AST (SGOT) PROCEDURE

Intended for the Quantitative Determination of Aspartate Aminotransferase in Serum

SUMMARY AND EXPLANATION

Serum Glutamic Oxalacetic Transaminase (SGOT), named Aspartate Aminotransferase (AST) 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 damage to or disease of the heart and liver. Decreased AST levels are noted in Vitamin B6 deficiency. In myocardial infraction, serum AST levels commence to rise in 6 - 8 hours, peak within two days and return to normal by the fourth to fifth day after the infraction. Lesser amounts of AST are found in the skeletal muscle, kidneys, pancreas, spleen, lungs and brain. Damage to these tissues results in a slight rise of serum AST levels.

AST methods using U.V. procedures were first described by Karmen (1) in 1955 using malate dehydrogenase and NADH. Optimized procedures were later presented by Henry (2) and Amador-Wacker (3). These modifications improved the accuracy and decreased effect of interfering substances. The Committee on Enzymes of the Scandanavian Society for Clinical Chemistry published the recommended method based on optimized modifications in 1974 (4). The EAGLE AST (SGOT) PROCEDURE is based on this recommended method.

PRINCIPLE

AST catalyzes the transfer of the amino group from L-aspartate to a-Ketoglutarate resulting in the formation of oxalacetate and L-glutamate. In the presence of malate dehydrogenase (MDH), oxalacetate undergoes reduction and simultaneous oxidation of NADH to form malate and NAD. LDH is added to prevent interference from endogenous pyruvate which is normally present in serum. This reagent does not contain P-5-P since the diagnostic significance of increading AST is still under study (5, 6).

L-Asparate + a-Ketoglutarate  AST Oxalacetate + L-Glutamate

Oxalacetate + NADH+ H+ MDH ------------L-Malate + NAD+ + H2 O

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set Cat. No. 2350 provides:

AST (SGOT) REAGENT - (Cat. No. 2351)

REACTIVE INGREDIENTS:

After reconstitution, the solution will have the following approximate concentration: a-Ketoglutarate, 12 mM; L-Aspartic Acid, 200 mM; LDH, 800 U/L MDH, 600 U/L; NADH, 0.2 mM; Buffer, stabilizer and fillers added.

STORAGE AND STABILITY:

Store at 2 - 8° C. Stable until the expiration date if vial remains sealed and unopened. Caking would indicate that moisture has entered the vial and the reagent should not be used. After reconstitution, the reagent is stable for 14 days stored at 2 - 8° C or for 8 hours stored at room temperature. Prolonged exposure to room temperature shortens usability of reagent even when stored 2 - 8° C. For maximum stability, store reagent at 2 - 8° C immediately after reconstitution.

DETERIORATION:

In a sealed vial, the reagent should be a dry, white powder. 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 DH2O at 340 nm, it should not be used.

INSTRUMENTS

Use a spectrophotometer or colorimeter calibrated at 340 nm and a heating block capable of maintaining 30° C or 37° C.

SPECIMEN COLLECTION

PRECAUTIONS:

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

SAMPLE STORAGE:

AST in serum is reported to be stable for four days at room temperature or for up to fourteen days when stored at 2 - 8° C.

ADDITIVES:

No special additives or preservatives are required.

INTERFERING SUBSTANCES:

Some drugs and substances will affect AST activity.Young et al (7) have reviewed drug effects on serum.

PROCEDURE

MATERIALS PROVIDED:

AST (SGOT) REAGENT (Cat. No. 2351)in a dry powder form.

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 30° C or 37°C

5. Test tubes, rack and timer

REACTION CONDITIONS:

Wavelength 340 nm

Reaction Type Kinetic

Incubation Temperature 30° C or 37° C

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 (37° C) <<40 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 37° C 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 37° C for two minutes.

4. After the two minutes preincubation, record the absorbance (A1 reading). Return the tube to the 37° C 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 insturment used is equipped with a temperature controlled cuvette, the reaction mixture may be left in the cuvette while the absorbance readings are taken.

PROCEDURAL NOTE:

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 1:1with 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:

IU/L =  

(A1 - A2) X 1.1 X 1000

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

1 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 n