SGPT (ALT)

(Mod. IFCC method)

For the determination of SGPT (ALT) activity in serum.
(For In vitro Diagnostic Use Only)

SUMMARY:

SGPT is found in a variety of tissues but is mainly found in the liver. Increased levels are found in hepatitis,
cirrhosis, obstructive jaundice and other hepatic diseases. Slight elevation of the enzymes is also seen in
myocardial infarction.

PRINCIPLE:

SGPT (ALT) catalyzes the transfer of amino group between L-Aspartate and a-Ketoglutarate to form Oxaloacetate
and Glutamate. The Oxaloacetate formed reacts with NADH in the presence of Malate Dehydrogenase to form
NAD. The rate of oxidation of NADH to NAD is measured as a decrease in absorbance which is proportional to the
SGPT (ALT) activity in the sample.
SGOT
L-Aspartate acid + a-Ketoglutarate Oxaloacetate+L-Glutamate
MDH
Oxaloacetate + NADH + H Malate + NAD

CONTENTS:
PACK SIZE SUBSTRATE REAGENT(A1) BUFFER REAGENT(A2)

6x10ml 6x10ml 60ml

5x20ml 5x20ml 100ml

REAGENT PREPARATION:

WORKING REAGENT : For sample start assays a single reagent

is required. Reconstitute one vial of enzyme reagent (A1) with equivalent
Volume of diluent (A2) (mentioned on the Enzyme Reagent (A1) labels
.

STORAGE AND STABILITY:

Working reagent: This working reagent is stable for at least 4 weeks when stored at 2-8°C.

SAMPLE MATERIAL:

Serum. Free from hemolysis. SGPT (ALT) is report to be stable in serum for 3 days at 2-8°C.

GENERAL SYSTEM PARAMETERS:

Reaction Mode U.V Kinetic Sample Volume 100 µl
Wavelength 340 nm Reagent volume 1000 µl
Blank Distilled Water Factor 1746
Incubation 37˚C Reaction Slope Decreasing
Delay Time 60 sec Linearity 450 IU/L
Read Time 180 sec Units IU/L

ASSAY PROCEDURE:
Wavelength/ Filter : 340 nm
Temperature : 37˚C
Light Path : 1 cm
Pipette into clean dry test tube labeled as Test(T)

Addition Sequence Test (T)

Working Reagent 1000µl

Sample 100µl

Mix well and read the initial absorbance A after 1 min. and repeat the absorbance reading after 1,2 & 3 minutes.
Calculate the mean absorbance change per min.(∆A/min).

CALCULATIONS:

SGOT (AST) Activity in IU/L 37°C = ∆ A/ min. x 1746 x tf

TEMPERATURE CONVERSION FACTORS:

Desired
Assay Reporting Temp
370C

250C 1.00

300C 0.73

370C 0.48

NORMAL REFERENCE VALUES:

Serum (males) : Up to 40 IU/L at 37°C

(females) : Up to 31 IU/L at 37°C

LINEARITY:

The procedure is linear up to 450 IU/L at 37°C, If the absorbance change (∆ A/ min.) exceeds 0.250, use only the
value of the first two minutes to calculate the result, or dilute the sample 1+9 with normal saline (NaCl 0.9%) and
repeat the assay (Results x 10).

NOTE:

Samples having a very high activity show a very low initial absorbance as most of the NADH is consumed prior to
the start of measurement. If this is suspected then dilute the sample and repeat the assay. The working reagent
or the combined reagent should have an absorbance above 1.0 against distilled water at 340 nm. Discard the
reagent if the absorbance is below 1.0.

It is recommended that each laboratory establish its own normal range representing its patient population.

REFERENCES:

IFCC methods for the measurements of catalytic concentrations of enzymes, J. Clin. Chem. Clin. Biochem. (1986)
24: 481