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BACKGROUND
Serum glutamic-oxaloacetic transaminase (SGOT/AST) is the enzyme present in the liver cells, heart muscles, skeletal muscles, kidneys. These enzymes are released in blood stream into the tissues, when there is an injury.
Serum glutamic pyruvic transaminase (SGPT/ ALT) is present in liver and heart cells and are released in to blood when there is damage.
Reitman and Frankel Method
The L-Aspartate and alpha Ketoglutarate are converted from SGOT forms glutamate and oxaloacetate. This oxaloacetate combines with 2,4-dinitrophenyl hydrazine to form 2,4-dinitrophenylhydrazone in alkaline medium. Turns brown colour by producing hydrozone derivative and pyruvate standard which is a calibration curve.1
There are two methods (1) Calorimetric Method (2) ultraviolet spectrophotometry.
Calorimetric method
The DNPH supports the intensity of colour limiting the error and is more in oxaloacetate and pyruvate with oxoglutarate.
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REQUIREMENTS
Reagents: Phosphate buffer, pH 7.4, O.1 M
Disodium hydrogen phosphate dihydrate (0.1M) : dissolving 8.9 g of it in 200 ml water in 500 ml volumetric flask.
Monopotassium phosphate anhydrous (0.1M): Dissolving 1.36 g of it in 50 ml of distilled water and dilute the solution to 100 ml.
Mix 420 ml disodiumphosphate solution with 80 ml with mono potassium dihydrogen solution.
Substrate for SGOT: DL aspartate (200 mM/L) and alpha ketoglutarate (2 mM/L): by dissolving 2.66 g DL – aspartic acid and 30 mg oxoglutaric acid in 20.5 ml of 1 N NAOH in a beaker adding drop wise and adjusting pH and transferring into 100 ml in volumetric flask with phosphate buffer solution. As a preservative add 1ml of chloroform and refrigerate. If turns turbid discard the substance.
Substrate for SGPT
Dissolve 1.78 g DL – alanine and 30 mg oxoglutaric acid in 20 ml phosphate buffer were 1.25 ml of 0.4 N NaOH taken in a small beaker made to 100 ml with buffer. Adding 1 ml chloroform as preservative.
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PROCEDURE
Pyruvate standard
Stock standard: 220 mg sodium pyruvate in phosphate buffer made upto 100 ml and discarded once working standard is prepared.
Working standard:
10 ml stock standard to 100 ml with phosphate buffer and dispence 2 ml in test tubes with stopper refrigerate it. Using one tube at a time for preparing the calibration curve and the rest are refrigerated. Remaining solution is discarded. Dissolve 200 mg DNPH in 1N HCL (diluting 90 ml concentration with water) and made to 1 L with distilled water.
Sodium hydroxide solution 1N: 40 g of sodium hydroxide in 500 ml of water made up to 1000 ml. 0.4 N: 40 ml of 100 ml.
The substrate is pipette out into the test tube (1 ml) and is kept in water bath of 400 C for ten min. 0.2 ml of serum is mixed and incubated for 60 min (GOT) and 30 min (GPT) then the test tubes are removed. Then DNPH is added to stop the reaction and all for 20 min at room temperature. A 110 ml of 0.4 N sodium hydroxide were added and rubber stopper is placed for mixing by inverting (A1). After completing all these, once the optical density is 500 mu ml. Again repeat at 400C the absorbance (A2)
Calculation
For SGPT:
(A1 – A2) X 857 U/L
For SGOT:
( A1 – A2 ) X 514 U/L
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CONCLUSION
When there is a change in optical density more than 500 mu ml against transaminase is noted and the normal value is SGOT, SGPT 0-50 U/L; moderate range 400 – 2000 U/L; high range – 2000 – 30,000 U/L
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REFERENCES
- Reiter And Franel. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases ; 2018.
- Joshi A. Rashmi A Textbook of Practical Biochemistry; 2002: pg.no141.
