VLD 421: Veterinary Clinical Biochemistry and Laboratory Diagnosis-II (0+1)

Estimation of AST

ESTIMATION OF AST (Enzymatic method – Kinetic mode)

Principle:

• Kinetic determination of AST (Aspartate amino transferase) is done as per the following reactions:

                                                                            _{SGOT (AST)}

(1) α -Ketoglutarate + L-Aspartate →             L-Glutamate + Oxaloacetate

                                                                       _MDH

(2) Oxaloacetate + NADH + H⁺ → L-Malate + NAD⁺

• The utilization of NADH is monitored at 340 nm, whose rate of disappearance is proportional to the activity of SGOT. The rate of decrease in absorbance is measured at 340 nm.

Specimen:

Serum / plasma

Reagents :

• Reagent 1 (Tris buffer pH 7.8, L-aspartate, LDH, MDH)
• Reagent 2 (α –Ketoglutarate, NADH)

Preparation of working reagent:

• Mix four Volumes of Reagent 1 with one volume of Reagent 2. This working reagent is stable for 30 days at 2 – 8 ⁰ C.

Procedure:

Mark one clean test tube as (T):

S.No	Reagents	T
1.	Working Reagent	1000 ml
2.	Serum	100 m l
3.	Distilled Water	2000 m l

• Mix well and incubate for 1 minute at 37^oC. Read the change in absorbance per minute (Δ OD / min.)
• during 3 minutes at 340 nm setting zero with D.W..

Calculation:

• SGOT activity (U/L) = (Δ OD / min.) x 1745 x 3

Result:

• The SGOT activity of given serum = ___________U / L.

Clinical Significance:

• Elevated levels of SGOT are found in myocardial infarction, in liver diseases, skeletal muscle trauma and some times in renal diseases.