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

Estimation of ALT

ESTIMATION OF SGPT  (Enzymatic method – Kinetic mode)

Principle:

• Kinetic determination of SGPT is done as per the following reactions:

                                                                  _{SGPT (ALT)}

(1) α -Ketoglutarate + L-Alanine → L-Glutamate + Pyruvate

                                                               _LDH

(2) Pyruvate + NADH + H⁺ → L-Lactate + NAD⁺

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

Specimen:

Serum / plasma

Reagents :

• Reagent 1 (Tris buffer pH 7.8, L-alanine, LDH,)
• 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 m l
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:

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

Result:

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

Clinical Significance :

• ALT is increased in hepatocellular injury in dog and cat. It is not useful in evaluating chronic liver disease. It may be elevated in corticosteriod treatment. This enzyme is not useful in evaluating hepatic disease in horse, cow, sheep, goat and pig.