuric acid and made up to 100ml
with the same.

4. Standard solution: Standard in the range of 4-20 µg/ml was prepared in5%
oxalic acid.

Procedure

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

centrifuged for 20 min at 3,500 g. 1.0 ml of the supernatant was mixed with 0.2 ml
of DTCS reagent and incubated for 3 hours at 37 0 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 min. Absorbance was determined at 520
nm. These results were expressed as µg/mg protein.

APPENDIX XXIX

ESTIMATION OF VITAMIN E

Principle

Tocopherol can be estimated using Emmerie-Engel reaction of ferric to

ferrous ions by tocopherols, which then forms a red color with 2,2’ dipyridyl.
Tocopherols and carotenese are first extracted with xylene and the extinction
readat 460 nm to measure carotenes. A correlation is made for these after adding
ferric chloride and reading at 520 nm.

Reagents

1. Absolute ethanol

2. Xylene

3. 2,2’ dipyridyl: 1.2 g/L n-propanol

4. Ferric chloride: 1.2g of FeCl3.6H2O or 720 mg of anhydrous ferric

chloride in one litre of ethanol.

5. Standard D, L-α-tocopherol: 10 mg /L in absolute ethanol .91 mg of

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

6. Same extraction: Weighed 1.0g the tissue and were homogenised in

a blender and transferred to a conical flask.Added 50ml of 0.1N
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 10-15ml of the filtrate. Aliquot of the filtrate was used for
the estimation.

Procedure

Into 3 stoppered centrifuge tubes (test,standard and blank) pipetted out

1.5ml of each tissue extract, 1.5ml of the standard and 1.5ml ml of water
respectively. To the test and blank added 1.5ml ethanol and to the standard added
1.5ml of water. Added 1.5ml of xylene to all the tubes,stoppered,mixed well and
centrifuged.

Transferred 1.0ml of xylene layer into another stoppered tube, 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 1.5ml of the mixtures into
spectrophotometer cuvettes and read the absorbance of test and standard against
the blank at 460nm. Then in turn beginning wit the blank, added 0.33ml of ferric
chloride solution. Mixed well and after exactly 1.5minutes read test and standard
against the blank at 520nm.The amount of vitamin E can be calculated using the
formula.

(∆A520nm~∆A450nm×conc [s]×0.29)×Total volume

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

APPENDIX XXX
ESTIMATION OF LIPID PEROXIDATION

Principle

Malondialdehyde has been identified as the product of lipid peroxidation

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

Reagents

1. 15%KCl

2. 1%Phophoric acid

3. n-butanol

4. 0.6%thiobarbituric acid

5. 10mM ferrous sulphate

6. 0.2mM ascorbate

To 1.0ml of the sample, 2.0ml of TCA-TBA-HCl reagent was added and

mixed thoroughly. The solution was heated for 15 min in a boiling water bath.
After colling, the flocculent precipitate was removed by centrifugation at 1,000g
for 10min. The absorbance was determined at 535 nm against a blank that
contained all the reagents minus the sample. The results were expressed as n
moles of MDA formed/mg protein using an extinction coefficient of the
chromophore 1.56x10-5M-1cm-1 and expressed as nmoles of MDA formed/mg
protein.

Ferrous sulphate and ascorbate induced lipid peroxidation:

Ferrous sulphate and ascorbate induced lipid peroxidation was carried out
as given above. The peroxidation system contained 10mM ferrous sulphate and
0.2mM ascorbate.

APPENDIX - XXXI

EXRACTION OF LIPIDS
Reagents

1. 0.1N KCl

2. Floch reagent – 0.1 N KCl: Methanol: Chloroform (10:10 v/v)

Procedure
The tissues were washed with saline and dried between filter paper. A
weighed amount of tissue (500 mg) was homogenized with 7.0ml of methanol in a
Potter-Elvehjem homogeniser and filtered through a Whatman No 1 filter paper
into a conical flask. The residue after filteration was scarped and homogenized in
14ml chloroform. The residue was once again scarped mixture and the resulting
filtrate was evaporated to dryness.
The dried lipid residue after evaporation was dissolved in 5ml of
chloroform-methanol mixture. The redissolved lipid extract was mixed with 1.0ml
of 0.1N KCl and the contents were shaken well. The upper aqueous phase
containing gangliosides and other water soluble compounds were separated. The
lower chloroform phase, containing neutral and phospholipids was again washed 3
times with 2.0ml of Folch’s reagent and the upper aqueous phase was aspirated.
The lower choloroform phase was made upto known volume (2.0ml) and aliquots
were taken for the analysis of cholesterol, triglycerides,free fatty acids and
phospholipids.
 APPENDIX XXXII

ESTIMATION OF CHOLESTEROL

Priciple

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 540 nm in a colorimeter.
Reagents

1. Stock ferric chloride: 840 mg of pure dry ferric chloride was weighed
and dissolved in 100ml of glacial acetic acid.
2. Ferric Chloride precipitation reagent: 10ml of stock ferric chloride
reagent was taken in 10 ml of standard flask and made up to the mark
with pure glacial acetic acid.
3. Ferric chloride diluting reagent: 8.5 ml of stock ferric chloride is diluted
to 100ml with pure glacial acid.
4. Standard cholesterol solution: 100 mg of cholesterol was dissolved in
100 ml of glacial acetic acid.
5. 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 microgram/ml.

Procedure

To 0.1 ml of plant extract added 4.9 ml of ferric chloride precipitating reagent.

Centrifuged and to 2.5ml of supernatant added 2.5 ml of ferric chloride diluting
agent. Added 4.0ml of concentrated sulphuric acid. A blank was prepared
simultaneously by taking 5.0 ml of diluting reagent and 4.0ml of concentrated
sulphuric acid. A set of standards (0.5-2.5ml) were taken and made up to 5.0 ml
with ferric chloride diluting reagent. Then added 4.0 ml of concentrated sulphuric
acid. After 30 minutes, the intensity of color developed was read at 540 nm against
a reagent blank. The amount of cholesterol in the sample is expressed as mg/dl.
 APPENDIX XXXIII

ESTIMATION OF PHOSPHOLIPIDS

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
660nm

Reagent

1. 70% perchloric acid

2. 3%ammonium molybdate

3. 3%ascorbic acid

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

contains 80μg of phosphorous/ml.

Procedure

To 0.1ml of serum or lioid extract, 1.0ml of perchloric acid was added and
digested on a sand bath until it become colorless. The volume was made up to
5.0ml with water was taken. Standards in the range 5-20μg were also taken and
0.8ml of perchloric acid was added and made up to 5.0 ml with water.

To all tubes, 0.5ml of ammonium molybdate was added followed by 0.5ml

of ascorbic acid solution and mixed well. The tubes were heated in a boiling water
bath for 6 min and the color developed was read immediately at 700nm.

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 XXXIV

ESTIMATION OF TRIGLYCERDIES

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 450nm.

Reagents

1. Chloroform-methanol mixture (2:1)

2. Activated silicic acid: It was activated by washing silicic acid wuth 4N or

2N HCl and then with water until the washings become natural. After
drying, ether was added in sufficient was discarred. Silicic acid was then
dried at 60˚c and activated at 100˚c over night prior to use.

3. 0.2 N H2SO4

4. Saponification reagent: Dissolved 5g of KOH in 60ml water and added

40ml of isopropanal.

5. Sodium-metaperiodate reagent: To 77g of anhydrous ammonium acetate in

700ml water, added 60ml acetic acid and 650mg of sodium metaperiodate.
Dissolved and diluted in 11 with distilled water.

6. Acetyl acetone reagent:Added 0.75ml of acetyl acetone to 20ml of

isopropanol and mixed well.Added 80ml of distilled water and mixed.

7. Tripalmitin standard was containing 100μg/ml in chloroform.

Procedure

Took 0.1ml of the serum or dried lipid extract. Made up the volume to
4.0ml with isopropanol. Mixed well and added 400mg of silicic acid. Placed them
in amechanical shaker and centrifuged.
 TO 2.0ml of the supernatant added o.6 ml of saponification reagent and
incubated at 60-70˚c for 15min.After cooling added 1.0ml of sodium
metaperiodate and mixed well. Then added 0.5ml of acetyl acetone reagent and
mixed again. Incubated the tubes at 50˚c for 30min.After cooling read the color at
405nm.Standard tripalmitin (20-100μg ) were taken in tubes and treated similarly.

Triglycerides are expressed as mg/100ml in serum and mg/g in tissue

APPENDIX XXXV

ESTIMATION OF FREE FATTY ACID

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
1. Chloroform –heptane-methanol mixture (CHM mixture), the mixture was
prepared in the ratio of 200:150:7(v/v)
2. Activated silicic acid
3. 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.
4. 0.1% diethyl dithiocarbamate in n-butanol
5. 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 XXXVI
HISTOPATHOLOGICAL EXAMINATION

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.