Flashcards in Serum Amylase Deck (15)
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.
Components of stock iodine solution (0.1 N)
24 g pure KI
13.5 g pure sublimed iodine
Amount of enzyme needed to hydrolyze 10 mg starch in 30 minutes
1 unit amylase
Standardized using 0.1 N sodium thiosulfate
Stock iodine solution (0.1 N)
Serum amylase activity in units/dL = (O.D. Control - O.D. Test/O.D. Control) x 800
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.
Photoelectric colorimeter (red filter)
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.
Serum (or plasma)
Components of phosphate buffer, pH 7, 0.02 M
1.736 g anhydrous disodium hydrogen phosphate (Na2HPO4)
1.059 g potassium dihydrogen phosphate (KH2PO4)
Serum : 25-130 U/L
Preserved with a few drops of chloroform
Phosphate buffer pH 7, 0.02 M