APPENDIX I

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.

APPENDIX II

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 III
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 IV
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 V
ESTIMATION OF 5’ NUCLEOTIDASE ACTIVITY

(Tietz,, 1994)

Principle
The sample is incubated with AMP at pH 7.5 at 37°C with and without added
nickel ions. After 30 minutes the amount of inorganic phosphate liberated is
determined. Phosphate produced in the absence of nickel represents the combined
activity of alkaline phosphatase and 5’ nucleotidase. Where as that produced in the
presence of nickel is due to the activity of alkaline phosphatase alone. Thus the
difference between these two values for liberated phosphate corresponds to the
activity of 5’ nucleotidase in the sample which is measured at 680nm. Manganese
ion is used as the activator of 5’ nucleotidase. The presence of copper ions or
acetate buffer accelerates the colour development.
Reagents
 Barbiturate buffer, 40 mM/L, and pH 7.5: Dissolved 8.25g of sodium
diethyl barbiturate in 140ml of 0.2M HCl and made up the volume to 1 litre
with water and adjusted the ph to 7.5 at 37°C is necessary.
 Substrate, 10 μM/L: Dissolved 365mg of adenosine 5’ phosphoric acid
monohydrate in 18ml of 0.1M/L NaOH and diluted to 100ml with water.
Alternatively used 391mg of disodium adenosine 5’ phosphoric acid and
diluted to 100ml with water.
 Manganese ion solution, 20 mM/L: Dissolved 338mg of manganese
sulphate (MnSO4. H2O) or 396mg of manganese chloride (MnCl2. 4H2O) in
100ml of water.
 Nickel chloride solution, 0.1 M/L: Dissolved 2.4g of NiCl2.6H2O in 100ml
of water.
 10%TCA
 Acetate buffer, 2.4 M/L, pH 4.0: Dissolved 2.5g of copper sulphate and 46g
of sodium acetate trihydride in 1 litre of 2.0 M/L acetic acid (115ml glacial
acetic acid diluted to 1 litre). Adjusted the pH to 4.0 if necessary.
 Ammonium molybdate solution: Dissolved 5.0g of ammonium molybdate
in 100ml of water.
 Reducing agent: Dissolved 2.0g metol in 80ml of water. Added 5.0g of
sodium sulphate and diluted to 100ml with water and filtered. Stored in a
brown bottle at 4°C.
 Stock phosphate solution: Dissolved 2.193g of anhydrous KH 2PO4 in
500ml water. Added 0.2ml of concentrated H2SO4 as a preservative before
diluting to the mark. Stored at 4°C.
 Working standard solution: Diluted 1.0ml of stock standard solution to
100ml in a standard flask with 10% TCA. 1.0ml of the solution contains
10μg of phosphate.
Procedure
To the test added 1.5ml of water and 0.1ml of manganese solution. To the
control added 1.3ml buffer, 0.1ml manganese solution and 0.2ml of sample and
0.2ml nickel chloride solution. Mix the contents well. Added 0.2ml of substrate
solution to both the tubes. Mixed and incubated at 37°C for 30 minutes. Stopped
the reaction by adding 2.0ml of 10% TCA to all the tubes. Mixed the contents
thoroughly and centrifuged at 3000g for 15 minutes. Pipetted out 2.0ml of the
clear supernatant from test and control mixtures into two clear test tubes. To all the
tubes added 3.0ml of acetate buffer,0.5ml of ammonium molybdate solution and
0.5ml of metol solution. Mixed well and read the colour developed at 680nm after
5 minutes. The colour was stable for atleast 30 minutes.
The enzyme activity was expressed as μmoles of phosphate liberated/minute/L
of sample in case of serum and urine. In tissue homogenate the activity was
expressed as μmoles of phosphate liberated/minute/mg protein.

APPENDIX VI
GAMMA GLUTAMYL TRANSFERASE
(Szasz Method)
Principle
Gamma-GT catalyzes the transfer of glutamic acid to acceptors like
glycyglycine. This process releases 5-amino-2-nitrobenzoate, which absorbs light
at 405 nm. The increase in absorbance at this wavelength is directly related to the
activity of gamma-GT.

Gamma-GT
L-Gamma-glutamyl-3-carboxy-4-nitroanilide + Glycyglycine
Gamma-glutamyl-glycylglycine + 5-Amino-2-nitrobenzoate
Reagents
Reagent 1: Enzyme reagent
Standard : 200 mg/dl

Procedure
Add 100μl of sample and 1000 μl of reagent 1 to all the tubes. Mix,
incubate for 5 min. then added 250 μl of reagent 2. Mix well, after 1min, read the
decrease in absorbance every minute for 3 minutes.

Calculation
Gamma-GT-activity[U/I] = ΔA/min x factor

APPENDIX VII

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 – VIII

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 – IX

ESTIMATION OF β-D-GLUCURONIDASE

(Kawai and Anno, 1971).

Reagents

 Citrate buffer 0.1 M pH 3.5: 1.04g of citric acid and 1.48g of sodium citrate
were dissolved in 100ml distilled water.
 Glycine buffer pH10.7: 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.
 Standard: 5mg of p- nitrophenol was dissolved in 100ml of distilled water.

Procedure

0.9 ml of substrate was incubated with 0.1ml of enzyme at 37°C for 60

minutes. The reaction was arrested by the addition of 3.0 ml of glycine buffer. To
the control tubes, 0.1 ml of enzyme was added. The colour developed was read at
400 nm against a reagent blank in a Photochem colorimeter. Standards (20 – 100
µg) were also run simultaneously.

The enzyme activity is expressed as µmoles of p-nitrophenol

liberated/hr/mg protein.
 APPENDIX – X
CATHEPSIN D
Sapolsky et al. (1973).

REAGENTS

 Sodium acetate buffer 0.1 M, pH 3.6:92.5 ml of 0.1 M acetic acid was

mixed with 7.5 ml of 0.1 M sodium acetate solution.

 Substrate 1.5 % : 1.5 gm of haemoglobin was dissolved in 100 ml of

sodium acetate buffer.

 TCA 5% : Five g of TCA was dissolved in100ml of distilled water.

 Folin’s phenol reagent.

 NaOH 5 % : Five g of NaOH was dissolved in100ml of distilled water.

 Alkaline copper reagent (Lowry’s Reagent) was prepared.

 Standard : A solution of tyrosine in the concentration of 10 mg / 100 ml

was prepared with 0.1 N HCL.

PROCEDURE

0.9 ml of buffered substrate was mixed with 0.1 ml of enzyme preparation

and incubated for 2 hours at 37 °C. The reaction was stopped with 1.0 ml of 5 %
TCA and the samples were centrifuged for 10 minutes. To the control tubes, the
enzymes preparation was added after the addition of TCA. To 1.0 ml of
supernatant, 1.0 ml of 5 % sodium hydroxide and 4.5 ml of alkaline copper
reagent were added. After 10 minutes, 0.5 ml of
Folin’s phenol reagent was added and the colour developed was read at 640 nm
after15 minutes. The standard were treated similarly.

Enzyme activity is expressed as µmoles of tyrosine liberated / hour / mg

protein at
37 °C.

APPENDIX XI
ESTIMATION OF SUCCINATE DEHYDROGENASE
(Slater and Bonner, 1952)

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 FDH2
and gets reduced. Thus by following the decrease in blue color of the dye .The rate
of oxidation of succinate of fumarate.
Reagents
 0.3M phosphate buffer, pH 7.6
 0.03 M EDTA
 0.03M potassium cyanide
 0.4M sodium succinate, pH 7.6
 3% BSA (w/v)
 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 under incubation condition.

APPENDIX XII
ESTIMATION OF MALATE DEHYDROGENASE
(Mehler et al., 1948)

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
 0.2M phosphate buffer, PH 7.4
 0.76 μm oxaloacetate (15.4 mg/5ml)
 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 was carried at 25˚c and was 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 –XIII
ESTIMATION OF HEXOKINASE
(Banstrup et al., 1957)
Principle
The enzyme was assayed by determining the concentration of glucose by
orthotoludine method.
Reagents
 0.005 M glucose solution
 0.72 M ATP
 0.05 M Mgcl2
 0.0125 M KH2PO4
 M KCl
 0.5 M Sodium fluoride
 M Tris Hcl buffer, pH 8.0
 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 –XIV
ESTIMATION OF ALDOLASE
(King, 1965a)

Reagents
 0.1M Tris-Hcl buffer, pH 8.6
 0.05 M substrate: 17.05mg of fructose-1, 6-diphosphate in 10ml of Tris Hcl
buffer were prepared just before use.
 0.56N Hydrazine Sulphate, pH 8.6
 10%TCA
 Coloring reagent: 0.1%2,4 dinitrophenylhydrazine
 0.75 N sodium Hydrazine
 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 color developed after the addition of 7.0ml of
0.75N NaOH solution was read at [Link] 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 – XV
ESTIMATION OF PHOSPHOGLUCOISOMERASE
(Horrocks, 1963)

Principle
The assay is based on the estimation of fructose by the resorcinol thiourea
reagent, which is measured at 410nm.
Reagent
 0.1M borate buffer,pH 7.8
 Buffered substrate: 3mg of sodium glucose-6-phosphate were dissolved in
1.0ml of buffer. This was prepared fresh before use.
 30%HCl
 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.
 Color reagent: 30% HCl, resorcinol-thiourea and water were mixed in the
proportion [Link] .The solution was used in the same day as prepared .
 Standard solution: 5.4 mg of pure fructose was dissolved in 100ml of
0.25%Benzoic acid.
Procedure

In to 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 [Link] 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 – XVI
ESTIMATION OF GLUCOSE-6-PHOSPHATASE
(King, 1965b)

Principle
Glucose-6-phasephatase catalyses the reaction
Glucose-6-phosphate + H2O…> Glucose +pi.
The phosphorus content was estimated by the method of Fiske and Subbarow
Reagent
 0.1M citrate buffer, pH 6.5
Solution A: 0.1-M citric acid (21.01 g/L)
SolutionB: 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.
 Substrate: Glucose-6-phosphate, 0.01m (20 mg in 6.5 ml distilled water)
  2.5% Ammonium molybdate solution.
 ANSA
 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 pi liberated/min/mg protein.

APPENDIX – XVII
ESTIMATION OF FRUCTOSE-1, 6-DIPHOSPHATASE
(Gancedo and Gancedo, 1971)

Principle
Fructose-1, 6-diphosphatase catalyzes the reaction

Fructose-1, 6-diphosephate + H2O… >Fructose-6-phosphate +pi

The phospharus content was estimated by the method of Gancedo and Gancedo.
Reagent
 0.1M 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.
 subatrate: 0.05 M fructose-1,6-diphosephate solution
 0.1 M MgCl2
  0.1 M KCl
 0.001M EDTA
 10%TCA
 2.5% Ammoniun molybdate solution
 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 mgcl2 .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 pi liberated/min/mg
protein.
APPENDIX XVIII
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 XIX
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 XX
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 XXI
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 – XXII
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 XXIII
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 XXIV
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 XXV
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 XXVI
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 XXVII
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 XXVIII
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 XXIX

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 XXX
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 XXXI
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 XXXII
ESTIMATION OF HEXOSE
(Niebes, 1972)
Reagents
 orcinol- sulphuric acid reagent
Reagent A: Sulphuric acid – Water mixture (3.2v/v)
Reagent B: 1.6g of orcinol in 100 ml of distilled water
Reagents A & B were mixed in the ratio of 15:2 (v/v) justbefore use.
 Standard hexose solution: Fifty mg of galactose and 50 mg of mannose
were dissolved in 100 ml of distilled water. This solution was diluted to
1:10 proportion which gave a concentration of hexose 100μg/ml.

Procedure
An aliquot of the neutralized sample (0.5ml) was made upto 1.0 ml with
distilled water. To this 8.5 ml of ice-cold orcinol-sulphuric acid was added. The
contents were mixed well and the tubes were heated at 80°C for 15 minutes. Then,
the colour developed in dark, after cooling was read at 540nm using a photochem
colorimeter. Standard solution containing 25-100 μg of hexose and blank
containing 0.5ml of water instead of neutralized sample were also treated in a
similar manner.
 APPENDIX XXXIII
ESTIMATION OF HEXOSEAMINE
(Wagner, 1979)
Reagents
 Acetyl acetone reagent 3.5%
Reagent A: Trisodium phosphate 1M- 38g of trisodium phosphate was dissolved
in 100ml of distilled water.
Reagent B: Potassium tetraborate 0.5M – 1.9g of borate was dissolved in 10ml of
distilled water.
3.5 ml of acetyl acetone was added to a mixture containing reagent A and B in the
ratio of 98:2 (v/v).
 Ehrlich’s reagent: 320mg of p- dimethylaminobenzaldehyde was dissolved
in 21 ml of isopropanol and 3.0ml of concentrated HCl was added to it.
 Standard: 10mg of galactosamine was dissolved in 100ml of distilled water
which gave a concentration of 100μg/ml.
Procedure
0.4ml of the neutralized sample was made up to 1.0ml with distilled water.
Standard galactosamine (in the range of 10-40μg) was also made up to 1.0ml.
Blank comprised of 1.0ml distilled water, 0.6 ml of acetylacetone reagents was
added to all the tubes and heated in a boiling water bath for 30 minutes. After
cooling, 2.0 ml of Ehrlich’s reagent was added and then contents were shaken
well. The pink colour developed was read at 540nm against a blank using a
Photochem colorimeter.
 APPENDIX XXXIV
ESTIMATION OF SIALIC ACID
(Warren,1959)
Reagents:
 Periodate 0.25 M: 53.4mg of sodium periodate was dissolved in 100mg of
0.1N H2SO4.
 Sodium Meta arsenite 4%: 4g of Sodium Meta arsenite was dissolved in
100ml of 0.1N HCl.
 Thiobarbituric acid (TBA) 0.1M: 140mg of TBA was dissolved in 10ml of
distilled water. The pH was adjusted to 9.0 with 6N NaOH and the reagent
was prepared fresh.
 Acidified butanol: 95ml of n- butanol was mixed with 5.0ml of conc. HCl.
 Standard: 10mg of N- acetyl neuraminic acid was dissolved in 100ml of
distilled water which has a concentration of 100μg/ml.
Procedure
To 0.5ml of neutralized sample, 0.25 ml of periodate was added and incubated
at 37°C for 30 minutes. After incubation, the reaction was arrested by the addition
of 0.25ml of arsenite. The tubes were shaken well and 2.0ml of TBA was added
and the tubes were heated in a boiling water bath for 6 minutes. After cooling,
5.0ml of acidified butanol was wdded and the butanol phase was separated after
shaking well. The absorbance was read at 540nm against a blank treated similarly
using a Photochem colorimeter. Standard solutions containing 10-50μg of N-
acetyl neuraminic acid were also treated similarly.
 APPENDIX XXXV
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 XXXVI
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 XXXVII
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 XXXVIII
CHOLESTEROL
Principle
Cholesterol and its esters are released from lipoproteins by detergents.
Cholesterol esterase hydrolyses the esters. In the subsequent oxidation by
cholesterol oxidase, H2O, is liberated. The colorimetric indicator is quinoneimine
is generated from 4-aminoantipyrine and phenol by H2O2 under the catalytic action
of peroxidase (Trinder’s reaction) [3].
CHO
Cholesterol ester + H2O Cholesterol + Fatty Acid
CHO
Cholesterol + O2 Cholesterol-3-one + H2O
POD
2H2O2 + 4-Aminoantipyrine + Phenol Quinoneimine + 4H2O

Reagents
Reagent 1 Reaction solution 4 x 20 ml
Reagent 2 Standard solution 1 x 3 ml
Procedure
Add 100μl of sample and 1000 μl of reagent 1 to all the tubes. Mix,
incubate for 5 min. then added 250 μl of reagent 2. Mix well, after 1min, read the
decrease in absorbance every minute for 3 minutes.

Calculation
ASample
Cholesterol [mg/dl] = x [Link]  mg / dl
AStd
 APPENDIX XXXIX
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 XXXX
TRIGLYCERIDES
Principle

Determination of triglycerides involves enzymatic splitting with lipoprotein

lipase. Indicator is quinoneimine, which is generated from 4-aminoantipyrine and
4-chlorophenol by hydrogen peroxide under the catalytic action of peroxidase.

Lipase
Triglycerides Glycerol + fatty acids
GK
Glycerol + ATP Glycerol-e-phosphate + ADP

GPO
Glycerol-3-phosphate + O2 Dihydroxyaceton phosphate + H2O2
POD
2H2O2 + Aminoantipyrine + 4-Chlorophenol Quinoneimine + HCl+4 H2O

Reagents
Reagent 1: Reaction solution
Reagent 2: Standard solution

Procedure
Add 100μl of sample and 1000 μl of reagent 1 to all the tubes. Mix,
incubate for 5 min. then added 250 μl of reagent 2. Mix well, after 1min, read the
decrease in absorbance every minute for 3 minutes.

Calculation
ASample
Triglycerides [mg/dl] = x [Link]  mg / dl
AStd
 APPENDIX XXXXI
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 [Link](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 XXXXII

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 XXXXIII
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.