GOT / GPT PROCEDURES

Intended for the Quantitative Determination of Glutamic-Oxalecetic

and Glutamic-Pyruvic Transaminases in Serum Using a Colorimetric Method

SUMMARY AND EXPLANATION

Serum transaminases consist of enzymes which reversibly exchange amino and keto groups on alpha carbon positions of serum organic acids. Serum Glutamic-Oxalacetic Transaminase (GOT) uses aspartic acid as a substrate. This enzyme is prevalent in heart, liver, muscle and kidney tissue. Its elevation in serum can be used for differential diagnosis involving these organs. Serum Glutamic-Pyruvic Transaminase (GPT) uses alanine as a substrate. GPT exists primarily in the liver (1).

PRINCIPLE

Reitman and Frankel' described a calorimetric procedure for assaying GOT and GPT activities. End products of the transaminations (oxalacetate or pyruvate) react with dinitrophenylhydrazine to form a hydrazone complex. This product yields a colored complex when alkaline diluent is added. The color intensity at 505 nm can be related to enzyme activity by reference to a

standard curve.

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set Cat. No. 2850 includes:

GOT SUBSTRATE - (Cat. No. 2851)

REACTIVE INGREDIENTS:

200 mM aspartic acid and 1.8 mM ketoglutaric acid buffered at pH 7.5. Preservative 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.

GPT SUBSTRATE - (Cat. No. 2852)

REACTIVE INGREDIENTS:

200 mM alanine and T.8 mM ketoglutaric acid buffered at pH 7.5. Preservative added.

PRECAUTIONS:

Do not ingest.

STORAGE AND STABILITY:

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

DETERIORATION:

The substrate should be a clear and colorless solution. Turbidity would indicate deterioration.

GOT / GPT CALIBRATION SOLUTION - (Cat. No. 2854)

REACTIVE INGREDIENTS:

1.5 mM pyruvate buffered at pH 7.5. Preservative added.

PRECAUTIONS:

Do not ingest.

STORAGE AND STABILITY:

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

DETERIORATION:

The calibration solution is a clear and colorless solution. Turbidity would indicate deterioration.

GOT / GPT COLOR REAGENT - (Cat. No. 2853)

REACTIVE INGREDIENTS:

1.0 mM 2,4-dinitrophenylthydrazine in dilute acid.

PRECAUTIONS:

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

STORAGE AND STABILITY:

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

DETERIORATION.

The color reagent is a clear yellow solution. Turbidity or a brown color would indicate deterioration.

INSTRUMENTS

Use a spectrophotometer or colorimeter calibrated at 490 - 520 nm.

SPECIMEN COLLECTION

PRECAUTIONS:

1. Use unhemolyzed serum separated from the blood clot as soon as possible.

2. Plasma may be used.

3. Grossly hemolyzed or lipemic samples should not be used.

SAMPLE STORAGE:

Serum transaminases appear stable for one week at 2-8ºC.

ADDITIVES:

No special additives or preservatives are needed.

INTERFERING SUBSTANCES:

Serum from patients receiving erythromycin may show elevated results (3). Young et al (4) have reviewed drug effects on serum transaminases.

PROCEDURE

MATERIALS PROVIDED:

GOT SUBSTRATE (Cat. No. 2851), GPT SUBSTRATE (Cat. No. 2852), GOT / GPT CALIBRATION SOLUTION (Cat. No. 2854) and GOT / GPT COLOR REAGENT (Cat. No. 2853).

MATERIALS REQUIRED BUT NOT PROVIDED:

1. 0.1 mL and 0.5 mL micropipettors

2. 5.0 mL pipettor

3. Incubator capable of maintaining 37+ 0.5ºC

REACTION CONDITIONS:

Wavelength 490-520 nm

Incubation Temperature 37ºC

Incubation Time GOT 60 min

GPT 30 min

Sample Volume 0.1 mL

Substrate Volume 0.5 mL

Color Reagent Volume 0.5 mL

Alkaline Diluent Volume 5.0 mL

Total Volume 6.1 mL

PREPARATION OF ALKALINE DILUENT:

Dissolve 16 grams of sodium hydroxide in 1.0 L D-H2O. Allow to cool before use.

PREPARATION OF CALIBRATION CURVE:

All reagents should be at room temperature.

1. Separate calibration curves must be prepared for GOT and GPT. Use the specific substrate and set up the following tubes:

2.

TUBE D-H2O (mL) SUBSTRATE (mL) cal. Ref. (mL) GOT/GGPT (U/mL)

1 0.2 1.0 0.0 0 0

2 0.2 0.9 0.1 22 25

3 0.2 0.8 0.2 55 50

4 0.2 0.7 0.3 95 83

5 0.2 0.6 0.4 150 126

6 0.2 0.5 0.5 215 ---

3. Add 1.0 mL color Reagent to each tube. Mix well and let stand for 5 minutes at room temperature.

4. Add 10.0 mL alkaline diluent to each tube. Mix well and let stand for 5 minutes at room temperature.

5. Zero instrument with D-H2O at 505 nm (490 - 520 nm acceptable).

6. Read and record absorbances.

7. Plot enzyme activity versus absorbance. A non-linear curve is obtained with decreasing slope as enzyme activity increases.

8. Linearity extends to 215 U.mL for GOT and 126 U/mL for GPT. If enzyme activity exceeds these levels, dilute the specimen X 10 with isotonic saline and repeat the assay. Multiply the result X 10 to correct for dilution.

PERFORMANCE OF TEST:

1. Dispense 0.5 mL substrate (GOT or GPT) into tubes labeled: REAGENT, BLANK, CAL. SOLN., CONTROL, SAMPLE, etc.

2. Prewarm at 37º C for 3 minutes.

3. At timed intervals, add 0.1 mL sample into appropriately labeled tube and mix well. Use D-H2O as specimen into appropriately labeled tube and mix well. Use D-H2O as specimen for Reagent Blank.

4. Incubate at 37º C for 60 minutes for GOT and 30 minutes for GPT.

5. At timed intercals, add 0.5 Color Reagent to each tube and mix well.

6. Allow to stand at room temperature for 20 minutes.

7. Add 5.0 mL Alkaline Diluent to each tube and mix well. Let stand for 5 minutes at room temperature.

8. Zero instrument with Reagent Blank at 505 nm (490 - 520 nm acceptable.

9. Read and record absorbances for CAL. SOLN., CONTROL, SAMPLE 1, etc.

10. Determine enzyme activity from calibration curve.

STABILITY OF FINAL REACTION PRODUCT:

The test samples should be read within 30 minutes.

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.

The calibration curve should be validated with each run. This is accomplished by using the calibration solution in the performance of test and obtaining the value from the curve. The calibration solution has a GOT value equal to 51 U/mL and GPT value equal to 132 U/mL.

CALCULATION OF RESULTS

Determine enzyme activities using the calibration curves generated with GOT / GPT CALIBRATION SOLUTION.

LIMITATIONS

Detergent-FREE glassware should be used.

EXPECTED VALUES (2)

GOT 8 - 40 U/mL

GPT 5 - 30 U/mL

PERFORMANCE CHARACTERISTICS

PRECISION:

Within Run - Normal and abnormal control sera were assayed 20 times each for within run precision:

MEAN / STD. DEV. / %cv

Normal

GOT 22 / 0.92 / 4.2

GPT 19 / 0.97 / 5.1

Abnormal

GOT 80 / 0.94 / 1.2

GPT 55 / 0.94 / 1.7

Run to Run - Normal and abnormal control sera were assayed 10 times each for within run precision:

MEAN STD. DEV. %cv

Normal

GOT 25 / 1.65 / 6.6

GPT 22 / 1.56 / 7.1

Abnormal

GOT 83 / 1.54 / 1.8

GPT 59 / 1.60 / 2.7

SPECIFICITY:

A comparison study of the GOT / GPT with another widely used commercial method show a 98% correlation for GOT and a 96% correlation for GPT.

SENSITIVITY:

The GOT / GPT PROCEDURE have a GOT sensitivity of 0.39 U/mL and a GPT sensitivity of 0.37 U/mL per 0.001 absorbance units.

REFERENCES

1. Davidson, I., and Henry, J.B., Clinical Diagnosis by Laboratory Methods, W.B. Saunders Co., Philadelphia, 1974, p. 848.

2. Reitman, S., and Frankel, S., Am. J. Clin. Path. 28, 56 (1957).

3. Sabath, L.D., Gerstein, D.A. and Finland, M., New Eng. J. Med. 21, 1137 (1968).

4. Young, D.S., Pestaner, L.C., and Gebberman, V. Clin. Chem. 21, 242D and 258D (1975).