GOT/AST

IFCC-SCE kinetic method for the quantitative

determination of Aspartate Aminotransferase (AST)
activity in serum and plasma

ORDER INFORMATION ANCILLARY EQUIPMENT

REF Kit size  Automatic pipettes
GA4910 00 5x40 + 1x20 ml  Photometer
GA4911 00 10x40 + 2x20 ml  Analysis cuvettes (optical path = 1 cm)
KL4910 00 4x50 + 4x5 ml  Temperature controlled water bath
BK4910 00 2x(80+8 ml)  NaCl solution 9 g/l

INDICATION SAMPLES
Measurement of the activity of serum aminotransferases Serum, (heparin or EDTA) plasma.
(formerly called transaminases) is indicated in the diagnosis Do not use haemolysed samples because haemolysis can
of acute hepatic disorders and in monitoring their evolution. cause falsely positive results. Do not use anticoagulants
Increased aspartate aminotransferase (AST) levels, containing ammonium salts (ex. ammonium heparin).
however, can occur in connection with damages of hearts or Loss of activity within 3 days: at 2-8 °C < 8%
skeletral muscle as well as of liver perenchyma. In patients at 15-25 °C < 10%
with myocardial infarction, there is an increased AST Stability at - 20 °C at least 3 months.
concentration in blood due to its rapid release into the by
the damaged cells; therefore it is an important clinical Specimen collection / Preanalytical factors
parameter for the evaluation of this pathology.
It is recommended that specimen collection should be
carried out in accordance with NCCLS Document H11-A3.
METHOD PRINCIPLE
Optimized UV test according to SCE (Scandinavian INTERNAL QUALITY CONTROL
Committee on Enzymes) recommendations.
It is recommended to use commercial Quality Control sera
The principle of the method is based on the following
with known GOT/AST activity. Check that the values
enzymatic reactions:
obtained are within the reference range provided.
AST
L-Aspartate + 2-Oxoglutarate  L-Glutammate + Oxalacetate
ANALYTICAL PROCEDURE
MDH Working temperature 37 °C
Oxalacetate + NADH + H+  L-Malate + NAD+ Wavelength 340 nm (334 nm, 365 nm)
Optical path 1 cm
Decrease in absorbance value at 340 nm, due to the Reaction Kinetic (decrease)
oxidation of NADH to NAD+, is directly proportional to the
AST activity in the sample. Allow the reagents to reach working temperature before
using.
COMPOSITION
REAGENT A: Bireagent procedure
TRIS 28 mmol/l
EDTA-Na2 5.68 mmol/l Pipette into disposable or well clean cuvettes:
L-Aspartate 284 mmol/l
Sample
MDH ≥ 800 U/l
Sodium Azide 2 g/l Reagent A 1000 l
Sample 100 l
REAGENT B: Mix and incubate at 37 °C for 5 minutes, then add:
2-Oxoglutarato 68 mmol/l Reagent B 100 l
NADH 1.12 mmol/l Mix and incubate at 37 °C. After 1 minute read the
Sodium Hydroxide ≤ 1% absorbance (A) at 340 nm.
Read absorbance again 1, 2, 3 minutes thereafter.
PREPARATION OF REAGENTS Calculate A/min.

Bireagent procedure: Monoreagent procedure

The reagents are liquids ready to use.
Pipette into disposable or well clean cuvettes:
Monoreagent procedure:
Sample
Mix 10 parts of Reagent A and 1 part of Reagent B to obtain
Working reagent 1000 l
the working reagent (ex. 20 ml of RA + 2 ml of RB).
Incubate at 37 °C for 5 minutes, then add:
Storage and stability Sample 100 l
Store at 2-8 °C. Do not freeze the reagents! The reagents Mix and incubate at 37 °C. After 1 minute read the
are stable up to the expiry date stated on the label, if absorbance (A) at 340 nm.
contamination and evaporation are avoided, protected from Read absorbance again 1, 2, 3 minutes thereafter.
light. The above conditions are valid if the vials are opened Calculate A/min.
just only for the time to take the reagent, closed
immediately with their cap and stored at the indicated Note
conservation temperature.  Reaction volumes can be proportionally changed.
 For values upper than 440 U/l dilute samples 1+9 with
Prepare the required quantity of working reagent for the saline solution and multiply result by 10.
analytical session.

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GA4910 00 – GA4911 00 – KL4910 00 – BK4910 00 GOT/AST

CALCULATION OF RESULTS PRECAUTIONS IN USE

The Reagent A (REF GB09100A) is not considered
Activity (U/l) = A/min x factor (f) indicated in the following harmful (GHS00) according to (CE) n. 1272/2008
table: directive about classification, packaging and labelling of
Bireaegent procedure dangerous substances.
340 nm f = 1905 Reagent A:
334 nm f = 1945 Supplemental Hazard statement Code:
365 nm f = 3529 EUH032 - Contact with acids liberates very toxic gas.
Monoreagent procedure
The Reagent B (REF GB09100B) is harmful (Warning -
340 nm f = 1746 GHS07: H315, H319).
334 nm f = 1780 Skin Irrit. 2, Eye Irrit. 2
365 nm f = 3235 Hazard statement Code(s): H315 - Causes skin irritation;
Note: H319 - Causes serious eye irritation.
As the factor "f" used to calculate results depends on several Precautionary statements:
variables (wavelength, temperature, sample volume, reaction Prevention P280 - Wear protective gloves/protective
volume…), it is recommended to use commercial calibration clothing/eye protection/face protection. Response
sera to asset the instruments. P305+P351+P338 - IF IN EYES: Rinse cautiously with water
for several minutes. Remove contact lenses, if present and
REFERENCE VALUES easy to do. Continue rinsing. P332+P313 - If skin irritation
occurs: Get medical advice/attention. P337+P313 - If eye
Male: 10÷50 U/l irritation persists: Get medical advice/attention. P362 -
Female: 10÷35 U/l Take off contaminated clothing.
However, the reagents should be handled with caution,
avoiding swallowing and contact with skin, eyes and
Each laboratory should establish reference ranges for its mucous membranes. The use of laboratory reagents
own patients population. according to good laboratory practice is recommended.

ANALYTICAL PERFORMANCES Waste Management

Precision Please refer to local legal requirements.
Within-run and between-run coefficients of variation have
been calculated on replicates of two samples at different BIBLIOGRAPHY
enzymatic activities. The obtained results are reported in 1. Thomas L. Alanine aminotransferase (ALT), Aspartate
aminotransferase (AST). In: Thomas L, editor. Clinical
the following table:
Laboratory Diagnostics. 1st ed. Frankfurt: TH-Books
Verlagsgesellschaft; 1998. p. 55-65.
Within Run Between Run 2. Moss DW, Henderson AR. Clinical enzymology. In: Burtis CA,
Mean Ashwood ER, editors. Tietz Textbook of Clinical Chemistry. 3rd
Sample SD %CV SD %CV ed. Philadelphia: W.B Saunders Company; 1999. p. 617-721.
(U/l)
3. The Committee on Enzymes of the Scandinavian Society for
Serum 1 44.7 0.69 1.5 2.23 5.0 Clinical Chemistry and Clinical Physiology. Recommended
Serum 2 132.4 2.00 1.5 6.74 5.1 methods for the determination of four enzymes in blood. Scand
J Clin Lab Invest 1974; 33:291-306.
4. Penttilä IM, Jokela HA, Viitala AJ, Heikkinen E, Nummi S,
Linearity Pystynen P et al.: Activities of aspartate and alanine
The assay is linear up to 440 U/l. aminotransferases and alkaline phosphatase in sera of healthy
subjects. Scand J Clin Lab Invest 1975; 35: 275-84.
Sensitivity 5. Chitto G, Fabi A, Franzini C, Galletta G, Leonardi A, Marelli M,
Test sensitivity, in terms of limit of detection, is 3 U/l. Morelli AM: Variabilità biologica intra-individuo: rassegna della
letteratura, contributo sperimentale e considerazioni critiche.
Biochimica Clinica, 1994; 18, 10:673.
Correlation 6. NCCLS Document, “Procedures for the collection of arterial
A correlation study comparing the present method an a blood specimens”, Approved Standard, 3rd Ed. (1999).
commercial one gave the following results: 7. EU-Dir 1999/11 Commission Directive of 8 March 1999
adapting to technical progress the principles of good laboratory
y = 1.0457x – 0.8281 U/l r = 0.9853 practice as specified in Council Directive 87/18/EEC.

Interferences
Bilirubin > 40 mg/dl NOTE: Changes Highlighted
Triglycerides > 2000 mg/dl
Ascorbic acid > 30 mg/dl
Hemoglobin The presence of hemoglobin in serum
indicates destruction of erythrocytes with
release of AST, producing high interference.

Pag. 2/2
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