Perform the amylase method via a manual kit by Stanbio
Label 4 tubes and then zero spectrophotometer with DI water (405 nm), add Amylase reagent to each tube and incubate at 37 C for 3 minutes. Add appropriate sample to each tube one at a time and then read immediately. Read absorbance at 30 seconds intervals for 2 minutes. Determine the mean absorbance difference per minute, and multiply that by 4824 to get result in U/L.
Identify interfering substances for amylase determination
Drugs and substances
reference range for amylase serum
reference range for amylase urine
principle of the amylase determination
The principle of this method is based on alpha-amylase enzyme which catalyzes the hydrolytic cleavage of starch and glycogen. The procedure is based on the Wallenfels modification which uses substrate p-Nitrophenyl-D-maltoheptaoside (PNPG7) with the terminal glucose blocked to reduce spontaneous degradation of the substrate by glucosidase and glycoamylase. Amylase hydrolyzes p-Nitrophenyl D-maltoheptaoside (PNPG7) to p-Nitrophenyl-maltotriose (PNPG3) and maltotetraose. Glucoamylase hydrolyzes PNPG3 to p-Nitrophenylglycoside (PNPG1) and glucose. Then PNPG1 is hydrolyzed by glucosidase to glucose and p-Nitrophenol, which produces a yellow color. The rate of increase in absorbance is measured at 405 nm and is proportional to the amylase activity in the sample.
concentration of amylase using change in absorbance over time.
U/L = ?Abs./min. x 4824
State classic reference method for amylase activity
Briefly describe the classic reference method for amylase activity
Wallenfrels method uses PNPG7 which is hydrolyzed by amylase in the specimen to PNPG3 and maltotetraose. PNPG3 is then is hydrolyzed by glucoamylase to PNPG1 and glucose. PNPG1 is hydrolyzed by glucosidase to glucose and p-Nitrophenol which produces a yellow color. The rate of increase in absorbance is proportional to the amylase activity in the sample.
Briefly describe the analytical methods which may be used to determine amylase isoenzymes.
The analytic methods for determining amylase isoenzymes are (1) electrophoresis (2) ion-exchange chromatography (3) isoelectric focusing (4) selective inhibition of the S-AMY by a wheat germ inhibitor (5) immunoprecipitation by a monoclonal antibody and (6) immunoinhibition.
List several sources of error for the assay of amylase activity.
The sources of error for the amylase assay are interfering drugs and substances.
What is the diagnostic significance of elevated serum and urine amylase measurement?
The diagnostic significance of elevated serum and urine amylase is that amylase rised with 6 hours of pancreatitis.