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Home , Alanine transaminase, Aspartate transaminase, DU spectrophotometer, Transaminase

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 assays: a reference method for aspartate and

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 (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 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 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- dinucleotide (NADH), (EC 2.6.1.2.). which is simultaneously oxidized. NADH has an

and 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 (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 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 lactate dehydrogenase (LD-5) and the 0 81 moles/litre L-alan While Laudahn et al (1970) ) causedmlte,littlefrecommendfurther enhance-ned 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 . 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- , 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 optimized test combiination. most British clinical laboratories. J Clin Pathol: first published as 10.1136/jcp.25.11.940 on 1 November 1972. Downloaded from 942 J. H. Wilkinson, D. N. Baron, D. W. Moss, and P. G. Walker The original IUB recommendation of 25'C has 3 NADH solution been criticized mainly because of the technical 2-5 mg per ml reagent 2. Best prepared fresh each difficulty of maintaining it in hot countries and day. The development of inhibitors of some de- especially in the USA, but during the past decade, hydrogenases in certain NADH solutions is not a several moderately priced refrigerated water baths critical factor when used for transaminase deter- or portable refrigerator units have been marketed mination as the dehydrogenase used as indicator which overcome this problem. We therefore con- enzyme is present in considerable excess. Slight sider that it is practicable to standardize the deter- variations between batches of good quality NADH mination of NAD- and NADP-dependent enzymes have negligible effects on the results of transaminase at 25°C. The procedures described are designed for assays. use at 25'C, but the conditions recommended may not be optimal at higher temperatures. The follow- 4 2-Oxoglutarate (01 moles/litre) ing conversion factors have been experimentally 0-73 g 2-oxoglutaric acid is dissolved in 35 ml water. determined with the suggested method at 37°C on Five ml reagent 1 is added and the pH adjusted to six sera with AST and ALT activities ranging from 7-4 at 25°C by the addition of NaOH (1 mole/litre), 47 to 184 U/litre and 46 to 139 U/litre respectively.3 after which the volume is made up to 50 ml with While these enable results obtained at 37°C to be water. May be stored up to one month at 4°C. compared with those measured at 25'C, such factors should be used with caution. 5 L-Aspartate (0375 moles/litre) 5 0 g L- is dissolved with the aid of heat in a mixture of 50 ml water and 35 ml NaOH Cuvette Temperature Factors to Convert to AST ALT (1 mole/litre). After cooling to room temperature, 25°C 37°C 2-18 196 10 ml reagent 1 is added and the pH adjusted to 7-4 37°C 25°C 0-46 0 51 at 25°C with NaOH (1 mole/litre). The volume is then made up to 100 ml. May be stored up to one

Factors for the interconversion of AST and ALT by copyright. activities determined at 250C or 370C month at 40C.

6 L-Alanine (075 moles/litre) The Working Party considers that individual 6-67 g L-alanine is dissolved in 75 ml water and 10 ml laboratories should use whatever temperature is reagent 1, and the pH is adjusted to 7-4 at 25°C with appropriate to their equipment, but should calibrate NaOH (1 mole/litre). The volume is then made up their procedures with the aid of a suitable reference to 100 ml. May be stored for one month at 4°C. method. Whatever temperature of estimation may be chosen in a particular laboratory, it is essential to use a spectrophotometer equipped with effective 7 Malate dehydrogenase S Uper ml http://jcp.bmj.com/ means of controlling the temperature of the sample A commercial preparation should be diluted with accurately and precisely. water to provide a day's supply. Each batch of malate dehydrogenase should be checked for the presence of AST activity by performing a blank re- Recommended Method action, ie, omitting , and also for apotrans- aminase by out a REAGENTS carrying blank reaction in which

serum is replaced by 0-2 ml on September 24, 2021 by guest. Protected Distilled or solution containing demineralized water should be used for 0-2 ,ug phosphopyridoxal (Rosalki and Wilkinson, the preparation of the following reagents: 1959). The presence of the apotransaminase must be excluded since it would be activated by the phos- 1 Phosphate buffer (1J0 moles/litre), pH 7.4 phopyridoxal of serum to give a spuriously high One hundred and thirty-six g KH2PO4 and 33 g NaOH dissolved in water and the volume adjusted transaminase activity. to 1 litre. 8 Lactate dehydrogenase 5 Uper ml 2 Phosphate buffer (01 moles/litre), pH 7-4 The rabbit-muscle enzyme should be diluted with One hundred ml of IM-phosphate buffer made up to water to provide a day's supply. I litre and adjusted to pH 7-4 at 25°C. PROCEDU RE 'A working party of the Association of Clinical Biochemists is currently investigating the reproducibility of temperature coefficients The following mixtures are prepared and incubated for AST and ALT determined by the methods described. for 15 min. at 25°C ± 0-2°C. J Clin Pathol: first published as 10.1136/jcp.25.11.940 on 1 November 1972. Downloaded from Standardization of clinical enzyme assays: a reference methodfor aspartate and alanine transaminases 943

AST (ml) ALT (ml) encountered in the case of dis- cussed in the first report of the Working Party. Phosphate buffer (0 1 moles/litre) 1 3 1-3 The most commonly used automated systems Serum 0-2 0-2 NADH solution 0-2 0-2 include the AutoAnalyzer methods with 2,4- L-aspartate 10 - dinitrophenylhydrazine (Fingerhut, Ferzola, Marsh, L-alanine - 1 0 Malate dehydrogenase 01 - and Levine, 1962) or a diazonium reagent (Schaffert, Lactate dehydrogenase - 01 Kingsley, and Getchell, 1964), but the fluorimetric Total volume 2-8 2-8 AutoAnalyzer method (Levine and Hill, 1965) also requires calibration. An ultra-violet spectrophotometer, preferably a For this purpose it is suggested that the trans- recording instrument, is switched on and set for aminase activities of a series of about five sera4 be recording changes in absorbance at 340 nm. The determined by the reference methods described in cuvette compartment should be thermostatically this report. The activities should span the effective range of the automated technique, ie, from about controlled so that the cuvette temperature is 25°C ± 0-2°C. When the instrument is ready the incubated 10-300 U/litre. The reference sera may be stored solutions should be transferred to cuvettes. In each frozen in small containers for periods of up to eight case, the reaction is started by the addition of 0-2 ml months, or at 4°C for up to one week without signi- 2-oxoglutarate solution followed by careful mixing. ficant loss of activity. Nevertheless we recommend Recordings for the purpose of calculation should that the activities of such sera be checked at least not be made until the reaction obeys zero-order twice a week. kinetics, ie, the decrease in absorbance becomes Non-automated manual versions of these colori- steady. (There is often a lag phase of 2 to 5 min.) metric or fluorimetric techniques can be calibrated The rate of change in absorbance per minute is in a similar manner using sera whose transaminase measured (a A/min), and the transaminase activity activities have been measured by the reference calculated in U per litre: method. by copyright. Transaminase = / activity 6A22 103 X o02 X 3000 Comments and Recommendations = a A/min x 2400 U/litre I Spectrophotometric methods are considered to The spectrophotometric unit introduced by be the most suitable reference procedures for trans- Karmen (I1955) is the enzyme activity in 3 ml reaction aminase determination. The rate of oxidation of mixture producing a change in absorbence at NADH, when measured by the change in the 340 nm of 0-001 per minute per ml of serum. Thus absorbance at 340 nm is a much more dependable Karmen units/ml can be converted into Inter- standard than any of the alternatives which have national units per litre by multiplying by 3/6 22 or been proposed, eg, pyruvate, 'standard' sera, etc. http://jcp.bmj.com/ 0-48. Since the indicator enzymes are in considerable It is important to check the absorbence scale of excess the presence of possible dehydrogenase the spectrophotometer, since the value of the method inhibitors in the NADH (Fawcett, Ciotti, and depends upon accurate measurement. Methods for Kaplan, 1961; Dalziel, 1961) has little or no effect checking accuracy with spectrophotometric stand- on the transaminase reaction which remains the ards have recently been discussed by Rand (1969). rate-determining step. 2 Transaminase activities determined by a colori- Normal Ranges for Serum Transaminase Activities metric technique should not be reported in spectro- on September 24, 2021 by guest. Protected photometric units, eg, Karmen units, unless the The normal ranges were obtained at 25°C on healthy method has been calibrated by reference to the blood donors and laboratory personnel by the recommended or other appropriate spectrophoto- methods described: metric procedure. Aspartate transaminase males 5-15 U/litre 3 The techniques recommended have been demon- females 5-12 strated to give maximal activities and to be repro- Alanine transaminase males 5-19 females 5-12 41f pools of human sera are used, care should be taken in case some specimens contain virus. Horse serum with added liver extracts free from Australia antigen may be used. See First Report of Calibration of Automated and Manual Procedures Working Party (Moss et al, 1971). If liver extracts are employed they should be centrifuged to remove mitochondria since mitochondrial might catalyse the reductive amination of The problems involved in the calibration of auto- 2-oxoglutarate by salts present in the malate dehydro- mated transaminase procedures are similar to those genase preparation. 2 J Clin Pathol: first published as 10.1136/jcp.25.11.940 on 1 November 1972. Downloaded from

944 J. H. Wilkinson, D. N. Baron, D. W. Moss, and P. G. Walker ducible, a day-to-day coefficient of variation of determination of glutamic oxalacetic and glutamic pyruvic transaminase in plasma. J. Lab. clin. Med., 46, 785-789. ±55% being obtained on sera with borderline and Henry, R. J., Chiamori, N., Golub, 0. J., and Berkman, S. (1960). moderately elevated transaminase activities. Revised spectrophotometric methods for the determination of glutamic oxalacetic transaminase, glutamic pyruvic trans- 4 The methods of standardization described in this aminase and dehydrogenase. Amer. J. clin. Path., report are applicable to other enzyme procedures 34, 38 1-398. dependent upon the oxidation of NADH or NADPH, Karmen, A. (1955). A note on the spectrophotometric assay of glutamic-oxalacetic transaminase in human blood serum. eg, lactate dehydrogenase, or the reduction of J. clin. Invest., 34, 131-133. NAD+ or NADP+, eg, glucose 6-phosphate de- Laudahn, G., Hartmann, M., Rosenfeld, B. M., Weyer, H., and Muth, H. W. (1970). Normalwerte der Serum-Transaminasen hydi ogenase. bei Verwendung substratoptimierter Testansatze zur Aktivitats- messung. Untersuchungsergebnisse bei 3979 Klinisch gesunden References Werktatigen beiderlei Geschlechts. Klin. Wschr., 48, 838-847. Laursen, T., and Hansen, P. F. (1958). A fluorimetric method for Arvan, D. A., and Coyle, M. (1969). Serum and tissue alanine amino- measuring the activity in serum of the enzyme glutamic transferase (GPT) based upon optimal substrate concentration. pyruvic transaminase. Scand. J. clin. Lab. Invest., 10, 53-58. C/in. Chem., 15, 785. Levine, J. B., and Hill, J. B. (1965). Automated fluorometric deter- Babson, A. L., Shapiro, P. O., Williams, P. A. R., and Phillips, G. M. minations of serum glutamic oxalacetic transaminase and (1962). The use of a diazonium salt for the determination of serum glutamic pyruvic transaminase. In Automation in glutamic-oxalacetic transaminase in serum. C/in. chim. Acta, Analytical Chemistry, p. 569. Technicon Symposium, New 7, 199-205. York. Boyd, J. W. (1961). The intracellular distribution, latency and electro- Moss, D. W., Baron, D. N., Walker, P. G., and Wilkinson, J. H. phoretic mobility of L-glutamate-oxaloacetate transaminase (1971). Standardization of clinical enzyme assays. First report from rat liver. Biochenm. J., 81, 434-441. of a Working Party with special reference to alkaline phos- Dalziel, K. (1961). An inhibitor of liver alcohol dehydrogenase in phatase estimation. J. clin. Path., 24, 740-743. preparations of reduced diphosphopyridine . Nature Rand, R. N. (1969). Practical spectrophotometric standards. Clin. (Lond.), 191, 1098-1099. Chem., 15, 839-863. Fawcett, C. P., Ciotti, M. M., and Kaplan, N. 0. (1961). Inhibition of Reitman, S., and Frankel, S. (1957). A colorimetric method for the dehydrogenase reactions by a substance formed from reduced determination of serum glutamic oxalacetic and glutamic diphosphopyridine nucleotide. Biochim. biophys. Acta, 54, pyruvic transaminases. Amer. J. clin. Path., 28, 56-63. 210-212. Rosalki, S. B., and Wilkinson, J. H. (1959). A source of error in serum Fingerhut, B., Ferzola, R., Marsh, W. H., and Levine, W. H. (1962). glutamic-oxaloacetic transaminase determination. J. clin. An automated method for a colorimetric determination of Path., 12, 138-139. serum glutamic oxalacetic transaminase. Ann. N. Y. Acad. Sci., Schaffert, R. R., Kingsley, G. R., and Getchell, G. (1964). Automated

102, 137-143. determination of serum glutamic oxalacetic and glutamicby copyright. Henley, K. S., and Pollard, H. H. (1955). A new method for the pyruvic transaminase. Clin. Chenm., 10, 519-532. http://jcp.bmj.com/ on September 24, 2021 by guest. Protected