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Flashcards in Serum Amylase Deck (15)
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0

Wavelength

660-800

1

Procedure

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.

2

Components of stock iodine solution (0.1 N)

24 g pure KI
13.5 g pure sublimed iodine

3

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

1 unit amylase

4

Standardized using 0.1 N sodium thiosulfate

Stock iodine solution (0.1 N)

5

Incubation period

15 minutes

6

Calculation

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

9

Principle

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.

9

Equipment

Spectrophotometer
Photoelectric colorimeter (red filter)

10

When should 0.1% starch solution be discarded?

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

11

Sample

Serum (or plasma)

12

Components of phosphate buffer, pH 7, 0.02 M

1.736 g anhydrous disodium hydrogen phosphate (Na2HPO4)
1.059 g potassium dihydrogen phosphate (KH2PO4)

13

Reference range

Serum : 25-130 U/L

13

Preserved with a few drops of chloroform

Phosphate buffer pH 7, 0.02 M

14

Reagents

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