UNIT: Enzymes II (Kinetic / Rate Reaction)
Amylase Procedure
Thermo Procedure 3.2.1.1
Summary
Determination of amylase activity in serum and urine is considered to be useful for the diagnosis
of acute pancreatitis. In acute pancreatitis, the amylase activity in urine samples remains
elevated for longer periods than in serum. Some authors recommend the determination of the
ratio of the amylase and creatinine clearance is important in following the course of the disease.
A decrease in pancreatic amylase is characteristic of pancreatic insufficiency.
Principle- see product insert for clarification
Amylase hydrolyzes the alpha - 1,4-glucan link in starch and certain other polysaccharides to
form maltose. Many of the methods described in the literature for the determination of amylase
activity are based on the hydrolytic activity of the enzyme. Procedures based on coupled
enzyme reactions have been described for the determination of the enzyme activity.
Methodologies which were developed recently, use p-nitrophenylglycosides as substrates for the
determination of alpha-amylase activity.
In the procedure alpha- amylase hydrolyzes a p-nitrophenylglycoside substrate forming pnitrophenyl and glucose. The p-nitrophenyl absorbs light at 405 nm and following a 2 minute
lag period the rate of increase in absorbance at 405 nm is directly proportional to the amylase
activity in the sample. Details of the reaction can be found on product insert.
Specimen Collection and Storage
Unhemolyzed clear serum is the specimen of choice. Heparinized plasma may be used, but
plasma from other anticoagulants such as citrate, oxalate and EDTA may inhibit amylase
activity. The pH of urine samples (random or timed specimens) should be adjusted to 7 before
storing at 2-8 o C. Amylase activity is stable for several weeks when the samples are stored
refrigerated 2-8 o C.
Materials Required But Not Provided
1.
2.
Waterbath or heating block set to assay temperature.
Temperature controlled spectrophotometer and cuvets with optical properties suitable for
use at 405 nm.
3.
Pipetting devices for accurate delivery of volumes required for the assay.
4.
Timer & test tubes.
Procedure
The temperature of the reaction mixture should be maintained at the desired assay temperature
of 37o C.
1.
Reagent preparation.
a.
Dissolve contents of each vial in volume of deionized water indicated on vial
label. Avoid pipetting by mouth to prevent contamination of the reagent by
salivary amylase.
b.
Stopper vial and immediately mix several times by inversion. DO NOT SHAKE.
c.
Reconstituted reagent is stable until expiration date when refrigerated.
MLT 2401 Lab Manual 1
UNIT: Enzymes II (Kinetic / Rate Reaction)
2.
Optimize the spectrophotometer at 405 nm, and cuvet compartment to desired
temperature. Use deionized water to zero instrument.
3.
Into test tubes marked to identify control(s) and patient(s), pipette 2.0 mL of amylase
substrate and place in 37o C waterbath for 5-10 minutes. Also prewarm a dry cuvette.
4.
To one (1) tube at a time***, add 0.050 mL of sample. Mix immediately by inversion and
return it to the waterbath for 240 seconds to allow reaction kinetics to become linear
(zero order). Transfer the mixture to the dry prewarmed cuvette.
5.
At the end of the 4 minutes, wipe away any water or marks on the outside of the cuvette
and place it in the spectrophotometer.
6.
Read and record the absorbance at 405 nm vs water as reference. Start the
stopwatch. This is INITIAL ABSORBANCE.
7.
Continue incubation by returning the cuvette containing the solution to the waterbath
swirling the contents gently.
8.
Record absorbance again after exactly 1 minute. Repeat the process and record
absorbance at 2 minutes. Repeat the process for all specimens.
9.
Calculate the change in absorbance per minute by subtracting INITIAL ABSORBANCE
from FINAL ABSORBANCE and dividing by 2. The change in absorbance should be at a
constant rate.
Calculations
The determination of amylase concentration is based on the molar absorptivity of p-nitrophenyl
at 405 nm. The complex formula can be viewed in the product insert. A reduction of the formula
will result in determination of amylase concentration by the following calculation:
Amylase Activity (U/L) at 37o C. = Averaged change in Absorbance per min. x 6406
Example:
When a serum sample was assayed for amylase activity, the following absorbance values were
obtained at 37o C.
INITIAL A
FINAL A
= 0.110
= 0.340
change in absorbance/min = (0.340 - 0.110) / 2 = 0.115
Amylase Activity (U/L) at 37o C = 0.115 x 6406 = 736 U/L
Name
Date
MLT 2401 Lab Manual 2
UNIT: Enzymes II (Kinetic / Rate Reaction)
Amylase Report Form
Reaction temperature
Spectrophotometer
Wavelength
Calculation factor
Absorbance
Initial
1 min
2 min
(Final A)
average
change/min
Concentration U/L
control 1
control 2
patient 1
patient 2
Example Calculation
MLT 2401 Lab Manual 3