EXPERIMENT NO.

: 25

Aim: Determination of serum amylase.

Reference: Godkar Praful B.; “Clinical Biochemistry, principles and practice”; Published by
Bhalani Publishing house, Bombay, India; Page No: - 162.

Requirement: Chemical: serum sample, glucose, disodium hydrogen phosphate,

benzoic acid, potassium iodide, potassium iodate, distilled water.

Apparatus: Test tube, water bath, volumetric flask, pipette, burner etc.

Principle: Amylase in the specimen act on the substrate starch. The products formed are
dextrin and maltose. After the incubation when the end products are treated with the colour
reagent (iodine reagent), decrease the blue colour is observed, comparing to that is produced
with the blank. This appearance of the blue colour is directly proportional to the amylase
concentration in the specimen and gives the measure of amylase present in the specimen.

Preparation of reagents:

Buffered substrate: It is prepared by mixing 2.66 g anhydrous disodium hydrogen

phosphate, 0.86 g of benzoic acid and 0.04 g of starch in 100 ml of distilled water.

2) Stock colour reagent: It is prepared by mixing 0.3567 g of potassium iodate, 4.5 g of

potassium iodide and
0.9 ml of concentrated HCl in 100 ml of distilled water.

3) Working colour reagent: Dilute stock colour reagent 1:10 in distilled water to get
the working colour reagent. It should be prepared fresh for the test.

Procedure:

Pipette in the tube labelled as follows

Test Blank
Buffered substrate, ml 2.5 2.5
0
Keep at 37 C for 5 minutes
Serum, ml 0.1 -
 0
Mix, incubate at 37 C for 7.5 min
Working colour reagent, 2.5 2.5
ml
Serum, ml - 0.1
Distilled water, ml 20 20
Mix thoroughly and read intensity of test and blank against distilled water at 660 nm.

Observation:
Sr no. Absorbance of Absorbance of test
Control
1.
2.
3.
Mean

40

Calculation:

Serum amylase, caraway units = (O.D. Blank-O.D. Test/O.D. Blank) X 400

Result:

Evidence based additional knowledge