Liquid AST (SGOT)

Reagent Set

Intended Use Reagent Storage

For the quantitative determination of Aspartate Aminotransferase (AST) in 1. Store reagents at 2-8°C.
human serum. 2. Working reagent is stable for 48 hours at room temp. (15-30°C) and for 14
days when refrigerated (2-8°C).
Clinical Significance
AST is widely distributed in tissues with the highest concentrations found in Reagent Deterioration
the liver, heart, skeletal muscle and kidneys. Diseases involving any of Do not use reagent if:
these tissues can lead to elevated levels of AST in serum. Following 1. The initial absorbance at 340nm is below 0.800.
myocardial infarction, AST levels are elevated and reach a peak after 48 to 2. The reagent fails to meet stated parameters of performance.
60 hours.
Hepatobiliary diseases such as cirrhosis, metastatic carcinoma and viral Precautions
hepatitis can show increased levels of AST. Other disorders which can lead 1. This reagent set is for in vitro diagnostic use only.
to an elevated level of AST are muscular dystrophy, dermatomyositis, acute 2. The reagent contains sodium azide (0.25%) as a preservative. Do not
pancreatitis and infectious mononucleosis.1 ingest. May react with lead and copper plumbing to form highly explosive
metal azides. Upon disposal, flush with a large volume of water to prevent
Method History azide build up.
Karmen2 developed a kinetic assay procedure in 1955 which was based
upon the use of malate dehydrogenase and NADH. Optimized procedures Specimen Collection and Storage11
were presented by Henry3 in 1960 and Amador and Wacker4 in 1962. These 1. Non-hemolyzed serum is recommended. Red cells contain AST which can
modifications increased accuracy and lowered the effect of interfering give falsely elevated results.
substances. The Committee on Enzymes of the Scandinavian Society for 2. AST in serum is reported stable for ten days when refrigerated (2-8°C), two
Clinical Chemistry and Clinical Physiology5 published a recommended weeks when frozen (-20°C), and four days when stored at room temperature
method based on optimized modifications in 1974. In 1976, the Expert Panel (15-30°C).
on Enzymes of the International Federation of Clinical Chemistry (IFCC)6
proposed the addition of pyridoxal-5-phosphate to the reaction mixture to Interferences
ensure maximum activity. The IFCC7 published a recommended method 1. A number of drugs and substances affect AST activity. See Young, et al.12
that included P-5-P in 1978. The present method is based on IFCC 2. Patients with severe vitamin B6 deficiency could have a decreased recovery
recommendations but does not contain P-5-P since most specimens contain of AST, presumably due to a lack of pyridoxal phosphate.13
adequate amounts of this cofactor for full recovery of AST activity.8,9,10 3. Bilirubin to at least 18 mg/dl, and hemoglobin to at least 300 mg/dl, have
been found to have a negligible effect on this procedure.
Principle
AST Materials Provided
L-Aspartate + α-Ketoglutarate ----------------¾ Oxalacetate + L-Glutamate AST (SGOT) Reagents R1 and R2.
MDH
Oxalacetate + NADH + H+ ---------------------------¾ L-Malate + NAD+ +H2O Materials Required but not Provided
1. Accurate pipetting devices.
Aspartate aminotransferase (AST) catalyzes the transfer of the amino group 2. Test tubes/rack.
from L-aspartate to α-Ketoglutarate to yield oxalacetate and L-glutamate. 3. Timer.
The oxalacetate undergoes reduction with simultaneous oxidation of NADH 4. Spectrophotometer able to read at 340 nm. (UV)
to NAD in the malate dehydrogenase (MDH) catalyzed indicator reaction. 5. Heating bath/block (37°C).
The resulting rate of decrease in absorbance at 340nm is directly
proportional to the AST activity. Lactate dehydrogenase (LDH) is added to Test Procedure (Automated)
prevent interference from endogenous pyruvate which is normally present in Wavelength: 340 nm
serum. Assay Type: Kinetic
Sample/Reagent Ratio: 1:11
Reagents Reaction Direction: Decreasing
After combining R1 and R2 the reagent contains: L-aspartic acid >200mM, Temperature: 37°C
α-ketoglutaric acid 12mM, LDH (microbial) > 1000U/L, MDH (microbial) Lag Time: 60 seconds
>800U/L, NADH >0.18mM, buffer, pH 7.8±0.1, sodium azide 0.25%, Read Time: 60 seconds
Stabilizers. Low Normal: 5 U/L
High Normal: 34 U/L
Reagent Preparation Application Parameters for various automated instruments are available. Please
The reagents are ready to use for systems capable of handling two reagents. contact the manufacturer’s Technical Service Department for specific information.
If a single reagent is required, prepare working reagent by mixing 5 parts of
R1 reagent with 1 part R2 reagent. (e.g. 250 ul R1 with 50 ul R2 reagent.) Test Procedure (Manual)
1. Prepare reagent according to instructions.
2. Pipette 1.0ml of reagent into appropriate tubes and pre-warm at 37°C for five
minutes.

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 Liquid AST (SGOT)
Reagent Set

3. Add 0.100ml (100ul) of sample reagent, mix and incubate at 37°C for Expected Values13
one minute. 8 to 22 IU/L (30°C)
4. After one minute, read and record absorbance at 340nm against a 5 to 34 IU/L (37°C)
water blank. Return tube to 37°C. Repeat readings every minute for Since the expected values are affected by age, sex, diet, and geographical
the next two minutes. location, each laboratory is strongly urged to establish its own reference range for
5. Calculate the average absorbance difference/minute (ΔAbs/Min.). this procedure.
6. The ΔAbs./Min. multiplied by the factor 1768 (See Calculation) will yield
results in IU/L. Performance
1. Linearity: 0-500 IU/L.
Procedure Notes 2. Comparison: Studies between the present method and a similar method
1. If the spectrophotometer being used is equipped with a temperature- yielded a correlation coefficient of 0.999 and a regression equation of
controlled cuvette, the reaction mixture may be left in the cuvette while y=0.98x + 1.6. (n=125, range=15-659 IU/L)
the absorbance readings are taken. 3. Precision:
2. Turbid or highly icteric samples may give readings whose initial
absorbance exceeds the capabilities of the spectrophotometer. More Within Day Day to Day
accurate results may be obtained by using 0.05ml (50 ul) of sample and Mean S.D. C.V.% Mean S.D. C.V.%
multiplying the final answer by two. 43 1.2 2.7 44 1.3 4.9
186 1.7 1.4 183 4.0 2.5
Limitations
4. Sensitivity: The sensitivity for this reagent was investigated by reading the
1. Samples with values above 500 IU/L should be diluted 1:1 with saline,
change in absorbance at 340nm for a saline sample and samples with known
re-assayed and the results multiplied by two.
concentrations. Ten replicates were performed. The results of this
2. Patients with severe vitamin B6 deficiency could have a decreased
investigation indicated that, on the analyzer used, the AST (SGOT) reagent
recovery of AST, presumably due to a lack of pyridoxal phosphate.13
showed little or no reagent drift on a zero sample. Under the reaction
conditions described, 1 U/L AST activity gives a ΔAbs/Min. of 0.0004.
Calibration
The procedure is standardized by means of the millimolar absorptivity of
NADH taken as 6.22 at 340nm under the test conditions described. References
1. Tietz, N.W., Fundamentals of Clinical Chemistry, W.B. Saunders co., p 674
(1982).
Calculation 2. Karmen, A., et al, J. Clin. Invest 34:126 (1955).
One international Unit (IU/L) is defined as the amount of enzyme that
3. Henry, R.J., et al, Am. J. Clin. Path. 34:381 (1960).
catalyzes the transformation of one micromole of substrate per minute under
4. Amador, E., Wacker, W., Clin. Chem. 8:343 (1962).
specified conditions.
5. The Committee on Enzymes of the Scandinavian Society for Clinical
Chemistry and Clinical Physiology, Scand. J. Clin. Lab. Invest 32:291 (1974).
AST (IU/L) = ΔAbs./Min. x 1.10 x 1000 = ΔAbs./min. x 1768
6. Expert Panel of Enzymes of the International Federation of Clinical
6.22 x 0.10 x 1.0
Chemistry, Clin. Chem. Acta. 70:F19 (1976).
Where ΔAbs./Min. = Average absorbance change per minute 7. Expert Panel of Enzymes of the International Federation of Clinical
1000 = Conversion of IU/ml to IU/L Chemistry, Clin. Chem. 24:720 (1978).
1.10 = Total reaction volume (ml) 8. Jung, K., Bohm, M., Enzyme 23:201 (1978).
6.22 = Millimolar absorptivity of NADH 9. Hafkenscheid, J.C.M., Dijit, C.C.M., Clin. Chem. 25/1:55 (1979).
0.10 = Sample Volume (ml) 10. Horder, M., Bowers, G.N., Jr., Clin. Chem. 23:551 (1977).
1.0 = Light path in cm 11. Henry, R.J., Clinical Chemistry: Principles and Technics, 2nd Ed.,
Hagerstown (MD), Harper & Row, P882 (1974).
Example: If the average absorbance change per minute = 0.12 then 0.12 x 12. Young, D.S., et al, Clin. Chem. 21:1D (1975).
1768 = 212 IU/L 13. Kaplan, L.A., Pesce, A.J., Clinical Chemistry, St. Louis, C.V. Mosby, p.911-
912 (1989).
NOTE: If test parameters are altered the factor has to be recalculated using
the above formula. Manufactured by Pointe Scientific, Inc.
5449 Research Drive, Canton, MI 48188
SI Units: To convert to SI Units (nkat/L) multiply IU/L by 16.67.
European Authorized Representative:
Quality Control Obelis s.a.
The validity of the reaction should be monitored using control sera with Boulevard Général Wahis 53
known normal and abnormal AST (SGOT) values. These controls should be 1030 Brussels, BELGIUM
run at least with every shift in which AST (SGOT) assays are performed. It is Tel: (32)2.732.59.54 Fax:(32)2.732.60.03 email: mail@obelis.net
recommended that each laboratory establish their own frequency of control
determination.
Rev. 1/10 P803-A7561-01