Substrate reagent (L1) 0.50 0.50
Incubate at 37°C for 3 minutes
Mix well and incubate at 37°C for 60 minutes
Mix well and allow to stand at RT for 20 minutes
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
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.
Reaction : End point Interval : —
Wavelength : 505 nm Sample volume : 0.10 mL
Zero setting : Reagent blank Reagent volume : 6.00 mL
Delay time : — React slope : Increasing
Read time : — Linerity : 190 U/mL
(Courtesy: Tulip Group of Companies)
For the determination of SGOT (AST) activity in serum
(For in vitro diagnostic use only).
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.
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
MDH Oxaloacetate + NADH + H+ Malate + NAD+
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.
L1 : Enzyme reagent 20 mL 60 mL
L2 : Starter reagent 5 mL 15 mL
Contents are stable at 2–8°C till the expiry mentioned on
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
Serum. Free from hemolysis. SGOT (AST) is reported to be
stable in serum for 3 days at 2–8°C.
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
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/
Pipette into a clean dry test tube labeled as Test (T):
Incubate at the assay temperature for 1 minute and add
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
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
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
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
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
Reaction : UV kinetic Interval : 60
Wavelength : 340 nm Sample volume : 0.10 mL
Zero setting : Distilled water Reagent volume : 1.00 mL
Delay time : 60 sec Reac. slope : Decreasing
Read time : 180 sec Linerity : 500 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).
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.
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
αKetoglutarate pH 7.4 L-Glutamate
Pyruvate Alkaline 2,4,Dinitrophenyl
It is recommended that each laboratory establish its
own normal range representing its patient population.
S : Pyruvate standard (2 mM) 5 mL
Contents are stable at 2–8°C till the expiry mentioned on
the labels. Sodium hydroxide can be stored at RT till the
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.
Serum. Free from hemolysis. SGPT (ALT) is reported to be
stable in serum for 3 days at 2–8°C.
Wavelength/filter : 505 nm (Hg 546 nm)/Green
Plotting of the Calibration Curve
Pipette into 5 clean dry test tubes labeled as 1, 2, 3, 4, and 5:
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
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.
Pipette into clean dry test tubes labeled as Blank (B) and
Substrate reagent (L1) 0.50 0.50
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