CONCEPT DIAGNOSTICS

ONTARIO, CALIFORNIA 91761 USA

AST (SGOT) REAGENT SET
CRESTLINE SCIENTIFIC CORPORATION (UV-KINETIC METHOD)
2902 Finlandia cor Copernico St.,
San Isidro Village, Makati City
Tel: 8875777 Fax: 8875778
STORAGE AND STABILITY
AST (SGOT) REAGENT SET 1. Store dry reagent at 2 - 8°C.
For the quantitative determination of Aspartate Aminotransferase (AST) 2. The reconstituted reagent is stable for eight (8) hours at room
in serum. temperature and for twenty one (21) days when refrigerate
immediately.
INTRODUCTION
Serum aspartate aminotransferase (AST) also known as serum glutamic REAGENT DETERIORATION
oxalacetic transaminase (SGOT) is a tissue enzyme that catalyzes the The reagent should be discarded if:
exchange of amino and keto groups between alpha-amino acids and 1. The initial absorbance, read against water at 340 nm, is below
alpha-keto acids. AST is widely distributed in tissue principally 0.800.
cardiac, hepatic, muscle and kidney. Injury to these tissues results in 2. The reagent fails to meet stated parameters of performance.
the release of the AST (SGOT) enzyme to general circulation.
Following a myocardial infarction, serum levels of AST (SGOT) are SPECIMEN COLLECTION
elevated and reach a peak 48 to 60 hours after onset. Hepatobiliary This assay is intended for use with serum. Reports indicate that AST in
diseases, such as cirrhosis, metastatic carcinoma, and viral hepatitis serum remains stable at 4°C for a minimum of 7 days.
also will increase serum AST levels. 1 Hemolyzed specimens should not be used as erythrocytes contain
Fifteen times the AST activity in serum. 5
The first kinetic assay of AST for diagnostic purposes was described by
Karmen et al. in 1955, using a coupled reaction of malate INTERFERING SUBSTANCES
dehydrogenase (MDH) and NADH2. This assay system was critically Pyridoxal phosphate can elevate AST values by activating the
evaluated and optimized in 1960 by Henry et al. 3 In 1977 the apoenzyme form of the transaminase. Pyridoxal phosphate may be
International Federation of Clinical Chemistry recommended a found in diluent water contaminated with microbial growth. 6 High
reference procedure for the measurement of AST activity based upon levels of um pyruvate may also interfere with assay performance.
Karmen's procedures.4 The AST reagent applies the formulation Young et al. give a list of drugs and other substances that interfere with
recommended by the IFCC. the determination of AST activity. 7 Refer also to N.E. Saris for a list
of references.8
PRINCIPLE
The enzymatic reaction sequence employed in the assay of MATERIALS REQUIRED BUT NOT PROVIDED
aspartate aminotransferase is as follows: 1. Pipetting devices.
AST 2. Test tubes/rack.
L-Aspartate + 2-Oxoglutarate ------------ Oxalacelate + L-Glutamate 3. Timing device.
MDH 4. Spectrophotometer capable of reading at 340 nm (UV).
Oxalacetate + NADH + H+ --------- L- Malate + NAD+ + H 0 5. Heating block or bath (37°C).

AST catalyzes the transfer of an amino group between L-aspartate and GENERAL INSTRUCTIONS
2-oxoglutarate. The oxalacetate formed in the first reaction is then The reagent for AST is intended for use either as an automated
reacted with NADH in the presence of malate dehydrogenase (MDH) to procedure on chemistry instruments or as a manual procedure on a
form NAD. AST activity is determined by measuring the rate of suitable spectrophotometer.
oxidation of NADH at 340nm. Lactate dehydrogenase is included in the
reagent to convert endogenous pyruvate in the sample to lactate during PROCEDURE (AUTOMATED)
the lag phase prior to measurement. Consult the appropriate instrument application guide available from us.

REAGENT COMPOSITION PROCEDURE (MANUAL)

When reconstituted as directed, the reagent for AST contains the 1.Reconstitute Reagent according to instructions on vial label.
following: (Concentrations refer to reconstituted reagent.)2- 2.Pipette 1.0 ml of reagent into a 1cm cuvette and allow to equilibrate
Oxoglutarate 12 mM, L-Aspartic Acid 200 mM, NADH 0.19 mM, to
LDH 800 U/L, MDH 600 U/L, Buffer 100 mM, pH 7.8 + 0.1. 37°C.
Nonreactive preservatives and fillers. 3.Add 0.10 ml (100 ul) of specimen to reagent and mix gently.
4.Maintain the solution at 37°C. After one (l) minute, measure the
WARNINGS AND PRECAUTIONS absorbance at 340 nm.
1. For in vitro diagnostic use. 5.Take two additional absorbance readings at 1 minute intervals.
CAUTION: In vitro diagnostic reagents may be hazardous. Handle Calculate the mean absorbance change per minute. ( A/min.)
in accordance with good laboratory procedures which dictate 6.Multiply the  A/min. by 1768 to calculate IU/L of AST.
avoiding ingestion, and eye or skin contact.
2 Serum specimens should be considered infectious and handled ALTERNATE VOLUMES
appropriately. If the spectrophotometer being used requires a final volume greater than
3. Use distilled or deionized water where indicated. 1.0 ml for accurate readings, follow the "ALTERNATE
PROCEDURE".

CALCULATIONS
One International Unit (IU) is defined as the amount of enzyme that either reagent deterioration, instrument malfunction, or procedural
catalyzes the transformation of one micromole of substrate per minute errors.
under specified conditions.
EXPECTED VALUES
 Abs./min.  TV  1000 =  Abs./min.  1.1  1000 Up to 28 IU/L (30°C) Up to 40 IU/L (37°C)
  SV  LD 6.22  0.1  1 It is strongly recommended that each laboratory establish its own
=  Abs./min.  176 normal range.
Where:  Abs./min. = Average absorbance change per minute
TV = Total reaction volume (ml) PERFORMANCE CHARACTERISTICS
1000 = Conversion of IU/ml to IU/L 1. Linearity: 500 IU/L
 = Millimolar absorptivity of NADH 2. Comparison: A comparison study between the present method with
SV = Sample volume in ml available commercial product using the same method on 22 fresh
LP = Light path in cm serum samples from 12 IU/L to 84 IU/L yielded a coefficient of
Example: If the average absorbance change per minute = 0.15 then 0.15 0.98 and a regression equation of
x 1768 = 265 IU/L Y = 1.04x - 1.25.
3. Sensitivity: Based on an instrument resolution of A= 0.001, this
SI UNIT: To convert to Sl Units (nkat/L) multiply IU/L by 16.67. procedure has a sensitivity of 2 IU/L
NOTE: If any of the test parameters are altered a new factor must be 4. Precision studies:
calculated using the above formula. Within Run precision: Two commercial serum controls were
assayed twenty times and the following Within Run precision was
ALTERNATE PROCEDURE obtained. Within Run
1. Reconstitute reagent according to instructions. Mean (lU/L) S.D. C.V.
2. Pipette 3.0 ml of reagent into a 1 cm cuvette and allow to 22.3 1.1 4.8
equilibrate to 37°C. 88.3 5.1 5.7%
3. Add 0.20 rnl (200 µl) of specimen and mix gently. Run to Run Run precision: Two commercial serum controls were
4. Maintain the solution at 37°C. After one (1) minute, measure assayed for five consecutive days (triplicate for each level), the
the absorbance (A ) at 340 nm following Run to Run precision was obtained.
 minutes, read and record absorbance (A ) Run to Run
5. After exactly five (5) 2
6. The difference in absorbance between readings (A - A ) Mean IU/L S.D. C.V.
  21.9 2.3 10.5%
multiplied by the factor 514 (see" ALTERNATE PROCEDURE
CALCULATIONS") will yield results in IU/L. 83.8 3.6 4.4%
7. Sample with values above 500 IU/L should be diluted 1:1
with saline, re-assayed and the results multiplied by two (2). TEMPERATURE CONVERSION FACTOR (Tf)3
Assay Mixture Tf 25°C Tf 30°C Tf 32°C Tf 37°C
PROCEDURE NOTES 25°C 1.00 1.37 1.57 1.96
Turbid or high icteric samples may give readings whose initial 30°C 0.78 1.00 1.13 1.43
absorbance exceeds the capabilities of the spectrophotometer used. 32°C 0.65 0.89 1.00 1.27
Run this kind of sample using 0.10 ml (100 µl), sample volume to 3.0 37°C 0.51 0.70 0.73 1.00
ml reagent and multiply result by two (2). Example: If the reaction is performed at 30°C but is to be reported at
37°C, simply multiply the result obtained at 30°C by the factor 1.43 to
ALTERNATE PROCEDURE CALCULATION obtain a correct value.
One International Unit (IU) is defined as the amount of enzyme that NOTE: Since temperature factors give only an approximate conversion,
catalyzes the transformation of one micromole of substrate per minute it is suggested that values be reported at the temperature of the
under specified conditions. measurement.
AST (IU/L) = (A1-A2) x TV x 1000 = (A1-A2) x 3.2 x 1000 REFERENCES
T x  x SV x LD 5 x 6.22 x 0.2 x 1 1. Henry, J.B.: Clinical Diagnosis and Management by
= (A - A )  514 Laboratory Methods., W.B. Saunders and Co., Philadelphia,
  change PA. p 361 (1974).
Where: (A -A ) = Absorbance
 TV 2. Karmen, A. et al.: J. Clin. Invest, 34:126 (1955).
= Total reaction volume (ml)
1000 = Conversion of IU/ml to IU/L 3. Henry, R.J., et al.: Am. J. Clin. Path, 34:381 (1960).
AT = Time interval between readings 4. The Committee on Enzymes of the Scandinavian Society for
LP = Light path in cm Clinical Chemistry and Clinical Physiology. Scand. J. Clin.
 = Millirnolar absorptivity of NADH Lab.Invest32:291(1974).
SV = Sample volume in ml 5. Henry, R.J.: Clinical Chemiatry Principles and Techniques,
Example: If A = 1.45 and A = 1.28 2nd Ed. Harper and Row, New York, p. 882 (1974).
 (1.45 - 1.28) = 0.17 x 514 = 87 IU/L 6. Reg R., et al.: Clin. Chem. 19:92 (1973).
then
SI UNIT: To convert to SI Units (nkat/L) multiply IU/L by 16.67 7. Young,D.S.etal.:Clin.Chcm.21:5(1975).
NOTE: If any of the test parameters are altered a new factor must 8. Saris,N.E.,(ed):Clin.Chem23:887(1977).
be calculated using the above formula. 9. Tietz, N.W.: Fundamentals of Clinical Chemistry, W.B.
Saunders Co., Phila., p. 682 (1976).
QUALITY CONTROL
It is recommended that control be included in each set of assays. Revised:07/96
Commercially available control material with established AST values
may be used for quality control. The assigned value of the control
material must be confirmed by the chosen application. Failure to obtain
the proper range of values in the assay of control material may indicate