SGPT
TEST PROCEDURE FOR SPECTROPHOTOMETER
The following is a general procedure for use on a manual
spectrophotometer.
Procedure Conditions
Wavelength 540
nm
Temperature 37°
C
Mode Endpoint
Sample to Reagent Ratio
TEST PROCEDURE
(1)
A. Label
reagent tubes, as "BLANK", "CONTROL" and
"PATIENT".
B. Incubate
reagent tubes at 37° C for 5 minutes.
C.
Add 0.8 ml of SGPT
reagent to each tube.
D.
Add 100 ml of normal control serum and to the "CONTROL" tube. Cap the tube and mix well by inversion.
E.
Add 100 ml of patient serum sample to the "PATIENT" tube. Cap the tube and mix well by inversion.
F.
Incubate reagent
tubes at 37° C for 30 minutes.
G.
Add 0.5 ml of color
developer A to each reagent tube. Cap
the tubes and mix well by inversion.
H.
Let reagent tubes
stand at room temperature for 20 minutes.
I.
Add 2 ml of color
developer B to each reagent tube. Cap
the tubes and mix well by inversion. Let
tube stand at room temperature of 5 minutes.
J.
Wipe reagent tubes
clean with a lint‑free tissue.
K.
Place the
"BLANK" tube in the test well and adjust the photometer to zero
absorbance.
L.
Place the
"CONTROL" and "PATIENT" tubes in the test well and record
the absorbance of the "CONTROL" and "PATIENT" samples.
Calculation Patient SGPT concentration =
absorbance of
"PATIENT" sample
‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑---------------------- X mean assay value of control
absorbance of
"CONTROL" sample
EXPECTED VALUES
Note 1. (a) Careful control of temperature and timing is
essential for the accuracy and precision of the test results.
(b)
The final reaction
color is stable for 30 minutes.
INTENDED USE
These reagents are for the quantitative determination of
Serum Glutamate‑Pyruvate Transaminase (SGPT) enzyme activity in serum.
SUMMARY AND EXPLANATION
Glutamate ‑ Pyruvate Transaminase (GPT) is one of
the amino transferase enzymes which catalyze the reversible reaction of amino
acids and alpha‑ketoglutaric acid by the transfer of the amino
group. GPT is found in large amounts in
the liver but in small amounts in other tissues. Consequently, the determination of serum GPT
could serve as a valuable aid in different diagnosis.
In 1957 Reitman and Frankel (1) presented a colorimetric
approach to measure GPT activity. An end
product of the transamination reaction, pyruvate is reacted with
dinitrophenylhydrazine (DNPH) to form the hydrazone complex. This hydrazone is reacted with an alkaline
diluent to form a colored complex which can be measured quantitatively in a
spectrophotometer or colorimeter. The
Biotron Diagnostics method is a modification of the Reitman and Frankel method.
TEST PRINCIPLE
The enzyme glutamate pyruvate transaminase (GPT)
catalyzes an exchange of an amino group of alanine for an alpha‑keto
group of alpha ketoglutarate. The end
products formed in this reaction are pyruvate and glutamate. Dinitrophenylhydrazine is added to form the
hydrazones of the pyruvate present.
These hydrazones are reacted with sodium hydroxide to form a color which
can be read by a spectrophotometer or colorimeter.
MATERIALS PROVIDED
SGPT (ALT) reagent 100 ml
Color developer A 60 ml
Color developer B 240 ml
Standard/Control/Calibrator
REAGENTS
For In Vitro Diagnostic use.
1. SGPT (ALT)
Reagent contains 0.15 mg/liter alpha‑ketoglutaric acid, 8.9 mg/liter dl‑alanine
in phosphate buffer and preservative.
2. Color
Developer A contains 0.02% w/v 2,4‑dinitrophenyl‑hydrazine, 8.6%
hydrochloric acid, and preservative.
3. Color
Developer B contains 1.6% w/v sodium hydroxide.
CAUTION! Do not
take these reagents internally or allow them to come in contact with the body.
STORAGE
Store reagents in refrigerator at 2‑8° C. All reagents are stable till
the expiration date stated on the label when stored in refrigerator.
ADDITIONAL MATERIALS REQUIRED
1. Blood
analyzer, spectrophotometer, or colorimeter.
2. Pipetting
devices.
3. Commercially
available assayed control serum.
SAMPLE AND PREPARATION
Freshly drawn serum sample.
EXPECTED VALUES
The normal range of SGPT is 5 to 30 U/L at 37° C.
The above range is intended as a guide. Each laboratory should establish its own
normal range.
PERFORMANCE
1. Precision ‑
The precision study was done by:
(a) repetitive assay (N=20) of normal
serum. The assay yielded a mean of 18
U/L and a standard deviation of 2 U/L and a coefficient of variation of 11.1%.
(b) 8 day reproducibility. A pool serum specimen yielded a mean of 25
U/L, a standard deviation of 4 U/L and a coefficient of variation of 16%.
2. Accuracy ‑
The accuracy study was done by running 25 specimens on Dade's (trademark of
Dade Diagnostics Inc.) SGPT kit and Biotron Diagnostics Kit on Starsar III
(registered trademark of Gilford Instruments).
The study yielded a regression equation of Biotron = 1.03 * reference method - 2.74 and
a correlation of 0.94.
LIMITATIONS OF THE PROCEDURE
This procedure is linear from 0 to 130 U/L at 37° C.
QUALITY CONTROL
Standard practice for quality control should be applied
to this system. Commercially available
lyophilized controls can be used. Daily quality control must fall within 2
standard deviations of the established value.
If correlation is not obtained and repetition of the assay excludes
error in technique, the following steps should be taken:
1. Calibrate
the instrument according to manufacturer's instructions.
2. Check the
cleanliness of the reagent tube.
3. Check the
expiration date of the reagent package.
4. Contact
Biotron Diagnostics Technical Services Department.
REFERENCES
1. Reitman, S.
and Frankel, S., American Journal of Clinical Pathology, 28:56‑63, 1957.
2. Henry, R.
J., Cannon, D. C. and Winkleman, J. W., "Clinical Chemistry, Principles
and Technics," 2nd Ed., 1974, Harper and Row,
3. Kin
Diagnostics Laboratory Data,