Serum Amylase

Question 0

Procedure

Answer 0

1 Prepare a 1:10 dilution of the serum or plasma by adding 0.1 mL to 1.8 mL of 0.85% NaCl then mix.
2 Set up 6x5/8” test tubes and mark them test and control
3 To both tubes add 0.5 mL buffer and 0.4 mL 0.1% starch solution
4 Warm both tubes for 5 minutes in the 37 Celsius water bath
5 To the “test” tube, add 0.1 mL of the serum dilution, replace the tube in the bath and set timer for 15 minutes
6 Place a small beaker of distilled water in the freezer.
7 At the end of the 15 minutes incubation, remove both tubes from the water bath and immediately add to both 8.6 mL of the ice-cold distilled water
8 To both tubes, add 0.4 mL of 1/100 iodine solution mixing well by inversion immediately after each addition
9 To the control, add 0.1 mL of the serum dilution and mix by inversion
10 Read the optical densities of both test and control on the spectrophotometer at 660-800 mu or in a photoelectric colorimeter using a red filter setting the zero point with distilled water in each case. Readings must be made within 5 minutes of adding the iodine and preferably without delay.

Question 1

Wavelength

Answer 1

660-800

Question 2

Components of stock iodine solution (0.1 N)

Answer 2

24 g pure KI

13.5 g pure sublimed iodine

Question 3

Amount of enzyme needed to hydrolyze 10 mg starch in 30 minutes

Answer 3

1 unit amylase

Question 4

Standardized using 0.1 N sodium thiosulfate

Answer 4

Stock iodine solution (0.1 N)

Question 5

Incubation period

Answer 5

15 minutes

Question 6

Calculation

Answer 6

Serum amylase activity in units/dL = (O.D. Control - O.D. Test/O.D. Control) x 800

Question 9

Principle

Answer 9

1 The enzyme in a fixed amount of serum is allowed to act on a fixed amount of starch under fixed conditions.
2 The amount of starch destroyed is measured by adding iodine.
3 The amount of color blue produced is compared with that of an iodine-treated central starch solution, which has not been exposed to amylase.

Question 9

Equipment

Answer 9

Spectrophotometer
Photoelectric colorimeter (red filter)

Question 10

When should 0.1% starch solution be discarded?

Answer 10

Discard after 1 week or sooner if O.D. of the control falls below the value obtained when the starch is fresh.

Question 11

Sample

Answer 11

Serum (or plasma)

Question 12

Components of phosphate buffer, pH 7, 0.02 M

Answer 12

1. 736 g anhydrous disodium hydrogen phosphate (Na2HPO4)

1. 059 g potassium dihydrogen phosphate (KH2PO4)

Question 13

Reference range

Answer 13

Serum : 25-130 U/L

Question 13

Preserved with a few drops of chloroform

Answer 13

Phosphate buffer pH 7, 0.02 M

Question 14

Reagents

Answer 14

1 0.85% NaCl
2 Phosphate buffer pH 7, 0.02 M
3 Stock iodine solution, 0.1 N
4 Working iodine solution
5 0.1% starch solution