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Kinetic measurement of serum glutamate dehydrogenase activity in the presence of oxamic acid and l -leucine
377
CLINICA CHIMICA ACT,4
KINETIC ACTIVITY
MEASUREMENT
OF SERUM GLUTAMATE
IN THE PRESENCE
DEHYDROGENASE
OF OXAMIC ACID AND L-LEUCINE
SUMMARY
During preincubation, NADH concentration will diminish in a number of cases below the range necessary for optimal serum glutamate dehydrogenase activity. The present paper describes experiments to overcome this problem either by inhibiting lactate dehydrogenase or by stabilizing serum glutamate dehydrogenase against NADH promoted inactivation.
INTRODUCTION
Elevations of serum glutamic acid dehydrogenase (GLDH) can be found in cases of liver diseases. We were especially interested in the diagnostic significance, of GLDH activities in serums of patients with metastatic liver. For that purpose it seemed interesting to make a comparison of GLDH activity and the activity of serum 5nucleotidase (5-Nu), known to be a sensitive monitor of liver metastasesl. In Fig. I the GLDH level of each patient is plotted against the 5-Nu value. The GLDH activities were estimated according to the method of Schmidt and Schmidt, normal range to 0.9 U/l (ref. z) ; the 5-Nu activities in accordance with the method of Persijn et d3, normal range to 10.5 U/l (ref. 4). In this method phenylphosphate is used to inhibit the nucleotidase effect of alkaline bone phosphatasc&. From the data of Fig. I it seemed justifiable to study the behaviour of these enzymes in a series of cases to obtain a clearer picture of the diagnostic significance of GLDH. Before this could be realised, however, several problems were encountered. In the Schmidt and Schmidt method, GLDH activity is measured by the decrease of absorbance at 340 nm caused by the oxidation of NADH according to NADH+ol-ketoglutaric
acid+NH,+
+ NAD++r,-glutamic
acid+H,O
The reaction is initiated by the addition of E-ketoglutaric acid to the assay mixture + Head Dr. J.-P. Per@. *+ Hea.d Drs. W. van der Slik.
Cl&. Chim. Acta, 39
(1930)
377-386.
PERSIJN
et ai.
39.0
at
22
I
20
i8 16
GLCi-I (U/l) .
I
14 12 70 8 6 4 2
0.1
0.2
0.3
0.4
0.5
0.6
NADH [m MOL/L] Fig. I. Comparison Fig. 2. Effect Schmidt5.
of serum 5-Nu activities
of NADH concentration
and serum GLDH
on GLDH
activity,
activities.
measured
according
to the method
of
after a preincubation time of 15 min. During preincubation NADH concentration diminishes as a consequence of short-lasting side reactions to be attributed to the presence of pyruvate and lactate dehydrogenase (LDH) in serum. There is unfortunately a very narrow range of NADH concentration for optimal activity; passing to either side of this range results in rapid decrease of GLDH activity (Fig. 2). Elevation of NADH concentration to compensate for loss of NADH during preincubation is not advisable, particularly since the diminution during preincubation is variable to high degree. In several cases we found that diminution of NADH concentration during preincubation was exceeding the range for optimal GLDH activity. This necessitated measurement of NADH diminution during preincubation in every assay and, if necessary, addition of a second quantity of NADH afterwards for adjustment to optimal concentration as has been already pointed out by Schrnidtb. This was felt to be a serious disadvantage for routine measurement of serum GLDH. In this paper are described several approaches to a solution for this problem by either (a) eliminating side reactions during the preincubation, by adding an agent inhibiting LDH but not GLDH, or (b) by broadening the optimal NADH range in order to apply higher NADH concentration without inactivating GLDH activity. In connexion with (a), we were interested in a report by Novoa and Schwert”. These authors observed that LDH binds oxamate more strongly than oxalate which inhibits LDH about 75%. Therefore in the present study, an attempt has been made to inhibit side reactions by an appropriate concentration of oxamate. It has been shown that beef liver GLDH can dissociate into subunits possessing less or no enzymatic activity. This dissociation of beef liver GLDH is promoted by higher concentrations of NADH’. Yielding and Tomkinss have found evidence that essential amino acids favour CEiw. Cl&z. Acta, 30 (1970) 377-386
SERUM
GLUTAMATE
DEHYDROGENASE
379
ACTIVITY
the reassociation of inactive subunits of bovine GLDH and, therefore, neutralize the action of NADH. According to Hershko and Kindle+‘, L-leucine has the greatest effect in this respect. Substances like L-leucine, that change catalytic properties while altering the configuration, are called “allosteric modifiers”lO. It seemed interesting to investigate whether L-leucine is an allosteric reagent to human serum GLDH, and if that is the case, whether L-leucine could thus achieve a broadening of the NADH optimal range. A second problem is the question of whether serum GLDH is partially inactivated during preincubation. This is not a groundless question since mammalian GLDH has been reported to be unstable in various medialI. The present paper describes experiments which allow us to make a few observations about this problem. METHODS
A brief description of the method will be given, as it has been carried out in our laboratory5. I. Trietkanolanrine-EDTA buffeer: 0.004 RI EDTA in 0.03 M triethanolamineHCl buffer, pH 8.0. 2. Ammoniuvn acetate solution: 3.2 M ammonium acetate in bidistilled water. 3. NADH solution: 0.012 M NADH in the above buffer. 4. a-Ketoglutarate solution: 0.41 M a-ketoglutaric acid in 0.82 N NaOH. A reaction mixture is prepared by combining 2.0 ml of Solution I, 1.0 ml of serum, 0.1 ml of Solution 2, and 0.05 ml of NADH solution. The mixture is kept for 15 min at 25” in a I.o-cm cuvette. During that time the absorbance at 340 nm is recorded. Then 0.05 ml of Solution 4 is added and incubation is continued for IO min, during which the decrease in absorbance is recorded. GLDH activity is calculated from the latter part of the rate of decrease of the absorbance over IO min to express the activity in International Units/l of serum. When variations of the NADH, a-ketoglutarate concentration or temperature etc. in the Schmidt method were used the details are described under RESULTS. RESULTS
LDH activity in the presence of oxamic acid was studied by following the decrease in absorbance at 340 nm in a 3.15-ml mixture containing 0.14 mM NADH, 0.30 mM sodium pyruvate and varying amounts of oxamic acid in the presence of either 0.031 M triethanolamine buffer (pH 8.0) (according to Schmidt) or 0.031 M phosphate buffer (pH 7.5). Fig. 3 illustrates the rapid loss of LDH activity in triethanolamine-HCl buffer with increasing concentration of oxamate. The curve was identical with the phosphate buffer. It will be seen that the LDH activity did not cease completely at high concentrations of oxamate but reached a limiting value which was not zero. This was observed with several serums with varying LDH activities. In the next experiment, oxamate in a final concentration of 3.6 mM was added to the assay by the method of Schmidt. During preincubation the absorbance reading showed different behaviour compared with the Schmidt method in two respects (Fig. 4) : the rate of decrease in absorbance was (a) considerably lower compared with the rate of decrease in the absence of oxamate and (b) approximately linear with time Cl&. Chim.
Acta,
30 (1970) 377-386
380
PERSIJN et al.
% LDH 100
1 60
I
40
25
\
j 20
i
%.
*-a-*
.
1
I
2
1
3
oxamate
Fig. 3. Effect of oxamate
4
[m Mot/ 11
concentration
I2
time
tn minutes
on serum LDH activity.
Fig. 4. Variation, with time, of absorbance at 340 nm of two samples during preincubation. The range o-r.0 of absorbance has been expressed as IOO scale units. The starting point of each curve (arrow) is calculated by extrapolation. e, with 3.7 mM oxamate. GLDH
I
GLDH
(U/L)
(U/l)
‘4 1
I
i
8-3
12 i
‘,
?’
‘4
2 Oi
012
a3
0.4
0.5
0.6
t .
*I
4 6
Ic
1
j
\
./
. \
I
NADH [m MOL/ I] Fig. 5. Effect of NADH concentration on GLDH activity. Curve I, with o.or M L-leucine, 8.5 mM ar-ketoglutarate at pH Curve 4, idem without L-leucine according to Schmidt; Curve 2, with 0.01 M L-leucine, 6.1 mM cc-ketoglutarate at pH Curve 3. with 0.01. M L-leucine, 6.1 mM cc-ketoglutarate at pH Curve 5, idem without L-leucine according to Schmidt. Curves I, 2, and 3 refer to different serum samples. Fig. 6. pH-activity curve for serum GLDH in the presence NADH added.
7.0
6.0
9.0
. t PH
7.8; 8.0; 8.0;
of
0.01
M
L-leucine
and 0.4 mM
(up to at least 25 min). Identical results were obtained with 5.6 m&I oxamate. Since residual LDH activity remains for a longer time compared with uninhibited LDH, the decrease in absorbance as a result of reduction of endogeneous pyruvate is, therefore, superimposed on the decrease in absorbance following the addition of a-ketoglutarate. C&z. Chinz. Acta, 30 (1970) 377-386
SERUM
GLUTAMATE
DEHYDROGENASE
381
ACTIVITY
Two pooled serums were assayed for GLDH activity with (0.0037 M) and without oxamate. In the former assay, correction was applied for residual LDH activity. The results-13.0 and 8.0 U/l without oxamate, 1z.5 and 8.0 U/l with oxamate-indicate that oxamate does not inhibit serum GLDH. Fig. 5 shows the effect of increasing concentration of NADH on the GLDH activity of three different serums measured in the presence of 0.01 M L-leucine (Curves I, 2 and 3). In these and subsequent experiments L-leucine was added as a component of the triethanolamine-EDTA buffer. For comparison, the course of GLDH activity of two serums when L-leucine is omitted is illustrated in Curves 4 and 5. This figure shows that at a NADH concentration between approximately 0.4 and 0.3 mM, the GLDH activity is independent of the NADH concentration. A second advantage of the use of L-leucine is the z-fold activation of the GLDH activity compared with the Schmidt method. Keeping final NADH concentration in the test mixture at 0.4 mM, the pH-activity relationship in the presence of 0.01 M L-leucine was investigated (Fig. 6). Variation of GLDH activity with substrate concentration in the presence of 0.01 M L-leucine at pH 7.8 was also studied. The results, shown in Fig. 7, indicate that maximal and constant activity was obtained when the reaction mixture contained more than 5 mM a-ketoglutarate. The rate of decrease in absorbance a.s a measure of L-leucine-activated serum GLDH is proportional to the amount of serum till about 40 U/l (Fig. 8). Table I presents data of eight experiments on the activity of L-leucine-activated serum GLDH after different times of preincubation. The experiments were performed both with and without oxamate. A preincubation time of zero minutes as indicated in Table I means that initial readings b/l]
GLDH GLDH
(U/l)
60
32 5
28
q
m-m-m-m
60
24
20 .
/ 40
16
w----o-o
---__o----0
12
8
i
/
/ 2
Fig.
b
20
0
4
1
7. GLDH
4 6 8 o:-Ketoglutarate activity
c
r
10 12 [m Mol/Lj
as a function
200
L-leucine at pH 7.8 with 0.4 mM NADH
400
600
600
1000
in the presence added. o = serum I; q = serum 2.
of cr-ketoglutarate
concentration
pL serum
of 0.01 M
Fig. 8. Relation of enzyme concentration to GLDH activity as measured in the presence of 0.01 M L-leucine and 0.4 mM NADH added. Sample a: with 6.2 mM c+Ketoglutarate at pH 8.0; Samples b and c with 8.5 mM cc-ketoglutarate at pH 7.8. Clin. Chim. Acta,
30 (1970)
377-386
382 TABLE
PERSIJN et &. T
STABILIZY
OF
SERUM
GLDH
DURING
PREINCU:BATION
IN
THE
PRESENCT3
OF
L-LEUCINE
(u/l)
Experiments with Samples I, 2, 3, 5, and 8 were done with 8.6 mX cc-Ketoglutarate at pH 7.8. The others with concentrations of ct-ketoglutarate and pH according to the method of Schmidt. Saqble NO.
I 2
Preincubation tinae (w&z)
0 15
3.7 3.7 4.5 4.8
0
15 3
0
26.2
26.8
15
4
0
5.X
5-I
I.5
5*
6
0
5.2
15
0.0037
M
Without
oxarnate 3.9 5.2 -
26.6 -
-
5-I
5.3
0 I5
4.8 4.4 4.3 4.1 3.7
5.0 -
20
3.8
-
8.6
8.7
0
15 20
7
With
oxanzatc:
8
-
0
I5
-
3.9 -
* Measured at 362 nm.
were taken within xo set after the addition of serum to the otherwise complete reaction mixture. In the other experiments, the reaction was started in the usual way, by the addition of ketoglutarate to the assay mixture after the time indicated. The measured rates of decrease of absorbance of the complete assay mixture in the presence of oxamate were corrected for rates of decrease caused by residual LDH activity to obtain real GLDH activities. In all. cases, the decrease of absorbance was linear with time for more than IO min. Results of similar experiments with oxamate, using the Schmidt procedure, are outlined in Table II. With Samples 4 and sz the following additional experiments were performed: After 8 min, measurement of GLDH activity was interrupted for I min to add L-leucine and XADH at final concentrations of 0.01 M and 0.4 mM, respectively. Thorough mixing after each of these additions was performed. The registration of absorbance was continued after this break which was kept as short as possible (approx. I min). Samples 4 and 5 showed immediately enhanced activity (Table II). Using the Schmidt method the mean ratio of activity at 35” and 25” was found to be 1.16, but several. samples of serums showed a temperature coefficient (Q1,Jof 1.0. The highest value of Qlo observed was 1.26. L-Leucine had an enhancing effect on the value of Qlo. In Fig. 9 the natural logarithms of the difference in absorbance over IO min are plotted against the inverses of the corresponding absolute temperatures over the range ~5-37~. There is a linear relationship, but the slopes are different. Curves a and b represent a serum measured within 6 and a4 h, respectively, after the clotting of the blood from which it was taken, Curve c a serum which had been stored for 4 days at 4O. Change of activity in the presence of L-leucine after storage was studied in the case of 27 samples. No difference was found between samples stored at 4O or at -20c. During storage at these temperatures, activity fell slowly. After 4 days the highest loss of activity was found to be IO%, after 7 days, 20%. Clin. Chiun.
Acta,
30 (1970) 377-386
SERUM GLUTAMATE TABLE
DEHYDROGENASE
ACTIVITY
383
II
STABILITY SCHMIDT
OF
GLDH
DURING
PREINCUBATION
IN
THE
MEDIUZvI
ACCORDING
TO
THE
METIXOD
OF
(u/l)
Pmincubation time (mix)
SLWW$le NO. I
9.5 9.8
0
I5 28
With 0.0037 M oxawtate
0
7.5
I5
3b
0
::I:
x5
4
6.5
0
5
?..PC
1.5
2.8d
0
8.Oe
I.5
8.@
Separate assay with L-leucine and 0.4 mM NADH: 8 17.0 U/l; b 15.5 U/l. After addition of L-leucine and NADH: C 5.8 U/l; d 5.3 U/l; e 20.0 U/l; f 20.3 U/l.
Ln
fAAx103f
52 -
5,o
I
3.2
Fig. g. Dependence
3.3
of GLDH
3.6
activity,
in the presence of 0.01 M r.-leucine upon temperature
for the range ~5-37~.plotted according to Arrhenius.On the ordinate: dJf = decreaseof absorbanceat 340 nm over IO min. DISCUSSION
The fact that oxamate reduces NADH consumption dnring preincubatioll to a large extent (Fig. 4) agrees with the findings presented in Fig. 3 and fits in with the theory of Karmen12 that this NADH consumption is caused by LDH activity of serum. Since in the presence of this inhibitor for LDH some residual LDH activity persists even after preincubation (Fig. 4), every assay of serum GLDH had to be corrected for this residual activity in a separate experiment. Addition of oxamate in Clilz.C&n, Acta, 30
(1970)
377-386
384
PERSIJN
et cd.
the assay seems therefore not adequate for the solution of the problem mentioned in IPU‘TRODUCTION.
The use of L-leucine bears more interesting features: GLDH activity in the presence of L-leucine is independent of NADH concentration over a broad range. It is not only NADH that has a biphasic action on_ GLDH activity. According to Schmidt5 the concentration of a-ketoglutarate is very critical. Optimal activity is obtained in the Schmidt medium with a cc-ketoglutarate concentration of 6.2 mM. Enhancement of the a-ketoglutarate concentration leads to rapid inactivation of the enzyme. This phenomenon could be interpreted in terms of substrate inhibition in the usual sense. The fact, however, that L-leucine prevents any inactivation at higher concentrations of cc-ketoglutarate (Fig, 7) strongly indicates that increasing concentration of a-ketoglutarate favours the inactivation of serum GLDH. Apparently r,-leucine is not only able to overcome the dissociation-promoting effect of NADH, but of cr-ketoglutarate as well. We judge the L-leucine-promoted independence of GLDH on cc-ketoglutarate concentration in the range of 5-12 mM, as an advantage if automatic pipetting devices are used in the GLDH assay. At a concentration of 8.5 mM, for example, occasional deviations in dispensed volume of as much as about 20% can be tolerated for micropipetting devices. It is our experience that such a possible range of variation is valuable. Remarkably, the inactivation of GLDH by a great number of conditions has been overlooked in clinical chemistry. This holds not only for the assay of serum GLDH itself. Bovine GLDH has been used as an aiding enzyme for the determination of NH, (ref. 13). From the work of the various authors cited above, it can be expected that in the conditions used for the dete~ination of NHs, rapid deterioration of the aiding enzyme GLDH may occur during the test. We have considered the possibility of using bovine GLDH in our method for the determination of 5-nucleotidase (5-Nu). In this method adenosine released from AMP by the action of 5-Nu is simultaneously converted by adenosine deaminase to NH, and inosine. The ammonia released is measured calorimetrically at 625 nm by means of the Berthelot reaction 894.Use of GLDH instead of the Berthelot reaction would enable us to estimate serum 5-Nu kinetically at 340 nm, provided that GLDH is prevented from inactivation under conditions optimal for 5-Nu activity. It appears that for this purpose allosteric modifiers protecting GLDH against inactivation will be indispensable. The inactivation of GLDH even in buffers without substances Iike NADH etc.‘l has led nonclinical biochemists to measure initial activities in their investigations of GLDH. Initial rates of decrease of absorbance should therefore be established immediately after the addition of the enzyme to the otherwise complete test medium. In the case of serum GLDH such a procedure would measure the combined action of serum LDH and serum GLDH. Since the action of serum LDH is revealed by a progressively diminishing decrease of absorbance (Fig. 4), initial activity of ,GLDH cannot be measured accurately. The question rises whether serum GLDH is inactivated to some degree during the preincubation. We thought to answer this question by measuring without preincubation the GLDH activity in the presence of oxamate. Under this condition there is a linear course of LDH-associated decrease of absorbance (Fig. 4). This makes it
SERUH GLUTAkIATE DEHYDROGENAS~
385
ACTIVITY
possible to correct accurately the total reaction rate for concomitant LDH activity in order to obtain true GLDH activity. Comparison of the results of these experiments with activities obtainedafter preincubation, with or without oxamate, enables us to answer the above question (Table I). From the findings given in Table I, it can be concluded that inactivation during preincubation does not take place. The data of Table II suggest that the partially inactivated GLDH as it is measured in the Schmidt procedure occurs in that form at the very beginning of the preincubation and remains at least r5 min in that form. In this connexion it seems interesting that addition of L-leucine during the meas~ement of initial rate or after preincubatio~l in the Schmidt medium immediately results in an enhanced rate of decrease in absorbance (see Table II). It seems reasonable to assume that the interaction of L-Ieucine with serum GLDH is instantaneous: there is no measurable lag phase. r,-Leucine does not change the pH-activity relationship to a great degree. Schmidt found optimal activity at pH 7.6-8.4. Fig. 6 illustrates that pH optimum for serum GLDH with L-leucine lies at 7.7-7.9. Comparison with data of bovine GLDH from literature is difficult since most investigations have been carried out at a definite pH and possible shifts in pH optimum by allosteric modifiers have not been taken in consideration. That such a shift is not purely hypothetical is illustrated by the findings of Bitensky et &.I4 who found, at least for the reverse reaction, that the pH optimum in the presence of I mM ADP is shifted from 8.0 to 9.0. For serum GLDH the pH-activity relationship with either z.-leucine or ADP (I mM) is identical in the forward reaction. GLDH activities are generally measured at z5”, as is done in the case of serum GLDH. The present paper is intended to modify the procedure so that a change of the NADH concentration can have no influence at this temperature. Nevertheless it seems of interest to mention briefly experiments on the determination of QIO, For the range 25535”, the value of Qlo for Samples a, b, and c (Fig. g) is 1.61, 1.84, and 1,43, respectively. The former two values of Qlo seem reasonable since Qlo varies from x.75 for aminotransferase to 2.50 for 5-nucleotidase. The behaviour of bovine GLDH at temperatures above 25” has seldom been investigated. Bitensky et al.l” have shown that above 26-27” small increases in temperature cause rapidly an unfolded and denaturated product. Probably the same phenomenon occurs with serum GLDH in the Schmidt medium where values of 1.0-1.25 for Qlo have been found.
Apparently
2537”,
although
I-Ieucine to a variable
is able extent.
to overcome
heat
For the present,
inactivation we would
in the range
propose
changing
as follows: to 2 ml of a solution containing 0.05 M triethanolamine buffer (pH 7.8), 0.004 M EDTA, 0.016 M I-leucine, and 0.18 M ammonium acetate are added 1.0 ml of serum and 0.1 ml of NADH solution (0.013 M). After equilibration at 25O for 15 min, 0.1 ml a-ketoglutarate solution (0.28 M) in 0.56 N NaOH is added. The decrease in absorbance is measured at 340 nm or 360 nm for IO min. the Schmidt
procedure
REFERENCES DER SLIK, J.-P. PERSIJX, E. ENGHLSMAN AND A. RIETHORST,~%Z. Biochem., 3 (1970) 59. E. SCKVIIDT AXD F. 1%'.SCHMIDT, E~~ywml. BioE. C&L, 3 (1963) I. J.-P. PERSIJN, W.VAX DER SLX, K. KRAMERAND C. ADE RUYTER,~. Kli~.Chenz. Biockenz., 6 (1968) 441.
W.VAN
Clin. Ckim. Acta. 30 (1970) 377-386
PERSIJN t?f d.
386
4 J.-P. PERSIJN, W. VAN DER SLIS ~SIJ A. W. 31. Box, 2. K&z. Chem. Biockem., 7 (1969) 493. 5 E. SCHMIDT, in H.-U. BERGXEYER (Ed.},~~I~th~~~n detere~~y~~~sc~e~z .ilndyse, Verlag Chemie, Weinheim, x962, p. 752. 6 TV. B. NOVOA AND B. W. SCHWERT, J. Biol. Chem., 236 (1961) 2150. 7 C. FKXEDEN, J. Biol. Che?%, 234 (1959) 809. 8 K. L. YIELDING AND G. M. TOMKINS. Proc. N&l. Acad. Sci. U.S., 47 (1961) 983. g A. KERSHKO AND S. H. KINDLER, Biochwc J., IOT (x966) 661. IO J. M~NOW, J. CHANGEUX AND E. JACOB, J. Mol.Bzal., 6 (1~63) 306. II G. DI PRXSCOAND H. J. STRECKER, Riochi~~.Biafihys. Acta, 122 (1966) 413. 12 A. KARMEN,]. CZin.Invest., 34 (1955) 131. 13 M. RUBIN AND L. KNOTT,CZ~~. Chins. Ada, 18 (1967) 409. 14 M. W. BITENSKY, K. L. YIELDING AND G. M. TOMKINS, J.BioZ. Chem., 240(1gh5) 663. 15 M. W. BITENSKY, K. L. YIELDING AND G. M. TOMKINS, J. BioE. Chena., 240 (1965) 1077. Cki?a. Chim.
Acta,
30 (1970) 377-386
CLINICA CHIMICA ACT,4
KINETIC ACTIVITY
MEASUREMENT
OF SERUM GLUTAMATE
IN THE PRESENCE
DEHYDROGENASE
OF OXAMIC ACID AND L-LEUCINE
SUMMARY
During preincubation, NADH concentration will diminish in a number of cases below the range necessary for optimal serum glutamate dehydrogenase activity. The present paper describes experiments to overcome this problem either by inhibiting lactate dehydrogenase or by stabilizing serum glutamate dehydrogenase against NADH promoted inactivation.
INTRODUCTION
Elevations of serum glutamic acid dehydrogenase (GLDH) can be found in cases of liver diseases. We were especially interested in the diagnostic significance, of GLDH activities in serums of patients with metastatic liver. For that purpose it seemed interesting to make a comparison of GLDH activity and the activity of serum 5nucleotidase (5-Nu), known to be a sensitive monitor of liver metastasesl. In Fig. I the GLDH level of each patient is plotted against the 5-Nu value. The GLDH activities were estimated according to the method of Schmidt and Schmidt, normal range to 0.9 U/l (ref. z) ; the 5-Nu activities in accordance with the method of Persijn et d3, normal range to 10.5 U/l (ref. 4). In this method phenylphosphate is used to inhibit the nucleotidase effect of alkaline bone phosphatasc&. From the data of Fig. I it seemed justifiable to study the behaviour of these enzymes in a series of cases to obtain a clearer picture of the diagnostic significance of GLDH. Before this could be realised, however, several problems were encountered. In the Schmidt and Schmidt method, GLDH activity is measured by the decrease of absorbance at 340 nm caused by the oxidation of NADH according to NADH+ol-ketoglutaric
acid+NH,+
+ NAD++r,-glutamic
acid+H,O
The reaction is initiated by the addition of E-ketoglutaric acid to the assay mixture + Head Dr. J.-P. Per@. *+ Hea.d Drs. W. van der Slik.
Cl&. Chim. Acta, 39
(1930)
377-386.
PERSIJN
et ai.
39.0
at
22
I
20
i8 16
GLCi-I (U/l) .
I
14 12 70 8 6 4 2
0.1
0.2
0.3
0.4
0.5
0.6
NADH [m MOL/L] Fig. I. Comparison Fig. 2. Effect Schmidt5.
of serum 5-Nu activities
of NADH concentration
and serum GLDH
on GLDH
activity,
activities.
measured
according
to the method
of
after a preincubation time of 15 min. During preincubation NADH concentration diminishes as a consequence of short-lasting side reactions to be attributed to the presence of pyruvate and lactate dehydrogenase (LDH) in serum. There is unfortunately a very narrow range of NADH concentration for optimal activity; passing to either side of this range results in rapid decrease of GLDH activity (Fig. 2). Elevation of NADH concentration to compensate for loss of NADH during preincubation is not advisable, particularly since the diminution during preincubation is variable to high degree. In several cases we found that diminution of NADH concentration during preincubation was exceeding the range for optimal GLDH activity. This necessitated measurement of NADH diminution during preincubation in every assay and, if necessary, addition of a second quantity of NADH afterwards for adjustment to optimal concentration as has been already pointed out by Schrnidtb. This was felt to be a serious disadvantage for routine measurement of serum GLDH. In this paper are described several approaches to a solution for this problem by either (a) eliminating side reactions during the preincubation, by adding an agent inhibiting LDH but not GLDH, or (b) by broadening the optimal NADH range in order to apply higher NADH concentration without inactivating GLDH activity. In connexion with (a), we were interested in a report by Novoa and Schwert”. These authors observed that LDH binds oxamate more strongly than oxalate which inhibits LDH about 75%. Therefore in the present study, an attempt has been made to inhibit side reactions by an appropriate concentration of oxamate. It has been shown that beef liver GLDH can dissociate into subunits possessing less or no enzymatic activity. This dissociation of beef liver GLDH is promoted by higher concentrations of NADH’. Yielding and Tomkinss have found evidence that essential amino acids favour CEiw. Cl&z. Acta, 30 (1970) 377-386
SERUM
GLUTAMATE
DEHYDROGENASE
379
ACTIVITY
the reassociation of inactive subunits of bovine GLDH and, therefore, neutralize the action of NADH. According to Hershko and Kindle+‘, L-leucine has the greatest effect in this respect. Substances like L-leucine, that change catalytic properties while altering the configuration, are called “allosteric modifiers”lO. It seemed interesting to investigate whether L-leucine is an allosteric reagent to human serum GLDH, and if that is the case, whether L-leucine could thus achieve a broadening of the NADH optimal range. A second problem is the question of whether serum GLDH is partially inactivated during preincubation. This is not a groundless question since mammalian GLDH has been reported to be unstable in various medialI. The present paper describes experiments which allow us to make a few observations about this problem. METHODS
A brief description of the method will be given, as it has been carried out in our laboratory5. I. Trietkanolanrine-EDTA buffeer: 0.004 RI EDTA in 0.03 M triethanolamineHCl buffer, pH 8.0. 2. Ammoniuvn acetate solution: 3.2 M ammonium acetate in bidistilled water. 3. NADH solution: 0.012 M NADH in the above buffer. 4. a-Ketoglutarate solution: 0.41 M a-ketoglutaric acid in 0.82 N NaOH. A reaction mixture is prepared by combining 2.0 ml of Solution I, 1.0 ml of serum, 0.1 ml of Solution 2, and 0.05 ml of NADH solution. The mixture is kept for 15 min at 25” in a I.o-cm cuvette. During that time the absorbance at 340 nm is recorded. Then 0.05 ml of Solution 4 is added and incubation is continued for IO min, during which the decrease in absorbance is recorded. GLDH activity is calculated from the latter part of the rate of decrease of the absorbance over IO min to express the activity in International Units/l of serum. When variations of the NADH, a-ketoglutarate concentration or temperature etc. in the Schmidt method were used the details are described under RESULTS. RESULTS
LDH activity in the presence of oxamic acid was studied by following the decrease in absorbance at 340 nm in a 3.15-ml mixture containing 0.14 mM NADH, 0.30 mM sodium pyruvate and varying amounts of oxamic acid in the presence of either 0.031 M triethanolamine buffer (pH 8.0) (according to Schmidt) or 0.031 M phosphate buffer (pH 7.5). Fig. 3 illustrates the rapid loss of LDH activity in triethanolamine-HCl buffer with increasing concentration of oxamate. The curve was identical with the phosphate buffer. It will be seen that the LDH activity did not cease completely at high concentrations of oxamate but reached a limiting value which was not zero. This was observed with several serums with varying LDH activities. In the next experiment, oxamate in a final concentration of 3.6 mM was added to the assay by the method of Schmidt. During preincubation the absorbance reading showed different behaviour compared with the Schmidt method in two respects (Fig. 4) : the rate of decrease in absorbance was (a) considerably lower compared with the rate of decrease in the absence of oxamate and (b) approximately linear with time Cl&. Chim.
Acta,
30 (1970) 377-386
380
PERSIJN et al.
% LDH 100
1 60
I
40
25
\
j 20
i
%.
*-a-*
.
1
I
2
1
3
oxamate
Fig. 3. Effect of oxamate
4
[m Mot/ 11
concentration
I2
time
tn minutes
on serum LDH activity.
Fig. 4. Variation, with time, of absorbance at 340 nm of two samples during preincubation. The range o-r.0 of absorbance has been expressed as IOO scale units. The starting point of each curve (arrow) is calculated by extrapolation. e, with 3.7 mM oxamate. GLDH
I
GLDH
(U/L)
(U/l)
‘4 1
I
i
8-3
12 i
‘,
?’
‘4
2 Oi
012
a3
0.4
0.5
0.6
t .
*I
4 6
Ic
1
j
\
./
. \
I
NADH [m MOL/ I] Fig. 5. Effect of NADH concentration on GLDH activity. Curve I, with o.or M L-leucine, 8.5 mM ar-ketoglutarate at pH Curve 4, idem without L-leucine according to Schmidt; Curve 2, with 0.01 M L-leucine, 6.1 mM cc-ketoglutarate at pH Curve 3. with 0.01. M L-leucine, 6.1 mM cc-ketoglutarate at pH Curve 5, idem without L-leucine according to Schmidt. Curves I, 2, and 3 refer to different serum samples. Fig. 6. pH-activity curve for serum GLDH in the presence NADH added.
7.0
6.0
9.0
. t PH
7.8; 8.0; 8.0;
of
0.01
M
L-leucine
and 0.4 mM
(up to at least 25 min). Identical results were obtained with 5.6 m&I oxamate. Since residual LDH activity remains for a longer time compared with uninhibited LDH, the decrease in absorbance as a result of reduction of endogeneous pyruvate is, therefore, superimposed on the decrease in absorbance following the addition of a-ketoglutarate. C&z. Chinz. Acta, 30 (1970) 377-386
SERUM
GLUTAMATE
DEHYDROGENASE
381
ACTIVITY
Two pooled serums were assayed for GLDH activity with (0.0037 M) and without oxamate. In the former assay, correction was applied for residual LDH activity. The results-13.0 and 8.0 U/l without oxamate, 1z.5 and 8.0 U/l with oxamate-indicate that oxamate does not inhibit serum GLDH. Fig. 5 shows the effect of increasing concentration of NADH on the GLDH activity of three different serums measured in the presence of 0.01 M L-leucine (Curves I, 2 and 3). In these and subsequent experiments L-leucine was added as a component of the triethanolamine-EDTA buffer. For comparison, the course of GLDH activity of two serums when L-leucine is omitted is illustrated in Curves 4 and 5. This figure shows that at a NADH concentration between approximately 0.4 and 0.3 mM, the GLDH activity is independent of the NADH concentration. A second advantage of the use of L-leucine is the z-fold activation of the GLDH activity compared with the Schmidt method. Keeping final NADH concentration in the test mixture at 0.4 mM, the pH-activity relationship in the presence of 0.01 M L-leucine was investigated (Fig. 6). Variation of GLDH activity with substrate concentration in the presence of 0.01 M L-leucine at pH 7.8 was also studied. The results, shown in Fig. 7, indicate that maximal and constant activity was obtained when the reaction mixture contained more than 5 mM a-ketoglutarate. The rate of decrease in absorbance a.s a measure of L-leucine-activated serum GLDH is proportional to the amount of serum till about 40 U/l (Fig. 8). Table I presents data of eight experiments on the activity of L-leucine-activated serum GLDH after different times of preincubation. The experiments were performed both with and without oxamate. A preincubation time of zero minutes as indicated in Table I means that initial readings b/l]
GLDH GLDH
(U/l)
60
32 5
28
q
m-m-m-m
60
24
20 .
/ 40
16
w----o-o
---__o----0
12
8
i
/
/ 2
Fig.
b
20
0
4
1
7. GLDH
4 6 8 o:-Ketoglutarate activity
c
r
10 12 [m Mol/Lj
as a function
200
L-leucine at pH 7.8 with 0.4 mM NADH
400
600
600
1000
in the presence added. o = serum I; q = serum 2.
of cr-ketoglutarate
concentration
pL serum
of 0.01 M
Fig. 8. Relation of enzyme concentration to GLDH activity as measured in the presence of 0.01 M L-leucine and 0.4 mM NADH added. Sample a: with 6.2 mM c+Ketoglutarate at pH 8.0; Samples b and c with 8.5 mM cc-ketoglutarate at pH 7.8. Clin. Chim. Acta,
30 (1970)
377-386
382 TABLE
PERSIJN et &. T
STABILIZY
OF
SERUM
GLDH
DURING
PREINCU:BATION
IN
THE
PRESENCT3
OF
L-LEUCINE
(u/l)
Experiments with Samples I, 2, 3, 5, and 8 were done with 8.6 mX cc-Ketoglutarate at pH 7.8. The others with concentrations of ct-ketoglutarate and pH according to the method of Schmidt. Saqble NO.
I 2
Preincubation tinae (w&z)
0 15
3.7 3.7 4.5 4.8
0
15 3
0
26.2
26.8
15
4
0
5.X
5-I
I.5
5*
6
0
5.2
15
0.0037
M
Without
oxarnate 3.9 5.2 -
26.6 -
-
5-I
5.3
0 I5
4.8 4.4 4.3 4.1 3.7
5.0 -
20
3.8
-
8.6
8.7
0
15 20
7
With
oxanzatc:
8
-
0
I5
-
3.9 -
* Measured at 362 nm.
were taken within xo set after the addition of serum to the otherwise complete reaction mixture. In the other experiments, the reaction was started in the usual way, by the addition of ketoglutarate to the assay mixture after the time indicated. The measured rates of decrease of absorbance of the complete assay mixture in the presence of oxamate were corrected for rates of decrease caused by residual LDH activity to obtain real GLDH activities. In all. cases, the decrease of absorbance was linear with time for more than IO min. Results of similar experiments with oxamate, using the Schmidt procedure, are outlined in Table II. With Samples 4 and sz the following additional experiments were performed: After 8 min, measurement of GLDH activity was interrupted for I min to add L-leucine and XADH at final concentrations of 0.01 M and 0.4 mM, respectively. Thorough mixing after each of these additions was performed. The registration of absorbance was continued after this break which was kept as short as possible (approx. I min). Samples 4 and 5 showed immediately enhanced activity (Table II). Using the Schmidt method the mean ratio of activity at 35” and 25” was found to be 1.16, but several. samples of serums showed a temperature coefficient (Q1,Jof 1.0. The highest value of Qlo observed was 1.26. L-Leucine had an enhancing effect on the value of Qlo. In Fig. 9 the natural logarithms of the difference in absorbance over IO min are plotted against the inverses of the corresponding absolute temperatures over the range ~5-37~. There is a linear relationship, but the slopes are different. Curves a and b represent a serum measured within 6 and a4 h, respectively, after the clotting of the blood from which it was taken, Curve c a serum which had been stored for 4 days at 4O. Change of activity in the presence of L-leucine after storage was studied in the case of 27 samples. No difference was found between samples stored at 4O or at -20c. During storage at these temperatures, activity fell slowly. After 4 days the highest loss of activity was found to be IO%, after 7 days, 20%. Clin. Chiun.
Acta,
30 (1970) 377-386
SERUM GLUTAMATE TABLE
DEHYDROGENASE
ACTIVITY
383
II
STABILITY SCHMIDT
OF
GLDH
DURING
PREINCUBATION
IN
THE
MEDIUZvI
ACCORDING
TO
THE
METIXOD
OF
(u/l)
Pmincubation time (mix)
SLWW$le NO. I
9.5 9.8
0
I5 28
With 0.0037 M oxawtate
0
7.5
I5
3b
0
::I:
x5
4
6.5
0
5
?..PC
1.5
2.8d
0
8.Oe
I.5
8.@
Separate assay with L-leucine and 0.4 mM NADH: 8 17.0 U/l; b 15.5 U/l. After addition of L-leucine and NADH: C 5.8 U/l; d 5.3 U/l; e 20.0 U/l; f 20.3 U/l.
Ln
fAAx103f
52 -
5,o
I
3.2
Fig. g. Dependence
3.3
of GLDH
3.6
activity,
in the presence of 0.01 M r.-leucine upon temperature
for the range ~5-37~.plotted according to Arrhenius.On the ordinate: dJf = decreaseof absorbanceat 340 nm over IO min. DISCUSSION
The fact that oxamate reduces NADH consumption dnring preincubatioll to a large extent (Fig. 4) agrees with the findings presented in Fig. 3 and fits in with the theory of Karmen12 that this NADH consumption is caused by LDH activity of serum. Since in the presence of this inhibitor for LDH some residual LDH activity persists even after preincubation (Fig. 4), every assay of serum GLDH had to be corrected for this residual activity in a separate experiment. Addition of oxamate in Clilz.C&n, Acta, 30
(1970)
377-386
384
PERSIJN
et cd.
the assay seems therefore not adequate for the solution of the problem mentioned in IPU‘TRODUCTION.
The use of L-leucine bears more interesting features: GLDH activity in the presence of L-leucine is independent of NADH concentration over a broad range. It is not only NADH that has a biphasic action on_ GLDH activity. According to Schmidt5 the concentration of a-ketoglutarate is very critical. Optimal activity is obtained in the Schmidt medium with a cc-ketoglutarate concentration of 6.2 mM. Enhancement of the a-ketoglutarate concentration leads to rapid inactivation of the enzyme. This phenomenon could be interpreted in terms of substrate inhibition in the usual sense. The fact, however, that L-leucine prevents any inactivation at higher concentrations of cc-ketoglutarate (Fig, 7) strongly indicates that increasing concentration of a-ketoglutarate favours the inactivation of serum GLDH. Apparently r,-leucine is not only able to overcome the dissociation-promoting effect of NADH, but of cr-ketoglutarate as well. We judge the L-leucine-promoted independence of GLDH on cc-ketoglutarate concentration in the range of 5-12 mM, as an advantage if automatic pipetting devices are used in the GLDH assay. At a concentration of 8.5 mM, for example, occasional deviations in dispensed volume of as much as about 20% can be tolerated for micropipetting devices. It is our experience that such a possible range of variation is valuable. Remarkably, the inactivation of GLDH by a great number of conditions has been overlooked in clinical chemistry. This holds not only for the assay of serum GLDH itself. Bovine GLDH has been used as an aiding enzyme for the determination of NH, (ref. 13). From the work of the various authors cited above, it can be expected that in the conditions used for the dete~ination of NHs, rapid deterioration of the aiding enzyme GLDH may occur during the test. We have considered the possibility of using bovine GLDH in our method for the determination of 5-nucleotidase (5-Nu). In this method adenosine released from AMP by the action of 5-Nu is simultaneously converted by adenosine deaminase to NH, and inosine. The ammonia released is measured calorimetrically at 625 nm by means of the Berthelot reaction 894.Use of GLDH instead of the Berthelot reaction would enable us to estimate serum 5-Nu kinetically at 340 nm, provided that GLDH is prevented from inactivation under conditions optimal for 5-Nu activity. It appears that for this purpose allosteric modifiers protecting GLDH against inactivation will be indispensable. The inactivation of GLDH even in buffers without substances Iike NADH etc.‘l has led nonclinical biochemists to measure initial activities in their investigations of GLDH. Initial rates of decrease of absorbance should therefore be established immediately after the addition of the enzyme to the otherwise complete test medium. In the case of serum GLDH such a procedure would measure the combined action of serum LDH and serum GLDH. Since the action of serum LDH is revealed by a progressively diminishing decrease of absorbance (Fig. 4), initial activity of ,GLDH cannot be measured accurately. The question rises whether serum GLDH is inactivated to some degree during the preincubation. We thought to answer this question by measuring without preincubation the GLDH activity in the presence of oxamate. Under this condition there is a linear course of LDH-associated decrease of absorbance (Fig. 4). This makes it
SERUH GLUTAkIATE DEHYDROGENAS~
385
ACTIVITY
possible to correct accurately the total reaction rate for concomitant LDH activity in order to obtain true GLDH activity. Comparison of the results of these experiments with activities obtainedafter preincubation, with or without oxamate, enables us to answer the above question (Table I). From the findings given in Table I, it can be concluded that inactivation during preincubation does not take place. The data of Table II suggest that the partially inactivated GLDH as it is measured in the Schmidt procedure occurs in that form at the very beginning of the preincubation and remains at least r5 min in that form. In this connexion it seems interesting that addition of L-leucine during the meas~ement of initial rate or after preincubatio~l in the Schmidt medium immediately results in an enhanced rate of decrease in absorbance (see Table II). It seems reasonable to assume that the interaction of L-Ieucine with serum GLDH is instantaneous: there is no measurable lag phase. r,-Leucine does not change the pH-activity relationship to a great degree. Schmidt found optimal activity at pH 7.6-8.4. Fig. 6 illustrates that pH optimum for serum GLDH with L-leucine lies at 7.7-7.9. Comparison with data of bovine GLDH from literature is difficult since most investigations have been carried out at a definite pH and possible shifts in pH optimum by allosteric modifiers have not been taken in consideration. That such a shift is not purely hypothetical is illustrated by the findings of Bitensky et &.I4 who found, at least for the reverse reaction, that the pH optimum in the presence of I mM ADP is shifted from 8.0 to 9.0. For serum GLDH the pH-activity relationship with either z.-leucine or ADP (I mM) is identical in the forward reaction. GLDH activities are generally measured at z5”, as is done in the case of serum GLDH. The present paper is intended to modify the procedure so that a change of the NADH concentration can have no influence at this temperature. Nevertheless it seems of interest to mention briefly experiments on the determination of QIO, For the range 25535”, the value of Qlo for Samples a, b, and c (Fig. g) is 1.61, 1.84, and 1,43, respectively. The former two values of Qlo seem reasonable since Qlo varies from x.75 for aminotransferase to 2.50 for 5-nucleotidase. The behaviour of bovine GLDH at temperatures above 25” has seldom been investigated. Bitensky et al.l” have shown that above 26-27” small increases in temperature cause rapidly an unfolded and denaturated product. Probably the same phenomenon occurs with serum GLDH in the Schmidt medium where values of 1.0-1.25 for Qlo have been found.
Apparently
2537”,
although
I-Ieucine to a variable
is able extent.
to overcome
heat
For the present,
inactivation we would
in the range
propose
changing
as follows: to 2 ml of a solution containing 0.05 M triethanolamine buffer (pH 7.8), 0.004 M EDTA, 0.016 M I-leucine, and 0.18 M ammonium acetate are added 1.0 ml of serum and 0.1 ml of NADH solution (0.013 M). After equilibration at 25O for 15 min, 0.1 ml a-ketoglutarate solution (0.28 M) in 0.56 N NaOH is added. The decrease in absorbance is measured at 340 nm or 360 nm for IO min. the Schmidt
procedure
REFERENCES DER SLIK, J.-P. PERSIJX, E. ENGHLSMAN AND A. RIETHORST,~%Z. Biochem., 3 (1970) 59. E. SCKVIIDT AXD F. 1%'.SCHMIDT, E~~ywml. BioE. C&L, 3 (1963) I. J.-P. PERSIJN, W.VAX DER SLX, K. KRAMERAND C. ADE RUYTER,~. Kli~.Chenz. Biockenz., 6 (1968) 441.
W.VAN
Clin. Ckim. Acta. 30 (1970) 377-386
PERSIJN t?f d.
386
4 J.-P. PERSIJN, W. VAN DER SLIS ~SIJ A. W. 31. Box, 2. K&z. Chem. Biockem., 7 (1969) 493. 5 E. SCHMIDT, in H.-U. BERGXEYER (Ed.},~~I~th~~~n detere~~y~~~sc~e~z .ilndyse, Verlag Chemie, Weinheim, x962, p. 752. 6 TV. B. NOVOA AND B. W. SCHWERT, J. Biol. Chem., 236 (1961) 2150. 7 C. FKXEDEN, J. Biol. Che?%, 234 (1959) 809. 8 K. L. YIELDING AND G. M. TOMKINS. Proc. N&l. Acad. Sci. U.S., 47 (1961) 983. g A. KERSHKO AND S. H. KINDLER, Biochwc J., IOT (x966) 661. IO J. M~NOW, J. CHANGEUX AND E. JACOB, J. Mol.Bzal., 6 (1~63) 306. II G. DI PRXSCOAND H. J. STRECKER, Riochi~~.Biafihys. Acta, 122 (1966) 413. 12 A. KARMEN,]. CZin.Invest., 34 (1955) 131. 13 M. RUBIN AND L. KNOTT,CZ~~. Chins. Ada, 18 (1967) 409. 14 M. W. BITENSKY, K. L. YIELDING AND G. M. TOMKINS, J.BioZ. Chem., 240(1gh5) 663. 15 M. W. BITENSKY, K. L. YIELDING AND G. M. TOMKINS, J. BioE. Chena., 240 (1965) 1077. Cki?a. Chim.
Acta,
30 (1970) 377-386