AST-GOT Cromatest Bio Chemistry Reagent

PRINCIPLE

Aspartate aminotransferase (AST/GOT) catalyzes the transfer of the amino group from aspartate to oxoglutarate with the formation of glutamate and oxalacetate. The latter is reduced to malate by malate dehydrogenase (MDH) in the presence of reduced nicotinamide adenine dinucleotide (NADH).

The reaction is monitored kinetically at 340 nm by the rate of decrease in absorbance resulting from the oxidation of NADH to NAD⁺, proportional to the activity of AST present in the sample.

AST/GOT

L-Aspartate + 2-Oxoglutarate              L-Glutamate + Oxalacetate

Oxalacetate + NADH + H⁺

MDH

L-Malate + NAD⁺

The method follows the proposed optimised formulation of the IFCC1.

                 REAGENT COMPOSITION

R1

AST substrate. TRIS buffer 121 mmol/L pH 7.8, L-aspartate

362 mmol/L, malate dehydrogenase > 460 U/L, lactate dehydrogenase > 600 U/L.

R2

AST coenzyme. NADH 1.3 mmol/L, 2-oxoglutarate 75

STORAGE AND STABILITY

Store at 2-8ºC.

The Reagents are stable until the expiry date stated on the label.

REAGENT PREPARATION
Working reagent. Mix 4 mL of R1 + 1 mL of R2. Stable for 4 weeks at 2-8ºC. Protect from light.

Discard the reagent if presents an absorbance below 1.200 at 340 nm against distilled water or if it fails to recover the declared values of control sera.

SAMPLES
Serum and EDTA or heparinized plasma free of hemolysis.

AST is stable in serum or plasma 24 hours at room temperature and for 1 week at 2-8ºC.

                  REFERENCE VALUE

Serum, plasma

Adults
37ºC
up to 40 U/L (0.67 mkat/L)
30ºC
up to 25 U/L (0.42 mkat/L)

Levels approximately twice the adult level are seen in neonates and infants; these decline to adult level by approximately 6 months of age.

It is recommended that each laboratory establishes its own reference range

                  QUALITY CONTROL

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To ensure adequate quality control (QC), each run should include a set of controls (normal and abnormal) with assayed values handled as unknowns.

REF

1980005 HUMAN MULTISERA NORMAL

Borderline level of AST. Assayed.

REF

1985005 HUMAN MULTISERA ABNORMAL

Elevated level of AST. Assayed.

CLINICAL SIGNIFICANCE
The group of enzymes called transaminase exist in tissues of many organs. Necrotic activity in these organs causes a release of abnormal quantitaties of enzyme into the blood where they are measured.

Since heart tissue is rich in AST increased serum levels appear in patients after myocardial infarction, as well as in patients with muscle disease, muscular dystrophy and dermatomyositis.

The liver is specially rich in ALT, being this enzyme measurement used primarily as a test for infectious and toxic hepatitis, although high levels of both ALT and AST may also be found in cases of liver cell damage and acute pancreatitis, suggesting that the obstruction of the biliary tree by the edematous pancreas and the presence of associate hepatic disease may contribute to elevated AST levels in these patients.

Slight or moderate elevations of AST and ALT activities may be observed after intake of alcohol and after administration of various drugs, such as salicylates, opiates and ampicillin.

ANALYTICAL PERFORMANCE
–  Linearity. Up to 500 U/L

–  Precision

U/L
Within-run
Mean
23
70
142
SD
0,28
0,61
1,3
CV%
1,22
0,87
0,97
N
10
10
10

Replicates: 10 for each level. Instrument: CECIL CE 2001

– Sensitivity. Using this reagent and method an DA/min of 0.00 read at 340 nm is equivalent to 2 U/L of GOT activity.

– Correlation. This assay (y) was compared with a similar commercial method (x). The results were:

N = 25      r = 0.996     y = 1.069x – 0.786

           INTERFERENCES

–     Samples from patients under hemodialysis, severe vitamine B deficiency or with related pathologies, lead to an underestimation of AST values.

–     As a result of the high levels of AST in red cells hemolyzed samples are not suitable for testing.

–     Lipemic samples (triglycerides up to 2 g/L) and icteric samples

(bilirubin >20 mg/dL) do not interfere.

–     Other drugs and substances may affect the AST values.2

              MATERIALS REQUIRED

–    Photometer or spectrophotometer with a thermostatted cell

compartment set at 30/37ºC, capable to read at 340 nm.

–     Stopwatch, strip-chart recorder or printer.

–     Cuvettes with 1-cm pathlength.

–     Pipettes to measure reagent and samples.

PROCEDURE

1.    Preincubate working reagent, samples and controls to reaction temperature.

2.    Set the photometer to 0 absorbance with distilled water.

Reaction temperature
37ºC
30ºC
Working reagent Sample or control
1.0 mL 50 mL
1.0 mL 100 mL
Pipette into a cuvette

1.   Mix gently by inversion. Insert cuvette into the cell holder and start stopwatch.

2.   Incubate for 1 minute and record initial absorbance reading.

3.   Repeat the absorbance readings exactly after 1, 2 and 3 minutes.

4.   Calculate the difference between absorbances.

5.   Calculate the mean of the results to obtain the average change in absorbance per minute (DA/min).

CALCULATIONS
U/L = DA/min x 3333 (37ºC) U/L = DA/min x 1746 (30ºC)

Samples with DA/min exceeding 0.160 at 340 nm should be diluted 1:10 with saline and assayed again. Multiply the results by 10.

If results are to be expressed as SI units apply: U/L x 0.01667 = mkat/L

REFERENCES
1.    Bergmeyer, H.V., HÆrder, M., Rej, R. Approved recommendati (1985) on IFCC methods for the measurement of cataly concentration of enzymes. Part 2. IFCC method for aspart aminotransferase, J. Clin. Chem. Clin. Biochem. 24 : 497 (1986

2.    Young, D.S. Effects of Drugs on Clinical Laboratory Tests. 4 th

Edition. AACC Press (1995).

1.    Tietz. N.W. Clinical Guide to Laboratory Tests, 3rd Edition. W.B. Saunders Co. Philadelphia, PA. (1995).