BIOCHEMICAL STUDIES OF FISH

Quantitative estimation of glucose, glycogen, protein, total lipid, cholesterol, acid

phosphatase and alkaline phosphatase were done in various tissues. Blood was collected
directly from the heart and analyzed for glucose, total protein and cholesterol. Muscle
tissues were analyzed for glycogen and total lipid.
Estimation of blood glucose Blood glucose was estimated using Folin and Wu (1919)
method.
Reagents
1 10% Sodium tungstate
2 2/3 N Sulphuric acid
3

Phosphomolybdic acid

4 0.1% Glucose stock

5 0.01% Glucose working standard
6 Alkaline copper reagent
Procedure
Deproteinisation of blood: Fresh blood without clot was collected. From this 1 ml was
pipetted out into 7 ml distilled water in a test tube so as to form a layer at the bottom. The
pipette was washed with the top layer of water in the test tube till it was cleared off the
blood. 1 ml of 10% sodium tungstate was added followed by 1 ml of 2/3 N sulphuric acid
to make the total volume to 10 ml. The contents were mixed well by vigorous shaking
and allowed to stand for a few minutes till the colour of the precipitate changed to
chocolate brown, and filtered through Whatman No. 41 filter paper. The protein free
filtrate was collected in a dry test tube.
Estimation of blood glucose: Three Folin Wu tubes were marked A, B and C for test,
standard and blank. 2 ml of protein free filtrate was taken in tube A, 2 ml of standard
glucose solution in tube B and 2 ml distilled water in tube C. To each tube 2 ml alkaline
copper reagent was added. The content of each tube was mixed well by tapping gently
between palms. The tubes were kept in boiling water bath for 8 minutes. After 8 minutes
the tubes were removed from the water bath and immediately cooled under running tap
water for 1 minute. 2 ml of phosphomolybdic acid was added without delay to each tube.
The contents were tapped between palms till the effervescence stopped. The tubes were

heated in boiling water bath for 1 minute to stabilize the colour. It was cooled and made
up to the mark with water. The contents were mixed well by inverting the tubes. Read test
and standard against the blank at 680 nm.
Estimation of glycogen
The liver and muscle glycogen was estimated using the anthrone reagent method (Seifter
et al., 1950).
Reagents :
1

60% Potassium hydroxide

30% KOH

Ethanol

4 Anthrone reagent
Procedure
The sample was homogenized by adding 0.5 ml of 60% KOH and 1 ml of 30% KOH,
both prepared in water. The mixture was incubated in a boiling water bath for 30 minutes.
4 ml of ethanol was added to the homogenate and it was kept in a refrigerator for 24
hours and then centrifuged at 3000 rpm. for 20 minutes. The pellet was resuspended in 1
ml of distilled water and from this 0.25 ml was taken and mixed with 1.75 ml of anthrone
reagent and kept in boiling water bath for 15 minutes. The colour developed was read at
620 nm spectrophotometrically.
Estimation of total protein
Total Protein was estimated using Biuret method (Gornall et al., 1949).
Reagents
1

Biuret reagent

0.85% Sodium chloride

2.5 N Sodium hydroxide

Standard protein(BSA)

Procedure

In a test tube 0.5 ml plasma was mixed with1.5 ml of 0.85% sodium chloride solution. 8
ml Biuret reagent was added to it. To prepare the standard, 1 ml of standard protein was
mixed with 1 ml of 0.85% sodium chloride solution. Then 8 ml of Biuret reagent was
added to this. To prepare the blank 2 ml of 0.85% sodium chloride solution was mixed
with 8 ml of Biuret reagent. All the three test tubes were shaken well and allowed to stand
for 30 minutes. Read test and standard against the blank at 520 nm.

Estimation of total lipids

Estimation of total lipid was carried out by Barnes and Blackstock (1973) method.
Reagents :
1

Chloroform methanol mixture (2:1)

Concentrated sulphuric acid

0.9% Sodium chloride solution

Phosphovanillin reagent

5 Chloroform
6

Cholesterol standard

Procedure 50 mg wet tissue was homogenized in 10 ml chloroform methanol mixture

using a glass homogenizer, filtered through Whatman No.1 filter paper and to this was
added 2 ml of 0.9% NaCl solution. This mixture was shaken well and transferred to a
separating funnel and was allowed to stand over night at 4 C. A clear biphasic layer was
formed with the lower phase containing all the lipids. It was removed and the volume was
made up to 10 ml by the addition of chloroform. This was transferred to a 50 ml beaker
and the solvent was allowed to evaporate at 50-60 0C for 5 hours. Then 5 ml of
concentrated sulphuric acid was added to it, mixed well, placed in boiling water bath for
ten minutes and was then cooled to room temperature. 0.2 ml of this was taken in a test
tube and 5 ml of phosphovanillin reagent was added. Mixed well and was allowed to
stand for half an hour. Standard was prepared by mixing 0.2 ml of standard cholesterol
and 5 ml of phosphovanillin reagent, allowing it to stand for half an hour. Blank was
prepared by taking 0.2 ml of chloroform and 5 ml of phosphovanillin reagent. Read test
and standard against the blank at 520 nm.

Estimation of cholesterol
Estimation of cholesterol was carried out by Zlatkis et al. (1953) method.
Reagents :
1

Glacial acetic acid

2 0.05 % Ferric chloride

3

Concentrated sulphuric acid

0.9 % Sodium chloride solution

Cholesterol standard

Working standard ( 0.04 mg/ml )

Procedure
0.1 ml of serum was taken and 10 ml of ferric chloride reagent was added. Mixed well
and kept for 10 minutes at room temperature. It was then centrifuged for 10 minutes at
3000 rpm. 5 ml of the supernatant was pipetted out into a test tube and 3 ml of
concentrated sulphuric acid was added and mixed well. To prepare the standard, 10 ml of
working standard was mixed with 0.1 ml of sodium chloride and kept for 10 minutes and
centrifuged. 5 ml of the supernatant was taken and to this added 3 ml concentrated
sulphuric acid. Both the tubes were kept for 30 minutes at room temperature. To prepare
the blank, 5 ml of ferric chloride was mixed with 3 ml of concentrated sulphuric acid.
This was also kept for 30 minutes. Read test and standard against the blank at 560 nm.
References:
Folin, O. and Wu, H. 1919. A system of blood analysis. J. Biol. Chem., 38: 81.
Seifter, S., Dayton, S., Novic, B. and Muntwyler, E. 1950. The estimation of glycogen

with the anthrone reagent. Arch. Biochem. Biophys., 50: 191 200.
Gornall, A. G., Bardawil, C.J. and David, M.M. 1949. Determination of serum proteins
by means of the Biuret reagent. J. Biol. Chem., 177: 751756.
Barnes, H. and Blackstock, J. 1973. Estimation of lipids in marine animals and tissues:
Detailed investigation of the sulphophosphovanillin method for total lipids. J. Exp.
Mar. Biol. Ecol., 12: 103 118.
Zlatkis, A., Zak, B. and Boyle, A. J. 1953. A New method for the direct determination of
serum cholesterol. J. Lab. Clin. Med., 41: 486 492.