INTRODUCTION
Serum aspartate aminotransferase (AST) also known as serum glutamic oxalacetic transaminase (SGOT) is a tissue enzyme that catalyzes the exchange of amino and keto groups between alpha--amino acids and alpha-keto acids. AST is widely distributed in tissue principally cardiac hepatic muscle and kidney. Injury to these tissues results in the release of the AST (SGOT) enzyme to general circulation. Following a myocardial infarction serum levels of AST (SGOT) are elevated and reach a peak 48 to 60 hours after onset. Hepatobiliary diseases such as cirrhosis metastatic carcinoma and viral hepatitis also will increase serum AST levels. (1)

The first kinetic assay of AST for diagnostic purposes was described by Kaman et al. in 1955 using a coupled reaction of malate dehydrogenase (MDH) and NADH2. This assay system was critically evaluated and optimized in 1960 by Henry et al. (3) In 1977 the International Federation of Clinical Chemistry recommended a reference procedure for the measurement of AST activity based upon Kaman's procedures. (4) The AST reagent applies the formulation recommended by the IFCC.

PRINCIPLE
The enzymatic reaction sequence employed in the assay of
aspirate aminotransferase is as follows:
AST
L-Aspirate + 2-Oxoglutarate ------------> Oxalacetate + L-Glutamate
MDH
Oxalacetate + NADH + H+ ---------> Malate + NAD+ + H*0

AST catalyzes the transfer of an amino group between L-aspartate and 2-oxoglutarate. The oxalacetate formed in the first reaction is then reacted with NADH in the presence of malate dehydrogenase (MDH) to form NAD. AST activity is determined by measuring the rate of oxidation of NADH at 340 nm. Lactate dehydrogenase is included in the reagent to convert endogenous pyruvate in the sample to lactate during the lag phase prior to measurement.

REAGENTS
After combining R1 (Buffer Reagent) and R2 (Co-Enzyme) the reagent contains:
L-Aspartate 240 mmol/L
MDH (porcine muscle) > 600 U/L
LDH (rabbit muscle) > 600 U/L
Tris Buffer pH 7.5 80 mmol/L
2 - Oxoglutarate 12 mmol/L
NADH 0.18 mmol/L
Stabilizers and Preservatives

PRECAUTIONS
The reagents are for "In Vitro Diagnostic Use". Normal precautions exercised in handling laboratory reagents should be followed. The reagents contain sodium azide which may be toxic if ingested. Sodium azide may also react with lead and copper plumbing to form highly explosive metal azides. Refer to Material Safety Data Sheet for any updated risk hazard or safety information.

REAGENT PREPARATION
The working reagent is prepared by mixing five (5) volumes of R1 with one (1) volume of R2 in a disposable container.
Example: 25ml R1 + 5ml R2

REAGENT STORAGE AND STABILITY
Reagents are stable until the expiration date on their respective labels when properly stored at 2-8°C and protected from light. Reagents should appear clear and colorless. Discard if either appears cloudy or contains particulate matter. The Working Reagent is stable for 4 weeks at 2-8°C. The Working Reagent should be discarded if the initial absorbance read against distilled water at 340 nm is below 1.100.

MATERIAL REQUIRED BUT NOT PROVIDED
1) Spectrophotometer capable of absorbance reading at 340 nm and
1 cm lightpath
2) Constant temperature block or bath 37°C or temperature controlled
cuvette well
3) Accurate pipetting devices
4) Test tubes
5) Interval timer

SPECIMEN COLLECTION AND STORAGE
Non-hemolyzed serum is the specimen of choice yet EDTA treated plasma or heparinized plasma can be used.5 Whenever possible specimens should be separated and analyzed on the day of collection. Store serum in stoppered tubes. The enzyme in serum is reportedly stable for a minimum of 7 days at 2-8°C. (6)

INTERFERING SUBSTANCES
Hemolysis must be avoided as the concentration of AST in red cells is roughly 10 times that of serum. (5) Bilirubin levels up to 40 mg/dL and triglyceride levels up to 2000 mg/dL show no interference in this test. Certain drugs and other substances are also known to affect AST values. (7)

MANUAL PROCEDURE
1. Prepare AST Working Reagent according to instructions.
2. Zero spectrophotometer at 340 nm with distilled water.
3. For each sample and control add 1.0 mL Working Reagent to cuvette or test tube and warm to 37°C for 3 minutes.
4. Add 100 µL (0.10 mL) serum to its respective tube and mix gently.
5. Read and record absorbance at 1 minute. Continue incubating at 37°C and record absorbance again at 2 and 3 minutes. Rate should be constant.
6. Determine the average absorbance per minute (DA/min) multiply by factor 1746 for results in U/L.

NOTE: If cuvette is not temperature controlled incubate samples at 37°C between readings.

QUALITY CONTROL
It is recommended that control be included in each set of assays. Commercially available control material with established AST values may be used for quality control. The assigned value of the control material must be confirmed by the chosen application. Failure to obtain the proper range of values in the assay of control material may indicate either reagent deterioration instrument malfunction or procedural errors.

CALIBRATION
AST activity is based on the "micromolar extinction coefficient" of NADH at 340 nm (see "Results" section). The instrument manufacturer's calibration guidelines should be followed to calibrate your analyzer. Assaying the AST contents of a control serum with known AST values can be used to assure instrument calibration has been performed correctly.

RESULTS
Values are derived based on the "absorptivity micromolar extinction coefficient" of NADH at 340 nm (0.0063). Units per liter (U/L) of AST/GOT activity is that amount of enzyme which oxidizes one µmol/L of NADH per minute.

DA/Min Total Volume
U/L = Absorptivity x Sample Volume .
DA/Min 1.10
U/L = 0.0063 x 0.10

U/L = DA/Min x 1746

LIMITATIONS
If the DA/min. is greater than 0.342 dilute 1 part sample with 9 parts isotonic saline and re-assay. Multiply the result by 10. AST values for neonatal patients have not been established with this procedure. Grossly icteric or turbid specimen may require the use of a sample blank.

EXPECTED VALUES (8)
Normal Range: 8 - 33 U/L (37°C)

This range should serve only as a guideline. It is recommended that each laboratory establish its own range of expected values since differences exist between instruments laboratories and local populations.

PERFORMANCE CHARACTERISTICS
Comparison: A group of 125 sera ranging in AST activity from 13 - 399 U/L was assayed by the described AST method and by a similar commercially available AST reagent. Comparison of the results yielded a correlation coefficient of 0.999 and the regression equation was y = 0.964x + 0.964. (Comparison studies were performed according to NCCLS Tentative Guideline EP9-T.)

Precision: Within-run precision was established by 30 assays on two different levels of commercial serum controls. Total Precision values were obtained by assaying the two commercial controls for 5 consecutive days.

Within-Run
Serum 1 Serum 2
Mean ALT (U/L) 41.7 115.2
Std. Deviation (U/L) 0.9 3.3
C.V. (%) 2.2 2.9

Run-to-Run
Serum 1 Serum 2
Mean ALT (U/L) 40.4 116.2
Std. Deviation (U/L) 0.87 4.88
C.V. (%) 2.14 4.19

Precision studies were performed according to NCCLS Tentative Guideline EP5-T.

Linearity: Linear to 500 U/L at 37°C.6 Performed according to NCCLS Guideline EP6-P.

Sensitivity: Based on an instrument resolution of A = 0.001 the method presented shows a sensitivity of 2.65 U/L.

REFERENCES
1. Henry J.B.: Clinical Diagnosis and Management by Laboratory Methods. W.B. Saunders and Co. Philadelphia PA. p 361 (1974).
2. Karmen A: A note on the spectrophotometric assay of glutamic-oxolacetic transaminase in human blood. J Clin Invest 34:131 1955
3. Henry R.J. et al.: Am. J. Clin. Path 34:381 (1960).
4. The Committee on Enzymes of the Scandinavian Society for
Clinical Chemistry and Clinical Physiology. Scand. J. Clin.
Lab.Invest32:291(1974).
5. Demetriou JA et al. In Clinical Chemistry - Principles and Techniques 2nd ed. RJ Henry et al. Eds. Harper & Row Hagerstown MD 1974 p 873.
6. International Federation of Clinical Chemistry. Provisional Recommendations on IFCC Methods for the Measurement of Catalytic Concentrations of Enzymes. Clin Chem 23: 887 1977.
7. Young D.S. Effects of drugs on clinical laboratory tests. AACC Press Washington D.C. 1990
8. Henry JB. Clinical Diagnosis and Management by Laboratory Methods 17th ed. WB Saunders Co. 1984 p 1437.