AMYLASE REAGENT SET
For quantitative
determination of amylase in serum.
INTRODUCTION
Most colorimetric method used
for assaying the activity of a-amylase are based on its
breakdown of starch. This reaction primarily yields intermediate dextrins and
maltose. The rate of the reaction is usually monitored by a) turbidimetric b)
iodometric c) reductometric methods.1 The
iodometric method as later modified by
Caraway is still considered to be one of the reference method for the amylase
determination.2 Our procedure is a slight-modification of the
original Caraway method
PRINCIPLE
Starch
is hydrolyzed in the presence of amylase to intermediate dextrins and
maltose. Serum is incubated with
buffered starch substrate at controlled temperature for 7 1/2 minutes and
subsequently reacted with Iodine to produce blue color with reacted starch. The decrease in color, compared with that
obtained in the absence of amylase, provides a measure of amylase activity.
CLINICAL SIGNIFICANCE
Serum amylase levels are
elevated in acute pancreatitis, obstruction of pancreatic ducts (carcinoma,
stone, stricture, duct sphincter spasm after morphine), mumps; occasionally
elevated in the presence of renal insufficiency, diabetic acidosis, and with
inflammation of the pancrease from a perforating peptic ulcer. Serum amylase
levels are decreased in acute and chronic hepatitis, pancreatic insufficiency,
occasionally in toxemia of pregnancy, and in barbiturate poising.3
REAGENTS(MATERIALS PROVIDED)
A. AMYLASE SUBSTRATE:
A solution of 0.04% starch, 0.85% sodium chloride, 0.86%
benzoic acid and 2.7% disodium phosphate, adjusted to
pH = 7.0.
B. AMYLASE COLOR REAGENT:
A solution of 193 mM
potassium iodide and 11.9 mM potassium iodate in diluted hydrochloric acid.
MATERIALS REQUIRED BUT NOT PROVIDED
1. Accurate pipetting
devices.
2. Timer.
3. Test tube rack.
4. Spectrophotometer with
temperature controlled cuvette.
5. Heating bath (37°).
6. Control serum.
WARNINGS AND PRECAUTIONS:
1. For in vitro diagnostic use.
2. Exercise the universal precautions required for the handling of all laboratory reagents. Pipetting by mouth is not recommended for any laboratory reagent.
REAGENT STORAGE
All reagents supplied with
this procedure may be stored at room temperature. The reagents can be used until the expiration
date indicated on the individual bottle.
REAGENT DETERIORATION
1. Physical Appearance:
Mold growth or the appearance of turbidity in the AMYLASE
SUBSTRATE may indicate reagent deterioration and the product should be
discarded.
2. Control Assays:
Failure to obtain accurate results in the assay of control
materials may indicate reagent deterioration.
SPECIMEN COLLECTION AND HANDLING
Serum, free of bacterial and
fungal contamination, is reportedly stable for one week at room temperature and
several months at
4 - 8°C, however, it is
generally recommended that the specimen be refrigerated if there is a delay
between acquisition of the specimen and analysis.
PROCEDURE (MANUAL ENDPOINT METHOD)
1. Place 0.5ml of AMYLASE SUBSTRATE into test tubes labeled
"blank", "control", "unknown", etc. and place in
37°C heating bath for 3-5 minutes.
2. Using timed intervals, add 0.010 ml (10 µl ) of sample to its
respective tube, mix by gently swirling and immediately return to heating bath
for exactly 7 1/2 minutes.
3. Remove tubes from 37°C heating bath and immediately add 4.0 ml
of distilled water, then add 0.5 ml of AMYLASE COLOR REAGENT. Mix by gently
inversion (4-5 times). DO NOT REVERSE
ORDER OF REAGENT ADDITION.
4. Let all tubes stand at room temperature for 15 minutes.
5. Set wavelength of spectrophotometer at 590 nm and zero
instrument with distilled water. Read and
record absorbance of all tubes.(Wavelength range:580-630).
* USE TC - MUTI PURPOSE CALIBRATOR TO
REPLACE STANDARD.
PROCEDURE NOTES
One amylase unit is defined
by Caraway (2) as the amount of enzyme that will hydrolyze 10 mg of starch in 30 minutes to a state at
which no color is produced by the addition of iodine; therefore, in 7 1/2
minutes with a 0.01 ml sample, 0.25 mg of starch is required per reaction
mixture.
STABILITY OF ENDPOINT REACTION
The final color produced in
the reaction is stable for about
30 minutes.
CALIBRATION
By definition, the Caraway
Unit for the amylase activity is that amount of enzyme that will digest 10 mg
starch in 30 minutes at 37°C. From the
above reaction conditions, complete digestion of starch would require:
1,000 m x 0.5 ml x
30 min. x 100
= l,000
U/dl
2,000 ml 10 mg 7 1/2 min. 0.01 ml
Calculation of the unknown
then is based on the fractional decrease in starch multiplied by l,000 U/dl,
the activity present if all starch is digested. Since the reaction becomes
non-linear when about one-half substrate is used, linearity is limited to 500
U/dl.
QUALITY CONTROL
Fresh control sera with known
normal and abnormal values should be run per each assay to monitor the validity
of the reaction.
CALCULATION OF RESULTS
Use the absorbance readings
of the UNKNOWN(S) and BLANK to
calculate the amylase as
follows: (A = Absorbance)
A(BLANK) - A(UNKNOWN) x 1,000 = AmyIase
Activity in
A(BLANK) UNKNOWN (U/dl)
EXAMPLE OF CALCULATION:
(blank) absorbance = 0.73
(unknown) absorbance = 0.62
0.73 - 0.62 x 1,000*
= 151 U/dl
0.73
*1,000 = maximum activity of
enzyme needed to reduce all the starch present under conditions of the
procedure (see Procedure Notes)
PROCEDURE LIMITATIONS
1. Highly icteric or lipemic sera may produce false values in this
method so that serum blanks are recommended (see Procedure Notes)
2. Most anticoagulants, i.e., EDTA, sodium fluoride, citrate, and
oxalates reportedly produce erratic results due to binding of calcium ions
which are required for maximum activation.l
3. Abnormally low concentrations of protein in the sample
reportedly produce erratic results.3
4. The injection of morphine reportedly causes a temporary rise in serum
amylase levels for up to 24 hours, as does the ingestion of large amounts of
alcohol or the administration of thiazide diuretics.4
EXPECTED VALUES4
40 - 180 U/dl
Since the expected values are
affected by age, sex, diet, and geographical location, each laboratory is
strongly urged to establish its own reference range for this procedure.
SPECIFIC PERFORMANCE
CHARACTERISTICS
1. Linearity: Amylase values exceeding 500 U/dl should be re-run on
dilution.
2. Sensitivity: Typically, a decrease of 0.001
A (i.e. from 0.730 to 0 729) would represent about 1.4 U/dl under the above
assay conditions.
3. Comparison: Studies done manually between this procedure and a
similar procedure using forty-seven patients samples yielded a correlation
coefficient of 0.97 with a regression equation of
Y = 0.98X - 4.0 (N= 47).
4. Precision:
Within Run (N= 20)
Mean (mg,./dl) S.D. C.V.(%)
83 6.7 8.1
290 12.5 4.3
Run
to Run (N= 20)
Mean (mg./dl) S.D. C.V.(%)
84 8.3 9.9
284 27.2 9.5
REFERENCES
1. Henry, R.J., Clinical
Chemistry: Principles and Technics, Harper & Row, NY, p 469 (1964).
2. Caraway, W.T., Am. J.
Clin. Path. 32:97-99 (1959).
3. Tietz, N.W., Text Book of
Clinical Chemistry, W.B. Saunders, Philadelphia. p. 726 (1986).
4. Faulkner, W.R. and Meites S., Selected Methods for the Small Clinical Chemistry Laboratory, Vol.
9, AACC. Washington,
p. 91 (1982).
revised: 6/95