ALT (SGPT) PROCEDURE

Intended for the Quantitative Determination of Alanine Aminotransferase in Serum

SUMMARY AND EXPLANATION

Serum Glutamic Pyruvic Transaminase (SGPT), named Alanine Aminotransferase (ALT) in compliance with IFCC standards, is one of several tissue enzymes that catalyze the exchange of amino and oxo groups between alpha-amino acids and alpha-oxo acids. Elevated levels are observed in hepatitis, muscle disease and trauma. Its major use is in the diagnosis of liver damage and when used in conjunction with AST (SGOT) it can aid in the diagnosis of myocardial infraction by its value remaining at a normal level in the presence of an elevated AST level.

ALT methods using U.V. procedures were first described by Henley (1) in 1955 and later by Wroblewski and LaDue (2) in 1956. The most current improved method has been recommended by the International Federation of Clinical Chemistry (IFCC) (3) utilizing the LDH-NADH coupled reaction. The EAGLE ALT PROCEDURE is based on this recommended method.

PRINCIPLE

ALT catalyzes the transfer of the amino group from L-Alanine to a-Ketoglutarate resulting in the formation of pyruvate and L-glutamate. Lactate dehydrogenase catalyzes the reduction of pyruvate and the simultaneous oxidation of NADH to NAD. The resulting rate of decrease in absorbance at 340 nm is directly proportional to ALT activity. This reagent does not contain P-5-P since the diagnostic significance of increasing ALT is still under study (5,6).

       L-alanine + a-Ketoglutarate      ALT   Pyruvate + L-Glutamate

        Pyruvate + NADH+ H +      LDH      L-Lactate + NAD + + H2 O

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set Cat. No.2250 provides:

ALT (SGPT) REAGENT - (Cat. No. 2251)

REACTIVE INGREDIENTS:

After reconstitution, the solution will have the following approximate concentration: a-Ketoglutarate, 12 mM; DL-Alanine, 400 mM; LDH, 1200 u/L NADH, 0.2 mM. Buffer, stabilizer and fillers added. pH = 7.8 + 0.1.

REAGENT PREPARATION:

Reconstitute the reagent with the amount of distilled water stated on the vial label. Reseal the vial. Swirl gently to dissolve. DO NOT SHAKE.

PRECAUTIONS:

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

STORAGE AND STABILITY:

Store at 2- 8ºC. Stable until expiration date if vial remains sealed and unopened. After reconstitution, the reagent is stable for 14 days stored at 2- 8ºC or for 8 hours stored at room temperature. Prolonged exposure to room temperature shortens the usability of the reagent even when stored at 2- 8ºC. For maximum stability, store the reagent at 2 - 8ºC immediately after reconstitution.

DETERIORATION:

In a sealed vial, the reagent should be a dry, white powder. Caking would indicate that moisture has entered the vial and the reagent should not be used. After reconstitution, the reagent should be a clear, colorless solution.Turbidity indicates deterioration and the reagent cannot be used. In addition, if the reconstituted reagent has an absorbance of less than 0.8 when read against DH20 at 340 nm, it should not be used.

INSTRUMENTS

Use a spectrophotomer or colorimeter calibrated at 340 nm and a heating block capable of maintaining 30ºC or 37ºC.

SPECIMEN COLLECTION

PRECAUTIONS:

Use fresh, unhemolyzed serum. Red cells contain large amounts of ALT.

SAMPLE STORAGE:

ALT in serum is reported not to be stable, therefore the sample should be assayed on the day of collection (7).

ADDITIVES:

No special additives or preservatives are required.

INTERFERING SUBSTANCES:

Some drugs and substances will affect ALT activity. Young et al (4) have reviewed drug effects on serum.

PROCEDURE

MATERIALS PROVIDED:

ALT REAGENT (Cat. No. 2251).

MATERIALS REQUIRED BUT NOT PROVIDED:

1. Instrument capable of reading at 340 nm

2. 0.1 mL micropipettor

3. 1.0 mL pipet or dispenser

4. Incubator capable of maintaining 30ºC or 37ºC

REACTION CONDITIONS:

Wavelength 340 nm

Reaction Type Kinetic

Incubation Temperature 30ºC or 37ºC

Prewarm Substrate 5 minutes

Preincubation Time 2 minutes

Incubation Time 1 minute

Sample Volume 0.1 mL

Reagent Volume 1.0 mL

Total Volume 1.1 mL

Normal (37ºC) << 38 IU/L

Calibration Factor 1768

PREPARATION OF WORKING REAGENT:

Reconstitute the reagent with the amount of distilled water specified on the vial label. Swirl to dissolve. DO NOT SHAKE.

AUTOMATED PROCEDURE:

Refer to specific instrument application for instructions.

MANUAL PROCEDURE:

1. Pipet 1.0 mL of reconstituted reagent in tubes labeled Control, Sample 1, etc. Prewarm tubes at 37ºC for five minutes.

2. Adjust instrument to zero absorbance at 340 nm using distilled water.

3. Place 0.1 mL specimen into appropriate tube, mix and incubate at 37ºC for two minutes.

4. After the two minute preincubation, record the absorbance (A1 reading). Return the tube to the 37ºC incubator.

5. After exactly one minute, read and record the absorbance (A2 reading). It is recommended that a second one minute reading be taken for better accuracy. Average the two readings to obtain change per minute.

NOTE: If the instrument used is equipped with a temperature controlled cuvette, the reaction mixture may be left in the cuvette while the absorbance readings are taken.

PROCEDURAL NOTES:

1. For instruments requiring a total volume 1.0 mL to read, increase Reagent Volume to 2.0 mL and Sample Volume to 0.2 mL and perform test as directed in MANUAL PROCEDURE.

2. Specimens with values greater than 500 IU/L should be diluted 1:1 with saline and reassayed multiplying the result by two.

STABILITY OF FINAL REACTION:

The reaction is ongoing, the timing of the readings must be accurate.

CALCULATION

One International Unit (IU/L) is defined as the amount of enzyme that catalyzes the transformation of one micromole of substrate per minute. Calculation factor = 1768 as derived from the following equation:

U/L =                                    

(A1 - A2) X 1.1 X 1000

---------------------------------   = (A1 - A2) X 1768

X 6.22 X 0.1

WHERE:

(A1 - A2) = Change in Absorbance

1.1 = Total reaction volume in mL

1000 = Conversion of IU/mL to IU/L

1 = Light path in cm

6.22 = Millimolar absorptivity of NADH

0.1 = Specimen volume in mL

EXAMPLE:

The initial reading (A1) abs. = 0.978, the second reading (A2) abs. = 0.822, the (A1 - A2) =  0.156, then 0.156 X 1768 (factor) = 275 IU/L.

Conversion to SI Units (nkat/L): IU/L X 16.67 = SI

CALIBRATION:

The reaction is standardized by means of the molar absorptivity of NADH taken as 6.22 at 340 nm under the test conditions outlined. Results are based on the change in absorbance per minute.

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 Unknowns. EAGLE DIAGNOSTICS makes available CHEM-TROL NORMAL (Cat. No. 8100) and CHEM-TROL ELEVATED (Cat. No. 8200) for this purpose. Failure to achieve assayed values of freshly prepared control sera should be thoroughly investigated before patient values are reported.

LIMITATIONS

1. Turbid or highly icteric specimens may give initial readings whose absorbance exceed the capabilities of the instrument being used. In this case, reduce sample volume to 0.05 mL and multiply the final answer by two.

2. Occasionally, the side reactions in the Pre-incubation stage may be quite extensive, thus consuming a large fraction of the available NADH. If the DAbs / minute in the test decreases with time (ie; the rate is non-linear), suboptimal NADH concentration is indicated. In this event, the assay must be repeated using a diluted specimen (8).

EXPECTED VALUES

Less than 26 IU/L (30ºC)

Less than 38 IU/L (37ºC)

This range represents the 95% confidence interval from a clinically normal population. Each laboratory should establish its own range of expected values.

PERFORMANCE CHARACTERISTICS

LINEARITY:

This method is linear to 500 IU/L.

PRECISION:

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

MEAN / ST. DEV. / %CV

Normal 22 / 1.2 / 4.1

Abnormal 93 / 0.7 / 1.2

Run to Run - Normal and abnormal control sera were assayed for 10 working days to establish run to run precision.

Abnormal 95 / 2.3 / 3.6

SPECIFICITY:

A comparison of this ALT PROCEDURE with another widely used commercial method showed a 99% correlation with 25 serum specimens in the normal and abnormal range.

SENSITIVITY:

This ALT PROCEDURE has a sensitivity of 1.8 IU/L per 0.001 absorbance unit.

REFERENCES

1. Henley, K.S., Pollard, H.M., J. Lab Clin. Med., Vol. 46, p. 785 (1955).

2. Wroblewski, F., LaDue, J.S., Pro. Soc. Exp. Biol. Med., Vol. 91, p. 569 (1956).

3. Clinica Chimica Acta,Vol. 105, p. 145F - 172F (1980).

4. Young, D.S., Pestaner, L.C., and Gibberman, V., Clin. Chem. Vol. 21, p. 272D (1975).

5. Expert Panel on Enzymes of the International Federation of Clinical Chemistry, Clin. Chem. Acta, Vol. 70, p. F19.

6. Expert Panel on Enzymes of the International Federation of Clinical Chemistry, Clin. Chem., Vol. 24, p. 720.

7. Tietz, N.W., Clinical Guide to Laboratory Tests, 2nd. ed., W.B. Saunders Co., Philadelphia 1990, p.26.

8. Tietz, N.W., Clinical Guide to Laboratory Tests, 2nd. ed., W.B Saunders Co., Philadelphia 1986, p. 676.