219

Clinica Chimica Acta, 66 (1976) 219-226 @ Elsevier Scientific Publishing Company,

Amsterdam

-

Printed

in The Netherlands

CCA 7500

ORNITHINE

CARBAMOYL

USE OF A BOVINE

G. MUSEUR, Centre 54500

A. BAGREL

de Me’decine Vandoeuvre

(Received

LIVER

August

TRANSFERASE EXTRACT

ACTIVITY

IN QUALITY

CONTROL

and G. SIEST

Preventive, 2, avenue les Nancy (France)

du Doyen

Jacques

Parisol,

1, 1975)

Summary To obtain a pure ornithine carbamoyltransferase for quality control in clinical chemistry, the authors compared human plasma and bovine liver ornithine carbamoyltransferase. The data show that, the K, of both enzymes are similar (respectively 1.03 and 1.33 mmol when ornithine is used as substrate; 1.02 and 1.06 mmol with carbamyl phosphate). The following properties are shown to be equivalent: linearity of the enzymatic reaction, inhibition by higher substrate concentrations, and temperature denaturation. The similarities between the two enzymes indicate that bovine liver ornithine carbamoyltransferase can be used in clinical chemistry quality control.

Introduction Ornithine carbamoyltransferase (OCT, EC 2.1.3.3) is an enzyme found almost exclusively in the hepatic cell. The measurement of the activity of this enzyme in plasma appears to be a sensitive and specific indicator of liver disease, and numerous authors, such as Reichard [l] consider its determination more sensitive than that of the transaminases. We have shown in a previous paper [2] that the determination of this enzyme’s activity seemed to be a discriminating factor for preventive medicine in the detection of individuals susceptible to hepatic disorders. However, experiments on OCT suffer from the absence of commercial preparations of the enzyme of sufficient purity for use in quality control. In France, a polyenzyme preparation was made available to clinicians: 22-26 rue du Capitaine Ferber, 75020 Preortan (Leurquin Laboratories,

220

Paris), containing ornithine carbamoyltransferase, aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2), traces of an aldolase (EC 4.1.2.7), a lactate dehydrogenase (EC 1.1.1.27) and acatalase (EC 1.11.1.6). Before using this bovine extract enzyme as a “control” in the measurement of human plasma OCT activity, a comparative study of the two different enzymes was necessary. This study allowed us to determine whether these two enzymes behaved similarly, especially with respect to their reaction kinetics. Materials and method OCT catalyzes the reversible reaction of ornithine into citrulline. The assays were carried out simultaneously on human plasma with high OCT activity, and the lyophilized extract (available commercially in vials and reconstituted with 1 ml of physiological solution, then diluted to 1 : 1000 in the experiments). The enzymatic activity is deduced from the level of citrulline formed. The citrulline is measured by making use of the colored reaction products which diacetylmonoxime yields with carbamide derivatives in an acidic medium in the presence of phenazone. Measurement of OCT activity has been described earlier 1101. Results Choice of buffer

Three buffers were tested: I. Phosphate buffer : 0.06 M, pH 7.0 (Ceriotti and Gazzaniga [3]) II. Tris buffer : 0.05 M, pH 7.0 (Mahuzier and Desmoulins [4]) III. Triethanolamine : 0.2 M, pH 7.7 (Snodgrass and Parry [5]).

Fig.

1. Michaelis

plot

for ornithine

F‘ig. 2. Michaelis

plot

for

carbamoyl

with

human

phosphate

~lasna with

OCT.

human

plasma

OCT.

221

O,“,Ihl”tZ

Fig.

3. Lineweaver-Burk

plot

for

p

1

mot

ornithine

with

human

plasma

OCT.

In triethanolamine buffer the most significant citrulline formation is obtained. In buffers I and III human plasma OCT gives colorations, 50% and 90% more intense than the coloration obtained in buffer II. A similar result is obtained for a dilution of Preortan, in which case the differences observed in intensity between the 3 buffers are 185% and 350%. We therefore used the triethanolamine buffer in the remaining assays. Comparative study of optimal substrate concentrations Figs. 1-8 demonstrate the inhibiting effect of excess substrate

p

Carbamoylphosphate

Fig.

4. Lineweaver-Burk

plot

for

carbamoyl

rnOlC’ phosphate

with

human

plasma

OCT.

on the ac-

35 :

G Z

7 .c E

30 25

Carbamoyl

phosphate l

50 Fig.

5. Michaelis

plot

for ornithine

Fig.

6. Michaelis

plot

for

with

c;lrbamoyl

bovine

phosphate

liver

OCT.

with

bovine

100

150

200

mmol

I

liter

11ver OC’r.

tivity of the two enzymes. We can establish the following table, Table I, which shows the optimal concentrations taken from Michaelian representations. Similarly, upon examining Lineweaver and Burk’s representations, we obtain the Khl values presented in Table II. The I(, values were checked by the method of least squares. It can be observed that Lineweaver and Burk’s representations lead to similar KM values for both enzymes.

0.060

Km

1000 Fig.

1,33mmol

75c

,A”

500

7. Lineweaver-Burk

plut

250

ti

for

ornithine

250 with

bovine

500 liver

750 OCT.

lL!PC:

223

Km:l.O6mmo

t-,

/./.'*

~1000 Fig.

-750

Carhamoy, phosphate ~_~~_~~_~~~_

I:

---A-500

-250

8. Lineweaver-Burk

0

plot

250

for carbamoyl

500

IO00

750

phosphate

prlw~

with

bovine

liver

OCT.

Comparative study of enzyme reaction kinetics The assays were carried out at 37°C for incubation periods varying from 5 to 60 minutes (Fig. 9). The kinetics observed for these two enzymes are linear for an incubation period of up to 30 min. A period of 20 minutes can therefore be chosen for a typical assay designed to determine the activity of these two enzymes. The same conclusions are reached when the influence of the quantity of bovine liver enzyme is studied (dilutions l/1000 and l/2000 for Preortan: Fig. 9).

Fig.

9.

plasma

Kinetics OCT;

-a,

of

the

enzymatic bovine

reaction liver

OCT

for

bovine

diluted

liver

l/1000;

and L-,

human

plasma bovine

OCT. liver

_,

OCT

Human diluted

1 f2000.

224

25 Fig.

10.

Denaturing

TABLE

of

bovine

37 liver

50

(w

) and

human

“C plasma

OCT

(A-)

by

Human

of

0.005

Ornithine

plasma

for

maximal

OCT

activity Bovine 0.015

mol/l

hepatic mol/l

optimum

Carbamoyl

phosphate

0.050

OCT (plateau

plateau

mol/l

(inhibition

TABLE

molil over

0.100

11

of

KM

(mmol)

Human Ornithine Carbamoyl

phosphate

plasma

at the

zone)

0.020-9.050

Excess

heat.

I

Concentration

Excess

60

OCT

Bovine

1.029

1.33

1.017

1.06

bepatic

OCT

mol/l)

225

Denaturing of enzymes by heat The assays were performed at temperatures ranging from 25°C to 60°C for 20 minutes (Fig. 10). Human plasma OCT activity increases up to 50°C after which it decreases. Bovine OCT behaves similarly. Discussion Seeking a preparation suitable for use in quality control, we were led to investigate Preortan. In 1961, Marshall and Cohen purified bovine OCT obtaining a purity of about 95%. Cohen and Marshall [6] give a molecular weight of 108 000, Grill0 and Bedino [7] have confirmed this value (mol. wt. 110 000). Few studies of this type have been undertaken on this human liver enzyme. We did a comparative study of OCT extracted from bovine liver, and human plasma. We observed similarities in the enzyme kinetic level, effects of incubation milieu, optimal substrate concentrations and denaturation by temperature. We demonstrated that the triethanolamine buffer gave more intense colorations than the Tris and phosphate buffers. The latter are known to inhibit both bovine [8] and human OCT activity [5]. Grill0 and Bedino showed that phosphate ions would inhibit OCT activity by competition with the carbamoyl phosphate [ 71. It seems possible to adopt the same optimum substrate concentrations for the measurement of both enzymes, because of the plateaux observed for bovine OCT in the case of a Michaelian representation (Figs. 2 and 6). The kinetics of the enzymatic reactions are linear for both enzymes for incubation periods between 5 and 30 minutes. A 20-min incubation period can therefore be chosen to determine the activity of these two enzymes. Finally, the denaturation by temperature of these two enzymes from different sources appears equivalent. In the case of calf liver, Grill0 and Bedino gave a denaturation temperature of 50°C [ 71. Lorentz and Wrabetz, in 1971, found an identical denaturation temperature for a human hepatic extract [9]. Due to the numerous similarities observed, we have contemplated using Preortan as a pure “internal standard” added to samples from the biological milieu during the measurement of OCT activity. However, introducing an enzyme of different origin into the assay is open to criticism because of its instability. Preortan can be used only as a “control preparation”. For each series of measurements a different dosage vial must be reconstituted, and then diluted to 1 : 1000 which represents a potential source of error. A study of the stability of bovine OCT allowed us to observe that the OCT activity at this dilution was extremely unstable. Therefore, we feel that a commercially available preparation of bovine OCT would be very useful in quality control, provided the enzyme concentration of the lyophilized product is decreased. Acknowledgement The authors wish to thank reading the manuscript.

Dr. Y. Landry

and B. Theobald

for critically

226

References 1 2 3 4 5 6 7 8 9 10

H. Reichard, Acta Med. Stand., 172 (1962) 723 A. Bagrel, These Pharmacie, Nancy, 1975 G. Ceriotti and A. Gazzaniga, Clin. Chin Acta, 16 (1967) 436 G. Mahuzier and R. Desmoulins, Ann. Biol. Clin., 27 (1969) 767 P..J. Snodgrass and D.J. Parry, J. Lab. Clin. Med.. 73 (1969) 940 P.P. Cohen and M. Marshall, In P.D. Bayer, H. Lady, K. Myrback (rds.) The enzymes, Academic Press, New York, 327 M.A. Grille and J. Bedino, Enzymologia, 35 (1968) 1 G.H. Burnett and P.P. Cohen, J. Biol. Chem.. 229 (1937) 337 K. Lorentz and W. Wrabetz, Z. Klin. Chem. Klin. Biochem., 9 (1971) 220 A. Bagrcl. G. Museur and G. Siest, Clin. Cbem., (1975) in press

Vol. 6, 1962,