CLINIC.4 CHIMICA ACTA 431

A NEW COLORTMETRIC ULTRAMICROMETHOD FOR SERUM GLUTAMIC-

OXALACETIC AND GT-UTA~IC-~YRU~IC TRA~SA~INASE DET~R~INA-

TION

In this paper an ultramicromethod for the determination of the serum glutamic- oxalacetic and glutamic-pyruvic transaminase activity is presented; this method is based on the use of glutamate dehydrogenase for the enzymatic estimation of the glutamate formed. The dehydrogenation of the glutamate gives rise to the reduction of a diazonium salt, and it is possible to perform a photometric reading of the colored compound at 520 nm.

20 ~1 of serum and an incubation time of only 45 min at the temperature of 37’ were necessary. The normal values never exceeded 54.5 I.U. for the serum glutamic-oxalacetic transaminase and 52 I.U. for the glutamic-pyruvic transaminase.

IJnder conditions of viral hepatitis values of 390 I.U. for glutamic-pyruv~c transaminase and 310 I.U. for serum glutamic-oxalacetic transaminase were obtained.

INTRODUCTION

The most widely used photometric method for the determination of the serum transaminase activity is that proposed by Reitman and Frankell.

The method of Babson et al.” can only be used for serum glutamic-oxalacetic transaminase determination.

However, there are several points on which the procedure of Reitman and Frankell may be criticized: (I) dinitrophenylhydrazine is a nonspecific reagenP; (2) the blank absorption in normal sera may be stronger than that of the colored sample, so that even small variations in the blank may cause large errors in the measure of the enzymatic activity*; (3) the calibration curve does not comply with the analytical conditions of the whole enzymatic reaction5 and it is linear only over a short range; (4) a disagreement between the dinitrophenylhydrazine method and the spectrophotometric method has been observed many times, so that many false- normal serum glutamic-oxalacetic transaminase activities have been measured in acute myocardial infarction-‘I.

czin. Chim. Acta, 28 (1970) /+3’--1_37

432 LIPPI, GUInI

Critical surveys on the phenylhydrazine method have been published by Fonty12 and by Amador et aL4.

The present study concerns a new ultramicromethod for the determination of the serum transaminase activity (glutamic-oxalacetic and glutamic-pyruvic trans- aminases) .

MATERIALS AND METHODS

The glutamate derived from the serum transaminases is dehydrogenated by glutamate dehydrogenase. The NADf-N-methylphenazonium methosulfate system is used as an Hf carrier and a diazonium salt as an H+ acceptor.

The formazan so formed is dissolved in 0.35 M HCl and photometric readings are performed at 52.0 nm.

The reaction sequence is as follows:

a-Oxoglutarate+aspartate SGoT ) oxalacetate+glutamate (1)

a-Oxoglutarate+alanine SGPT ) glutamate-tpyruvate COOH COOH

CH, CH,

CH, I

+ H,O+NAD’- GDH , CH, + NH, + NADH

I CHNH, c=o

I I coo- coo- Glutamate cc-Oxoglutarate

(2)

NADH+INT PEE , NAD+ + INT formazan (3)

SGOT = serum glutamic-oxalacctic transaminase ; SGI”L : strum glutamic-pyruvic transaminasc : GDH = glutamate dehydrogenase; INT = r-p-iodophcnyl-3-~~itroph~n~l-5-ph””yl tctrazolium chloride; PZ!IS = X-methyl phenazonium methosulfate.

Reagents (I) Glutamic-oxalacetic transaminase buffered substrate : 0.002 M a-oxoglu-

taric acid and 0.10 M r_(+)-aspartic acid in 0.10 M phosphate buffer (pH 7.4). (2) Glutamic-pyruvic transaminase-buffered substrate : 0.002 lK a-oxoglutaric

acid and 0.2 Jt rn-alanine in 0.10 M phosphate buffer (pH 7.4). (3) Color reagentI : 0.0039 M z+iodophenyl-3-nitrophenyl-5-phenyl tetrazo-

lium chloride (INT), 0.0075 M NAD+, 0.00162 M n’-methylphenazonium methosulfate (PMS).

(4) 20 mg/ml glutamate dehydrogenase (45 units/mg; C. I;. Boehringer and. Soehne GmbH, Mannheim).

(5) 0.35 M HCl.

Clin. China. Acta, 28 (1970) 431-437

COLORIMETRIC DETERMINATION OF SGOT AND SGPT 433

Glutamic-oxalacetic OY C&V glutamic-pyruvic transaminase reagent substrate (ml) (flzl)

..~___. _

0.25 0.2j

0.25 0.25

Procedure The reagents are preheated at 37O, mixed according to the scheme outlined in

Table I and then incubated at 37’ in a water bath for 45 min. The reaction is then stopped by adding 5 ml of HCl. The samples are measured at 520 nm against the corresponding blank.

Just before use the glutamic-oxalacetic or glutamic-pyruvic transaminase sub- strate solution and the color reagent are mixed in equal parts; then 0.5 ml of this mixture is placed in each test tube. Constriction pipettes were used for the addition of glutamate dehydrogenase. An excess of this latter enzyme does not compromise the course of the reaction.

The incubation time is 45 min because at this moment the optical absorption of the reaction product reaches an optimal value.

Calibration cu~vc The calibration curve was obtained in the following way: samples were in-

cubated, which contained color reagent together with a known amount of IKT and serum of a very high transaminase activity. The color reagent we used contained 0.09 pmole/ml INT.

Several samples containing different amounts of INT were prepared by dilu- tion. Taking into account the amount of serum involved in the reaction and the incu- bation time, the value we got from the measurements of the absorbance of the un- diluted sample, with respect to the blank which lacked serum, corresponds to IOO I.U., where an I.U. is 0.0009 (50000/45) j,6mole.

A plot of the absorbance measured at 520 nm of the samples containing different concentrations of INT, against the concentration, gives a straight line passing through zero (Fig. I).

05°C. 040

030 ./

A-*’

020~

010 ./

0 0 50 100 150 200

I u.

Fig. I. Calibration curve (see text)

Clin. Chim. Acta, 28 (1970) 431-437

434 LIPPI, C;CIDI

Basal glutamate

The amount of glutamate originally present in serum is extremely low, and its value is not modified in normal or pathologic sera. The quantity of the basal gluta- mate has been determined by performing the same measurements as before with samples in which the substrate solutions were replaced by an equal volume of 0.05 M phosphate buffer (pH 7.4).

The difference between the formed glutamate and the basal glutamate lies within such values that it does not effect the enzymatic activity.

Kim3tics of the enzyme reaction

A pool of normal sera and some sera with quite a high transaminase activity were incubated during periods ranging from 20- to r2o-min duration (Fig. 2). In every case the colored products of the reaction increased linearly with increasing incubation time.

Correlation between the enzymatic concentration and the progress qf the reaction A pool of normal sera was diluted I : IO-I : 6-1: 3-1: 2 ; 20 ,~l of the solutions

were incubated for 45 min and the reaction was stopped by adding 2.5 ml of 0.35 M HCl. The values of the absorbance could be plotted against the concentration in a straight line passing through zero. Another serum with very high transaminase activity was similarly diluted and incubated for 45 min. The reaction was stopped with IO ml of 0.35 M HCl. .4gain the values fitted a straight line, showing zero-order kinetics for the reaction (Fig. 3).

A

070 SGPT

060.

cm- A

.

040. .

. .

a30. . 0

. . . l

cl20 - . l . l .

010 - l . l . l

( : l

0 010 m 50 70 93 110120

A

070

t

SGOT

060 A

.

I . 040 .

g/::,,“ B ox) 30 50 70 90 no 120

ltwhation time (min)

Fig. 2. Kinetics of the enzymatic reaction at various incubation times at 37O. Data concern sera with quite high (A) and normal (B) glutamic-oxalacetic transaminase (SGOT) and glutamic- pyruvic transaminase (SGPT) activities.

Clin. Chim. Acta, 28 (1970) 431-437

COLORIIUETRIC DETERMINATION OF SGOT AND SGPT 435

0.40. SGOT

044. 040. 036. 032. 028. 024

Fig. 3. Kinetics of the enzymatic reaction at various serum glutamic-pyruvic transaminase (SGPT) and serum glutamic-oxalacetic transaminase (SGOT) contents. The reaction of a pool of normal sera has been stopped by adding 2.5 ml of 0.35 M HCl. The reaction of a high enzymatic activity serum has been stopped with IO ml of 0.35 M HCl.

SGPT

08 1 ,’

,/

07 ,,-’

Fig. 4. “Scalen” quantities of a serum with a very high serum glutamic-pyruvic transaminase (SGPT) activity have been incubated under standard conditions. The enzymatic reaction has been stopped with 20 ml of 0.35 M HCl.

In another experiment, different quantities of a serum with very high trans- aminase activity (0.010, 0.020, 0.040, 0.050, 0.060, 0.080 ml) were incubated under standard conditions. The absorbance remained constant for the samples containing 0.050 ml of serum (Fig. 4). Important is the observation that an excess of NAD+ in the reaction mixture slows down the enzymatic reaction.

We performed simultaneously 20 determinations using a pool of normal sera and some sera with very high enzymatic activity.

The absorbance values of the colored samples were always reproducible within every group of sera.

Particularly, it may be interesting and very useful to point out that the 1:2

dilution of the colored solution with 0.35 M HCl gives rise to absorbance readings of half values.

Normal values and pathological values 92 determinations of the transaminase activity on sera of healthy individuals

(blood donors and young soldiers) were performed. The values obtained never ex- ceeded 54.5 I.U. for serum glutamic-oxalacetic transaminase and 52 I.U. for serum glutamic-pyruvic transaminase, with a great incidence of 43-48 I.U. and of 43.5-48

Clin. Chim. Acta, 28 (1970) 431-437

LIPPI, GCIDI

SGPT IU. SGOT IU.

13 3 %

l-

356 (

L I 37-40 41-44 45-4c

8.8 %

1 4.4 %

4452 53-545

Fig. 5. Xormal distribution of serum glutamic-pyruvic transaminase (SGPT) and serum glutamic- oxalacetic transminase (SGOT) values.

In some pathological cases (viral hepatitis) serunl glutamic-oxalacetic and -pyruvic transaminase values of up to 310 and 390 I.ll., respectively, were found.

Srra

Sormal

Pathological (viral hepatitis)

I.U., respectively (Fig. 5). Table II lists the values of serum glutamic-oxalacetic transaminase and serum glutamic-pyruvic transaminase in normal and pathological sera.

REFERENCES

I S. KE1Tnl.w AND 5. FRAKKEL, z4wz. ,I. Clin. Pathol., 28 (1957) 56. L A. L. BABSON, P. 0. SHAPIRO, I’. A. R. WILLIAMS ASD G. IX. PHILLIPS, Clix. Chim. Acta, 7

(1962) 199. 3 H. SAIKAWA, Okayama Igakkai Zasshi, 76 (1964) 559. 4 E. AMADOR, M. F. Rbssoo AND R. J, FRA;UEY, Am. ,I. Clin. Pathol., 47 (1967) 479.

Clin. Ckim. Acta, 2X (1970) 431-437

COLORIMIETRIC DETERMINATION OF SGOT AND SGPT 437

5 E. ARIADOR, H. HFISSTEIX ASD N. BENOTTI, Am. J. Clis. Pathnl., .++ (1965) 62. 6 Ii. F. W'ITTER AND L. 31. GRUBRS, Cli?z. Chinz. Acta, 13 (1966) 524. 7 J. BURSTEIN ASD A. HARJANNE, Acta Md. Stand., 163 (1959) 175. Y H. I\. DEWAR, ?;. K. ROWELL ASD .4. J. SMITH, Hvit. Xrd. J., 2 (1958) 1121.

9 P. Y. GRIFFITHS, Bait. Hcavt J., 28 (1966) Igg.

ICI T. 1%‘. STEWART AXD F. G. WARBURTON, lint. Heavt J., ~3 (1961) 236. II J. STUART, T. C. CRAWFORD, J. FORSHALL ASD J. A. OWEN, Brit. JIrd. ,f,, I (1965) 4~3.

I2 P. L'OKTU, _!Tn~ymZo~i~, nlonographie .\IInuclle de la Sm. I;ran$. dc Rid. Clin., Paris, 1964.

‘3 A. I<. 13\~son- ASD G. Ji. PHILLIPS, Clin. Chim. 4cta. I2 (1965) 210.