AMYLASE PROCEDURE

Intended for the Quantitative Determination of Amylase in Serum and Urine

SUMMARY AND EXPLANATION

Amylase in normal serum derives mainly from the pancreas and salivary gland. Dramatic increases occur with acute pancreatitis, and this enzyme activity is used primarily for evaluation of pancreatic function and disease. Elevations are seen also with salivary gland lesions, cystitis, cholelithiasis, perforated peptic ulcer, intestinal obstructions, diabetic ketoacidosis, and pregnancy (1).

The EAGLE AMYLASE PROCEDURE is based on a modification of the Caraway amyloclastic method (2). This procedure is still recommended as a reference method for amylase assay since results agree closely with those obtained by the Somogyi saccharogenic method.

PRINCIPLE

The amyloclastic procedure measures amylase activity by determining starch hydrolysis as evidenced by decreased starch - iodine formation. The enzyme activity is inversely proportional to the amount of blue - colored complex, which absorbs maximally at 590 nm.

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set Cat. No. 2300 provides:

AMYLASE SUBSTRATE - (Cat. No. 2301)

REACTIVE INGREDIENTS:

500 mg/dL starch buffered at pH = 7.0. Activator and stabilizer added.

PRECAUTIONS:

Do not ingest.

STORAGE AND STABILITY:

Store at 2 - 8° C. Stable until expiration date if sealed tightly.

DETERIORATION:

The substrate should be a clear, colorless solution. Turbidity would indicate deterioration. Failure to achieve assayed values of freshly prepared control sera also indicates deterioration.

IODINE REAGENT - (Cat. No. 2302)

REACTIVE INGREDIENTS:

5.4 mM potassium iodine in dilute HCI. Stabilizer added.

PRECAUTIONS:

Causes irritation. Avoid contact with eyes, skin and clothing. In case of contact, wash with large amounts of water.

STORAGE AND STABILITY:

Store at 2 - 8° C. Stable until expiration date if sealed tightly. PROTECT FROM LIGHT.

DETERIORATION:

The reagent should be a clear, orange solution. Discoloration or turbidity would indicate deterioration. When combined with the Amylase Substrate and diluted with D-H2O, this "Blank" should have an absorbance greater than 0.50, if not, the reagent set should not be used.

INSTRUMENTS

Use a spectrophotometer or colorimeter calibrated at 590 nm.

SPECIMEN COLLECTION

PRECAUTIONS:

1. Serum separated from the blood clot as soon as possible. Heparinized plasma may be used also.

2. EDTA, citrate and oxalate should not be used as anti-coagulants.

3. Urine may be analyzed by this method. Prepare a 5 X dilution prior to assay.

SAMPLE STORAGE:

Amylase appears stable in serum and urine for two weeks at 2 - 8° C.

ADDITIVES:

No special additives or preservatives are needed, although urine should be diluted 5 X with 0.9% sodium chloride solution prior to assay.

INTERFERING SUBSTANCES:

Some patient samples contain materials which inhibit starch - iodine formation and lead to falsely high results. The nature of this material is not known. Injection of morphine reportedly increases amylase levels. Young et al (3) have reviewed drug effects on serum and urinary amylase.

PROCEDURE

MATERIALS PROVIDED:

AMYLASE SUBSTRATE (Cat. No. 2301) and IODINE REAGENT (Cat. No. 2302).

MATERIALS REQUIRED BUT NOT PROVIDED:

1. 0.02 micropipettor

2. 0.5 mL and 4.0 mL pipettors

3. Incubator capable of maintaining 37° C

REACTION CONDITIONS:

Wavelength 590 nm

Filter Selection 570 - 610 nm

Incubation Temperature 37° C

Incubation Time 5 minutes

Sample Volume 0.02 mL

Substrate Volume 0.50 mL

Iodine Reagent Volume 0.50 mL

D-H2 O Volume 4.0 mL

Total Volume 5.02 mL

Low Normal (Serum) 40 U/dL

High Normal (Serum) 180 U/dL

Calibration Factor 750 U/dL

PERFORMANCE OF TEST:

1. Dispense 0.5 mL AMYLASE SUBSTRATE into tubes labeled Blank, Control, Sample 1, etc.

2. Prewarm for 5 minutes at 37° C.

3. At timed intervals, add 0.02 mL Specimen into appropriately labeled tube. Use D-H2O as sample for Blank.

4. Incubate for exactly 5 minutes at 37° C.

5. Following the same sequence as in Step 3, add 0.5 mL IODINE REAGENT at timed intervals and mix well.

6. After the incubation is completed, add 4.0 mL D-H2O to all tubes and mix well.

7. Adjust the instrument to zero absorbance at 590 nm using D-H2O.

8. Read and record absorbance values for Blank, Control and Unknowns.

PROCEDURAL NOTE:

Linearity extends to 400 U/dL. Specimens exceeding this value should be diluted 5 X with 0.9% sodium chloride solution and reassayed. Multiply the test result by the dilution factor to obtain the final answer.

STABILITY OF FINAL REACTION PRODUCT:

The test samples should be read within 15 minutes of color development.

CALIBRATION:

By definition, the Caraway Unit for 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:

500 mg 0.5 mL   30 min   100 mL/dL

---------------- x -------------- x ---------------- x ---------------------- = 750 U/dL

1000 mL 10 mg/U 5 min 0.02 mL  

Calculation of the Unknown then is based on the fractional decrease in starch multiplied by 750 U/dL, the activity present if all starch is digested. Since the reaction becomes non-linear when about one-half the substrate is used, linearity is limited to 400 U/dL

QUALITY CONTROL:

The reliability of test results should be monitored routinely using suitable quality control materials (normal and abnormal) analyzed in the same manner used for the Unknowns. EAGLE DIAGNOSTICS offers CHEM-TROL NORMAL (Cat. No. 8100) and CHEM-TROL ELEVATED (Cat. No. 8200) for this purpose.

CALCULATION OF RESULTS

The following equations are used to determine Unknown activities (see secion on CALIBRATION):

SERUM:

Unknown (U/dL) =

Abs. Blank - Abs. Unk.

--------------------------------

 X 750 U/dL Abs. Blank

EXAMPLE:

The Blank Abs. = 0.715 while the Unknown Abs. = 0.372. The Unknown activity is:

0.715 - 0.372  

------------------- X 750 U/dL = 360 U/dL

0.715

URINE:

Unknown (U/hr)   =

Abs. Blank - Abs. Unk.

------------------------------- X 750 U/dL

Abs. Blank

X 5 (dilution) X 0.01 dL/mL

Collection Volume (mL)

-------------------------------------

Collection Time (hr)

EXAMPLE:

The Blank Abs. = 0.715 while the Unknown Abs. = 0.572 for a 5 X dilution of a 2 hour collection that had 185 mL volume. The Unknown excretion rate is:

0.715 - 0.572

------------------ X 750 U/dL  X 5  X 0.01  X  

0.715

85 mL

--------- = 694 U/hr

2 hr

LIMITATIONS

1. Incubation temperature should be maintained at + 0.1° C

2. Contamination of Specimen or Reagents with saliva will lead to erroneously high results.

EXPECTED VALUES

Serum: 40 - 180 U/dL

Urine: less than 300 U/hr

The ranges represent the 95% confidence interval for 34 specimens obtained from a clinically normal population. The values are not age or sex dependent. It is recommended that each laboratory set its own range of expected values.

PERFORMANCE CHARACTERISTICS

PRECISION:

Normal and abnormal control sera were assayed 20 times each for within run precision and for 10 working days to establish run to run precision.

MEAN / ST. DEV. / %CV

WITHIN RUN

Normal 126 / 3.6 / 2.9

Abnormal 342 / 6.5 / 1.9

RUN TO RUN

Normal 122 / 4.4 / 3.6

Abnormal 348 / 7.7 / 2.2

SPECIFICITY:

A comparison of this AMYLASE PROCEDURE with another widely used commercial method showed a 99% correlation with 31 serum samples in the normal and abnormal range.

SENSITIVITY:

This AMYLASE PROCEDURE has a sensitivity of 1.05 U/dL per 0.001 absorbance unit.

REFERENCES

1. Caraway, T., Selected Methods for the Small Clinical Chemistry Laboratory, (ed. by Faulkner, W.R., and Meites, S.), Vol. 9, AACC, Washington, 1982, p. 91.

2. Caraway, W.T., Amer. J. Clin. Path. 32, 97 (1959).

3. Young, D.S., Pestaner, L.C., and Gibberman, V., Clin. Chem. 21, 255D (1975).