CHEMISTRY REAGENT KITS --->     [ASAT] ASPARTATE AMINOTRANSFERASE (SGOT)                                         COLORIMETRIC DNPH                             DOWNLOAD PDF

CLINICAL SIGNIFICANCE
A high activity of AsAT is the Clinical indication of myocardial infarctions, noticeable after a few hours of onset and lasting for 4-5 days High levels are also found in case of liver cell damage, muscular dystrophy and dermatomyositis.

PRINCIPLE
Aspartate  aminotransferase  catalyses  the following reaction:
L-aspartate                   oxaloacetate
     +                   GOT               +
                          -------
 oxo-2-glutarate        * L-glutamate

GOT activity is shown by the quantity of oxalacetic formed. This ketoacid may be assayed by the formation of 2, 4-dinitro phenylhydrazone which yields a brown colour in alkaline medium. The intensity of this colour is a function of the quantity of oxalocacetate formed, and thus the catalytic activity of aspartate aminotransferase.

REAGENTS SUPPLIED 1

Buffer-substrate

Phosphate buffer

(pH7.4) 100 mMol/L
L-alanine

100 mMol/L

Oxo-2-glutarate

    2 mMol/L

2..Chromogen

2,4-Dinitro phenylhydrazine     1 mMol/L
HydrochloricAcid    1 Mol/L
3.Standard
Sodium pyruvate

    2 mMol/L

4.Sodium hydroxide
Sodium hydroxide     4 Mol/L

STABILITY
18 months at 2-8°C
Avoid any bacterial contamination. Reagent 4 when diluted and kept in polyethylene vials is stable for at least 8 days between 18 and 25°C.

SAMPLE
Use fresh serum which has no signs of haemolysis. No prior patient preparation is needed.

LIMITATIONS
This method is not linear. Each kit should be quality controlled and calibrated for its activities corresponding to the enzyme. For more accuracy a control must be run along with the test adding serum after Step 4. Any colour shows above the blank of the standard (Tube no 1 of calibration graph) must be deducted from the test reading.

RESULTS
Compare the Optical Density of the Test against the standard calibration graph, the activity greater than 100 Karmen Units/mL must be diluted with 0.9% saline and be confirmed by reassay. (Multiply the result by dilution factor)

METHOD
1         Dilute reagent 4, 1 mL to 10 mL
           Reagent 4......................................... 1 volume
           Distilled Water.....................................9 Volume

2         Into a test tube, introduce :
           Reagent 1.............................0.5 mL
           Incubate at 37°C for 5 minutes.

3         Add : serum...................................0.1 mL
           Mix carefully, leave for exactly 60 minutes at 37°C.

4         Add : Reagent 2.......................................0.5mL
           well. Let stand 20 minutes at 18-25°C.

5         Add rapidly whilst mixing :
           Reagent 4, Diluted...........................   5 mL

6         After exactly 5 minutes, read the optical density against distilled water at
           530Nm (510 - 550 nM Green filter)

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CALIBRATION
1         Into 5 test tubes labelled as under introduce :

;

4.       Draw the standard curve, expressing GOT activities on the x-axis and           optical densities on the y-axis of a graph.
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EXPECTED VALUES
Male : 4.2 to 28.9 Karmen Units/ml
Female : 2.6 to 27.8 Karmen Units/ml

BIBLIGRAPHY
ACKARMANN and TORO Practical Clin. Chem. Little Brown and Co Boston

BERGMEYER H.U. Methods of Enzymatic analysis 2nd Ed. Vol. LI (1974) Academic Press N.Y.