J Clin Pathol: first published as 10.1136/jcp.25.11.940 on 1 November 1972. Downloaded from J. clin. Path., 1972, 25, 940-944 Standardization of clinical enzyme assays: a reference method for aspartate and alanine transaminases J. H. WILKINSON, D. N. BARON, D. W. MOSS, AND P. G. WALKER From Charing Cross Hospital Medical School, Royal Free Hospital School ofMedicine, the Royal Postgraduate Medical School, and the Institute of Orthopaedics, London This is the second report produced by the Working such as those proposed in this report. The principles Party set up under the auspices of the Standards Sub- of calibration were discussed in the first report of the group of the Laboratory Development Advisory Working Party. Group of the Department of Health and Social Security. The Working Party includes representatives Principle of the Association of Clinical Biochemists, the Asso- ciation of Clinical Pathologists, and the Royal The reactions involved in the determination of College of Pathologists. Its first report, devoted to aspartate transaminase (AST) activity are as follows: the standardization of the assay of alkaline phos- Aspartate + 2-oxoglutarate glutamate + phatase, has recently been published (Moss, Baron, oxaloacetate .. .. .. *- (1) Walker, and Wilkinson, 1971). malate by copyright. Aspartate and alanine transaminases have long Oxaloacetate + NADH + H + been known in clinical biochemistry as glutamate- dehydrogenase oxaloacetate transaminase and glutamate-pyruvate malate + NAD+ .. (2) transaminase respectively, but are officially recog- The oxaloacetate produced by the transaminase nized by the International Union of Biochemistry as serves as substrate for malate dehydrogenase by L-aspartate: 2-oxoglutarate aminotransferase (EC which it is reduced to malate in the presence of 2.6.1.1.) and L-alanine: 2-oxoglutarate amino- dihydronicotinamide-adenine dinucleotide (NADH), transferase (EC 2.6.1.2.). Aspartate transaminase which is simultaneously oxidized. NADH has an and alanine transaminase are approved trivial names absorbence peak at 340 nm which is not shown by http://jcp.bmj.com/ and in this report these are abbreviated to AST and the oxidized form, and the decrease in absorbence ALT respectively. at this wavelength provides a means for the measure- Several colorimetric, fluorimetric, and spectro- ment of the transaminase activity. The methods photometric techniques have been devised for their suggested are based upon those of Henry, Chiamori, determination but for the purposes of standardiza- Golub, and Berkman (1960) who re-investigated and tion a spectrophotometric procedure is recom- improved the technique originally introduced by mended as a reference method. Karmen (1955). The measurement of aspartate and alanine trans- The spectrophotometric procedure has an im- on September 24, 2021 by guest. Protected aminase activities, described in this report, are portant advantage over colorimetric procedures in examples of reaction-rate techniques in which the which the oxaloacetate produced in reaction 1 progress of the enzyme reaction is followed through- is allowed to react with a chromogenic reagent out the period of observation. Absorbence readings such as 2,4-dinitrophenyl-hydrazine (Reitman are taken at intervals timed with a stopwatch or, and Frankel, 1957) or a diazonium reagent preferably, with the aid of a recording spectropho- (Babson, Shapiro, Williams, and Phillips, 1962). tometer. By such means it can be verified that the Oxaloacetate is a potent inhibitor of AST (Boyd, reaction rate is constant. Such verification is clearly 1961) and in the spectrophotometric procedure is impossible with a two-point assay system. Neverthe- removed as fast as it is formed, whereas in the colori- less, within certain limits methods of the latter type metric techniques it is allowed to accumulate. can be used satisfactorily, provided they are cali- The spectrophotometric procedure is standardized brated with the aid of a suitable reference method, by reference to the molar absorbance of NADH Received for publication 20 September 1972. (6-22 x 103) at 340 nm. The decrease in unit time 940 J Clin Pathol: first published as 10.1136/jcp.25.11.940 on 1 November 1972. Downloaded from Standar-dizationt of clinical enzyme assays: a referenice mtiethod for aspartate amid alamiine tr-ansamninases 941 (1 min) during the rectilinear (zero order) phase of substrate for ALT and in our experience does not the reaction provides a means for the calculation of appear to inhibit the enzyme, DL-alanine may be the enzyme activity in intcrnational units (micro- used in place of L-alanine. The necessary two-fold moles NADH oxidized per minute) per litre' under increase in concentration, however, complicates the the reaction conditions defined. use of the DL-form, which is liable to crystallize Alternatively the NADH may be measured from stock solutions during storage in a refrigerator. fluorimetrically (Laursen and Hansen, 1958), and The reference methods recommended in this this procedure has been adapted to an end-point report are those of Henry et al (1960), slightly system for use in the AutoAnalyzer (Levine and Hill, modified in that measurements are made at 25°C, 1965) but it is unsuitable for use as a reference and a final concentration of 025 moles/litre L- method. alanine is used for the ALT reaction. Similar principles are cmployed in the measure- ment of alanine transaminase (ALT) activity. In the Temperature spectrophotometric procedure (Henley and Pollard, 1955), the pyruvate producAed in the transaminase Karmen (1955) performed his reaction at 'room reaction (3) is reduced to lactate by the NAD+- temperature' (23°C) and Henry et al (1960) specified dependent lactate dehydrogenase (4): 32°C, the temperature reached in the cell compart- Alanine + 2-oxoglutaratte glutamate + ment of the Beckman DU spectrophotometer. The pyruvate .. (3) International Union of Biochemistry originally lactate recommended 25°C, but later 30CC, for enzyme Pyruvate + NADH + H-+ - determination, but few clinical laboratories in dehydrogenase (4) Britain carry out enzyme measurements at 30CC, lactate + NAD+ .. - since in this country it is quite practicable to main- tain a temperature of 25°C in a thermostatically Substrate Concentrations controlled cuvette compartment of a spectropho- tometer for most of the year without the aid of an Henry et al (1960) modified the AST technique by by copyright. adjusting the substrate concentrations to give con external cooling unit other than tap water. The development of semi-automated enzyme rate siderably higher enzyme acd tivitiestivities. ThemLasparttedThe L-aspartatei analyzers, such as the LKB, or spectrophotometers concentration (0-033 moles/litre) employed in specially adapted for microanalysis, such as the Karmen's original spectrophi procedue otometric Eskalab, which operate at or has neces- the determination of AST (Karmen,l1955) was foundford sitated further review of 35°Coperating37CC,temperatures. to be suboptimal, and Heniry et al(1960) recoi- There is much to be gained by performing all NAD- mended increasing this to 0h125 molesitrehi i or NADP-dependent enzyme measurements at a aspartate cannot be used as Ithe D- form inhibits this single temperature, but the Working Party has not transaminase. http://jcp.bmj.com/ Although Henry et al (1'960) increased the final been able to recommend a universally satisfactory concentration of L-alanine to 0.167 moles/litre in temperature. The choice appears to lie between the measurement of ALT activity experience has 250C, 30'C, 35°C, and 37 C. We at first considered as the most suitable, since the phosphatases since shown that this conct entration is suboptimal and37°Cother enzymes are regularly determined at this (Arvan and Coyle, 1969; Laudahn, Hartmann temperature and every clinical laboratory is equip- Rosenfeld, Weyer, and Mtuth 1970). 0Arvan and ped a 37°C water bath. However, doubts were the use of 25 moles/ with Coyle (1969) recommended about the stability of certain enzymes at 37°C, on September 24, 2021 by guest. Protected litre L-alanine, since higher thnentuseiofns asfelt cinenrecommendeds2 byb in particular liver lactate dehydrogenase (LD-5) and the 0 81 moles/litre L-alan While Laudahn et al (1970) ) causedmlte,littlefrecommendfurther enhance-ned glucose 6-phosphate dehydrogenase (GPD). in the Working Party has been unable to obtain evi- ment of activity and led to difficulty preparing dence of loss of activity of the former at 37°C, the stock solutions, owing to t]he limited solubility of latter loses about 15% of its activity during pre- this amino acid. Since D-alI mnine does not act as a incubation for 20 minutes at 37°C. 'The international unit of enzyme a(ctivity (U or iu) has not been The Working Party has some reservations about adapted for use with the SI system (S5 ysteme Internationale d'Unites). proposing a temperature other than that recom- The IUPAC/1UB Commission on Biiochemical Nomenclature have instead recommended the 'katal' as the unit of 'catalytic amount', mended by the International Union of Biochemistry defined as the amount of a catalyst (irncluding an enzyme) that brings and endorsed by the International Federation of about the transformation of one molte of substrate per second under defined conditions. Thus 1 katal perr litre (kat/l) = 60 10' inter- Clinical Chemistry, namely 30'C, but the fact has to national units per litre (U/I) or I U/I -- 1667 nkat/l. be faced that this figure has not proved acceptable hn 2The Boehringer