Guidelines on Standard Operating Procedures for CLINICAL CHEMISTRY

Transaminases - Colorimetric End-Point Method

     Introduction

The two transaminases of diagnostic importance are:

     serum glutamic oxaloacetate transaminase (SGOT) or aspartate amino transferase (AST), and

     serum glutamic pyruvate transaminase (SGPT) or alanine amino transferase (ALT). While AST is found in every tissue of the body, including red blood cells, and is particularly high in the cardiac muscle, ALT is present in moderately high concentration in liver and low in cardiac, skeletal muscle and other tissues. Both AST and ALT measurements are useful in the diagnosis and monitoring of patients with hepatocellular disease.

     Principle of the method

Transamination is the process in which an amino group is transferred from amino acid to an a -keto acid. The enzymes responsible for transamination are called transaminases. The substrates in the reaction are a -ketoglutaric acid (a KG) plus L-aspartate for AST, and a KG plus L-alanine for ALT. The products formed by enzyme action are glutamate and oxaloacetate for AST and glutamate and pyruvate for ALT. Addition of 2,4, dinitrophenyl hydrazine results in the formation of hydrazone complex with the ketoacids. A red colour is produced on the addition of sodium hydroxide. The intensity of colour is related to enzymic activity.

     Specimen type, collection and storage

Serum or EDTA/heparinized plasma can be used in this assay.

Transaminases are stable in serum for 6 hours at 25-35⁰C, 7 days at 2-8⁰C and for one month when stored at -20⁰C.

     Reagents

All chemicals must be Analar grade

     Phosphate buffer, pH 7.4 :

Dissolve 14.9 g disodium hydrogen phosphate dehydrate (Na₂HPO4 2H₂0) and 2.2g anhydrous potassium dihydrogen phosphate (KH₂PO_H) in distilled water and make up to one litre. Check the pH, and, if necessary, adjust to 7.4 using small amounts of either KH₂PO₄ or Na₂HPO₄- Stable for 3 months when stored at 2-8° C.

     AST Substrate

Dissolve 2.66 g DL- aspartic acid and 30 mg a -keto glutarate in 20.5 ml of 1 M NAOH. Adjust the pH to 7.4 by adding I M NAOH drop wise while stirring. Make up to 100 ml with phosphate buffer. Add 1 ml of chloroform as preservative. Stable for 2 months when stored at 2-8° C. Discard if it becomes turbid.

     ALT Substrate

Dissolve 1.78 g DL-alanine and 30 mg a -keto glutarate in 20 ml of phosphate buffer containing 1.25 ml of 0.4 M NAOH. Make up to 100 ml with buffer and adjust to pH 7.4 if necessary. Add 1 ml chloroform as preservative. Stable for 2 months when stored at 2-8° C. Discard if it becomes turbid.

     Pyruvate standard 2 m mol/ml

Dissolve 220 mg sodium pyruvate inphosphate buffer and make up to 100 ml. Dilute 10 ml of this solution to 100 ml with phosphate buffer to obtain the working standard containing 2 m mol pyruvate per ml. The remaining 90 ml of the first solution should be discarded. The working standard should be stored in small aliquots of 2 ml in the freezer. One aliquot of working standard should be used for preparing a calibration graph. Discard the leftover standard in the vial.

     Colour reagent

Dissolve 200 mg 2,4 dinitro-phenylhydrazine (2,4 DNPH) in hot 1M HCI and make up to 1 litre with 1M HCI. Stable for 6 months when stored at 2-8⁰C.

     0.4 M Sodium hydroxide

Dissolve 16 g sodium hydroxide in about 800 ml of distilled water and make up to 1 litre with distilled water. Store in a polyethylene container at 25-35⁰C. Stable for 6 months.

     Equipment, glassware and other accessories

Refer to Section A (2), Introduction to SOP.

     Procedure

     AST

The protocol of the procedure is described below.

Pipette the following into appropriately labelled 18 x 150mm tubes

TBK = Test Blank & QCBK = QC Blank

Set the spectrophotometer/filter photometer to zero using distilled water at 5 1 0 nm/yellow green filter and measure the absorbance of TBK, QCBK, Test and QC in the order.

     ALT

Pipette the following into appropriately labelled 18 x 150 mm tubes.

Set the spectrophotometer/filter photometer to zero using distilled water at 5 1 0 nm/yellow green filter and measure the absorbance of TBK, QCBK, Test and QC in the order.

     Calculation and calibration graph

In the measurement of both serum AST & ALT, only pyruvate is used as the standard. Theoretically speaking, oxaloacetate should be used as the standard for AST assay and pyruvate as the standard for ALT assay. Oxaloacetate formed in the AST assay is unstable and immediately gets converted into pyruvate; hence the use of pyruvate standard for AST assay. One unit/L of AST or ALT is defined as the liberation of 1m mol of pyruvate per minute at 37⁰C incubation per litre of serum. As a -keto glutarate-cosubstrate in the assay contributes to the final absorbance, the change in absorbance is not linearly related to the theoretical value of pyruvate produced and hence the enzyme activity. This is evident from the sample calibration graph shown.

Construct a calibration curve by plotting the corresponding absorbance of standards against their respective AST/ALT activities. The measurable ranges with these graphs are from 5.0 to 150 U/L for ALT and 5.0 to 190 U/L for AST.

Plot the difference in the absorbance between test and TBK as well as QC and QCBK for AST & ALT and read off the enzyme activities on the calibration graphs; otherwise refer to the Table recommended by standard clinical chemistry textbooks like Tietz or Varley relating pyvurate values to AST/ALT activities (if it is available).

ALT

AST

     Analytical reliabilities

Refer to pages 7-9 of section 1 (General Introduction) on the use of internal QC and interpretation of daily QC data (for releasing patients' results).

Include one internal QC in every batch of samples analysed each day irrespective of the number of samples in a batch. Since AST or ALT is analysed in a single batch in a day in an intermediate laboratory, it will not be possible to analyse several QC samples and calculate within-day precision. However, even if only a single QC sample is analysed in a day, this value can be pooled with the preceding 10 or 20 values obtained in the previous days and between-day precision can be calculated and expressed as %CV. Ensure that this is well within the acceptable limit, i.e, 10%.

At least once a week analyse another QC serum from either a low QC or high QC pool.

"Assayed" QC sera with stated values (ranges) are available from several commercial sources, viz. Boehringer Mannheim, BioRad & Randox.

If a laboratory uses QC sera from a commercial source, it is important that the company certifies that their QC materials are traceable to international reference materials.

     Hazardous materials

This procedure uses NaOH, which is caustic. Do not swallow, and avoid contact with the skin and mucous membranes.

     Reference range and clinical interpretation

The reference ranges by this method are:

AST 8 - 40 U/L

ALT 5 - 35 U/L

High levels of serum AST activity are seen in the heart, liver, skeletal muscle and kidney tissues. Increased activity of AST in serum is observed in myocardial infarction after 20-36 hours of onset and hence used as a supporting evidence in the diagnosis of myocardial infarction. Values are usually less than 10 times the upper limit of normal (ULN). Increased activities are also observed in viral/toxic hepatitis, muscular dystrophy and in pulmonary embolism.

ALT is distributed mainly in the liver and to a lesser extent in the kidney and muscles. Increased ALT activity is observed in hepatitis and cirrhosis. Values may be increased to >10 times – 100 times ULN in hepatitis.

     Limitations

For samples with enzyme activity greater than 150 U/L for ALT and greater than 190 U/L for AST, dilute the specimen 1 in 10 with 0.9 % saline. Some specimens may require a further 1 in 10 dilution to give a final dilution of 1 in 100. Multiply the final result by the dilution factor.

Avoid using the haemolysed sample as this will cause falsely elevated values. In this case inform the requesting physician and ask for another specimen.

     Reference

1.      Reitman, S & Frankel, S. (1957) Am J Clin Pathol., 28, 56-63.