Addition

Sequence

B

mL

T

mL

Substrate reagent (L1) 0.50 0.50

Incubate at 37°C for 3 minutes

Sample - 0.10

Mix well and incubate at 37°C for 60 minutes

DNPH reagent (L2) 0.50 0.50

Mix well and allow to stand at RT for 20 minutes

Distilled water 0.10 -

Working NaOH reagent (L3) 5.00 5.00

Mix well and allow to stand at RT for 10 minutes.

Measure the absorbance of the test (T) against blank

(Blank) and read the activity of the test from the calibration

curve plotted earlier.

Note

One sample blank is sufficient for each assay series.

If enzyme activity exceeds 190 U/mL dilute the sample

with distilled water and repeat the assay. Multiply the

value with the proper dilution factor.

High concentration of aldehydes and ketones in the sample

or icteric or lipemic, samples may cause slightly elevated

results. It is recommended to run a sample blank for these

samples using serum instead of distilled water in the blank.

High levels of serum pyruvate may interfere with results.

System Parameters

Reaction : End point Interval : —

Wavelength : 505 nm Sample volume : 0.10 mL

Zero setting : Reagent blank Reagent volume : 6.00 mL

Incubation

temperature

: 37°C Standard : Calib curve

Incubation

time

: 80 min Factor : —

Delay time : — React slope : Increasing

Read time : — Linerity : 190 U/mL

No. of read : — Units : U/mL

SGOT (AST) (Mod. IFCC Method)

(Courtesy: Tulip Group of Companies)

For the determination of SGOT (AST) activity in serum

(For in vitro diagnostic use only).

Summary

SGOT is an enzyme found mainly in heart muscle, liver

cells, skeletal muscle and kidneys. Injury to these tissues

results in the release of the enzyme in blood. Elevated levels

are found in myocardial infarction, Cardiac operations,

hepatitis, cirrhosis, acute pancreatitis, acute renal diseases,

primary muscle diseases. Decreased levels may be found

in pregnancy, beri beri and diabetic ketoacidosis.

Principle

SGOT (AST) catalyzes the transfer of amino group between

L-aspartate and α 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 SGOT

(AST) activity in the sample.

532 Concise Book of Medical Laboratory Technology: Methods and Interpretations  SGOT L-Aspartate +α Ketoglutarate Oxaloacetate +

L-Glutamate

MDH Oxaloacetate + NADH + H+ Malate + NAD+

Normal Reference Values

Serum (males) : Up to 37 U/L at 37°C

(females) : Up to 31 U/L at 37°C.

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 25 mL 75 mL

L1 : Enzyme reagent 20 mL 60 mL

L2 : Starter reagent 5 mL 15 mL

Storage/stability

Contents are stable at 2–8°C till the expiry mentioned on

the labels.

Reagent Preparation

Reagents are ready to use.

Working reagent : For sample, start assays a single reagent

is required. Pour the contents of 1 bottle of L2 (Starter

Reagent) into 1 bottle of L1 (Enzyme Reagent). This working

reagent is stable for at least 3 weeks when stored at 2–8°C.

Alternatively for flexibility as much of working reagent

may be made as and when desired by mixing together

4 parts of L1 (Enzyme Reagent) and 1 part of L2 (Starter

Reagent). Alternatively, 0.8 mL of L1 and 0.2 mL of L2 may

also be used instead of 1 mL of the working reagent directly

during the assay.

Sample Material

Serum. Free from hemolysis. SGOT (AST) is reported to be

stable in serum for 3 days at 2–8°C.

Procedure

Wavelength/filter : 340 nm

Temperature : 37°C/30°C/25°C

Light path : 1 cm

Substrate Start Assay

Pipette into a clean dry test tube labeled as Test (T):

Addition Sequence (T) 25°C / 30°C (T) 37°C

Enzyme reagent ( L1 ) 0.8 mL 0.8 mL

Sample 0.2 mL 0.1 mL

Incubate at the assay temperature for 1 minute and add

Starter reagent ( L2 ) 0.2 mL 0.2 mL

Mix well and read the initial absorbance A0 and repeat

the absorbance reading after every 1, 2, and 3 minutes.

Calculate the mean absorbance change perminute (∆A/

min).

Sample Start Assay

Pipette into a clean dry test tube labeled as Test (T):

Addition (T) (T)

Sequence 25°C / 30°C 37°C

Working reagent 1.0 mL 1.0 mL

Incubate at the assay temperature for 1 minute and add

Sample 0.2 mL 0.1 mL

Mix well and read the initial absorbance A0 after

1 minute and repeat the absorbance reading after every 1,

2, and 3 minutes. Calculate the mean absorbance change

per minute (∆A/min).

Calculations

Substrate/sample start

SGOT (AST) activity in U/L 25°C/30°C = ∆A/min × 952

SGOT (AST) activity in U/L 37°C = ∆A/min × 1746

Temperature Conversion Factors

Assay Desired Reporting Temperature

Temperature 25°C 30°C 37°C

25°C 1.00 1.37 2.08

30°C 0.73 1.00 1.54

37°C 0.48 0.65 1.00

Linearity

The procedure is linear up to 500 U/L at 37°C. If the

absorbance change (∆A/min) exceeds 0.250, use only the

value of the first 2 minutes to calculate the result, or dilute

the sample 1+9 with normal saline (NaCL 0.9%) and repeat

the assay (Results × 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 1.000 against distilled water

at 340 nm. Discard the reagent if the absorbance is below

1.000.

Enzymology 533

System Parameters

Reaction : UV kinetic Interval : 60

Wavelength : 340 nm Sample volume : 0.10 mL

Zero setting : Distilled water Reagent volume : 1.00 mL

Incubation

temperature

: 37°C Standard : —

Incubation

time

: — Factor : 1746

Delay time : 60 sec Reac. slope : Decreasing

Read time : 180 sec Linerity : 500 U/L

No. of read : 4 Units : U/L

SGPT (ALT) (Reitman and Frankel’s Method)

(Courtesy: Tulip Group of Companies)

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 converts L-alanine and α ketoglutarate to pyruvate

and glutamate. The pyruvate formed reacts with 2, 4,

Dinitrophenyl hydrazine to produce a hydrazone derivative,

which in an alkaline medium produces a brown colored

complex whose intensity is measured. The reaction does

not obey Beer’s law and hence a calibration curve is plotted

using a pyruvate standard. The activity of SGPT (ALT) is read

off this calibration curve.

L - Alanine SGPT Pyruvate

 + +

αKetoglutarate pH 7.4 L-Glutamate

Pyruvate Alkaline 2,4,Dinitrophenyl

 + hydrazone

2,4,DNPH

Medium (Brown colored

complex)

Normal Reference Values

Serum = 5–35 Units/mL

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 40 assays

L1 : Substrate reagent 25 mL

L2 : DNPH reagent 2 × 12.5 mL

L3 : NaOH reagent (4 N) 25 mL

S : Pyruvate standard (2 mM) 5 mL

Storage/stability

Contents are stable at 2–8°C till the expiry mentioned on

the labels. Sodium hydroxide can be stored at RT till the

expiry mentioned.

Reagent Preparation

All reagents are ready to use except NaOH Reagent (4N)

which has to be diluted 1:10 with distilled/deionized water.

Working NaOH reagent: Dilute the sodium hydroxide

to 250 mL or for every 1.0 mL of NaOH reagent (4N) add

9.0 mL of water. The working sodium hydroxide reagent is

stable at RT till the expiry mentioned, in a plastic bottle.

Sample Material

Serum. Free from hemolysis. SGPT (ALT) is reported to be

stable in serum for 3 days at 2–8°C.

Procedure

Wavelength/filter : 505 nm (Hg 546 nm)/Green

Temperature : 37°C and RT

Light path : 1 cm

Plotting of the Calibration Curve

Pipette into 5 clean dry test tubes labeled as 1, 2, 3, 4, and 5:

Addition

Sequence

Enzyme

1

0

(mL)

2

28

(mL)

3

57

(mL)

4

97

(mL)

5

150

(mL)

Activity (U/mL)

Substrate reagent (L1) 0.50 0.45 0.40 0.35 0.30

Pyruvate standard (S) - 0.05 0.10 0.15 0.20

Distilled water 0.10 0.10 0.10 0.10 0.10

DNPH reagent (L2) 0.50 0.50 0.50 0.50 0.50

Mix well and allow to stand at

RT for 20 minutes

Working NaOH Reagent (L3) 5.00 5.00 5.00 5.00 5.00

Mix well and allow to stand at RT for 10 minutes. Measure

the absorbances of the tubes 2–5 against tube 1 (Blank). Plot

a graph of the absorbances of tubes 2–5 on the Y-axis versus

the corresponding enzyme activity on the ‘X’-axis.

Assay

Pipette into clean dry test tubes labeled as Blank (B) and

Test (T).

Addition

Sequence

(B)

(mL)

(T)

(mL)

Substrate reagent (L1) 0.50 0.50

Incubate at 37°C for 3 minutes

Contd...