28.4 Reagents........................................
1.Starch substrate solution: Dissolve 75 mg of starch in about 20 ml of distilled
water with the aid of heat. Add 250 mg of NaCl, dissolve, and make thefinal
volume 100 ml with distilled water.
2.Stock iodine solution (0.1 M): Dissolve 40 g of potassium iodide in about 100 ml
distilled water, and add 12.7 g of iodine. Mix well to dissolve, and make thefinal
volume 1000 ml with distilled water.
3.Working iodine solution (0.05 M): 5 ml of stock iodine solution is diluted to
100 ml with distilled water. Prepare fresh before use. Store in a brown bottle.
32.5 Procedure
Pipette out 2 ml starch substrate in a test tube, and keep at 37C in water bath for
3 – 5 min. Add 0.5 ml serum and mix and then note time. After 5 min, remove 0.2 ml
of the above mixture from the incubator and add to 0.2 ml of working iodine solution
kept outside and mix and observe the color of iodine solution. If the color of iodine
solution is purple brown, this indicates that the starch present in that tube is not
completely hydrolyzed by the action of amylase enzyme. Again remove 0.2 ml of
starch serum mixture at the end of sixth min of incubation time, and add 0.2 ml
iodine solution. The procedure is repeated at every 1 min interval until the iodine
solution gives yellow color with serum starch mixture. Note the time for calculating
enzyme activity.
Urinary Amylase (Diastase) Estimation Procedure for the estimation of urinary
amylase is same as that of serum amylase. Report the amylase activity in terms of per
hour urine sample.
Note If the activity of urinary amylase and serum amylase is very high, dilute the
urine and serum and repeat the test and calculate the value and multiply the value
obtained with dilution factor.
28.6 Calculation
One Somogyi unit (SU) is defined as 1.5 mg of starch (2 ml substrate in present case)
hydrolyzed by the action of amylase present in 100 ml serum. If time taken for
hydrolysis is“t”min, then amylase activity (SU/100 ml serum) can be calculated as
¼
8 100
tvolof sample used114 28 To Determine Serum and Urinary Amylase Activity