Pointe Liquid AST (SGOT)

Reagent Set

Intended Use Reagent Deterioration

For the quantitative determination of Aspartate Aminotransferase (AST) in Do not use reagent if:
human serum. Rx Only 1. The initial absorbance at 340nm is below 0.800.
2. The reagent fails to meet stated parameters of performance.
Clinical Significance
AST is widely distributed in tissues with the highest concentrations found in Precautions
the liver, heart, skeletal muscle and kidneys. Diseases involving any of 1. This reagent set is for in vitro diagnostic use only.
these tissues can lead to elevated levels of AST in serum. Following 2. The reagent contains sodium azide (0.25%) as a preservative. Do not
myocardial infarction, AST levels are elevated and reach a peak after 48 to ingest. May react with lead and copper plumbing to form highly explosive
60 hours. metal azides. Upon disposal, flush with a large volume of water to prevent
Hepatobiliary diseases such as cirrhosis, metastatic carcinoma and viral azide build up.
hepatitis can show increased levels of AST. Other disorders which can lead
to an elevated level of AST are muscular dystrophy, dermatomyositis, acute Specimen Collection and Storage11
pancreatitis and infectious mononucleosis.1 1. Non-hemolyzed serum is recommended. Red cells contain AST which can
give falsely elevated results.
Method History 2. AST in serum is reported stable for ten days when refrigerated (2-8°C), two
Karmen2 developed a kinetic assay procedure in 1955 which was based weeks when frozen (-20°C), and four days when stored at room temperature
upon the use of malate dehydrogenase and NADH. Optimized procedures (15-30°C).
were presented by Henry3 in 1960 and Amador and Wacker4 in 1962. These Interferences
modifications increased accuracy and lowered the effect of interfering
1. A number of drugs and substances affect AST activity. See Young, et al.12
substances. The Committee on Enzymes of the Scandinavian Society for
2. Patients with severe vitamin B6 deficiency could have a decreased recovery
Clinical Chemistry and Clinical Physiology5 published a recommended
of AST, presumably due to a lack of pyridoxal phosphate.13
method based on optimized modifications in 1974. In 1976, the Expert Panel
3. Bilirubin to at least 18 mg/dl, and hemoglobin to at least 300 mg/dl, have
on Enzymes of the International Federation of Clinical Chemistry (IFCC)6
been found to have a negligible effect on this procedure.
proposed the addition of pyridoxal-5-phosphate to the reaction mixture to
ensure maximum activity. The IFCC7 published a recommended method
that included P-5-P in 1978. The present method is based on IFCC
Materials Provided
AST (SGOT) Reagents R1 and R2.
recommendations but does not contain P-5-P since most specimens contain
adequate amounts of this cofactor for full recovery of AST activity.8,9,10
Materials Required but not Provided
Principle 1. Accurate pipetting devices.
2. Test tubes/rack.
AST
3. Timer.
L-Aspartate + α-Ketoglutarate ---------------- Oxalacetate + L-Glutamate
4. Spectrophotometer able to read at 340 nm. (UV)
MDH
5. Heating bath/block (37°C).
Oxalacetate + NADH + H+ --------------------------- L-Malate + NAD+ +H2O
Aspartate aminotransferase (AST) catalyzes the transfer of the amino group Test Procedure (Automated)
from L-aspartate to α-Ketoglutarate to yield oxalacetate and L-glutamate. Wavelength: 340 nm
The oxalacetate undergoes reduction with simultaneous oxidation of NADH Assay Type: Kinetic
to NAD in the malate dehydrogenase (MDH) catalyzed indicator reaction. Sample/Reagent Ratio: 1:11
The resulting rate of decrease in absorbance at 340nm is directly Reaction Direction: Decreasing
proportional to the AST activity. Lactate dehydrogenase (LDH) is added to Temperature: 37°C
prevent interference from endogenous pyruvate which is normally present in Lag Time: 60 seconds
serum. Read Time: 60 seconds
Low Normal: 5 U/L
Reagents High Normal: 34 U/L
After combining R1 and R2 the reagent contains: L-aspartic acid >200mM, Application Parameters for various automated instruments are available. Please
contact the manufacturer’s Technical Service Department for specific information.
α-ketoglutaric acid 12mM, LDH (microbial) > 1000U/L, MDH (microbial)
>800U/L, NADH >0.18mM, buffer, pH 7.8±0.1, sodium azide 0.25%, Test Procedure (Manual)
Stabilizers. 1. Prepare reagent according to instructions.
2. Pipette 1.0ml of reagent into appropriate tubes and pre-warm at 37°C for five
Reagent Preparation minutes.
The reagents are ready to use for systems capable of handling two reagents. 3. Add 0.100ml (100ul) of sample reagent, mix and incubate at 37°C for one
If a single reagent is required, prepare working reagent by mixing 5 parts of minute.
R1 reagent with 1 part R2 reagent. (e.g. 250 ul R1 with 50 ul R2 reagent.) 4. After one minute, read and record absorbance at 340nm against a water
blank. Return tube to 37°C. Repeat readings every minute for the next two
Reagent Storage minutes.
1. Store reagents at 2-8°C. 5. Calculate the average absorbance difference/minute (∆Abs/Min.).
2. Working reagent is stable for 48 hours at room temp. (15-30°C) and for 6. The ∆Abs./Min. multiplied by the factor 1768 (See Calculation) will yield
14 days when refrigerated (2-8°C). results in IU/L.

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 Pointe Liquid AST (SGOT)
Reagent Set

Procedure Notes 3. Precision:

1. If the spectrophotometer being used is equipped with a temperature- Within Day Day to Day
controlled cuvette, the reaction mixture may be left in the cuvette while Mean S.D. C.V.% Mean S.D. C.V.%
the absorbance readings are taken. 43 1.2 2.7 44 1.3 4.9
2. Turbid or highly icteric samples may give readings whose initial 186 1.7 1.4 183 4.0 2.5
absorbance exceeds the capabilities of the spectrophotometer. More
accurate results may be obtained by using 0.05ml (50 ul) of sample and 4. Sensitivity: The sensitivity for this reagent was investigated by reading the
multiplying the final answer by two. change in absorbance at 340nm for a saline sample and samples with known
concentrations. Ten replicates were performed. The results of this
Limitations investigation indicated that, on the analyzer used, the AST (SGOT) reagent
1. Samples with values above 500 IU/L should be diluted 1:1 with saline, showed little or no reagent drift on a zero sample. Under the reaction
re-assayed and the results multiplied by two. conditions described, 1 U/L AST activity gives a ∆Abs/Min. of 0.0004.
2. Patients with severe vitamin B6 deficiency could have a decreased
recovery of AST, presumably due to a lack of pyridoxal phosphate.13 References
1. Tietz, N.W., Fundamentals of Clinical Chemistry, W.B. Saunders co., p 674 (1982).
Calibration 2. Karmen, A., et al, J. Clin. Invest 34:126 (1955).
3. Henry, R.J., et al, Am. J. Clin. Path. 34:381 (1960).
The procedure is standardized by means of the millimolar absorptivity of 4. Amador, E., Wacker, W., Clin. Chem. 8:343 (1962).
NADH taken as 6.22 at 340nm under the test conditions described. 5. The Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and
Clinical Physiology, Scand. J. Clin. Lab. Invest 32:291 (1974).
Calculation 6. Expert Panel of Enzymes of the International Federation of Clinical Chemistry, Clin.
One international Unit (IU/L) is defined as the amount of enzyme that Chem. Acta. 70:F19 (1976).
catalyzes the transformation of one micromole of substrate per minute under 7. Expert Panel of Enzymes of the International Federation of Clinical Chemistry, Clin.
specified conditions. Chem. 24:720 (1978).
8. Jung, K., Bohm, M., Enzyme 23:201 (1978).
9. Hafkenscheid, J.C.M., Dijit, C.C.M., Clin. Chem. 25/1:55 (1979).
AST (IU/L) = ∆Abs./Min. x 1.10 x 1000 = ∆Abs./min. x 1768 10. Horder, M., Bowers, G.N., Jr., Clin. Chem. 23:551 (1977).
6.22 x 0.10 x 1.0 11. Henry, R.J., Clinical Chemistry: Principles and Technics, 2nd Ed., Hagerstown (MD),
Harper & Row, P882 (1974).
Where ∆Abs./Min. = Average absorbance change per minute 12. Young, D.S., et al, Clin. Chem. 21:1D (1975).
1000 = Conversion of IU/ml to IU/L 13. Kaplan, L.A., Pesce, A.J., Clinical Chemistry, St. Louis, C.V. Mosby, p.911-912 (1989).
1.10 = Total reaction volume (ml)
6.22 = Millimolar absorptivity of NADH Symbol Key
0.10 = Sample Volume (ml)
Use by (YYYY-MM-DD) Lot and batch code
1.0 = Light path in cm
Catalog number Manufacturer
Example: If the average absorbance change per minute = 0.12 then 0.12 x
1768 = 212 IU/L In vitro diagnostic medical device Temperature limitation

NOTE: If test parameters are altered the factor has to be recalculated using Consult instructions for use Rx Only: Prescription Use Only
the above formula.

SI Units: To convert to SI Units (nkat/L) multiply IU/L by 16.67. CE mark Authorized representative in the European Community

Quality Control
The validity of the reaction should be monitored using control sera with Manufactured by MedTest Dx: Pointe Brand

known normal and abnormal AST (SGOT) values. These controls should be
A7561 5449 Research Drive
Canton, MI 48188
run at least with every shift in which AST (SGOT) assays are performed. It is
recommended that each laboratory establish their own frequency of control
determination. Manufactured by MedTest Dx: Pointe Brand
5449 Research Drive, Canton, MI 48188
Expected Values13
8 to 22 IU/L (30°C) European Authorized Representative:
5 to 34 IU/L (37°C) Obelis s.a.
Since the expected values are affected by age, sex, diet, and geographical Boulevard Général Wahis 53
location, each laboratory is strongly urged to establish its own reference 1030 Brussels, BELGIUM
range for this procedure. Tel: (32)2.732.59.54 Fax:(32)2.732.60.03 email: [email protected]

Performance Certified Performance Guarantee

1. Linearity: 0-500 IU/L. MedTest Dx certifies that all of our products are manufactured according to
2. Comparison: Studies between the present method and a similar specified parameters. Any product not meeting specifications through their listed
method yielded a correlation coefficient of 0.999 and a regression expiration date will be remedied immediately without charge.
equation of y=0.98x + 1.6. (n=125, range=15-659 IU/L)
Rev. 08/19 P803-A7561-01