A simple procedure for the routine determination of aspartate aminotransferase and alanine aminotransferase with pyridoxal phosphate

Clinica Chimica Acta, 153 (1985) 241-247 Elsevier

241

CCA 03349

Brief technical note

A simple procedure for the routine determination of aspartate aminotransferase and alanine aminotransferase with pyridoxal phosphate F. Javier Gella, Teresa Olivella, M. Cruz Pastor, Joaquin Arenas, Rosendo Moreno, Rafael Durban and J. Antonio Gomez Departamento de Bioquimica, Fact&ad de Medicina (Hospital Sta Creu i S Pau), Universidad Autbnoma de Barcelona, S Anionio M Claret 167, 08025 Barcelona (Spain) (Received

March 25th. 1985; revision July 4th. 1985)

Key words: Aspartaie aminotransferase; Alanine aminotransferase; Pyridoxal phosphate; IFCC recommended procedures

Introduction The preferred techniques for determination of the catalytic activity of aspartate aminotransferase (AST; EC 2.6.1.2) are those first described by Karmen, Wroblewski and La Due [1,2] and later improved by several Societies of Clinical Chemistry [3-51, in which oxaloacetate or pyruvate formed are enzymatically reduced to malate or lactate with concomitant oxidation of NADH. More recently, it has been found that serum aminotransferases may be undersaturated with the coenzyme pyridoxal phosphate [6-Q which should be included in the reaction mixture to improve the accuracy and the diagnostic sensitivity of the aminotransferases determination. The proposed IFCC methods for AST [9,10] and ALT [ll] saturate serum aminotransferases with pyridoxal phosphate during a lo-mm period of preincubation and the reaction is then initiated by addition of 2-oxoglutarate. The IFCC recommendations, that can be considered ‘reference methods’ [12], are inappropriate for use as routine methods in many laboratories because the use of two reagents and the long preincubation period of 10 min are serious inconveniences. We have studied a modification of the IFCC methods for AST and ALT that simplifies the procedure. The proposed procedure can use commercially available reagent kits and can be easily mechanised. Material and methods Samples

Serum samples 0009-8981/85/$03.30

were from hospitalized

and from apparently

Q 1985 Elsevier Science Publishers

B.V. (Biomedical

healthy

Division)

individuals.

242 TABLE Catalytic

I activity

of the erythrocyte

aspartate

aminotransferase

Units

Before dialysis After dialysis

- PyrP

+ PyrP

3260 42

3 462 2 791

The enzyme activity was measured phosphate (PyrP) 0.1 mmol/l.

at 37°C by the IFCC method

before and after dialysis

treatment

% of apoenzyme 5.8 98.5 in the presence

and absence

of pyridoxal

Aspartate aminotransferase was partially purified from recently outdated bloodbank human erythrocytes as described by Rej et al [13] up to the ammonium sulfate precipitation. In order to obtain the apoenzyme form, the AST preparation was dialyzed against phosphate buffer as described by Bergmeyer et al [14]. The enzyme, that was mainly in the holo form after purification, was transformed into the apo form by the dialysis treatment (Table I). Reagents Commercially available kits following the IFCC recommended reagent concentrations were used to measure AST and ALT activities (Knickerbocker Laboratories, Barcelona, Spain, kits Cod. E534 and E535; and Boehringer Mannheim GmbH, Barcelona, Spain, kits Cod. 487350 and 487386. The following reagents were prepared from the kits components. Reagent A for AST 96 mmol/l Tris buffer, 288 mmol/l r_-aspartate, 500 U/l malate dehydrogenase, 720 U/l lactate dehydrogenase, 0.216 mmol/l NADH (pH 7.8). Reagent also contained 0.11 mmol/l pyridoxal phosphate when specified in the text. Reagent

B for AST

144 mmol/l

2-oxoglutarate.

Reagent A for ALT 120 mmol/l Tris buffer, 600 mmol/l r_-alanine, 1440 U/l LDH, 0.216 mmol/l NADH, 12 mmol/l sodium bicarbonate (pH 7.5). Reagent also contained 0.11 mmol/l pyridoxal phosphate when specified in the text. Reagent

B for ALT

180 mmol/l

Reagent C for AST or ALT mixed with 0.2 vol of Reagent

2-oxoglutarate.

2 vol of Reagent B.

Measurements of catalytic activities AST and ALT catalytic activities

were measured

A with pyridoxal

phosphate

were

at 37°C as follows.

IFCC and Bergmeyer et al [9-111 procedure 1 vol of serum was mixed with 10 vol of prewarmed Reagent A and the mixture was preincubated for 10 min. Reaction

243

was started by adding 1 vol of Reagent B to the preincubation mixture. Absorbance at 340 nm was followed over a period of 3 min. No corrections for sample blanks were made. Proposed simplified ‘routine’ procedure 1 vol of serum was mixed with 20 vol of prewarmed Reagent C. After a 2-min period of preincubation, the absorbance at 340 nm was followed over a further period of 3 min. Results Effect of pyridoxal phosphate on the aminotransferase activity during incubation with a camp Iete reagent

Samples used in this experiment were especially selected sera for which a substantial pyridoxal phosphate effect in the AST activity had been previously

0.12 D

L[ I

0.10

C

I

:;

c.i

0.08

5 9 B 2

I I I

0.06

z

z

E

3

I I I

0.04

0.02

I

MINUTES

OF INCUBATION

I

AT 37W

Fig. 1. Activation of serum apo-AST by pyridoxal phosphate during incubation with the proposed reagent. Serum samples (A, C, D) and erythrocyte apo-AST (B) were mixed with the proposed AST reagent (Reagent C) as described in methods, and the reaction rate at 37°C was followed for 8 min. For comparison purposes, it is also represented the rate obtained using the IFCC procedure without (0) and with (arrow) pyridoxal phosphate.

244

observed with the IFCC procedure. Samples were mixed with Reagent C at 37°C and the reaction mixture was immediately inserted into the. photometer. Reaction rate was followed for 8 min. The increase in absorbance change with pyridoxal phosphate supplementation was almost instantaneous. After 1-2 min, the reaction rate was constant and practically coincident with the absorbance change determined by the IFCC method. A preparation of AST from human erythrocytes with 98.5%

AST

0

im

aJ0

900

4ce

so0

4m

500

IFCC PROCEDURE

ALT

0

1m

2m

am

IFCC PROCEDURE

Fig. 2. Comparison of the proposed concentrations are expressed in U/l.

procedure

with

the IFCC

reference

method.

Enzyme

catalytic

245

TABLE

11

Relationships of results with various incubation obtained with the reference IFCC procedure (y) Enzyme

Incubation

interval

time intervals

in the proposed

procedure

(x)

r

Intercept

Slope

n

to those

(mm) AST

0.5-3.5 2-5 3-6 4-l 5-8

0.998 0.998 0.998 0.998 0.999

+ 9.0 + 1.7 +1.2 +0.6 -0.7

1.027 1.031 1.026 1.022 1.012

58 110 108 107 55

ALT

0.5-3.5 2-5 3-6 4-l 5-8

0.997 0.998 0.996 0.998 0.998

- 0.4 -0.6 +0.7 -1.9 - 1.8

1.024 1.020 1.007 1.028 1.023

91 91 88 88 81

apoenzyme form (Fig. 1B) was also fully activated by the reagent containing pyridoxal phosphate after about 2 min incubation. Similar results were obtained for ALT, although the activation effect by pyridoxal phosphate observed in human sera was always small (lo-20%). Methods comparison

The AST and ALT catalytic activities of about 100 human sera and several commercial lyophilized control sera were estimated by the IFCC assay procedure and by the proposed ‘routine’ procedure. In the proposed procedure, the change in absorbance at 340 nm was followed for 8 min. The catalytic activity was calculated for several time intervals. Table II shows the correlation between results for the tested human sera, The figure illustrates the individual data for the time interval of 2-5 min. Proportional differences between the reference and the proposed procedure were 2-356 and were not notably reduced by increasing the preincubation period up to 5 min. The interval period of 2-5 min of incubation was chosen for the measurement of the AST and ALT catalytic concentration in the proposed routine procedure. TABLE

III

Relationship of results obtained by the proposed of sera from ‘ hepatic’ and from ‘heart’ patients Enzyme

Patients

(x) and the reference

IFCC (y) procedures

Intercept

Slope

n

group AST

Hepatic Heart

0.999 0.996

+4.5 - 2.0

1.008 1.037

16 55

ALT

Hepatic Heart

0.997 0.998

- 5.6 -6.9

1.046 1.033

31 12

for groups

246 TABLE

IV

Effect of pyruvate

on the AST and ALT determination

Serum 1: No additions Pyruvate 1 mmol/l Serum 2: No additions Pyruvate 1 mmol/l

by the proposed

procedure

AST (U/I)

ALT (U/l)

239 240

144 139

69 68

28 21

When sera from patients with a provisional diagnosis of myocardial infarction or with liver disease were examined separately from the individuals that could not be classified in one of these categories, a similar correlation between the proposed method (preincubation period: 2 min) and the IFCC method was found than for all the studied sera (Table III). Interference

of pyruvate

During the preincubation period of 10 min recommended by the IFCC reaction to completion of NADH with endogenous ketoacids (mainly serving as susbstrate for lactate dehydrogenase. In order to evaluate if the tion period of 2 min in the proposed procedure was long enough to reaction to go to completion, portions of serum were added with 1 mmol/l (about 8 times the upper normal limit) and the AST and ALT catalytic tions were measured by the proposed procedures. The obtained results demonstrated that the period needed to consume pyruvate was < 2 min.

occurs the pyruvate) preincubaallow this pyruvate concentra(Table IV)

Discussion A simplified procedure for AST and ALT determination at 37°C is proposed. The procedure can be easily mechanised even in those analyzers that can pipette only one reagent or that cannot preincubate the reaction mixture for relatively extended periods of time (Technicon SMAC and others). The main factor that imposed a lo-min preincubation period in the IFCC methods was the need of completely saturate the apo-AST or apo-ALT with pyridoxal phosphate prior to measurement of the enzyme catalytic activity. In the present studies, we have found that a practically maximum activation of AST and ALT by pyridoxal phosphate can be obtained in a 2-min preincubation period with a reagent containing all the reaction components and the coenzyme. The results obtained with the proposed method were not significantly different from the IFCC reference procedure. The same was true for groups of sera from patients with myocardial infarction or with liver disease and for several lyophilized control sera supplemented with aminotransferases of non-human origin. A partially purified apo-AST obtained from human erythrocytes was also found to be completely

247

reactivated in < 2 min with the proposed assay conditions. It has been also demonstrated that the 2-min period of preincubation is long enough to allow complete consumption of endogenous interfering pyruvate up to a concentration of 1 mmol/l. Acknowledgement

We thank M. Carmen Sanchez for her technical assistance in this study. References 1 Karmen A. A note on the spectrophotometric assay of glutamic-oxalacetic transaminase in human blood serum. J Clin Invest 1955; 34: 131-133. 2 Wroblewski F, La Due JS. Serum glutamic pyruvic transaminase in cardiac and hepatic disease. Proc Sot Exp Biol Med 1956; 91: 569-571. 3 Wilkinson JH, Baron DN, Moss DW, Walker PG. Standardization of clinical enzyme assays: a reference method for aspartate and alanine transaminases. J Clin Path01 1972; 25: 940-944. 4 Recommendations of the German Society for Clinical Chemistry. Standardisation of methods for the estimation of enzyme activities in biological fluids. Z Klin Chem Klin Biochem 1972; 10: 182-191. 5 The Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical Physiology. Recommended methods for the determination of four enzymes in blood. Stand J Clin Lab Invest 1974; 33: 291-306. 6 Rej R, Fasce CF. Vanderlinde RE. Increase aspartate aminotransferase activity of serum after in vitro supplementation with pyridoxal phosphate. Clin Chem 1973; 19: 92-98. 7 Rosalki SB, Bayoumi RA Activation by pyridoxal 5-phosphate of aspartate transaminase in serum of patients with heart and liver disease. Clin Chim Acta 1975; 59: 357-360. 8 Moss DW. Reactivation of the apoenzyme of aspartate aminotransferase in serum. Clin Chim Acta 1976; 67: 169-174. 9 Bergmeyer HU, Bowers GN, Horder M, Moss DW. Provisional recommendations on IFCC methods for the measurement of catalytic concentrations of enzymes. Part 2. IFCC method for aspartate aminotransferase. Clin Chim Acta 1976; 70: F19-F42. 10 Bergmeyer HU, Horder M, Moss DW. Provisional recommendations on IFCC methods for the measurement of catalytic concentrations of enzymes. Part 3. Revised IFCC method for aspartate aminotransferase. Clin Chim Acta 1977; 80: F21-F22. 11 Bergmeyer HU, Horder M. IFCC methods for the measurement of catalytic concentrations of enzymes. Part 3. IFCC method for alanine aminotransferase. Clin Chim Acta 1980; 105: 147F-154F. 12 Haeckel R, Horder M, Zender R. International Federation of Clinical Chemistry. Report on the survey of opinions concerning preferred incubation temperatures for measurement of enzymes (and possibly other components) in clinical chemistry. J Clin Chem Clin Biochem 1982; 20: 947-958. 13 Rej R, Vanderlinde RE, Fasce CF. An t_-aspartate: 2-oxoglutarate aminotransferase reference material from human erythrocytes: preparation and characterization. Clin Chem 1972; 18: 374-383. 14 Bergmeyer HU, Scheibe P, Wahlefeld AW. Optimization of methods for aspartate aminotransferase and alanine aminotransferase. Clin Chem 1978; 24: 58-73.